Cristina BENATTI - personale UniMoRe

Nuova ricerca

Cristina BENATTI

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche
Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

Home | Curriculum(pdf) | Didattica |

Pubblicazioni

2024 - Low TGF-β1 plasma levels are associated with cognitive decline in Down syndrome [Articolo su rivista]
Grasso, Margherita; Fidilio, Annamaria; L’Episcopo, Francesca; Recupero, Marilena; Barone, Concetta; Bacalini, Maria Giulia; Benatti, Cristina; Giambirtone, Maria Concetta; Caruso, Giuseppe; Greco, Donatella; Di Nuovo, Santo; Romano, Corrado; Ferri, Raffaele; Buono, Serafino; Cuello, A. Claudio; Blom, Johanna Maria Catharina; Tascedda, Fabio; Piazza, Pier Vincenzo; De La Torre, Rafael; Caraci, Filippo
abstract

2024 - Snails go on a fast when acetylsalicylic acid comes along with heat stress: A possible effect of HSPs and serotonergic system on the feeding response [Articolo su rivista]
Batabyal, A.; Rivi, V.; Benatti, C.; Blom, J. M. C.; Tascedda, F.; Lukowiak, K.
abstract

: A novel food followed by sickness, causes a taste-specific conditioned aversion, known as the 'Garcia effect'. We recently found that both a heat shock stressor (30 °C for 1 h - HS) and the bacterial lipopolysaccharide (LPS) can be used as 'sickness-inducing' stimuli to induce a Garcia effect in the pond snail Lymnaea stagnalis. Additionally, if snails are exposed to acetylsalicylic acid (ASA) present in aspirin tablets before the LPS injection, the formation of the Garcia effect is prevented. Here, we hypothesized that exposing snails to crushed aspirin before the HS (ASA-HS) would prevent the HS-induced 'sickness state' and - therefore -the Garcia effect. Unexpectantly, the ASA-HS procedure induced a generalized and long-lasting feeding suppression. We thus investigate the molecular effects underlying this phenomenon. While the exposure to the HS alone resulted in a significant upregulation of the mRNA levels of the Heat Shock Protein 70 (HSP 70) in snails' central ring ganglia, the ASA-HS procedure induced an even greater upregulation of HSP70, suggesting that the ASA-HS combination causes a severe stress response that inhibits feeding. Additionally, we found that the ASA-HS procedure induced a significant downregulation of the mRNA levels of genes involved with the serotoninergic system which regulates feeding in snails. Finally, the ASA-HS procedure prevented HS-induced upregulation of the mRNA levels of key neuroplasticity genes. Our study indicates that two sickness-inducing stimuli can have different physiological responses even if behavioral outcomes are similar under some learning contexts.

2023 - A Novel Behavioral Display in Lymnaea Induced by Quercetin and Hypoxia [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Tascedda, F.; Blom, J. M. C.; Lukowiak, K.
abstract

The pond snail Lymnaea stagnalis employs aerial respiration under hypoxia and can be operantly conditioned to reduce this behavior. When applied individually, a heat shock (30 7C for 1 h) and the flavonoid quercetin enhance long-term memory formation for the operant conditioning of aerial respiration. However, when snails are exposed to quercetin before the heat shock, long-term memory is no longer enhanced. This is because quercetin prevents the heat-induced upregulation of heat-shock proteins 70 and 40. When we tested the memory outcome of operant conditioning due to the simultaneous exposure to quercetin and 30 7C, we found that Lymnaea entered a quiescent survival state. The same behavioral response occurred when snails were simultaneously exposed to quercetin and pond water made hypoxic by bubbling nitrogen through it. Thus, in this study, we performed six experiments to propose a physiological explanation for that curious behavioral response. Our results suggest that bubbling nitrogen in pond water, heating pond water to 30 7C, and bubbling nitrogen in 30 7C pond water create a hypoxic environment, to which organisms may respond by upregulating the heat-shock protein system. On the other hand, when snails experience quercetin together with these hypoxic conditions, they can no longer express the physiological stress response evoked by heat or hypoxia. Thus, the quiescent survival state could be an emergency response to survive the hypoxic condition when the heat-shock proteins cannot be activated.

2023 - ADOLESCENCE AS A CRITICAL TIME-WINDOW FOR NEUROINFLAMMATION IN THE MOUSE: WHY SEX MATTERS [Abstract in Rivista]
Toscano, Ylenia; Benatti, Cristina; Alboni, Silvia; Ciani, Miriam; Rigillo, Giovanna; Tascedda, Fabio; Blom, Johanna Maria Catharina; Brunello, Nicoletta
abstract

2023 - Behavioral and Transcriptional Effects of Short or Prolonged Fasting on the Memory Performances of Lymnaea stagnalis [Articolo su rivista]
Rivi, Veronica; Benatti, Cristina; Actis, Pietro; Tascedda, Fabio; Blom, Johanna Maria Catharina
abstract

Introduction: The Garcia effect, a solid learning paradigm, was used to investigate the molecular and behavioral effects induced by different lengths of fasting on the cognitive functions in the pond snail Lymnaea stagnalis, a valid model systemMethods: Three experimental groups were used: Moderately hungry snails, food-deprived for 1 day (D1 snails), severely hungry snails (D5 snails), fasting for 5 days, and satiated snails with ad libitum access to food (AL snails). In the Garcia effect, a single pairing of an appetitive stimulus with a heat stressor results in a learned taste-specific negative hedonic shift. D5 snails were injected with bovine insulin and D1 snails with the insulin receptor antibody (Ab). As a control group, AL snails were injected with saline. Gene expression analyses were performed by Real-time PCR in snails' central nervous system (CNS).Results: AL snails are 'average learners', D1 snails are the best performers, whereas the D5 ones do not show the Garcia effect. Severely fasting snails injected with insulin 3h before the training procedure, show the Garcia effect, whereas injecting 1-day fasting snails with insulin receptor Ab blocks their ability to express memory. The differences in memory performances are associated with changes in the expression levels of selected targets involved in neuronal plasticity, energy homeostasis, and stress response.Discussion: Our results suggest that short-term fasting creates an optimal internal state in L. stagnalis' CNS, allowing a spike in insulin release and an upregulation of genes involved in neuroplasticity. Long-term fasting, instead, upregulates genes involved in energy homeostasis and animal survival.

2023 - Comparison of behavioural and transcriptional responses to a heat stressor between freshly collected and an inbred strain of Lymnaea [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Tascedda, F.; Blom, J. M. C.; Lukowiak, K.
abstract

Different populations of organisms occurring across varying thermal regimes show diversity in responses to heat stress. We use a "common garden experimental" approach designed to deal with phenotypic plasticity to study in Lymnaea stagnalis (Linnaeus, 1758) the behavioural and molecular responses to a heat shock in laboratory-inbred snails (W-strain) and freshly collected snails (Stony strain) from ponds. In the W-strain, which has been reared under standardized temperatures for generations, the exposure to 30 degrees C for 1 h (heat shock, HS) when experienced after a novel "taste" results in a taste-specific aversion known as the "Garcia effect". This learned avoidance requires the upregulation of heat shock proteins (HSPs). In contrast, freshly collected Stony strain, which experiences temperature fluctuations regularly, does not exhibit a Garcia effect. Here, we found that (1) Stony-strain snails have higher basal mRNA levels of HSPs than W-strain ones; (2) in the W-strain, the training procedure to cause the Garcia effect upregulates the mRNA levels of HSPs and key neuroplasticity-related genes such as CREB1 and GRIN1; (3) in Stony-strain snails, the same training procedure fails to alter the mRNA levels of those targets. These data suggest that Stony-strain snails do not perceive the HS as a stressor because of the higher HSP basal mRNA levels, which may confer a higher thermal tolerance.

2023 - Dysbindin, D3 receptors, and their genetic interaction differently regulate neuroplasticity genes in the mouse brain [Abstract in Atti di Convegno]
Rivi, V.; Benatti, C.; Blom, J. M. C.; Pani, L.; Brunello, N.; Drago, F.; Papaleo, F.; Torrisi, S.; Leggio, G.; Tascedda, F.
abstract

2023 - Environmental enrichment influences mouse hippocampal neuroinflammatory response [Abstract in Rivista]
Rigillo, Giovanna; Benatti, Cristina; Toscano, Ylenia; Tascedda, Fabio; Pani, Luca; Brunello, Nicoletta; Alboni, Silvia
abstract

Background: Many studies have shown the positive effects of environmental enrichment on brain plasticity with significant implications for development, behavior, learning, memory, and recovery from brain damage (1). Experimental and clinical studies report that one’s living environment can modulate cellular and molecular responses in the brain, counteracting cognitive decline, alleviating anxiety, and depressive behaviours, as well as moderating the outcome of pharmacological treatments (2). Neuroinflammation has been well established as an important factor in the aetiopathogenesis and progression of brain disorders. It can affect neural development and alters hippocampal plasticity thus resulting in cognitive impairments. Neuroinflammation is characterized by a dysregulation of the NLRP3 inflammasome activation, an increase in the expression of inflammatory cytokines, and a decrease of neurotrophic factors (3). Behavioral and neurochemical changes, caused by neuroinflammation, have been most frequently investigated through peripheral administration of lipopolysaccharide (LPS), which can, directly and indirectly, affect the central nervous system (4). Based on these premises, the aim of this study was to explore the molecular effects of the quality of the living environment in modulating the LPS-induced neuroinflammatory response in the hippocampus of wild-type mice. Methods: Male C57BL6J mice (13 weeks-old) were randomly housed in Impoverished (IE) or Enriched Environment (EE) condition for 28 days, then exposed to LPS (0.830 mg/Kg, i.p.) or saline (SAL). Twenty-four hours after injection hippocampi were removed for gene expression analysis performed by means of qRT-PCR. Data from groups were analyzed by Two-way ANOVA followed by Tukey’s post hoc test. Results: The analysis of the environmental effects on the LPS signaling system highlighted the downregulation of the membrane-bound protein LBP, the receptor TLR4 and the co-receptor cluster CD14 expression levels in EE-housed animals compared to their counterparts. The exposure to an EE condition was able to attenuate the LPS-induced increase of TLR4 and NLRP3 inflammasome mRNA levels. Gene expression analysis revealed a significant downregulation of the pro-inflammatory cytokines IL-1β and TNFα levels in EE-housed mice while LPS exposure strongly increased IL-1β and TNFα mRNA levels irrespective of the housing conditions. Moreover, EE-exposed mice showed a significant upregulation of BDNF hippocampal mRNA levels, although no effects were observed after LPS treatment in both conditions. Conclusions: Our results displayed the beneficial effect of EE in regulating the expression of inflammatory mediators involved in the LPS-induced response in the hippocampus, a key area for learning, memory, and emotion. These data suggest that living environment may exert a positive and protective role on the brain by reducing susceptibility toward neurodegenerative or neuropsychiatric disorders.

2023 - Farmaci antidepressivi parte II: SNRI e farmaci multimodali [Capitolo/Saggio]
Benatti, Cristina; Alboni, Silvia; Brunello, Nicoletta; Pani, Luca; Tascedda, Fabio
abstract

2023 - Invertebrates as models of learning and memory: investigating neural and molecular mechanisms [Articolo su rivista]
Rivi, Veronica; Benatti, Cristina; Rigillo, Giovanna; Blom, Johanna M C
abstract

: In this Commentary, we shed light on the use of invertebrates as model organisms for understanding the causal and conserved mechanisms of learning and memory. We provide a condensed chronicle of the contribution offered by mollusks to the studies on how and where the nervous system encodes and stores memory and describe the rich cognitive capabilities of some insect species, including attention and concept learning. We also discuss the use of planarians for investigating the dynamics of memory during brain regeneration and highlight the role of stressful stimuli in forming memories. Furthermore, we focus on the increasing evidence that invertebrates display some forms of emotions, which provides new opportunities for unveiling the neural and molecular mechanisms underlying the complex interaction between stress, emotions and cognition. In doing so, we highlight experimental challenges and suggest future directions that we expect the field to take in the coming years, particularly regarding what we, as humans, need to know for preventing and/or delaying memory loss. This article has an associated ECR Spotlight interview with Veronica Rivi.

2023 - Investigating the interactions between multiple memory stores in the pond snail Lymnaea stagnalis [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Blom, J. M. C.; Tascedda, F.; Lukowiak, K.
abstract

The pond snail Lymnaea stagnalis exhibits various forms of associative learning including (1) operant conditioning of aerial respiration where snails are trained not to open their pneumostome in a hypoxic pond water environment using a weak tactile stimulus to their pneumostome as they attempt to open it; and (2) a 24 h-lasting taste-specific learned avoidance known as the Garcia effect utilizing a lipopolysaccharide (LPS) injection just after snails eat a novel food substance (carrot). Typically, lab-inbred snails require two 0.5 h training sessions to form long-term memory (LTM) for operant conditioning of aerial respiration. However, some stressors (e.g., heat shock or predator scent) act as memory enhancers and thus a single 0.5 h training session is sufficient to enhance LTM formation lasting at least 24 h. Here, we found that snails forming a food-aversion LTM following Garcia-effect training exhibited enhanced LTM following operant condition of aerial respiration if trained in the presence of the food substance (carrot) they became averse to. Control experiments led us to conclude that carrot becomes a ‘sickness’ risk signal and acts as a stressor, sufficient to enhance LTM formation for another conditioning procedure.

2023 - LPS-Induced Garcia Effect and Its Pharmacological Regulation Mediated by Acetylsalicylic Acid: Behavioral and Transcriptional Evidence [Articolo su rivista]
Rivi, V.; Batabyal, A.; Lukowiak, K.; Benatti, C.; Rigillo, G.; Tascedda, F.; Blom, J. M. C.
abstract

Lymnaea stagnalis learns and remembers to avoid certain foods when their ingestion is followed by sickness. This rapid, taste-specific, and long-lasting aversion—known as the Garcia effect—can be formed by exposing snails to a novel taste and 1 h later injecting them with lipopolysaccharide (LPS). However, the exposure of snails to acetylsalicylic acid (ASA) for 1 h before the LPS injection, prevents both the LPS-induced sickness state and the Garcia effect. Here, we investigated novel aspects of this unique form of conditioned taste aversion and its pharmacological regulation. We first explored the transcriptional effects in the snails’ central nervous system induced by the injection with LPS (25 mg), the exposure to ASA (900 nM), as well as their combined presentation in untrained snails. Then, we investigated the behavioral and molecular mechanisms underlying the LPS-induced Garcia effect and its pharmacological regulation by ASA. LPS injection, both alone and during the Garcia effect procedure, upregulated the expression levels of immune- and stress-related targets. This upregulation was prevented by pre-exposure to ASA. While LPS alone did not affect the expression levels of neuroplasticity genes, its combination with the conditioning procedure resulted in their significant upregulation and memory formation for the Garcia effect.

2023 - Narrative Review of the Complex Interaction between Pain and Trauma in Children: A Focus on Biological Memory, Preclinical Data, and Epigenetic Processes [Articolo su rivista]
Rivi, V.; Rigillo, G.; Toscano, Y.; Benatti, C.; Blom, J. M. C.
abstract

The incidence and collective impact of early adverse experiences, trauma, and pain continue to increase. This underscores the urgent need for translational efforts between clinical and preclinical research to better understand the underlying mechanisms and develop effective therapeutic approaches. As our understanding of these issues improves from studies in children and adolescents, we can create more precise preclinical models and ultimately translate our findings back to clinical practice. A multidisciplinary approach is essential for addressing the complex and wide-ranging effects of these experiences on individuals and society. This narrative review aims to (1) define pain and trauma experiences in childhood and adolescents, (2) discuss the relationship between pain and trauma, (3) consider the role of biological memory, (4) decipher the relationship between pain and trauma using preclinical data, and (5) examine the role of the environment by introducing the importance of epigenetic processes. The ultimate scope is to better understand the wide-ranging effects of trauma, abuse, and chronic pain on children and adolescents, how they occur, and how to prevent or mitigate their effects and develop effective treatment strategies that address both the underlying causes and the associated physiological and psychological effects.

2023 - No food for thought: An intermediate level of food deprivation enhances memory in Lymnaea [Articolo su rivista]
Kagan, Diana; Rivi, Veronica; Benatti, Cristina; Tascedda, Fabio; Blom, Johanna M C; Lukowiak, Ken
abstract

: Nutritional status plays an important role in cognitive functioning but there is disagreement on the role that food deprivation plays in learning and memory. In this study, we investigated the behavioral and transcriptional effects induced by different lengths of food deprivation: 1 day, which is a short-time period of food deprivation, and 3 days, which is an 'intermediate' level of food deprivation. Snails were subjected to different feeding regimens and then trained for operant conditioning of aerial respiration where they received a single 0.5h training session followed by a long-term memory (LTM) test 24h later. Immediately after the memory test, snails were sacrificed and the expression levels of key genes for neuroplasticity, energy balance, and stress response were measured in the central ring ganglia. We found that 1 day of food deprivation was not sufficient to enhance snails' LTM formation and subsequently did not result in any significant transcriptional effects. However, 3 days of food deprivation resulted in enhanced LTM formation and caused the upregulation of neuroplasticity and stress-related genes and the downregulation of serotonin-related genes. These data provide further insight into how nutritional status and related molecular mechanisms impact cognitive function.

2023 - Novel taste, sickness, and memory: Lipopolysaccharide to induce a Garcia-like effect in inbred and wild strains of Lymnaea stagnalis [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Blom, J. M.; Tascedda, F.; Lukowiak, K.
abstract

Food is not only necessary for our survival but also elicits pleasure. However, when a novel food is followed sometime later by nausea or sickness animals form a long-lasting association to avoid that food. This phenomenon is called the ‘Garcia effect’. We hypothesized that lipopolysaccharide (LPS) could be used as the sickness-inducing stimulus to produce a Garcia-like effect in inbred and wild populations of Lymnaea stagnalis. We first demonstrated that the injection of 25 μg (6.25 µg/mL) of Escherichia coli-derived LPS serotype O127:B8 did not by itself alter snails’ feeding behavior. Then we showed that the presentation of a novel appetitive stimulus (i.e., carrot slurry) and LPS resulted in a taste-specific and long-lasting feeding suppression (i.e., the Garcia-like effect). We also found strain-specific variations in the duration of the long-term memory (LTM). That is, while the LTM for the Garcia-like effect in W-strain snails persisted for 24h, LTM persisted for 48h in freshly collected Margo snails and their F1 offspring. Finally, we demonstrated that the exposure to a non-steroidal anti-inflammatory drug, aspirin (acetylsalicylic acid) before the LPS injection prevented both the LPS-induced sickness state and the Garcia-like effect from occurring. The results of this study may pave the way for new research that aims at (1) uncovering the conserved molecular mechanisms underlying the Garcia-like effect, (2) understanding how cognitive traits vary within and between species, and (3) creating a holistic picture of the complex dialogue between the immune and central nervous systems.

2023 - Prey populations with different predation histories show differences in behavioral and transcriptional effects under acute predation threat [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Tascedda, F.; Blom, J. M. C.; Lukowiak, K.
abstract

Predator detection induces both behavioral and physiological responses in prey organisms. Our model organism, the pond snail Lymnaea stagnalis, shows multiple defensive behaviors in response to predator cues. In this study, we investigated and compared the transcriptional effects induced by the exposure to a predator scent (i.e., crayfish effluent - CE) in a strain of lab-inbred snails (i.e., W snails), which have been raised and maintained under standardized laboratory conditions for generations and a strain of freshly collected snails (i.e., Margo snails), which live in a crayfish-free pond. Neither the W- strain nor the Margo Lake snails used in this study have actually experienced crayfish. However, the W strain innately recognizes crayfish as a threat. We found that, following the exposure to CE, both strains showed significantly higher mRNA levels of serotonin-related genes. This is important, as the serotonergic system modulates predator detection and vigilance behaviors in pond snails. However, the expression levels of CREB1 and HSP70 were only upregulated in CE-exposed W snails but not in Margo ones. As CREB1 plays a key role in learning and memory formation, whereas HSP70 is involved in stress response, we investigated whether these differences in CREB1 and HSP70 mRNA levels would reflect differences in predator-induced learning (e.g., configural learning). We found that only W snails formed configural learning memory, whereas Margo snails did not. Thus, while both the strains molecularly respond to the CE by upregulating the serotoninergic system, only W snails behaviorally recognize CE as a threat and, therefore, form configural learning.

2023 - Role of dopamine D3 receptors, dysbindin, and their functional interaction on the expression of key genes for neuroplasticity in the mouse brain [Abstract in Rivista]
Rivi, Veronica; Benatti, Cristina; Blom, Johanna; Pani, Luca; Brunello, Nicoletta; Drago, Filippo; Papaleo, Francesco; Torrisi, Sebastiano; Salomone, Salvatore; Leggio Gian, Marco; Tascedda, Fabio
abstract

2023 - The Role of Dopamine D3 Receptors, Dysbindin, and Their Functional Interaction in the Expression of Key Genes for Neuroplasticity and Neuroinflammation in the Mouse Brain [Articolo su rivista]
Rivi, Veronica; Benatti, Cristina; Blom, Johanna; Pani, Luca; Brunello, Nicoletta; Drago, Filippo; Papaleo, Francesco; Caraci, Filippo; Geraci, Federica; Torrisi, Sebastiano Alfio; Leggio, Gian Marco; Tascedda, Fabio
abstract

2023 - VORTIOXETINE ATTENUATES NEUROINFLAMMATION BY MODULATING THE NOD-LIKE RECEPTOR FAMILY PYRIN DOMAIN CONTAINING 3 INFLAMMASOME ACTIVATION IN MICROGLIA: IMPLICATIONS FOR COGNITIVE FUNCTION [Abstract in Rivista]
Rigillo, G.; Ciani, M.; Benatti, C.; Blom, J. M. C.; Tascedda, F.; Pani, L.; Alboni, S.; Brunello, N.
abstract

Vortioxetine (VTX) is a multimodal antidepressant with an extensive pharmacological profile that includes modulation of various neurotransmitter systems, neuroprotective activity, and beneficial effects on cognitive functions. Recent research has revealed a novel aspect of VTX's activity - its antiinflammatory effects - that suggests an intriguing molecular mechanism may underpin its therapeutic benefits. Neuroinflammation, dysfunctional neurogenesis and neurotransmission, and dysregulation of the hypothalamus– pituitary–adrenal (HPA) axis are all pivotal in the onset and progression of depression. One particular immune-inflammatory pathway overactivated in brain disorders is the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex. This complex's activation is mediated by NF-kB and reactive oxygen species (ROS) signalling pathways, leading to caspase-1-dependent release of the proinflammatory cytokines, IL-1β and IL-18. Mounting evidence implicates NLRP3 inflammasome in neuroinflammation- related disorders, with its activation associated with cognitive function impairment. Of note, microglia, the resident immune cells crucial for brain plasticity, express high levels of the NLRP3-inflammasome components. Our initial findings indicate that VTX exerts a region-dependent modulatoryeffect on the NLRP3-inflammasome system in a LPS-induced memory impairment in vivo model. Furthermore, VTX's ability to modulate immune response suggests that microglia could be a direct target of the drug. AIM: In light of the compelling evidence surrounding the role of the NLRP3 inflammasome in cognitive dysfunctions and the recent discovery of VTX's anti-inflammatory activity, we aimed to investigate the molecular effects induced by VTX pre-treatment in the presence or absence of the inflammasome-inducer LPS in a well-established in vitro model of mouse microglia: BV2 cells. METHODS: To dissect the influence of VTX pre-treatment (24h) on the NLRP3 inflammasome signaling pathway and microglial polarization, we analyzed gene and protein expression in BV2 cells stimulated with LPS or vehicle for 6h. We also scrutinized the activation/translocation of NF-kB and ROS release under these conditions. We applied one-way or two-way ANOVA followed by Tukey’s post hoc test for statistical analysis based on the experimental design. RESULTS: Our data demonstrate that short-term exposure to LPS significantly induces the activation/translocation of NF-kB signaling and ROS release in BV2 cells. We observed a time-dependent transcriptional upregulation of the inflammasome complex, IL-1β and IL-18, and microglial pro-inflammatory targets post-LPS stimulation, alongside a downregulation of the anti-inflammatory factors. Interestingly, a pre-treatment with VTX (10 nM) for 24h effectively modulated the LPS-induced NF-kB translocation and ROS production compared to control cells. Cells pre-treated with VTX exhibited lower levels of LPS-induced NLRP3 inflammasome- and microglia pro-inflammatory-related targets. However, VTX did not influence the expression of anti-inflammatory factors in both unstimulated and LPS-stimulated BV2 cells. CONCLUSIONS: Our findings reinforce the emerging evidence that supports VTX's anti-inflammatory activity. This activity is mediated via modulation of the inflammasome signaling pathway, which plays a pivotal role in the inflammatory response of microglia cells.

2022 - A flavonoid, quercetin, is capable of enhancing long-term memory formation if encountered at different times in the learning, memory formation, and memory recall continuum [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Blom, J. M.; Tascedda, F.; Lukowiak, K.
abstract

A major extrinsic factor influencing memory and neuro-cognitive performances across taxa is diet. Studies from vertebrates have shown the effects of a flavonoid rich diet on cognitive performance, but the mechanism underlying this action is still poorly understood. A common and abundant flavonoid present in numerous food substances is quercetin (Q). The present study provides the first support for Q-modulated enhancement of cognitive function in an invertebrate model, the pond snail Lymnaea stagnalis, after an operant conditioning procedure. We found that when snails were exposed to Q 3 h before or after a single 0.5 h training session, which typically results in memory lasting ~ 3 h, they formed a long-term memory (LTM) lasting for at least 24 h. Additionally, we assessed the effects of the combined presentation of a single reinforcing stimulus (at 24 h post-training or 24 h before training) and Q-exposure on both LTM formation and reconsolidation. That is, when applied within 3 h of critical periods of memory, Q regulates four different phases: (1) acquisition (i.e., a learning event), (2) consolidation processes after acquisition, (3) memory recall, and (4) memory reconsolidation. In all these phases Q-exposure enhanced LTM persistence.

2022 - Aspirin reverts lipopolysaccharide-induced learning and memory impairment: first evidence from an invertebrate model system [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Tascedda, F.; Blom, J. M. C.; Lukowiak, K.
abstract

By employing a reductionistic (but not simplistic) approach using an established invertebrate model system, the pond snail Lymnaea stagnalis, we investigated whether (1) lipopolysaccharide (LPS)-induced inflammation would cause a sickness state and impair cognitive function, and-if so-(2) would aspirin (acetylsalicylic acid-ASA) restore the impaired cognition. To test our hypotheses, we first determined if the injection of 25 mg (6.25 μg/mL) of Escherichia coli-derived LPS serotype O127:B8 altered homeostatic behavior, aerial respiration, and then determined if LPS altered memory formation when this behavior was operantly conditioned. Next, we determined if ASA altered the LPS-induced changes in both aerial respiration and cognitive functions. LPS induced a sickness state that increased aerial respiration and altered the ability of snails to form or recall long-term memory. ASA reverted the LPS-induced sickness state and thus allowed long-term memory both to be formed and recalled. We confirmed our hypotheses and provided the first evidence in an invertebrate model system that an injection of LPS results in a sickness state that obstructs learning and memory, and this impairment can be prevented by a non-steroidal anti-inflammatory.

2022 - Comprehensive Pain Management Using Opioids for Children and Adolescents: Still a Wild Goose to Chase? [Articolo su rivista]
Blom, J. M. C.; Benatti, C.
abstract

2022 - Deciphering the central immunomodulatory effects of a vortioxetine pretreatment on the LPS-induced inflammatory cascade [Abstract in Atti di Convegno]
Ciani, M.; Toscano, Y.; Benatti, C.; Blom, J. M. C.; Tascedda, F.; Alboni, S.; Brunello, N.
abstract

2022 - Editorial: Digital biomarkers in testing the safety and efficacy of new drugs in mental health: A collaborative effort of patients, clinicians, researchers, and regulators [Articolo su rivista]
Blom, Johanna Maria Catharina; Benatti, Cristina; Mascalzoni, Deborah; Tascedda, Fabio; Pani, Luca
abstract

2022 - Fluoride affects memory by altering the transcriptional activity in the central nervous system of Lymnaea stagnalis [Articolo su rivista]
Rivi, V.; Batabyal, A.; Wiley, B.; Benatti, C.; Tascedda, F.; Blom, J. M. C.; Lukowiak, K.
abstract

Fluoride (F-), has been found to affect learning and memory in several species. In this study, we exposed an F--naïve, inbred strain of Lymnaea stagnalis to a concentration of F- similar to that naturally occurring in wild ponds. We found that the exposure to F- before the configural learning procedure obstructs the memory formation and blocks the configural learning-induced upregulation of CREB1, GRIN1, and HSP70 in snails' central ring ganglia. Along with altering the mRNA levels of these key genes for memory formation, a single acute F- exposure also upregulates Cytochrome c Oxidase, a major regulatory enzyme of the electron transport chain, which plays direct or indirect roles in reactive oxygen species production. As the central nervous system is sensitive to oxidative stress and consistent with previous studies from mammals, our results suggest a potential role of oxidative stress in memory impairment. To our knowledge, this is the first study investigating the neuronal mechanism of memory impairment in an invertebrate species that is exposed to natural F- levels.

2022 - Gut microbiota alterations promote traumatic stress susceptibility associated with p-cresol-induced dopaminergic dysfunctions [Articolo su rivista]
Laudani, Samuele; Torrisi, Sebastiano A; Alboni, Silvia; Bastiaanssen, Thomaz F S; Benatti, Cristina; Rivi, Veronica; Moloney, Rachel D; Fuochi, Virginia; Furneri, Pio M; Drago, Filippo; Salomone, Salvatore; Tascedda, Fabio; Cryan, John F; Leggio, Gian Marco
abstract

Mounting evidence suggests a link between gut microbiota abnormalities and post-traumatic stress disorder (PTSD). However, whether and how the gut microbiota influences PTSD susceptibility is poorly understood. Here using the arousal-based individual screening model, we provide evidence for pre-trauma and post-trauma gut microbiota alterations in susceptible mice exhibiting persistent PTSD-related phenotypes. A more in-depth analysis revealed an increased abundance of bacteria affecting brain processes including myelination, and brain systems like the dopaminergic neurotransmission. Because dopaminergic dysfunctions play a key role in the pathophysiological mechanisms subserving PTSD, we assessed whether these alterations in gut microbiota composition could be associated with abnormal levels of metabolites inducing dopaminergic dysfunctions. We found high levels of the l-tyrosine-derived metabolite p-cresol exclusively in the prefrontal cortex of susceptible mice. We further uncovered abnormal levels of dopamine and DOPAC, together with a detrimental increase of dopamine D3 receptor expression, exclusively in the prefrontal cortex of susceptible mice. Conversely, we observed either resilience mechanisms aimed at counteracting these p-cresol-induced dopaminergic dysfunctions or myelination-related resilience mechanisms only in the prefrontal cortex of resilient mice. These findings reveal that gut microbiota abnormalities foster trauma susceptibility and thus it may represent a promising target for therapeutic interventions.

2022 - Identification and characterization of the kynurenine pathway in the pond snail Lymnaea stagnalis [Articolo su rivista]
Benatti, Cristina; Rivi, Veronica; Alboni, Silvia; Grilli, Andrea; Castellano, Sara; Pani, Luca; Brunello, Nicoletta; Blom, Johanna Maria Catharina; Bicciato, Silvio; Tascedda, Fabio
abstract

2022 - Microglial diversity along the hippocampal longitudinal axis impacts synaptic plasticity in adult male mice under homeostatic conditions [Articolo su rivista]
De Felice, E; Gonçalves de Andrade, E; Golia, M T; González Ibáñez, F; Khakpour, M; Di Castro, M A; Garofalo, S; Di Pietro, E; Benatti, C; Brunello, N; Tascedda, F; Kaminska, B; Limatola, C; Ragozzino, D; Tremblay, M E; Alboni, S; Maggi, L
abstract

The hippocampus is a plastic brain area that shows functional segregation along its longitudinal axis, reflected by a higher level of long-term potentiation (LTP) in the CA1 region of the dorsal hippocampus (DH) compared to the ventral hippocampus (VH), but the mechanisms underlying this difference remain elusive. Numerous studies have highlighted the importance of microglia-neuronal communication in modulating synaptic transmission and hippocampal plasticity, although its role in physiological contexts is still largely unknown. We characterized in depth the features of microglia in the two hippocampal poles and investigated their contribution to CA1 plasticity under physiological conditions. We unveiled the influence of microglia in differentially modulating the amplitude of LTP in the DH and VH, showing that minocycline or PLX5622 treatment reduced LTP amplitude in the DH, while increasing it in the VH. This was recapitulated in Cx3cr1 knockout mice, indicating that microglia have a key role in setting the conditions for plasticity processes in a region-specific manner, and that the CX3CL1-CX3CR1 pathway is a key element in determining the basal level of CA1 LTP in the two regions. The observed LTP differences at the two poles were associated with transcriptional changes in the expression of genes encoding for Il-1, Tnf-α, Il-6, and Bdnf, essential players of neuronal plasticity. Furthermore, microglia in the CA1 SR region showed an increase in soma and a more extensive arborization, an increased prevalence of immature lysosomes accompanied by an elevation in mRNA expression of phagocytic markers Mertk and Cd68 and a surge in the expression of microglial outward K+ currents in the VH compared to DH, suggesting a distinct basal phenotypic state of microglia across the two hippocampal poles. Overall, we characterized the molecular, morphological, ultrastructural, and functional profile of microglia at the two poles, suggesting that modifications in hippocampal subregions related to different microglial statuses can contribute to dissect the phenotypical aspects of many diseases in which microglia are known to be involved.

2022 - Nature versus nurture in heat stress induced learning between inbred and outbred populations of Lymnaea stagnalis [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Blom, J. M.; Lukowiak, K.
abstract

Changing environmental conditions often lead to microevolution of traits that are adaptive under the current selection pressure. Currently, one of the major selection pressures is the rise in temperatures globally that has a severe impact on the behavioral ecology of animals. However, the role of thermal stress on neuronal plasticity and memory formation is not well understood. Thermal tolerance and sensitivity to heat stress show variation across populations of the same species experiencing different thermal regimes. We used two populations of the pond snail Lymnaea stagnalis: one lab-bred W-snails and the other wild Delta snails to test heat shock induced learning and memory formation for the Garcia effect learning paradigm. In Garcia effect, a single pairing of a heat stressor (30 °C for 1h) with a novel taste results in a taste-specific negative hedonic shift lasting 24h as long-term memory (LTM) in lab bred W-snails. In this study we used a repeated heat stress procedure to test for increased or decreased sensitivity to the heat before testing for the Garcia effect. We found that lab-bred W-snails show increased sensitivity to heat stress after repeated heat exposure for 7days, leading to enhanced LTM for Garcia effect with only 15min of heat exposure instead of standard 1h. Surprisingly, the freshly collected wild snails do not show Garcia effect. Additionally, F1 generation of wild snails raised and maintained under laboratory conditions still retain their heat stress tolerance similar to their parents and do not show a Garcia effect under standard learning paradigm or even after repeated heat stressor. Thus, we found a differential effect of heat stress on memory formation in wild and lab bred snails. Most interestingly we also show that local environmental (temperature) conditions for one generation is not enough to alter thermal sensitivity in a wild population of L. stagnalis.

2022 - Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-κB-mediated signaling [Articolo su rivista]
Borgonetti, V.; Benatti, C.; Governa, P.; Isoldi, G.; Pellati, F.; Alboni, S.; Tascedda, F.; Montopoli, M.; Galeotti, N.; Manetti, F.; Miraldi, E.; Biagi, M.; Rigillo, G.
abstract

Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and β-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

2022 - Too Hot to Eat: Wild and Lab-Bred Lymnaea stagnalis Differ in Feeding Response Following Repeated Heat Exposure [Articolo su rivista]
Rivi, V.; Batabyal, A.; Benatti, C.; Tascedda, F.; Blom, J. M.; Lukowiak, K.
abstract

AbstractAcute extreme heat events are increasing in frequency and intensity. Understanding their effects on ectothermic organisms' homeostasis is both important and urgent. In this study we found that the exposure to an acute heat shock (30 °C for 1 hour) repeated for a seven-day period severely suppressed the feeding behavior of laboratory-inbred (W-strain) Lymnaea stagnalis, whereas the first-generation offspring of freshly collected wild (F1 D-strain) snails raised and maintained under similar laboratory conditions did not show any alterations. The W-strain snails might have inadvertently been selected against heat tolerance since they were first brought into the laboratory many (∼70) years ago. We also posit that the F1 D-strain snails do not perceive the heat shock as a sufficient stressor to alter their feeding response because their parental populations in wild environments have repeatedly experienced temperature fluctuations, thus becoming more tolerant and resilient to heat. The different responses exhibited by two strains of the same species highlight the importance of selecting the most appropriate strain for addressing questions about the impacts of global warming on organisms' physiology and behavior.

2021 - Carnosine Protects Macrophages against the Toxicity of Aβ1-42 Oligomers by Decreasing Oxidative Stress [Articolo su rivista]
Caruso, Giuseppe; Benatti, Cristina; Musso, Nicolò; Fresta, Claudia G.; Fidilio, Annamaria; Spampinato, Giorgia; Brunello, Nicoletta; Bucolo, Claudio; Drago, Filippo; Lunte, Susan M.; Peterson, Blake R.; Tascedda, Fabio; Caraci, Filippo
abstract

2021 - Digital Phenotyping and Dynamic Monitoring of Adolescents Treated for Cancer to Guide Intervention: Embracing a New Era [Articolo su rivista]
Blom, J. M. C.; Colliva, C.; Benatti, C.; Tascedda, F.; Pani, L.
abstract

2021 - Long term memory of configural learning is enhanced via CREB upregulation by the flavonoid Quercetin in Lymnaea stagnalis [Articolo su rivista]
Batabyal, Anuradha; Rivi, Veronica; Benatti, Cristina; Blom, Johanna M. C.; Lukowiak, Ken
abstract

2021 - Serum metabolic signature of binge-like palatable food consumption in female rats by nuclear magnetic resonance spectroscopy [Articolo su rivista]
Cifani, Carlo; Alboni, Silvia; Mucci, Adele; Benatti, Cristina; Botticelli, Luca; Brunello, Nicoletta; Micioni Di Bonaventura, Maria Vittoria; Righi, Valeria
abstract

Maladaptive eating behavior is a growing public health problem and compulsively eating excessive food in a short time, or binge eating, is a key symptom of many eating disorders. In order to investigate the binge-like eating behavior in female rats, induced by intermittent food restrictions/refeeding and frustration stress, we analyzed for the first time the metabolic profile obtained from serum of rats, through nuclear magnetic resonance (NMR) spectroscopy. In this experimental protocol, rats were exposed to chow food restricting/refeeding and frustration stress manipulation. This stress procedure consists of 15 min exposure to the odor and sight of a familiar chocolate paste, without access to it, just before offering the palatable food. In this model, a "binge-eating episode" was considered the significantly higher palatable food consumption within 2 h in restricted and stressed rats (R + S) than in the other three experimental groups: rats with no food restriction and no stress (NR + NS), only stressed rats (NR + S) or only restricted rats (R + NS). Serum samples from these four different rat groups were collected. The statistical analysis of the 1 H NMR spectral profiles of the four sets of samples pointed to O- and N-acetyl glycoproteins as the main biomarkers for the discrimination of restriction effects. Other metabolites, such as threonine, glycine, glutamine, acetate, pyruvate and lactate, showed trends that may be useful to understand metabolic pathways involved in eating disorders. This study suggested that NMR-based metabolomics is a suitable approach to detect biomarkers related to binge-eating behavior.

2021 - To eat or not to eat: a Garcia effect in pond snails (Lymnaea stagnalis) [Articolo su rivista]
Rivi, Veronica; Batabyal, Anuradha; Juego, Karla; Kakadiya, Mili; Benatti, Cristina; Blom, Johanna M C; Lukowiak, Ken
abstract

Taste aversion learning is universal. In animals, a single presentation of a novel food substance followed hours later by visceral illness causes animals to avoid that taste. This is known as bait-shyness or the Garcia effect. Humans demonstrate this by avoiding a certain food following the development of nausea after ingesting that food ('Sauce Bearnaise effect'). Here, we show that the pond snail Lymnaea stagnalis is capable of the Garcia effect. A single 'pairing' of a novel taste, a carrot slurry followed hours later by a heat shock stressor (HS) is sufficient to suppress feeding response elicited by carrot for at least 24 h. Other food tastes are not suppressed. If snails had previously been exposed to carrot as their food source, the Garcia-like effect does not occur when carrot is 'paired' with the HS. The HS up-regulates two heat shock proteins (HSPs), HSP70 and HSP40. Blocking the up-regulation of the HSPs by a flavonoid, quercetin, before the heat shock, prevented the Garcia effect in the snails. Finally, we found that snails exhibit Garcia effect following a period of food deprivation but the long-term memory (LTM) phenotype can be observed only if the animals are tested in a food satiated state.

2021 - Vortioxetine Prevents Lipopolysaccharide-Induced Memory Impairment Without Inhibiting the Initial Inflammatory Cascade [Articolo su rivista]
Alboni, S.; Benatti, C.; Colliva, C.; Radighieri, G.; Blom, J. M. C.; Brunello, N.; Tascedda, F.
abstract

Vortioxetine is a novel multimodal antidepressant that modulates a wide range of neurotransmitters throughout the brain. Preclinical and clinical studies have shown that vortioxetine exerts positive effects on different cognitive domains and neuroprotective effects. Considering the key role of microglial cells in brain plasticity and cognition, we aimed at investigating the effects of pretreatment with vortioxetine in modulating behavioral and molecular effects induced by an immune challenge: peripheral injection of lipopolysaccharide (LPS). To this purpose, C57BL/6J male mice were first exposed to a 28-day standard diet or vortioxetine-enriched diet, which was followed by an acute immune challenge with LPS. Sickness symptoms and depressive-like behaviors (anhedonia and memory impairment) were tested 6 and 24 h after exposure to LPS, respectively. Moreover, the expressions of markers of immune activation and M1/M2 markers of microglia polarization were measured in the dorsal and ventral parts of the hippocampus. The pretreatment with vortioxetine did not affect both LPS-induced sickness behavior and anhedonia but prevented the deficit in the recognition memory induced by the immune challenge. At the transcriptional level, chronic exposure to vortioxetine did not prevent LPS-induced upregulation of proinflammatory cytokines 6 h after the immune challenge but rather seemed to potentiate the immune response to the challenge also by affecting the levels of expression of markers of microglia M1 phenotype, like cluster of differentiation (CD)14 and CD86, in an area-dependent manner. However, at the same time point, LPS injection significantly increased the expression of the M2 polarization inducer, interleukin 4, only in the hippocampus of animals chronically exposed to vortioxetine. These results demonstrate that a chronic administration of vortioxetine specifically prevents LPS-induced memory impairment, without affecting acute sickness behavior and anhedonia, and suggest that hippocampal microglia may represent a cellular target of this novel antidepressant medication. Moreover, we provide a useful model to further explore the molecular mechanisms specifically underlying cognitive impairments following an immune challenge.

2021 - What can we teach Lymnaea and what can Lymnaea teach us? [Articolo su rivista]
Rivi, Veronica; Benatti, Cristina; Lukowiak, Ken; Colliva, Chiara; Alboni, Silvia; Tascedda, Fabio; Blom, Johanna M. C.
abstract

2020 - Lymnaea stagnalis as model for translational neuroscience research: from pond to bench [Articolo su rivista]
Rivi, Veronica; Benatti, C; Colliva, C; Radighieri, G; Brunello, N; Tascedda, F; Blom, Johanna
abstract

The purpose of this review is to illustrate how a reductionistic, but sophisticated, approach based on the use of a simple model system such as the pond snail Lymnaea stagnalis (L. stagnalis), might be useful to address fundamental questions in learning and memory. L. stagnalis, as a model, provides an interesting platform to investigate the dialog between the synapse and the nucleus and vice versa during memory and learning. More importantly, the "molecular actors" of the memory dialogue are well-conserved both across phylogenetic groups and learning paradigms, involving single- or multi-trials, aversion or reward, operant or classical conditioning. At the same time, this model could help to study how, where and when the memory dialog is impaired in stressful conditions and during aging and neurodegeneration in humans and thus offers new insights and targets in order to develop innovative therapies and technology for the treatment of a range of neurological and neurodegenerative disorders.

2020 - P.304 Executive functioning and genetic variation in pediatric patients with cancer [Abstract in Rivista]
Colliva, C.; Dalla Porta, M. F.; Ferrari, M.; Benatti, C.; Cellini, M.; Brunello, N.; Blom, J. M.
abstract

2020 - Psychosocial assessment of families caring for a child with acute lymphoblastic leukemia, epilepsy or asthma: Psychosocial risk as network of interacting symptoms [Articolo su rivista]
Colliva, C.; Cellini, M.; Porta, F. D.; Ferrari, M.; Bergamini, B. M.; Guerra, A.; Di Giuseppe, S.; Pinto, A.; Capasso, R.; Caprino, D.; Ferrari, M.; Benatti, C.; Tascedda, F.; Blom, J. M. C.
abstract

The purpose of this study is to assess psychosocial risk across several pediatric medical conditions and test the hypothesis that different severe or chronic pediatric illnesses are characterized by disease specific enhanced psychosocial risk and that risk is driven by disease specific connectivity and interdependencies among various domains of psychosocial function using the Psychosocial Assessment Tool (PAT). In a multicenter prospective cohort study of 195 patients, aged 5-12, 90 diagnosed with acute lymphoblastic leukemia (ALL), 42 with epilepsy and 63 with asthma, parents completed the PAT2.0 or the PAT2.0 generic version. Multivariate analysis was performed with disease as factor and age as covariate. Graph theory and network analysis was employed to study the connectivity and interdependencies among subscales of the PAT while data-driven cluster analysis was used to test whether common patterns of risk exist among the various diseases. Using a network modelling approach analysis, we observed unique patterns of interconnected domains of psychosocial factors. Each pathology was characterized by different interdependencies among the most central and most connected domains. Furthermore, data-driven cluster analysis resulted in two clusters: patients with ALL (89%) mostly belonged to cluster 1, while patients with epilepsy and asthma belonged primarily to cluster 2 (83% and 82% respectively). In sum, implementing a network approach improves our comprehension concerning the character of the problems central to the development of psychosocial difficulties. Therapy directed at problems related to the most central domain(s) constitutes the more rational one because such an approach will inevitably carry over to other domains that depend on the more central function.

2020 - Redefining operant conditioning of escape behaviour in lymnaea stagnalis [Articolo su rivista]
Benatti, C.; Rivi, V.; Colliva, C.; Radighieri, G.; Tascedda, F.; Blom, J. M. C.
abstract

The escape behaviour is one of the many behavioural responses that can be operantly conditioned in a stimulus-dependent manner in both vertebrates and invertebrates. By exposing the pond snail Lymnaea stagnalis repeatedly to a negative reinforcement its natural tendency to explore its surroundings can be operantly conditioned in both adult and aged snails. When adult snails were trained with 100 mM of KCl their number of escapes was significantly decreased and the latency to first escape was significantly increased. Our behavioural protocol allowed us to investigate memory acquisition, consolidation, and retrieval in pre-and post-training sessions over different days. From the 3rd day of training the learned response was strengthened: the number of the escapes in the post-test session remained significantly reduced even when animals were presented with distilled water. Moreover, adult snails exposed to the negative reinforcement for at least 4 days started to escape significantly less than the control group also in the pre-test session. This effect became more pronounced in the following days and was accompanied by a significant increase in the latency to first escape at the beginning of the pre-test on day 6 and 7. Aged snails, instead, showed selective deficiencies when operantly conditioned: memory retention appeared only after 7 days, while memory retrieval could not be induced. This redefined paradigm can help unravelling a variety of sophisticated cognitive phenomena in L. stagnalis and could be employed also to study the basis of memory impairment occurring during neuro-aging.

2019 - Cannabidiol-enriched Cannabis sativa L. extract modulates inflammatory-induced human peripheral mononuclear cells response [Abstract in Atti di Convegno]
Rigillo, G; Borgonetti, V; Benatti, C; Governa, P; Tascedda, F; Biagi, M
abstract

2019 - Executive functioning in children with epilepsy: Genes matter [Articolo su rivista]
Colliva, Chiara; Ferrari, Marta; Benatti, Cristina; Guerra, Azzurra; Tascedda, Fabio; Blom, Johanna Maria Catharina
abstract

Pediatric epilepsy has emerged as a chronic medical disease with a characteristic behavioral and cognitive phenotype, which includes compromised executive functioning (EF) and attention-related deficits. However, considerable interindividual variability exists; children often display very different or even opposite outcomes, and some children are more likely than others to develop neurocognitive problems in the face of similar individual and disease-related problems. The factors responsible for this interindividual variability are still largely unknown, but we do know that some genetic factors render the developing brain more susceptible to damage or traumatic experiences than others. Dopamine availability has a neuromodulatory function in the prefrontal cortex (PFC) and especially affects EF. Dopamine availability relates to polymorphisms in the gene encoding catechol-O-methyltransferase (COMT Val158Met), which in turn is affected by the methylation state of its promoter. Allelic variation of the methylenetetrahydrofolate reductase (MTHFR C677T) gene, alters methylation and may influence the methylation state of the COMT promoter. Given this, we tested the hypothesis that these polymorphisms interact in children with epilepsy, and that variability in allelic expression is associated with variability in cognitive phenotype. Executive function was tested directly and indirectly (parent-rated) in 42 children between 5 and 12 years of age. The MTHFR T allele carriers performed worse than MTHFR homozygous CC carriers on indirect EF, and a significant decline was observed when T allele carriers had at least one met allele of the COMT gene, especially on Working Memory. Direct EF was significantly compromised in COMT Val/Val carriers where reduced dopamine availability seems to confer a higher risk in a test that requests a high degree of executive attention and planning. This finding suggests that in children with epilepsy, genes that influence methylation and dopamine availability affect PFC-related EF. Therefore, we should consider genetic vulnerability as a polygenic risk, which might predispose for a particular phenotype and include specific genetic signatures as part of each patient's behavioral and cognitive profile from the moment that we start to take care of the child.

2019 - Modulation of neuroplasticity-related targets following stress-induced acute escape deficit [Articolo su rivista]
Benatti, C.; Radighieri, Giulia; Alboni, S.; Blom, J. M. C.; Brunello, N.; Tascedda, F.
abstract

Understanding resilience is a major challenge to improve current pharmacological therapies aimed at complementing psychological-based approaches of stress-related disorders. In particular, resilience is a multi-factorial construct where the complex network of molecular events that drive the process still needs to be resolved. Here, we exploit the acute escape deficit model, an animal model based on exposure to acute unavoidable stress followed by an escape test, to define vulnerable and resilient phenotypes in rats. Hippocampus and prefrontal cortex (PFC), two of the brain areas most involved in the stress response, were analysed for gene expression at two different time points (3 and 24 h) after the escape test. Total Brain-Derived Neurotrophic Factor (BDNF) was highly responsive in the PFC at 24-h after the escape test, while expression of BDNF transcript IV increased in the hippocampus of resistant animals 3 h post-test. Expression of memory enhancers like Neuronal PAS Domain Protein 4 (Npas4) and Activity-regulated cytoskeleton-associated protein (Arc) decreased in a time- and region-dependent fashion in both behavioural phenotypes. Also, the memory inhibitor Protein Phosphatase 1 (Ppp1ca) was increased in the hippocampus of resilient rats at 3 h post-test. Given the importance of neurotrophic factors and synaptic plasticity-related genes for the development of appropriate coping strategies, our data contribute to an additional step forward in the comprehension of the psychobiology of stress and resiliency.

2019 - P.1.04 Expression of histone variants H3.3 and H2a.z in the rat brain: Physiopathological and pharmacological implications [Abstract in Rivista]
Radighieri, G.; Benatti, C.; Zoli, M.; Blom, J. M. C.; Brunello, N.; Tascedda, F.
abstract

In the overall context of epigenetic modifications in charge of managing genome plasticity and dynamics [1, 2], the role of nucleosomal loading of histone variants is becoming increasingly captivating. Two replication-independent isoforms of histones H3 and H2A, namely H3.3 and H2A.Z, have caught attention because of their involvement in neuronal plasticity processes, cognitive functions and behavioral outcomes. In fact, their incorporation/eviction in nucleosomes and their turnover in neurons influence chromatin accessibility and therefore transcription. H3.3 enrichment at gene bodies and promoters of genes involved in synaptic plasticity was proved to be positively correlated with their expression, while learning-induced H2A.Z eviction in specific genes promotes gene transcription, intervening in memory consolidation processes. H3.3 is encoded by H3f3a and H3f3b independent genes, generating identical proteins, namely H3.3A and H3.3B. Notably, H3f3b gene, but not H3f3a, was proved responsive to neuronal activating stimuli as well as environmental triggers and stressful procedures [3]. H2A.Z hypervariants H2A.Z.1 and H2A.Z.2, encoded respectively by H2afz and H2afv genes, regulate both basal and stimulus-induced neuronal gene expression of independent gene sets [4]. Starting from this evidence, the purpose of this study was to characterize basal expression levels of all genes encoding for the histones variants above mentioned in rodent hippocampus and prefrontal cortex (PFC), two brain regions closely related to brain plasticity, cognition and behavior. Adult male rats (n=7) were sacrificed, their brains removed and dissected. Total RNA extraction was performed, followed by total RNA reverse transcription and Real Time PCR, where specific forward and reverse primer were used for each gene encoding for H3.3 (H3f3a and H3f3b), H2A.Z (H2afz and H2afv) and endogenous control GAPDH. Statistical analysis was performed by means of one-way ANOVA; p<0.05 was considered as a threshold for statistically significant difference. Molecular analyses revealed that, for both hippocampus and PFC, H3f3a mRNA was more expressed at the steady-state compared to H3f3b (p<0.001), as happens for H2afz mRNA, which displays higher levels than H2afv (p<0.001). Moreover, comparing hippocampal and PFC mRNA levels for each variant, H3f3a and H3f3b expression was increased in the hippocampus with respect to the prefrontal cortex (p<0.001), and a comparable outcome was showed for H2afv (p<0.001) but not for H2afz (p>0.05). Our results suggest that 1) differential basal expression levels of genes encoding for H3.3 and H2A.Z may underlie unique gene responsiveness following different stimuli, as previously hypothesized by others [3,4], and this may be crucial in highly-responsive, pathological- and environment-related tissues like hippocampus and PFC; 2) striking lower steady-state expression of H3f3b and H2afv genes might imply a major sensitivity to neuronal inputs compared to their correspondent counterparts; 3) higher expression levels in the hippocampus with respect to the PFC might underpin brain-region specific expression and function for histone variants and their isoforms. Together, these data clear the way for further studies meant at investigating stimulus-dependent regulation of H3.3 and H2A.Z gene isoforms expression and their putative involvement in the physiopathology of brain and its diseases [5]. References [1] Rigillo, G., Vilella, A., Benatti, C., Schaeffer, L., Brunello, N., Blom, J.M.C., Zoli, M., Tascedda, F., 2018. LPS-induced histone H3 phospho(Ser10)-acetylation(Lys14) regulates neuronal and microglial neuroinflammatory response. Brain Behav Immun. https://doi.org/10.1016/j.bbi.2018.09.019. [2] Ottaviani, E., Accorsi, A., Rigillo, G., Malagoli, D., Blom, J.M., Tascedda, F., 2013. Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challe

2019 - The many faces of mitochondrial dysfunction in depression: From pathology to treatment [Articolo su rivista]
Caruso, G.; Benatti, C.; Blom, J. M. C.; Caraci, F.; Tascedda, F.
abstract

Introduction The last years of neurobiological research have transformed the way we consider mental illnesses. We have gone from a deterministic genetic view to a broader vision that includes the involvement of non-cerebral systems. This is especially true for major depression (MD). Historically, MD has been perceived as a multifactorial disorder correlated to various neurobiological changes like neurotransmitter deficits, endocrine disturbances, impaired plasticity, and neural adaptation (Benatti et al., 2016). Indeed, the development and progression of depressive disorders has been conceived as the disruption of body allostasis, defined as the process of achieving stability of physiological and mental processes through dynamic change (Wang et al., 2019). The main player in the “allostatic game” is the brain, an organ designed to integrate signals from the periphery that anticipate fluctuations, changes, and needs and coordinates allostatic mediators in order to develop successful coping mechanisms that ultimately lead to an adaptative strategy and resilience (de Kloet et al., 2005). The establishment and maintenance of these mechanisms requires large amounts of energy from the organism. Without energy, or in a partial lack of energy, the biological mechanisms necessary to respond appropriately to stimuli may not occur or be established incorrectly or abnormally. Human and animal studies suggest an intriguing link between our body’s ability to produce energy and the brain’s ability to correctly perform the complex cellular and molecular processes involved in allostatic processes. In eukaryotic cells, mitochondria are the powerhouse that produces and distributes energy to all other components. Functional or quantitative alterations of the ability of mitochondria to adequately supply energy can have important repercussions primarily on cellular processes and cascades of serial events (Herst et al., 2017) as well as on the correct functioning of the organism including mechanisms of brain plasticity, mood, and behavior in general (Allen et al., 2018). In this framework, it is particularly intriguing to think of the mitochondria as an active regulator of many of the biological phenomena involved in depression and in the efficacy of or resistance to the most widely used pharmacological treatments. Once the energetic equilibrium is compromised, the body becomes more “vulnerable.” This is especially true for stress-related disorders, such as depression. In fact, depression is often associated with energetic imbalance leading to profound effects on the disease (Zuccoli et al., 2017). The driving questions then are as follows: What happens to the brain in the presence of an energetic imbalance? Does depression or depression-related symptoms impact mitochondrial energetic efficiency? Is antidepressant efficacy mediated by mitochondrial functionality?

2018 - LPS-induced histone H3 phospho(Ser10)-acetylation(Lys14) regulates neuronal and microglial neuroinflammatory response [Articolo su rivista]
Rigillo, Giovanna; Vilella, Antonietta; Benatti, Cristina; Schaeffer, Laurent; Brunello, Nicoletta; Blom, Johanna M. C.; Zoli, Michele; Tascedda, Fabio
abstract

Epigenetic modifications of DNA and histone proteins are emerging as fundamental mechanisms by which neural cells adapt their transcriptional response to environmental cues, such as, immune stimuli or stress. In particular, histone H3 phospho(Ser10)-acetylation(Lys14) (H3S10phK14ac) has been linked to activation of specific gene expression. The purpose of this study was to investigate the role of H3S10phK14ac in a neuroinflammatory condition. Adult male rats received a intraperitoneal injection of lipopolysaccharide (LPS) (830 μg/Kg/i.p., n = 6) or vehicle (saline 1 mL/kg/i.p., n = 6) and were sacrificed 2 or 6 h later. We showed marked region- and time-specific increases in H3S10phK14ac in the hypothalamus and hippocampus, two principal target regions of LPS. These changes were accompanied by a marked transcriptional activation of interleukin (IL) 1β, IL-6, Tumour Necrosis Factor (TNF) α, the inducible nitric oxide synthase (iNOS) and the immediate early gene c-Fos. By means of chromatin immunoprecipitation, we demonstrated an increased region- and time-specific association of H3S10phK14ac with the promoters of IL-6, c-Fos and iNOS genes, suggesting that part of the LPS-induced transcriptional activation of these genes is regulated by H3S10phK14ac. Finally, by means of multiple immunofluorescence approach, we showed that increased H3S10phK14ac is cell type-specific, being neurons and reactive microglia, the principal histological types involved in this response. Present data point to H3S10phK14ac as a principal epigenetic regulator of neural cell response to systemic LPS and underline the importance of distinct time-, region- and cell-specific epigenetic mechanisms that regulate gene transcription to understand the mechanistic complexity of neuroinflammatory response to immune challenges.

2018 - Molecular changes associated with escitalopram response in a stress-based model of depression [Articolo su rivista]
Benatti, Cristina; Alboni, Silvia; Blom, Johanna Maria Catharina; Mendlewicz, Julien; Tascedda, Fabio; Brunello, Nicoletta
abstract

Converging evidence points at hypothalamus-pituitary-adrenal (HPA) axis hyperactivity and neuroinflammation as important factors involved in the etiopathogenesis of major depressive disorder (MDD) and in therapeutic efficacy of antidepressants. In this study, we examined the molecular effects associated with a response to a week-long treatment with escitalopram in the chronic escape deficit (CED) model, a validated model of depression based on the induction of an escape deficit after exposure of rats to an unavoidable stress. We confirmed our previous result that a treatment with escitalopram (10 mg/kg) was effective after 7 days in reverting the stress-induced escape deficit in approximately 50% of the animals, separating responders from non-responders. Expression of markers of HPA axis functionality as well as several inflammatory mediators were evaluated in the hypothalamus, a key structure integrating signals from the neuro, immune, endocrine systems. In the hypothalamus of responder animals we observed a decrease in the expression of CRH and its receptors and an increase in GR protein in total and nuclear extracts; this effect was accompanied by a significant decrease in circulating corticosterone in the same cohort. Hypothalamic IL-1Î² and TNFÎ± expression were increased in stressed animals, while CXCL2, IL-6, and ADAM17 mRNA levels were decreased in escitalopram treated rats regardless of the treatment response. These data suggest that efficacy of a one week treatment with escitalopram may be partially mediated by a decrease HPA axis activity, while in the hypothalamus the drug-induced effects on the expression of immune modulators did not correlate with the behavioural outcome.

2018 - Neither all anti-inflammatory drugs nor all doses are effective in accelerating the antidepressant-like effect of fluoxetine in an animal model of depression [Articolo su rivista]
Alboni, Silvia; Benatti, Cristina; Capone, Giacomo; Tascedda, Fabio; Brunello, Nicoletta
abstract

Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) have been studied as possible adjunctive therapy in the treatment of depression. However, administering NSAIDs to increase the effectiveness of antidepressant has yielded inconsistent results. Methods: We evaluated the effect of the co-administration of fluoxetine (5 mg/kg) and flurbiprofen (5 mg/kg) or fluoxetine (5 mg/kg) and celecoxib (5 mg/kg) in the chronic escape deficit (CED) model of depression after 7 days of treatment. The co-administration of fluoxetine plus acetylsalicylic acid (ASA, 45 mg/kg i.p.) was used as a positive control. Moreover, we tested the behavioral effect of different doses (45, 22.5, and 11.25 mg/Kg i.p.) of ASA as potentiating agent of the effect of fluoxetine in the same paradigm. Results: Our study showed that only the co-administration of ASA with fluoxetine was able to revert the stress-induced condition of escape deficit after 7 days of treatment, and that the amplitude of the antidepressant-like effect of ASA was dose dependent. In the same experimental conditions, celecoxib with fluoxetine only partially resolved the stress-induced impaired behavior while flurbiprofen/fluoxetine cotreatment was ineffective. Limitations: Our study is still exploratory, more doses, longer treatment regimens, and different behavioral outcomes must be investigated to draw a clear conclusion. Conclusion: Our results further stress the importance of the type and dose when NSAIDs are associated with antidepressants to ameliorate a clinical response.

2017 - Hypothalamic expression of inflammatory mediators in an animal model of binge eating [Articolo su rivista]
Alboni, Silvia; Micioni di Bonaventura, Maria Vittoria; Benatti, Cristina; Giusepponi, Maria; Elena, ; Brunello, Nicoletta; Cifani, Carlo
abstract

Binge eating episodes are characterized by uncontrollable, distressing eating of a large amount of highly palatable food and represent a central feature of bingeing related eating disorders. Research suggests that inflammation plays a role in the onset and maintenance of eating-related maladaptive behavior. Markers of inflammation can be selectively altered in discrete brain region where they can directly or indirectly regulate food intake. In the present study we measured expression levels of different components of cytokine systems (IL-1, IL-6, IL-18, TNF- and IFN-ɣ) and related molecules (iNOS and COX2) in the preoptic and anterior-tuberal parts of the hypothalamus of a validated animal model of binge eating. In this animal model, based on the exposure to both food restriction and frustration stress, binge-like eating behavior for highly palatable food is not shown when animals are exposed to the frustration stress during the estrus phase. We found a characteristic down-regulation of the IL-18/ IL-18 receptor system (with increased expression of the inhibitor of the pro-inflammatory cytokine IL18, IL-18BP, together with a decreased expression of the binding chain of the IL-18 receptor) and a three-fold increase in the expression of iNOS specifically in the anterior- tuberal region of the hypothalamus of animals that develop a binge-like eating behavior. Differently, when food restricted animals were stressed during the estrus phase IL-18 expression increased while iNOS expression was not significantly affected. Considering the role of this region of the hypothalamus in controlling feeding related behavior, this can be relevant in eating disorders and obesity. Our data suggest that by targeting centrally selected inflammatory markers, we may prevent that disordered eating turns into a full blown eating disorder.

2017 - Transcriptional effect of serotonin in the ganglia of Lymnaea stagnalis [Articolo su rivista]
Benatti, Cristina; Colliva, Chiara; Blom, Johanna Maria Catharina; Ottaviani, Enzo; Tascedda, Fabio
abstract

The serotonin system (5HT) is highly conserved in both vertebrates and invertebrates, and numerous evidence supports a biological link between 5HT and numerous animal function. In the present paper we evaluated the transcriptional effects of a serotonergic stimulation on selected targets involved in 5HT signalling and neurotransmission in the central nervous system of the great pond snail Lymnaea stagnalis. Adult snails were treated acutely (6 h) or chronically (48 h) with either 5-hydroxytrypthophan (5-HTP 1mM), the immediate precursor of serotonin, fluoxetine (FLX 1Î¼M), a selective serotonin reuptake inhibitor, or a combination of two. The central ring ganglia were dissected and used for q-PCR gene expression analysis. Transcription was strongly induced following a chronic, but not an acute, exposure to 5-HTP in the ganglia of Lymnaea. In particular, LymCREB1 and LymP2X mRNA levels were decreased following a 6 h exposure and increased in snails receiving 5-hydroxytryptophan for 48 h. Interestingly, this effect was reduced when snails were exposed chronically to both 5-HTP and FLX, suggesting a role for SERT in mediating the effect of 5-hydroxytryptophan. These data suggest that L. stagnalis is suited to unravel the complexity of the serotonin signaling pathway.

2016 - Disease-induced neuroinflammation and depression [Articolo su rivista]
Benatti, Cristina; Blom, Johanna Maria Catharina; Rigillo, Giovanna; Alboni, Silvia; Zizzi, Francesca; Torta, Riccardo; Brunello, Nicoletta; Tascedda, Fabio
abstract

Progression of major depression, a multifactorial disorder with a neuroinflammatory signature, seems to be associated with the disruption of body allostasis. High rates of comorbidity between depression and specific medical disorders, such as, stroke, chronic pain conditions, diabetes mellitus, and human immunodeficiency virus (HIV) infection, have been extensively reported. In this review, we discuss how these medical disorders may predispose an individual to develop depression by examining the impact of these disorders on some hallmarks of neuroinflammation known to be impaired in depressed patients: altered permeability of the blood brain barrier, immune cells infiltration, activated microglia, increased cytokines production, and the role of inflammasomes. In all four pathologies, blood brain barrier integrity was altered, allowing the infiltration of peripheral factors, known to activate resident microglia. Evidence indicated morphological changes in the glial population, increased levels of circulating pro-inflammatory cytokines or increased production of these mediators within the brain, all fundamental in neuroinflammation, for the four medical disorders considered. Moreover, activity of the kynurenine pathway appeared to be enhanced. With respect to the inflammasome NLRP3, a new target whose role in neuroinflammation is emerging as being important, accumulating data suggest its involvement in the pathogenesis of brain injury following stroke, chronic pain conditions, diabetes mellitus or in HIV associated immune impairment. Finally, data gathered over the last 10 years, indicate and confirm that depression, stroke, chronic pain, diabetes, and HIV infection share a combination of underlying molecular, cellular and network mechanisms leading to a general increase in the neuroinflammatory burden for the individual.

2016 - Fluoxetine prevents Aβ1-42-induced toxicity via a paracrine signaling mediated by transforming-growth-factor-β1 [Articolo su rivista]
Caraci, Filippo; Tascedda, Fabio; Merlo, Sara; Benatti, Cristina; Spampinato, Simona F.; Munafò, Antonio; Leggio, Gian Marco; Nicoletti, Ferdinando; Brunello, Nicoletta; Drago, Filippo; Sortino, Maria Angela; Copani, Agata
abstract

Selective reuptake inhibitors (SSRIs), such as fluoxetine and sertraline, increase circulating Transforming-Growth-Factor-β1 (TGF-β1) levels in depressed patients, and are currently studied for their neuroprotective properties in Alzheimer's disease. TGF-β1 is an anti-inflammatory cytokine that exerts neuroprotective effects against β-amyloid (Aβ)-induced neurodegeneration. In the present work, the SSRI, fluoxetine, was tested for the ability to protect cortical neurons against 1 μM oligomeric Aβ1-42-induced toxicity. At therapeutic concentrations (100 nM-1 μM), fluoxetine significantly prevented Aβ-induced toxicity in mixed glia-neuronal cultures, but not in pure neuronal cultures. Though to a lesser extent, also sertraline was neuroprotective in mixed cultures, whereas serotonin (10 nM-10 μM) did not mimick fluoxetine effects. Glia-conditioned medium collected from astrocytes challenged with fluoxetine protected pure cortical neurons against Aβ toxicity. The effect was lost in the presence of a neutralizing antibody against TGF-β1 in the conditioned medium, or when the specific inhibitor of type-1 TGF-β1 receptor, SB431542, was added to pure neuronal cultures. Accordingly, a 24 h treatment of cortical astrocytes with fluoxetine promoted the release of active TGF-β1 in the culture media through the conversion of latent TGF-β1 to mature TGF-β1. Unlike fluoxetine, both serotonin and sertraline did not stimulate the astrocyte release of active TGF-β1. We conclude that fluoxetine is neuroprotective against Aβ toxicity via a paracrine signaling mediated by TGF-β1, which does not result from a simplistic SERT blockade.

2016 - The proinflammatory cytokine interleukin 18 regulates feeding by acting on the bed nucleus of the stria terminalis [Articolo su rivista]
Francesconi, Walter; Sánchez Alavez, Manuel; Berton, Fulvia; Alboni, Silvia; Benatti, Cristina; Mori, Simone; Nguyen, William; Zorrilla, Eric; Moroncini, Gianluca; Tascedda, Fabio; Conti, Bruno
abstract

The proinflammatory cytokine IL-18 has central anorexigenic effects and was proposed to contribute to loss of appetite observed during sickness. Here we tested in the mouse the hypothesis that IL-18 can decrease food intake by acting on neurons of the bed nucleus of the stria terminalis (BST), a component of extended amygdala recently shown to influence feeding via its projections to the lateral hypothalamus (LH). We found that both subunits of the heterodimeric IL-18 receptor are highly expressed in the BST and that local injection of recombinant IL-18 (50 ng/ml) significantly reduced c-fos activation and food intake for at least 6 h. Electrophysiological experiments performed in BST brain slices demonstrated that IL-18 strongly reduces the excitatory input on BST neurons through a presynaptic mechanism. The effects of IL-18 are cell-specific and were observed in Type III but not in Type I/II neurons. Interestingly, IL-18-sensitve Type III neurons were recorded in the juxtacapsular BST, a region that contains BST-LH projecting neurons. Reducing the excitatory input on Type III GABAergic neurons, IL-18 can increase the firing of glutamatergic LH neurons through a disinhibitory mechanism. Imbalance between excitatory and inhibitory activity in the LH can induce changes in food intake. Effects of IL-18 were mediated by the IL-18R because they were absent in neurons from animals null for IL-18Rα (Il18ra-/-), which lack functional IL-18 receptors. In conclusion, our data show that IL-18 may inhibit feeding by inhibiting the activity of BST Type III GABAergic neurons.

2015 - P.2.a.012 Co-administration of fluoxetine with acetylsalicylic acid, but not flurbiprofen or celecoxib, for one week shows an antidepressant-like effect [Abstract in Rivista]
Alboni, Silvia; Benatti, Cristina; Tascedda, Fabio; Brunello, Nicoletta
abstract

Increasing evidence is now demonstrating the involvement of the immune system and in particular of their effectors, cytokines, in the development and progression of depression. In particular, it is worth underlying how pro-inflammatory cytokines appear to be increased in blood or brain of patients with major depression (MD) and that pharmacological use of pro-inflammatory cytokines (i.e. interferon alpha) may induce MD. These data suggest a role for inflammation in the pathogenesis of depression and that anti-inflammatory drugs may be used as an adjunctive therapy in the treatment of MD. However, some studies reported contradictory results and suggest that adverse effects may contraindicate the use of anti-inflammatory agents in the treatment of depression. Nevertheless, non-steroidal anti-inflammatory drugs (NSAIDs) can have different mechanisms of action also depending on the dose. This is true for the therapeutic effects as well as for the unwanted side effects. On this basis, the present study aimed at evaluating the behavioural effect of the co-administration of fluoxetine (FLX, 5 mg/kg i.p.) with different NSAIDs in the chronic escape model of depression (CED). The CED model of depression possesses face, construct and pharmacological validity and is based on the induction, and maintenance, of an escape deficit upon exposing rats to unavoidable stressors. We previously demonstrated that, in this model, the stress-induced impaired behaviour can be resolved by one week of treatment with the co-administration of FLX (5 mg/kg/i.p.) plus acetylsalicylic acid (ASA, 45 mg/kg i.p.) but not FLX alone [1]. Here we evaluated the effect of the co-administration of FLX and flurbiprofen (FLB, an inhibitor of Cox-1 and Cox-2, 5 mg/kg, p.o.) or celecoxib (CLX, a selective COX2 inhibitor, 5 mg/kg, p.o.) in the CED model after 7 days of treatment. The co-administration FLX plus ASA (45mg/kg i.p.) was used as a positive control. Morever we tested the behavioral effect of different doses (45, 22.5 and 11.25 mg/Kg i.p.) of ASA as potentiating agent of the effect of fluoxetine. Our study shows that only the co-administration of ASA with FLX reverted the stress-induced condition of escape deficit after 7 days of treatment. Moreover, the amplitude of the antidepressant-like effect was dose dependent. The percentage of the antidepressant response was about 90%, 60% and 40% for animals receiving FLX (5 mg/kg/i.p.) plus ASA at the dose of 45, 22.5 or 11.25 mg/Kg i.p. respectively. Both flurbiprofen and celecoxib, when administered together with FLX for 7 days, failed to induce an antidepressant-like effects in the CED model. Higher dose of FLB (50 mg/Kg p.o.) and CLX (20 mg/Kg p.o.) were also tested, but they were associated to high mortality rate (80% and 25% respectively). These data demonstrated that neither all NSAIDs, nor all doses, may be useful in the treatment of depression while adverse effects can be potentiated or induced by the co-administration with antidepressants. Unraveling the cellular and molecular mechanisms behind the dissimilar behavioral response elicited by different anti-inflammatory drugs can contribute to understand the role of inflammation in the etiopathogensis of MD and to improve patient care. [1] Brunello, N., Alboni, S., Capone, G., Benatti, C., Blom, J.M., Tascedda, F., Kriwin, P., Mendlewicz, J., 2006 Shortened onset of action of antidepressants in major depression using acetylsalicylic acid augmentation: a pilot open-label study. Int Clin Psychopharmacol. 21:227-31.

2015 - RESILIENCY OF CHILDREN AND ADOLESCENTS DURING TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA: THE USE OF 5-HTT AND BDNF POLYMORPHISMS AS BIOMARKERS [Abstract in Rivista]
Blom, Johanna Maria Catharina; Pomicino, L; Migliozzi, C; Montanari, L; Rigillo, Giovanna; Zanazzo, G; Cellini, M; Benatti, Cristina; Tascedda, Fabio
abstract

Background/Objectives: Why are some children more likely than others to develop resilience in the face of similar levels of trauma exposure as compared to others who do not. It is increasingly clear that there are critical roles for predisposing genetic and environmental influences in differentially mediating psychological risk. Resilience differs from traditional concepts of risk and protection in its focus on individual variations in response to comparable experiences. Here, we tested the hypothesis that anxiety and depression as well as neural repair and plasticity related polymorphisms may partly account for the difference in resilience observed during treatment for acute lymphoblastic leukemia (ALL). Design/Methods: Forty-five patients (1-18 yrs old) diagnosed with ALL were enrolled in two centers (protocol AIEOP-BFM-2009) and genotyped for 5HTT and BDNF (val66met). Patients and their family were subjected to a short screening battery, psychosocial testing (PAT2.1) and a specific assessment of their resiliency during treatment. The resiliency scale was composed of three subscales: Sense of Mastery (MAS), Sense of Relatedness (REL), and Emotional Reactivity (REA). Results: Patients with the SL allele of 5HTTLPR had a more compromised score in some areas of resiliency than patients with the LL allele; the presence of the S allele most affected emotional reactivity REA and sense of mastery MAS. Furthermore, age was an important factor, as younger children displayed a reduced trust and tolerance versus their surroundings. This then contributed importantly to an overall reduction in their overall resiliency. Also, resiliency was reduced one year into therapy while vulnerability was significantly enhanced. Conclusion: Genes regulating susceptibility to stress, such as 5HTTLPR and BDNF, may help to predict susceptibility towards the development of resiliency in children and adolescents treated for cancer, and may play a critical role as a predisposing factor in differentially dealing efficiently with the emotional risks related to cancer and its treatment.

2014 - Behavioural and transcriptional effects of escitalopram in the chronic escape deficit model of depression [Articolo su rivista]
Benatti, Cristina; Alboni, Silvia; Blom, Johanna Maria Catharina; Gandolfi, Francesco; Mendlewicz, Julien; Brunello, Nicoletta; Tascedda, Fabio
abstract

The study of depression is facing major challenges: first, the need to develop new drugs with a faster onset of action and second, fulfilling the unmet needs of treatment resistant patients with more effective compounds. The chronic escape deficit (CED) is a valid and useful model of depression and is based on the induction of an escape deficit after exposure of rats to an unavoidable stress. This behavioural model provides a method for evaluating the capacity of a treatment to revert the escape deficit. The majority of antidepressant drugs need to be administered for at least 3-4 weeks in order to revert the escape deficit. A 7-day treatment with escitalopram reverted the stress-induced escape deficit in approximately 50% of the animals. Escitalopram treatment decreased anxiety-related behaviours in stressed animals, by increasing the time spent in the central part of the arena with respect to saline treated stressed animals, without affecting exploratory related behaviours. Gene expression profiling was carried out in the hippocampus to identify new targets associated with the effects of stress or with the different response to escitalopram. By combining a well-validated animal model with gene expression analysis we demonstrated that the CED model may represent a perfect tool for studying treatment-resistant depression.

2014 - Interleukin 18 activates MAPKs and STAT3 but not NF-κB in hippocampal HT-22 cells [Articolo su rivista]
Alboni, Silvia; Montanari, C; Benatti, Cristina; Sanchez Alavez, M; Rigillo, Giovanna; Blom, Johanna Maria Catharina; Brunello, Nicoletta; Conti, B; Pariante, Mc; Tascedda, Fabio
abstract

Interleukin (IL)-18 is a cytokine previously demonstrated to participate in neuroinflammatory processes. Since the components of the IL-18 receptor complex are expressed in neurons throughout the brain, IL-18 is also believed to directly influence neuronal function. Here we tested this hypothesis on mouse hippocampal neurons by measuring the effects of IL-18 on three pathways previously shown to be regulated by this cytokine in non-neuronal cells: the MAPK pathways, p38 and ERK1/2 MAPKs, STAT3 and NF-κB. Experiments were carried out in vitro using the immortalized hippocampal neuronal line HT-22 or in vivo following i.c.v. injection with recombinant mouse IL-18. We showed that IL-18 did not activate NF-κB in HT-22 cells whereas it induced a rapid (within 15min) activation of the MAPK pathways. Moreover, we demonstrated that IL-18 treatment enhanced P-STAT3 (Tyr705)/STAT3 ratio in the nucleus of HT-22 cells after 30-60min of exposure. A similar increase in P-STAT3 (Tyr705)/STAT3 ratio was observed in the whole hippocampus one hour after i.c.v. injection. These data demonstrate that IL-18 can act directly on neuronal cells affecting the STAT3 pathway; therefore, possibly regulating the expression of specific genes within the hippocampus. This effect may help to explain some of the IL-18-induced effects on synaptic plasticity and functionality within the hippocampal system.

2014 - Successful treatment of HIV-1 infection increases the expression of a novel, short transcript for IL-18 receptor α chain [Articolo su rivista]
Nasi, Milena; Alboni, Silvia; Pinti, Marcello; Tascedda, Fabio; Benatti, Cristina; Benatti, Stefania; Gibellini, Lara; DE BIASI, Sara; Borghi, Vanni; Brunello, Nicoletta; Mussini, Cristina; Cossarizza, Andrea
abstract

The importance of interleukin (IL)-18 in mediating immune activation during HIV infection has recently emerged. IL-18 activity is regulated by its receptor (IL-18R), formed by an α and a β chain, the IL-18-binding protein, and the newly identified shorter isoforms of both IL-18R chains. We evaluated gene expression of the IL-18/IL-18R system in peripheral blood mononuclear cells from HIV+ patients. Compared with healthy donors, IL-18 expression decreased in patients with primary infection. The IL-18Rα short transcript expression was strongly upregulated by successful highly active antiretroviral therapy. HIV progression and its treatment can influence the expression of different components of the complex IL-18/IL-18R system.

2013 - Chronic antidepressant treatments resulted in altered expression of genes involved in inflammation in the rat hypothalamus [Articolo su rivista]
Alboni, Silvia; Benatti, Cristina; Montanari, Claudia; Tascedda, Fabio; Brunello, Nicoletta
abstract

To gain insight into the possible immune targets of antidepressant, we evaluated the expression of several inflammatory mediators in the hypothalamus of rats chronically (28 days) treated with the serotonin selective reuptake inhibitor fluoxetine (5mg/kg, i.p.) or the tricyclic compound imipramine (15 mg/kg, i.p.). We focused our attention on the hypothalamus as it plays a key role in determining many of the somatic symptoms experienced by depressed patients. This brain region, critical also for expression of motivated behaviours, participates in the control of the hypothalamic-pituitary-adrenal axis activity and in stress response as well as coordinates physiological functions such as sleep and food intake that have been found altered in a high percentage of depressed patients. Notably, hypothalamus is a key structure for brain cytokine expression and function as it integrates signals from the neuro, immune, endocrine systems. By means of quantitative Real Time PCR experiments we demonstrated that a chronic treatment with either fluoxetine or imipramine resulted in a reduction of IL-6 and IFN-γ mRNAs and increased IL-4 mRNA expression in the rat hypothalamus. Moreover, we demonstrated that hypothalamic expression of members of IL-18 system was differentially affected by chronic antidepressant treatments. Chronically administered fluoxetine decreased IL-8 and CX3CL1 hypothalamic expression, while a chronic treatment with imipramine decreased p11 mRNA. Our data suggest that a shift in the balance of the inflammation toward an anti-inflammatory state in the hypothalamus may represent a common mechanism of action of both the chronic treatments with fluoxetine and imipramine.

2013 - Interferon alpha exposure increases the expression of the enzymes belonging to the kynurenine pathway in an in vitro model of human neurons: SH-SY5Y cells [Abstract in Rivista]
Alboni, Silvia; Benatti, Cristina; Claudia, Montanari; Benatti, Stefania; Tascedda, Fabio; Cannazza, Giuseppe; Pariante Carmine, M; Brunello, Nicoletta
abstract

The past two decades have witnessed a burgeoning area of pre-clinical and clinical research linking psychiatric illnesses – particularly major depression (MD) – to activation of the inflammatory immune system. One of the stronger evidence supporting a causal role for inflammation in leading MD comes from reports indicating that depressive symptoms frequently develop in patients undergoing immunotherapy with cytokines, such as interferon (IFN)-α, for the treatment of malignancies or chronic viral infection. Although INF-alpha- induced effects on the brain made of IFN-α a model to study the influence of pro-inflammatory cytokines in the CNS and behavior the molecular mechanisms underlying these effects are far from being fully understood. It has been proposed that IFN-α may contribute to the etiology of MD by inducing indolamine 2,3-dioxygenase (IDO) expression and thus unbalancing in the tryptophan/kynurenine metabolism toward the production of neurotoxic metabolites and\or reducing serotonin (5-HT) availability. IDO catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway (KP). Kynurenine, the initial product of tryptophan degradation, is further catalysed into neurotoxic end-products through steps catalyzed by kynurenine 3-monooxygenase (KMO) and kynureninase (Kynu). However, Kynurenine can also be catabolised by kynurenine aminotransferase (KAT), into kynurenic acid, a potentially neuroptotective agent. A role for a disturbance in the equilibrium between neurotoxic/ neuropoptective end KP endproducts producing an alteration in the neuroprotective–neurodegenerative balance in the brain of patients with MD, has been proposed in the neurodegeneration hypothesis of depression. Given that we previously demonstrated that IFN-α induces toxic effects in an in vitro model of human neurons (human SH-SY5Y neuroblastoma cells) we were aim to investigate the effects of IFN-α on KP in these cells. Our studies show that IFN-α exposure increased the expression of all the kynurenergic enzymes investigated (IDO, KMO, Kynu and KAT). More particularly strongly induced the expression of IDO mRNA (more than 900 –fold) in SH-SY5Y cells. Similar effects on kynurenergic enzyme expression were also observed when SH-SY5Y cells where differentiated with all-trans retinoic acid (in presence of neurotrophic support and in serum deprived conditions). We also demonstrated that INF-α decreased 5-HT levels whereas increased the kynurenine levels in the medium of both differentiated as well not differentiated SH-SY5Y cells.

2013 - N-acetyl-cysteine prevents toxic oxidative effects induced by IFN-α in human neurons. [Articolo su rivista]
Alboni, Silvia; Gibellini, Lara; Montanari, Claudia; Benatti, Cristina; Benatti, Stefania; Tascedda, Fabio; Brunello, Nicoletta; Cossarizza, Andrea; Pariante, Carmine M.
abstract

Currently IFN-α is widely used for effective treatment of viral infections and several malignancies. However, IFN-α can cause neuropsychiatric disturbances and mental impairments, including fatigue, insomnia, depression, irritability and cognitive deficits. Molecular and cellular mechanisms leading to such side-effects are still poorly understood. Neurons seem to be an important target in mediating cellular effects induced by exposure to this cytokine, but so far little is known about IFN-α-induced effects on these cells. We have investigated the ability of IFN-α (2-100 ng/ml) to induce damage and toxicity to the human neuroblastoma SH-SY5Y cell line, commonly used for studying such phenomena, and the mechanisms underlying these effects. After 24 h treatment, IFN-α increased mitochondrial activity, whereas cell density was reduced in a dose- and time-dependent manner. This effect did not depend on reduced cell proliferation, but rather the activation of apoptosis, as revealed by an increased Bax:Bcl-2 mRNA ratio after 72-h IFN-α exposure. At this time-point, IFN-α also reduced the expression of the brain-derived neurotrophic factor gene, and induced an increase in reactive oxygen species (ROS). A co-treatment with N-acetyl-cysteine (NAC; 5 mm), a potent antioxidant and mitochondrial modulator, was able to counteract all of these IFN-α-induced effects. These findings demonstrated that IFN-α induces neurotoxicity and apoptosis that is, in part, very likely due to mitochondrial damages and production of ROS. We suggest that NAC, already tested for the treatment of psychiatric disorders, may be useful to prevent IFN-α-induced central side-effects in a safe and effective way.

2012 - Interferon-alpha exposure increases the expression of enzymes of the kynurenine pathway and induces apoptosis in a model of human neurons [Abstract in Rivista]
Alboni, Silvia; Benatti, Cristina; Montanari, Claudia; Tascedda, Fabio; C. M., Pariante; Brunello, Nicoletta
abstract

Major depression (MD) is associated with a profound unbalance between the nervous-, the endocrine- and the immune- systems. This suggests the possibility that molecules that regulate the homeostasis of these systems may contribute to the development of MD. For instance, some cytokines, important neuro-endocrineimmuno modulators, have been proposed to have a role in MD as supported by the observation that activation of immune system with therapeutically used cytokines may induce MD. IFN-a is an innate immune cytokine with potent antiviral and anti-proliferative properties that is used to treat viral infections, such as hepatitis C, and certain cancers. Despite therapeutic efficacy in these illnesses, it has been observed that IFN-a exposure may be associated with important side effects including neuropsychological and behavioural changes that overlap with MD. Although IFN-a- induced effects on the brain make IFN-alpha a model to study the influence of pro-inflammatory cytokines in the CNS and behavior, the molecular mechanisms underlying these effects are far from being fully understood. It has been proposed that cytokines may contribute to the etiology of MD by inducing indolamine 2,3-dioxygenase (IDO) expression. IDO catalyzes the initial rate-limiting step in tryptophan (TPR) degradation along the kynurenine pathway (KP). Kynurenine, the initial product of TPR degradation, is further catalysed into neurotoxic end-products through steps catalyzed by kynurenine 3-monooxygenase (KMO) and kynureninase (Kynu). However, Kynurenine can also be catabolised by kynurenine aminotransferase (KAT), into kynurenic acid, a potentially neuroprotective agent. A role for a disturbance in the KP in the neuroprotective–neurodegenerative balance in the brain of patients with MD, has been proposed in the neurodegeneration hypothesis of depression. This prompted us to investigate the effects of IFN-a on the expression of the IDO, KMO, Kynu and KAT mRNAs in an in vitro model of human neurons: SH-SY5Y (human neuroblastoma) cells. We also evaluated the IFN-a-induced effect on cell viability and number in these cells. Finally, because cell number results from the balance between cell proliferation and cell elimination, we measured cell proliferation and apoptosis in SH-SY5Y cells after IFN-a exposure. The same experiments were performed in differentiated SH-SY5Y cells with retinoic acid (RA) and Brain- Derived Neurotrophic Factor (BDNF). Our studies show that IFN-a exposure increased the expression of all the kynurenergic enzymes investigated (with an unbalance of the KP toward the synthesis of neurotoxic end-products) and in more particularly strongly induced the expression of IDO mRNA (more than 900-fold) in SH-SY5Y cells. We also demonstrated that IFN-a reduced in a dose- (2, 20, 50 and 100 ng/ml) and time- (24, 48 and 72 hrs) dependent manner the cell number and induces apoptosis in SH-SY5Y cells. Similar results were obtained in SHSY5Y RA/BDNF differentiated cells. Together our results clearly enlighten the cytotoxic effects of IFN-a in this in vitro model of human neurons. Moreover, our findings provide further information on the molecular pathways involved in cytokine-induced effects in the brain and add a piece to the puzzle of what and how these factors or pathways may contribute to the pathogenesis of MD.

2012 - Transcriptional profiles underlying vulnerability and resilience in rats exposed to an acute unavoidable stress [Articolo su rivista]
Benatti, Cristina; Valensisi, Cristina; Blom, Johanna Maria Catharina; Alboni, Silvia; Montanari, Claudia; Ferrari, Francesco; Tagliafico, Enrico; Julien, Mendlewicz; Brunello, Nicoletta; Tascedda, Fabio
abstract

A complex interplay between gene and environment influences the vulnerability or the resilience to stressful events. In the acute escape deficit (AED) paradigm, rats exposed to an acute unavoidable stress (AUS) develop impaired reactivity to noxious stimuli. Here we assessed the behavioral and molecular changes in rats exposed to AUS. A genome-wide microarray experiment generated a comprehensive picture of changes in gene expression in the hippocampus and the frontal cortex of animals exposed or not to AUS. Exposure to AUS resulted in two distinct groups of rats with opposite behavioral profiles: one developing an AED, called “stress vulnerable,” and one that did not develop an AED, called “stress resilient.” Genome-wide profiling revealed a low percentage of overlapping mechanisms in the two areas, suggesting that, in the presence of stress, resilience or vulnerability to AUS is sustained by specific changes in gene expression that can either buffer or promote the behavioral and molecular adverse consequences of stress. Specifically, we observed in the frontal cortex a downregulation of the transcript coding for interferon-β and leukemia inhibitory factor in resilient rats and an upregulation of neuroendocrine related genes, growth hormone and prolactin, in vulnerable rats. In the hippocampus, the muscarinic M2 receptor was downregulated in vulnerable but upregulated in resilient rats. Our findings demonstrate that opposite behavioral responses did not correspond to opposite regulatory changes of the same genes, but resilience rather than vulnerability to stress was associated with specific changes, with little overlap, in the expression of patterns of genes.

2011 - Central effects of a local inflammation in three commonly used mouse strains with a different anxious phenotype [Articolo su rivista]
Benatti, Cristina; Alboni, Silvia; Montanari, Claudia; Caggia, Federica; Tascedda, Fabio; Brunello, Nicoletta; Blom, Johanna Maria Catharina
abstract

As in humans, genetic background in rodents may influence a peculiar set of behavioural traits such as sensitivity to pain and stressors or anxiety-related behaviours. Therefore, we tested the hypothesis that mice with different genetic backgrounds [outbred (CD1), inbred (C57BL/6J) and hybrid (B6C3F1) adult male mice] display altered reactivity to pain, stress and anxiety related behaviours. We demonstrated that B6C3F1 mice displayed the more anxious phenotype with respect to C57BL/6J or CD1 animals, with the latter being the less anxious strain when tested in an open field and on an elevated plus maze. No difference was observed across strains in thermal sensitivity to a radiant heat source. Mice were then treated with a sub-plantar injection of the inflammatory agent Complete Freund's Adjuvant (CFA), 24h later they were hyperalgesic with respect to saline exposed animals, irrespective of strain. We then measured intra-strain differences and CFA-induced inter-strain effects on the expression of various genes with a recognized role in pain and anxiety: BDNF, IL-6, IL-1β, IL-18 and NMDA receptor subunits in the mouse thalamus, hippocampus and hypothalamus. The more anxious phenotype observed in B6C3F1 hybrid mice displayed lower levels of BDNF mRNA in the hippocampus and hypothalamus when compared to outbred CD1 and C57BL/6J inbred mice. CFA led to a general decrease in central gene expression of the evaluated targets especially in CD1 mice, while BDNF hypothalamic downregulation stands out as a common effect of CFA in all three strains evaluated

2011 - Constitutive and LPS-regulated expression of interleukin-18 receptor beta variants in the mouse brain [Articolo su rivista]
Alboni, Silvia; Montanari, Claudia; Benatti, Cristina; Blom, Johanna Maria Catharina; Simone, Ml; Brunello, Nicoletta; Caggia, Federica; Guidotti, G; Marcondes, Mc; Sanchez Alavez, M; Conti, B; Tascedda, Fabio
abstract

Interleukin (IL)-18 is a pro-inflammatory cytokine that is proposed to be involved in physiological as well as pathological conditions in the adult brain. IL-18 acts through a heterodimer receptor comprised of a subunit alpha (IL-18Rα) required for binding, and a subunit beta (IL-18Rβ) necessary for activation of signal transduction. We recently demonstrated that the canonical alpha binding chain, and its putative decoy isoform, are expressed in the mouse central nervous system (CNS) suggesting that IL-18 may act on the brain by directly binding its receptor. Considering that the co-expression of the beta chain seems to be required to generate a functional receptor and, a short variant of this chain has been described in rat and human brain, in this study we have extended our investigation to IL-18Rβ in mouse. Using a multi-methodological approach we found that: (1) a short splice variant of IL-18Rβ was expressed in the CNS even if at lower levels compared to the full-length IL-18Rβ variants, (2) the canonical IL-18Rβ is expressed in the CNS particularly in areas and nuclei belonging to the limbic system as previously observed for IL-18Rα and finally (3) we have also demonstrated that both IL-18Rβ isoforms are up-regulated in different brain areas three hours after a single lipopolysaccharide (LPS) injection suggesting that IL-18Rβ in the CNS might be involved in mediating the endocrine and behavioral effects of LPS. Our data highlight the considerable complexity of the IL-18 regulation activity in the mouse brain and further support an important central role for IL-18.

2011 - Stress induces altered CRE/CREB pathway activity and BDNF expression in the hippocampus of glucocorticoid receptor-impaired mice [Articolo su rivista]
Alboni, Silvia; Tascedda, Fabio; Corsini, Daniela; Benatti, Cristina; Caggia, Federica; Capone, Giacomo; Barden, N; Blom, Johanna Maria Catharina; Brunello, Nicoletta
abstract

The gene coding for the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) is a stress-responsive gene. Changes in its expression may underlie some of the pathological effects of stress-related disorders like depression. Data on the stress-induced regulation of the expression of BDNF in pathological conditions are rare because often research is conducted using healthy animals. In our experiments, we used transgenic mice with glucocorticoid receptor impaired (GR-i) expression in the hypothalamus created as a tool to study the neuroendocrine changes occurring in stress-related disorders. First, under basal condition, GR-i mice displayed lower levels of BDNF exons IX and IV and decreased CRE(BDNF) binding activity with respect to wild-type (WT) mice in the hippocampus. Then, we exposed GR-i and WT mice to an acute restraint stress (ARS) to test the hypothesis that GR-i mice display: 1] different ARS induced expression of BDNF, and 2] altered activation of signaling pathways implicated in regulating BDNF gene expression in the hippocampus with respect to WT mice. Results indicate that ARS enhanced BDNF mRNA expression mainly in the CA3 hippocampal sub-region of GR-i mice in the presence of enhanced levels of pro-BDNF protein, while no effect was observed in WT mice. Moreover, ARS reduced CREB signaling and binding to the BDNF promoter in GR-i mice but enhanced signaling and binding, possibly through ERK1/2 activation, in WT mice. Thus, life-long central GR dysfunction resulted in an altered sensitivity at the transcriptional level that may underlie an impaired response to an acute psycho-physical stress.

2010 - Time-dependent effects of escitalopram on brain derived neurotrophic factor (BDNF) and neuroplasticity related targets in the central nervous system of rats [Articolo su rivista]
Alboni, Silvia; Benatti, Cristina; Capone, Giacomo; Corsini, Daniela; Caggia, Federica; Tascedda, Fabio; Mendlewicz, J; Brunello, Nicoletta
abstract

Chronic treatment with antidepressants affects several proteins linked to neuroplasticity, particularly brain derived neurotrophic factor (BDNF): this leads eventually to their therapeutic effects. It is possible that also for putative early therapeutic onset, antidepressants may act by promoting cellular adaptations linked to neuroplasticity. Escitalopram, known to be already effective in preclinical models of depression after 7 days, allowed us to investigate whether two effective treatment regimens (7 and 21 days) may contribute to synaptic plasticity by acting on BDNF signalling. We focused our attention on two regulators of BDNF transcription, CREB and CaRF (calcium responsive factor), and on kinases, CaMKII, ERK1/2 and p38 MAPK, linked to BDNF that play a distinctive role in synaptic plasticity. We evaluated whether the effects of escitalopram on these targets may be different in brain areas involved in the depressive symptomatology (hippocampus, frontal and prefrontal cortex). Here we demonstrate that escitalopram regulates intracellular pathways linked to neuroplasticity at both the time points evaluated in an area-specific manner. While the two escitalopram-treatment regimens failed to affect gene expression in the rat frontal cortex, 7days of treatment with escitalopram activated intracellular pathways linked to BDNF and increased the levels of Pro-BDNF in the rat prefrontal cortex. Moreover, 21 days of treatment with escitalopram decreased CREB/BDNF signalling while increasing p38 levels in the rat hippocampus. Even if further experiments with different antidepressant strategies will be needed, our data suggest that escitalopram efficacy may be mediated by early and late effects on synaptic plasticity in selective brain areas.

2009 - Early neonatal inflammation affects adult pain reactivity and anxiety related traits in mice: genetic background counts [Articolo su rivista]
Benatti, Cristina; Alboni, Silvia; Capone, Giacomo; Corsini, Daniela; Caggia, Federica; Brunello, Nicoletta; Tascedda, Fabio; Blom, Johanna Maria Catharina
abstract

Protracted or recurrent pain and inflammation in the early neonatal period may cause long-lasting changes in central neural function. However, more research is necessary to better characterize the long-term behavioral sequelae of such exposure in the neonatal period. Objectives: (1) to study whether timing of postnatal exposure to persistent inflammation alters responsiveness to thermal pain in the adult animal; (2) to assess whether animals experiencing early postnatal chronic inflammation display altered anxiety related behavior; (3) to study the importance of genetic background. Newborn mice (outbred strain, CD1 and F1 hybrid strain, B6C3F1) received an injection of Complete Freund's Adjuvant (CFA) or saline on either postnatal day 1 or 14 (PND1; PND14) into the left hind paw. Pain to radiant heat and anxiety were examined in 12-week-old adult animals. Adult baseline PWL was significantly decreased in CD1 mice exposed to CFA on PND 1 and 14 as compared to their saline treated counterparts. B6C3F1 mice exposed to CFA on PND14 showed markedly reduced baseline PWL compared to the PND14 saline group. Persistent inflammation experienced by B6C3F1 mice on PND1 failed to affect baseline adult thermal responsiveness. Adult mice, CD1 and B6C3F1, displayed low anxiety traits only if they had been exposed to persistent inflammation on PND1 and not on PND14. Our research suggests a role for genetic background in modulating long-term behavioral consequences of neonatal persistent inflammation: the data support the hypothesis that pain experienced very early in life differentially affects adult behavioral and emotional responsiveness in outbred (CD1) and hybrid mice (B6C3F1).

2009 - Gene expression profile of the hippocampus of a behavioural model of depression [Abstract in Rivista]
Valensisi, Cristina; Caggia, Federica; Alboni, Silvia; Benatti, Cristina; Ferrari, F; Mendlewicz, J; Blom, Johanna Maria Catharina; Brunello, Nicoletta; Tascedda, Fabio
abstract

Although the neurobiological basis of depression has not been fully elucidated, numerous studies have emphasized that in the etiology of depression stress may be the most significant cause, together with genetic vulnerability. Stress induces a coordinated and complex response that is adaptive and integral to survival. The brain's ability to adapt and change over time is refered to neuroplasticity and long-term plasticity in the brain requires changes in gene expression. However, exposure to intense or chronic stressors leads to an increased risk for the development of stress-related disorders including major depression. Numerous studies demonstrate that neuronal atrophy and loss of plasticity occur in hippocampus in response to stress and depression. Therefore, the hippocampal region may play a central role in depressive illness. Likewise changes in gene expression underlying the plasticity of hippocampal structures appear to be relevant in undenstanding the molecular and cellular mechanisms involved in the etiology as well as the treatment of depression, and the mechanisms leading vulnerability or resilience to stress. In fact, humans display a remarkable heterogeneity in their responses to stress and adversity. Although we are beginning to understand how maladaptive neurobiological changes may contribute to depression, relatively little is known about the molecular mechanisms that may underlie stress resilience. Here we set out to investigate the changes in the gene expression profile underlying the effects of stress on the hippocampus using a behavioural paradigm of depression, the chronic escape deficit model [1], which is based on the modified reactivity of rats to external stimuli, the escape deficit, induced by exposure to intense and unavoidable stress. The chronic escape deficit model starts as an acute escape deficit which can be indefinitely sustained by repeated administration of mild stressors. This approach has proved to be a valid and useful model of depression because it consider depressive symptoms like behavioural despair. We performed gene expression profiling in the rat hippocampus, using GeneChip Rat Exon Array (Affymetrix). Using this new platform we carried out analyses of gene expression on three different levels: gene, transcript and exon level analyses. The behavioural results showed that exposure to intense and unavoidable stressful procedure induced escape deficit only in 60% of them. Whereas the animals remaining display a behaviour apparently identical to control animals which did not undergo the stressful procedure. Comparing gene expression profiles and performing functional analysis on differently expressed genes we have indicated multiple pathways that may be involved in the underlying mechanisms of stress condition associated with escape deficit. Moreover we identified possible cellular functions and biological processes that could represent targets that may contribute to mediate the effects of stress on the hippocampal plasticity. Such as, gene expression profiling of stress-vulnerable and stress-resilient animals revealed distinct transcriptional profiles, suggesting that resilient behaviour represents an active neurobiological process and not simply the absence of vulnerability.

2009 - Microarray analysis in hippocampus of rats treated with escitalopram in the chronic escape deficit model of depression [Abstract in Rivista]
Caggia, Federica; Valensisi, Cristina; Alboni, Silvia; Benatti, Cristina; Corsini, Daniela; F., Ferrari; Tagliafico, Enrico; J., Mendlewicz; Tascedda, Fabio; Brunello, Nicoletta
abstract

Currently, the biological bases of depression and the molecular mechanisms underlying antidepressant action are not completely understood. Valuable tools to better understand the pathophysiology of this disease are behavioural models of depression eventually combined with genome-wide gene expression analysis. The Chronic Escape Deficit (CED) is a validated behavioural model of depression, based on the induction of an escape deficit after exposure of rats to an unavoidable stress. This model allows to evaluate the capacity of a treatment to revert the escape deficit. The antidepressant drugs tested in CED model need to be administered for at least 3−4 weeks in order to revert the escape deficit [1,2]. In this study, we demonstrated that already after one week of treatment with Escitalopram, a widely used SSRI, 50% of the animals responded reverting the escape deficit induced. Moreover, the other 50% of treated animals did not respond also after 3−4 weeks of treatment. Since in the CED model the behavioural alteration is induced by stress application and reverted by escitalopram treatment in only half of animals, the aims of our study were two fold: (i) to investigate transcriptional changes activated by stress; (ii) to study the different gene expression pattern involved into mechanisms of the response and not response to the pharmacological treatment. To address these issues we performed a microarray experiment in the rat hippocampus using Affymetrix GeneChip Rat Exon 1.0 ST evaluating both gene-level and exon-level expression profiling on the whole genome. Total RNA extracted from hippocampus of each animal was utilized to chip a single array using the Affymetrix protocols. 20 single arrays were utilized for data analysis and divided into five replicates for each experimental group (control, stress, stress-escitalopram responders and stress escitalopram-not responders). Using two parallel analyses (gene level and exon level) of raw data files carried out in Expression Console software using iterPLIER algorithms, we identified genes and exons that were differentially regulated in each pairwise comparison considered. The exons identified in this study were examinated for their biological association to gene ontology (GO) categories using eGOn software. Moreover, all exons differentially expressed were also uploaded into Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) in order to identify molecular pathways and functions related to stress and escitalopram response. Our results suggest that stress may exert a negative effect on gene transcription since the largest number of genes was downregulated. Moreover from our data it seems that a different pattern of gene expression exhibits between animals that respond and that did not respond to escitalopram treatment. Functional analysis of exon dataset, arising from stress protocol and escitalopram treatment, reflects interesting different biological features. More specifically, the biological functions regard both molecular and cellular functions, such as cellular growth and proliferation, gene expression and signal transduction, as well as involvement of central neurotransmission and immune response. We believe that this pharmacogenomic approach will be helpful to understand the molecular mechanisms involved in the pathogenesis of depression as well as in the response to antidepressant drugs.

2008 - Impaired stress-induced regulation of brain derived neurotrophic factor expression in hippocampus of glucocorticoid receptor impaired mice: Model of depression [Abstract in Rivista]
Alboni, Silvia; Corsini, Daniela; Caggia, Federica; Benatti, Cristina; Capone, Giacomo; Barden, N; Blom, Johanna Maria Catharina; Tascedda, Fabio; Brunello, Nicoletta
abstract

Objective: The gene codifying for the neurotrophin Brain Derived Nurotrophic Factor (BDNF) is a stress-responsive gene and alteration in its expression may be important in producing some of the pathophysiological effects of stress in the hippocampus as seen in stress-related pathologies like depression. While the effects of stress procedures on the regulation of BDNF expression was widely investigated in hippocampus of healthy control animals, the stress-induced effects on BDNF hippocampal expression in “pathological” condition are still lacking. In order to deepen our knowledge in the understanding of the effects of an acute stressful procedure on molecular targets of synaptic plasticity we used transgenic mice with impaired glucocorticoid receptor (GR-i) expression that represent an animal model of depression. The hypothesis was tested that a single period of restraint stress (6 hours) affects BDNF mRNA expression in the hippocampus of GR-i mice differently than in wild-type (WT) mice. Methods: Using real time RT-PCR we evaluated the effects of a 6 hours acute stress on the levels of BDNF coding exon VIII and the activity regulated BDNF exon IV mRNA. In the WT and in the GR-i animals, the hippocampal levels of the two BDNF exons, immediately after the stress ended, were significantly lower in stressed animals with the respect to respective control unstressed animals. However, for the BDNF exon IV mRNA the reduction is most pronounced inWT animals and two-way ANOVA followed by Bonferroni posttest revealed a significant interaction between stress response and genotype at the level of BDNF exon IV mRNA expression. Results: The BDNF gene is a very complex gene regulated by a wide array of stimuli and signalling pathways. An electophoresis mobility shift assay (EMSA) was used to study DNA-binding activity of two transcription factors with an important role in controlling synaptic plasticity most likely trough an involvement BDNF: the cAMP responsive element binding (CREB) protein and the nuclear factor kB (Nf-kB). Taken together, our results show a different binding activity of these transcription factors in GR-i mice with respect to WT mice following acute stressful conditions. Conclusion: The identification of the molecular mechanisms activated by stress in GR-i mice model of depression may contribute to the development of new strategies that reducing neuron vulnerability to stress and prevent neurophatologocal alteration in the hippocampus.

2008 - Microarray analysis of the chronic escape deficit model of depression: Effects of escitalopram treatment in hippocampus [Abstract in Rivista]
Caggia, Federica; Valensisi, Cristina; Alboni, Silvia; Benatti, Cristina; Corsini, Daniela; Ferrari, F; Tagliafico, Enrico; Mendlewicz, J; Brunello, Nicoletta; Tascedda, Fabio
abstract

Objective: Currently, the biological bases of depression and the molecular mechanisms underlying antidepressant action are not completely understood. Behavioural models of depression and genome-wide gene expression analysis can be relevant to better understand the pathophysiology of this disease. Chronic escape deficit is a valid and useful model of depression and is based on the induction of an escape deficit after exposure of rats to unavoidable stress. This behavioural model allows to evaluate the capacity of a treatment to revert the escape deficit. The majority of antidepressant drugs need to be administered for at least 3−4 weeks in order to revert the escape deficit. In this study, we demonstrated that only one week of treatment with Escitalopram, a widely used SSRI, is effective in the chronic escape deficit model of depression. Also, our study demonstrated that only 50% of the animals receiving ESC responded to the treatment. The mechanisms underlying the action of escitalopram are still poorly understood and the molecular targets and pathways involved remain to be identified. In order to identify the biological target involved in the response to escitalopram, we performed a microarray experiment using the chronic escape deficit model of depression after a 7 day treatment with escitalopram. Methods: Gene expression patterns in the rat hippocampus were analyzed using Affymetrix GeneChip Rat Exon 1.0 ST evaluating both gene-level and exon-level expression profiling on the whole genome. Total RNA extracted from hippocampus of each treated animal was utilized to chipping a single array using the Affymetrix protocols. 20 single arrays were utilized for data analysis and divided into five replicates for each experimental group (naive, stress, escitalopram responders and not responders). With two parallel analyses (gene level and exon level) of raw data files carried out in Expression Console software using iterPLIER algorithms, we identified various transcripts that were differentially regulated in each pairwise comparison. In order to identify biological processes and signalling networks regulated by escitalopram response, we performed a functional analysis using Ingenuity web tool. Results: Functional annotation of selected genes reflected interesting different biological features between escitalopram responders and not responders. More specifically, the biological functions regard cellular growth and proliferation, gene expression and signal transduction. Conclusion: We believe that this pharmacogenomic approach will be helpful to understand the molecular mechanisms involved in the pathogenesis of depression as well as in the response to antidepressant drugs.

2008 - Molecular effects of subchronic andchronic treatment with escitalopram inthe rat central nervous system [Abstract in Rivista]
Benatti, Cristina; Alboni, Silvia; Capone, Giacomo; Corsini, Daniela; Caggia, Federica; Blom, Johanna Maria Catharina; Tascedda, Fabio; Brunello, Nicoletta
abstract

A clear understanding of the mechanisms behind depressionand its treatment is a critical issue for amelioratethe effectiveness of existing antidepressants. Acutely,antidepressant drugs increase synaptic concentrations ofmonoamines, but clinical efficacy requires several weeksof continuous treatment, proposing a key role for timedependentneural adaptations, perhaps induced by acutesynaptic actions, in their therapeutic efficacy.Escitalopram is the S(+) enantiomer of citalopram, oneof the most widely prescribed serotonin selective reuptakeinhibitor (SSRIs) antidepressants. In the chronic mildstress model of depression sucrose intake was alreadynormalized after one week of treatment.We evaluated the effects of a subchronic or a chronictreatment with escitalopram on expression levels of someof the main targets of antidepressant drugs such as theneurotrophin Brain Derived Neurotrophic Factor (BDNF),the transcription factors cAMP Response Element Binding(CREB) [1] Protein and Calcium Responsive Factor(CaRF).Sprague-Dawley rats were treated for 7 days (subchronic)or 21 days (chronic) with either escitalopram(10 mg/kg die i.p) or saline (1 mL/kg die); BDNF, CREBand CaRF mRNAs were evaluated using RNAse ProtectionAssay in hippocampus and prefrontal cortex.No difference was observed on BDNF, CREB andCaRF expression in the hippocampus of rats treatedsubchronically with escitalopram with respect to the grouptreated with saline. In contrast a significant inductionof BDNF mRNA was observed in prefrontal cortexof escitalopram-treated animals with respect to salinetreated ones. CaRF expression patterns were similar.Escitalopram for 7 days caused a significant induction ofCaRF mRNA with respect to the group treated with saline(p<0.05; Dunnett t-test), on the other hand CREB mRNAremained unaffected. Following a chronic treatment withEscitalopram, BDNF, CREB and CaRF mRNA levels weresignificantly decreased with respect to the group treatedwith saline in hippocampus (p<0.05; Dunnett t-test),while a 21 day treatment with escitalopram failed toproduce changes in gene expression in prefrontal cortex.These results showed that escitalopram is able todifferentially affect BDNF, CREB and CaRF expressionwith respect to treatment duration and that the observedeffects are area-specific.Consequently, to further investigate the possiblemolecular mechanisms underlying the observed effectson gene expression we evaluated by western blottingsome of the main signalling pathways regulating CREB aswell as BDNF expression, such as p38 MAPK (Mitogen-Activated Protein Kinase), CaMKII (Calcium CalmodulineKinase), ERK 1/2 (Extracellular Signal-Regulated Kinase)and CREB itself [1].Our study demonstrates that:1. A subchronic treatment with escitalopram inducesBDNF and CaRF expression in prefrontal cortexprobably through activation of p38 MAPK signallingpathway.2. A 21 day escitalopram treatment reduces hippocampalBDNF, CaRF, CREB gene expression and also CREBphosphorylated nuclear levels.Spatially distinct signalling pathways may be involvedin mediating the differential effect on gene expressionobserved following either a subchronic or a chronictreatment with escitalopram.

2007 - Combined effect of antidepressant and anti-inflammatory drugs in an animal model of depressio [Abstract in Rivista]
Brunello, Nicoletta; Alboni, Silvia; Benatti, Cristina; Corsini, Daniela; Capone, Giacomo; Tascedda, Fabio; J., Mendlewicz
abstract

The delay in the onset of action of antidepressants remains one of the unsolved issues in the treatment of depression despite the availability of safe and effective drugs. It has been known for years that depression may share identical symptoms as those in inflammatory reaction and that immune function and inflammation markers are altered in psychiatric patients. This suggests to use anti-inflammatory drugs as an adjunctive therapy for depression. We previously demonstrated that the combination of acetylsalicylic acid (ASA) with Fluoxetine (FLX) accelerated and potentiated the effect of the antidepressant in the chronic escape model of depression (Brunello et al., 2006). These results, together with preliminary clinical data in major depressed nonresponder patients, suggest that ASA might accelerate the onset of action of SSRIs (Mendlewicz et al., 2006). Preclinical and clinical studies have recently reported that Escitalopram (ESC), the active enantiomer of citalopram, shows a faster onset of action compared to other antidepressants. The aim of our study was to compare the effect of one week of combined treatment with ESC plus ASA vs. ESC alone in the chronic escape deficit model of depression. Significant response after one week of treatment was present in about 50% of the animals receiving ESC (10 mg/kg/day) alone and in about 75% of the rats receiving ESC plus ASA (45 mg/kg/day). These results suggest that the co-administration of ASA with ESC increased the response to treatment in reverting the behavioural despair induced by stress in rats, thus confirming the specific effect of combined therapy.

2007 - Farmacologia e neurobiologia nei disturbi d'ansia e nella depressione [Articolo su rivista]
Brunello, Nicoletta; Benatti, Cristina
abstract

2006 - Acetylsalicylic acid accelerates the antidepressant effect of fluoxetine in a rat model of depression [Abstract in Rivista]
Capone, Giacomo; Alboni, Silvia; Benatti, Cristina; Tascedda, Fabio; Blom, Johanna Maria Catharina; J., Mendlewicz; Brunello, Nicoletta
abstract

Depression currently ranks fourth among the major causesof disability worldwide and by 2020, it is estimated thatunipolar major depression will rank second as a sourceof lost disability-adjusted life years (DALYs) worldwide(Murray and Lopez, 1997). To date however no singleagent is effective in all patients treated, probably due tothe different neurobiological alterations occurring for thedisorder and to individual differences in pharmacogeneticand pharmacodynamic parameters.Different therapies have been proposed to amelioratethe clinical responses of antidepressant drugs throughaugmentation or combination strategies.Another achievement in the development of newtreatments is to reduce the latency of clinical effect ofantidepressant drugs known to be characterized by 4-6weeks lag phase.Evidence has accumulated suggesting that majordepression is associated with dysfunction of inflammatorymediators and that psychiatric symptoms may occur duringinflammatory diseases. Moreover antidepressants show ananti-inflanmlatory action possibly related to their clinicalefficacy. In fact, an improvement in psychiatric symptomshas been recently reported in patients treated with antiinflammatorydrugs for other indications.These data imply that inflammation may be involved inthe pathogenesis of depression and that anti-inflanm~atorydrugs may be used as an adjunctive therapy.Among anti-inflammatory drugs acetylsalicylic acid(ASA) has been shown to act on the brain serotonergicsystem and to have a neuroprotective effect in vitro aswell as in vivo.Aim of the present study was to evaluate the effectsof combined treatment fluoxetine (FLX) plus ASA andASA alone in a behavioural model of depression:the chronic escape deficit (Gambarana et al., 2001).The chronic escape deficit model is based on themodified reactivity of rats to external stimuli inducedby exposure to unavoidable stress and allows evaluatingthe capacity of a treatment to revert the condition ofescape deficit. In this model, FLX alone needs to beadministered for at least 3 weeks in order to revert thiscondition.Our results showed that ASA (45 mg/kg) did not possessantidepressant properties in the chronic escape deficitmodel at any time tested. A combined treatment of FLX(5 mg/kg) and ASA (45 mg/kg) completely reverted thecondition of escape deficit as early as after 7 days, theeffect being already partially present after 4 days. Theeffect was maintained after 14 and 21 days of treatment.In the same experimental condition the effect of FLX(5 mg/kg) was significant only at 21 days, as previouslydemonstrated by other groups.The exact nature of the mechanisms underlying theabove behavioural effects are still unknown, neverthelessseveral hypotheses can be formulated. Further biochemicaland genetic researches could help to clarify the targets ofaction of the combined treatment FLX plus ASA for thedevelopment of more active and faster acting molecules.

2006 - Acetylsalicylic acid accelerates the antidepressant effect of fluoxetine in the chronic escape deficit model of depression [Articolo su rivista]
Brunello, Nicoletta; Alboni, Silvia; Capone, Giacomo; Benatti, Cristina; Blom, Johanna Maria Catharina; Tascedda, Fabio; Kriwin, P; Mendlewicz, J.
abstract

Evidence has accumulated suggesting that major depression is associated with dysfunction of inflammatory mediators. Moreover, antidepressants show an antiinflammatory action possibly related to their clinical efficacy. An improvement in psychiatric symptoms has been recently reported in patients treated with antiinflammatory drugs for other indications. These data imply that inflammation may be involved in the pathogenesis of depression and that anti-inflammatory drugs may be used as an adjunctive therapy. The aim of the present study was to evaluate the behavioural effect of the co-administration of acetylsalicylic acid (ASA, 45 mg/kg or 22.5 mg/kg) and fluoxetine (FLX, 5 mg/kg) in the chronic escape deficit model of depression. The chronic escape deficit model is based on the modified reactivity of rats to external stimuli induced by exposure to unavoidable stress and allows evaluation of the capacity of a treatment to revert the condition of escape deficit. In this model, FLX alone needs to be administered for at least 3 weeks to revert this condition. Our results show that combined treatment of fluoxetine and ASA completely reverted the condition of escape deficit by as early as 7 days, the effect being already partially present after 4 days. The effect was maintained after 14 and 21 days of treatment. ASA alone was ineffective at any time tested and the effect of fluoxetine was significant only at 21 days. These results, together with clinical data from preliminary results, suggest that ASA might accelerate the onset of action of selective serotonin reuptake inhibitor antidepressants.

2006 - Behavioural and molecular effects of the combined treatment fluoxetine plus acetylsalicylic acid in a rat model of depression [Abstract in Rivista]
Alboni, Silvia; Capone, Giacomo; Benatti, Cristina; Tascedda, Fabio; Blom, Johanna Maria Catharina; Mendlewicz, J; Brunello, Nicoletta
abstract

Current treatments for depression are inadequate for many patients, and different therapies have been proposed to ameliorate the clinical responses of antidepressant drugs through augumentation or combination strategies. Another achievement in the development of new treatments is to reduce the latency of clinical effect of antidepressant drugs known to be characterized by 4−6 weeks lag phase. Evidence has accumulated suggesting that major depression is associated with dysfunction of inflammatory mediators and that psychiatric symptoms may occur during inflammatory diseases. Moreover antidepressants show an anti-inflammatory action possibly related to their clinical efficacy. In fact, an improvement in psychiatric symptoms has been recently reported in patients treated with anti-inflammatory drugs for other indications. These data imply that inflammation may be involved in the pathogenesis of depression and that anti-inflammatory drugs may be used as an adjunctive therapy. Among the anti-inflammatory drugs the Acetylsalicylic acid has been shown to act on the brain serotonergic system and to have a neuroprotective effect toward brain damage. Aim of the present study was to evaluate the effects of combined treatment fluoxetine (FLX) plus acetylsalicylic acid (ASA) in a behavioural model of depression: the chronic escape deficit. The chronic escape deficit model is based on the modified reactivity of rats to external stimuli induced by exposure to unavoidable stress and allows evaluating the capacity of a treatment to revert the condition of escape deficit. In this model, FLX alone needs to be administered for at least 3 weeks in order to revert this condition. Our results showed that ASA (45 mg/kg) did not possess antidepressant properties in the chronic escape deficit model at any time tested. A combined treatment of FLX (5 mg/kg) and ASA (45 mg/kg) completely reverted the condition of escape deficit as early as after 7 days, the effect being already partially present after 4 days. The effect was maintained after 14 and 21 days of treatment. In the same experimental condition the effect of FLX (5 mg/kg) was significant only at 21 days, as previously demonstrated by other groups. The exact nature of the mechanisms underlying the above behavioural effects are still unknown, nevertheless several hypotheses can be formulated. Moreover, because a role for the neurotrophin BDNF was proposed in the clinical response to antidepressant treatment, we have determined the effect of combined treatment FLX plus ASA on hippocampal BDNF mRNA and protein in the same behavioural model. Ours data demonstrated that the hippocampal levels of BDNF mRNA were significantly increased with respect to control groups (naive and stressed) only in the animals responding (number of escape 10 out of 30 trials) to the combined treatment. Further biochemical and genetic researches could help to clarify the targets of action of the combined treatment FLX plus ASA for the development of more active and faster molecules.

2006 - Early postnatal chronic inflammation produces long-term changes in pain behavior and N-methyl-D-aspartate receptor subtype gene expression in the central nervous system of adult mice [Articolo su rivista]
Blom, Johanna Maria Catharina; Benatti, Cristina; Alboni, Silvia; Capone, Giacomo; Ferraguti, Chiara; Brunello, Nicoletta; Tascedda, Fabio
abstract

The objective of this study was to test whether postnatal chronic inflammation resulted in altered reactivity to pain later in life when reexposed to the same inflammatory agent and whether this alteration correlated with brain-region-specific patterns of N-methyl-D-aspartate (NMDA) receptor subtype gene expression. Neonatal mouse pups received a single injection of complete Freund's adjuvant (CFA) or saline into the left hind paw on postnatal day 1 or 14. At 12 weeks of age, both neonatal CFA- and saline-treated animals received a unilateral injection of CFA in the left hind paw. Adult behavioral responsiveness of the left paw to a radiant heat source was determined in mice treated neonatally with saline or CFA before and after receiving CFA as adults. Twenty-four hours later, brains were dissected and NMDA receptor subunit gene expression was determined in four different brain areas by using an RNase protection assay. The results indicated that NMDA receptor subtype gene expression in adult mice exposed to persistent neonatal peripheral inflammation was brain region specific and that NMDA gene expression and pain reactivity differed according to the day of neonatal CFA exposure. Similarly, adult behavioral responsiveness to a noxious radiant heat source differed according to the age of neonatal exposure to CFA. The data suggest a possible molecular basis for the hypothesis that chronic persistent inflammation experienced early during development may permanently alter the future behavior and the sensitivity to pain later in life, especially in response to subsequent or recurrent inflammatory events

2006 - Effects of acute stress on brain-derived neurotrophic factor in the hippocampus of transgenic mouse model of depression [Abstract in Rivista]
Alboni, Silvia; Blom, Johanna Maria Catharina; Corsini, Daniela; Benatti, Cristina; Capone, Giacomo; Ferraguti, Chiara; N., Barden; Tascedda, Fabio; Brunello, Nicoletta
abstract

Brain-Derived Neurotrophic Factor (BDNF) is a member of the neurotrophin family which includes a group of molecules important for the development and the maintenance of the nervous system. Since BDNF is highly expressed in the hippocampus, the action and regulation of this neurotrophin in this area has become subject of intense study. The gene codifying for BDNF is a stress responsive gene and alterations in its expression may be important in regulating some of the physiological and pathophysiological effects of chronic and acute stress in the hippocampus. Different studies show that several types of stress reduce BDNF expression in the hippocampus of control animals [Smith et al., 1995] and these works led to a neurotrophic hypothesis of depression [Nestler et al., 2002]. Nevertheless, the effect of stress on BDNF gene expression may differ between a "normal" and a "pathological" brain. In our study, we used transgenic mice with glucocorticoid receptor impaired (GRi) expression created [Pepin et al., 1992], as a tool to study the neuroendocrine changes observed in stress-related disorders, such as major depression. This GRi mouse model is characterized by dysfunctional glucocorticoid inhibitory feedback and an excessive activation of the hypothalamic pituitar~adrenal (HPA) axis, that can be restored by antidepressant drugs' treatment. The hypothesis was tested that a single period of 30 minutes of restraint stress affects BDNF expression in the hippocampus of GRi mice differently than in wildtype (WT) mice. Using RNase protection assay and in situ hybridization we had assessed the BDNF mRNA hippocampal levels, while the levels of BDNF and its precursor, pro-BDNF were analyzed by western blotting. Our results indicated that 30 minutes of restraint enhanced BDNF mRNA expression in the CA3 hippocampal subregion of GRi mice; the same stress procedure induced also a statistically significant increase of pro- BDNF level in hippocampus of GRi mice. No effect of acute stress was observed in the WT at the level of the expression of BDNE Moreover, we evaluated the effects of restraint on signalling pathways implicated in the regulation of BDNF expression (mitogen-activated protein kinase and calcium/calmodulin-dependent kinase cascades) that converge on the phosphorylation of CREB that we found down-regulated in the hippocampus of GRi mice and up-regulated in WT mice. Our data suggest that, in the presence of psychophysiological stress (restraint stress), GRi mice display altered hippocampal regulation in BDNF gene expression. Thus, life-long central GR dysfunction may negatively affects neural functioning by limiting the capacity to cope with change or acute stress, which could be a predisposing or determining factor in depression. Understanding the mechanisms underlying the induction of BDNF mRNA and accumulation of pro-BDNF in the hippocampus of GRi mice, may help to clarify the molecular basis of action of this neurotrophin and contribute to the development of new strategies reducing the vulnerability of neurons to stress, thus preventing neuropathological alterations in the hippocampus.

2005 - Detection of levodopa, dopamine and its metabolites in rat striatum dialysates following peripheral administration of L-DOPA prodrugs by mean of HPLC-EC [Articolo su rivista]
Cannazza, Giuseppe; A., Di Stefano; B., Mosciatti; Braghiroli, Daniela; Baraldi, Mario; F., Pinnen; P., Sozio; Benatti, Cristina; Parenti, Carlo
abstract

A high performance liquid chromatography (HPLC) method was developed to detected simultaneously L-dihydroxyphenylalanine (L-DOPA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum dilaysates following oral administration of L-DOPA or its prodrugs. The chromatographic system uses a reversed-phase C-IS column with electrochemical detection at +0.30 V. Mobile phase consisted of 0.05 M citric acid. sodium EDTA 50 muM, sodium octylsulphonate 0.4 nM at pH of 2.9 and 8%, methanol (v/v) at a flow rate of 1 ml/min. The calibration curves were linear over the concentration range of 10 nm to 100 muM and the lower limits of detections were 125 fmol for L-DOPA. 50 fmol for DOPAC, 250 fmol for DA and 150 fmol for HVA at signal noise to ratio of 3. The repeatability (or intra-day precision), expressed by the relative standard deviation. were better than 4%. The construction of microdialysis probes has been described. The in vitro relative recoveries of each microdialysis probe were. evaluated and the results show that they are similar and reproducible for all the analytes with CVs from 1 to 4%. The HPLC-EC method was applied to detect the extracellular levels Of L-DOPA. DA. DOPAC and HVA in the striatum dialysates of freely moving rats after oral administration of six new potential L-DOPA prodrugs.

2005 - Neonatal persistent inflammation alters pain response and NMDA receptor expression in adult mice [Abstract in Rivista]
Benatti, Cristina; Alboni, Silvia; Ferraguti, Chiara; Tascedda, Fabio; Blom, Johanna Maria Catharina; Brunello, Nicoletta
abstract

Infant pain is of critical interest, especially with respect to premature infants and other high-risk neonates that experience many invasive and traumatic procedures early in development. The early neonatal period is characterized by great plasticity and reorganization. Sustained activation of central nervous circuits, caused by protracted and recurrent pain, may cause long-lasting changes in central neural function thus affecting developmental outcome and behavioural responsiveness to pain or stress later in life. However little is known about the neurobiological substrates underlying this ``memory'' process. The aim of our study was twofold:to study whether timing of postnatal exposure to a persistent inflammatory insult alters the responsiveness to thermal pain in the adult animal;given the role of the NMDA receptor in pain processing as well as in learning and memory, to examine if NMDA receptor subtype gene expression in specific areas of the cns is influenced by neonatal inflammation.Methods: Newborn mice received a single injection of Complete Freund's Adjuvant (CFA) or saline on either postnatal day 1, 3 or 14 (P1, P3 and P14) into the left hind paw. At twelve weeks of age paw withdrawal latency (PWL) of each animal was tested both in basal condition and 24h after an unilateral injection of 100 μL of CFA in the left hind paw. Mice were then killed by cervical dislocation and cerebral areas were removed. Using a sensitive RNAse protection assay, NMDA receptor subunit (NR1, NR2A, NR2B, NR2C) gene expression was evaluated in different brain areas; all data were processed by one-way ANOVA (p < 0.05).Results: Baseline paw withdrawal latency was significantly decreased in animals exposed to CFA at day 1 and 14 as compared to their saline exposed counterparts. Animals exposed to CFA at postnatal day 3 showed a significant increase in paw withdrawal latency with respect to saline injected animals. Twenty-four hours later a unilateral injection of CFA into the left hind paw, a significant decrease in paw withdrawal latency was observed in all experimental groups with respect to baseline values. PWL of P1 saline treated animals after CFA exposure was significantly higher than P3 and P14 saline treated mice. Adult mice exposed to an injection with CFA on postnatal day 1 exhibited reduced expression of the NMDA receptor subtype NR1 and NR2C in the hippocampus while mRNA levels for NR2A and NR2B did not differ between CFA treated and untreated mice. Exposure to CFA on postnatal day 3 and 14 did not affect adult expression levels of NMDA receptor subunits in the hippocampus. NMDA receptor subunit expression displayed a different profile in the thalamus. Exposure to CFA at P1 and P3 did not alter NMDA receptor subunit expression while exposure to CFA at P14 resulted in enhanced expression of the NR2A and NR2B subunits.Conclusions: These findings indicate that changes in NMDA receptor subtype gene expression in adult mice exposed to persistent neonatal peripheral inflammation are brain region specific and that NMDA gene expression and pain reactivity differ according to the day of neonatal exposure to CFA.

2005 - New combination therapies from animal to human [Abstract in Rivista]
Brunello, Nicoletta; Alboni, Silvia; Benatti, Cristina; Capone, Giacomo; Tascedda, Fabio; Blom, Johanna Maria Catharina; J., Mendlewicz
abstract

Evidence has accumulated suggesting that major depression is associated with dysfunction of inflammatory mediators. Antidepressants interfere with the synthesis and release of cytokines and do not exert behavioral effects in animal models of depression when hippocampal neurogenesis is blocked, a phenomenon which is occurring in the presence of inflammation. The anti-inflammatory drug acetylsalicylic acid (ASA), besides inhibiting the cyclooxigenase pathway, interacts with central serotonergic system, by increasing serotonin levels in cortex and reducing the density of different serotonin receptor subtypes. These neurochemical effects suggest a role of ASA in the treatment of depression. Therefore we studied the effect of ASA and fluoxetine combined treatment in a behavioral model of depression. The chronic escape deficit model is based on the modified reactivity of rats to external stimuli induced by exposure to unavoidable stress and allows evaluating the capacity of a treatment to revert the condition of escape deficit. Any kind of antidepressant drug needs to be administered for at least 3 weeks in order to revert this condition. The combined treatment of fluoxetine and ASA completely reverted the condition of escape deficit as early as after 7 days, the effect being already partially present after 4 days. The effect was maintained after 14 and 21 days of treatment. ASA alone was ineffective at any time tested and the effect of fluoxetine was significant only at 21 days. Given these preclinical results, an open clinical study has been started using the combination SSRI-ASA in treatment resistant depressed patients. Preliminary results suggest a potential accelerating effect of ASA in combination to SSRI.

2004 - Restraint stress increases the expression of brain derived neurotrophic factor in the hippocampus of a mouse model of depression [Abstract in Rivista]
Alboni, Silvia; Benatti, Cristina; Blom, Johanna Maria Catharina; Ferraguti, Chiara; Tascedda, Fabio; Barden, N; Brunello, Nicoletta
abstract

Statement of the study: Brain-Derived Neurotrophic Factor (BDNF) is a member of the neurotrophin family which includes a group of molecules important for the development and the maintenance of the nervous system. Since BDNF is highly expressed in the hippocampus, the action and regulation of BDNF in this particular area has become subject of intense study. Single or repeated immobilization stress markedly reduces both BDNF mRNA and protein levels in rat hippocampus. Consequently, BDNF is considered a stress-responsive gene, and it has been recently suggested that alterations in the expression of this growth factor may be important in regulating some of the physiological and pathophysiological effects of stress on the hippocampus. Stress-induced changes observed in the hippocampus of experimental animals resulted in a novel hypothesis attributing a central role to neurotrophic factors in both the etiology of depression and as well as in its treatment. However, the effects of stress on neurotrophic factors in the hippocampus of depressed patients remain unknown. In fact, the expression pattern of a large array of genes affected by depression or antidepressant drugs, such as BDNF, may differ between a normal and a pathological brain. Methods: In these experiments, we used transgenic (TG) mice deficient in glucocorticoid receptor (GR) functioning. This TG mouse was created as a model to study the neuroendocrine changes occurring in stress-related disorders, such as major depression. We evaluated the hypothesis that a single period of 30 minutes of restraint stress affects BDNF mRNA expression in the hippocampus of TG mice differently than in WT mice. Summary of results: BDNF mRNA was significantly increased by the stress procedure only in the hippocampus of TG mice, the induction being specific for the CA3 subregion as revealed by in situ hybridization. Moreover, we found that stress down-regulated CREB phosphorylation in the hippocampus of TG mice whereas it upregulated the level of phosphorylated CREB Ser133 in WT mice. Conclusion: These data suggest that, in the presence of emotional stress, lifelong central glucocorticoid receptor dysfunction results in altered hippocampal sensitivity, with respect to the level of neurotrophic gene expression. The understanding of the mechanisms through which psychological stress (such as restraint stress) induces BDNF mRNA in the hippocampus of TG mice, may help to clarify the biological and molecular basis of the action of neurotrophic factors and may contribute to the development of new strategies that will ultimately reduce the vulnerability of neurons and prevent neuropathological alterations in the hippocampus.

Università degli studi di Modena e Reggio Emilia

Pubblicazioni