The association between serotonin transporter availability and the neural correlates of fear bradycardia.

Susceptibility to stress-related psychopathology is associated with reduced expression of the serotonin transporter (5-HTT), particularly in combination with stress exposure. Aberrant physiological and neuronal responses to threat may underlie this increased vulnerability. Here, implementing a cross-species approach, we investigated the association between 5-HTT expression and the neural correlates of fear bradycardia, a defensive response linked to vigilance and action preparation. We tested this during threat anticipation induced by a well-established fear conditioning paradigm applied in both humans and rodents. In humans, we studied the effect of the common 5-HTT-linked polymorphic region (5-HTTLPR) on bradycardia and neural responses to anticipatory threat during functional magnetic resonance imaging scanning in healthy volunteers (n = 104). Compared with homozygous long-allele carriers, the 5-HTTLPR short-allele carriers displayed an exaggerated bradycardic response to threat, overall reduced activation of the medial prefrontal cortex (mPFC), and increased threat-induced connectivity between the amygdala and periaqueductal gray (PAG), which statistically mediated the effect of the 5-HTTLPR genotype on bradycardia. In parallel, 5-HTT knockout (KO) rats also showed exaggerated threat-related bradycardia and behavioral freezing. Immunohistochemistry indicated overall reduced activity of glutamatergic neurons in the mPFC of KO rats and increased activity of central amygdala somatostatin-positive neurons, putatively projecting to the PAG, which-similarly to the human population-mediated the 5-HTT genotype's effect on freezing. Moreover, the ventrolateral PAG of KO rats displayed elevated overall activity and increased relative activation of CaMKII-expressing projection neurons. Our results provide a mechanistic explanation for previously reported associations between 5-HTT gene variance and a stress-sensitive phenotype.

Own or dam's genotype? Classical colony breeding may bias spontaneous and stress-challenged activity in DAT-mutant rats.

There is considerable interest in understanding what makes an individual vulnerable or resilient to the deleterious effects of stressful events. From candidate genes, dopamine (DA) and dopamine transporter (DAT) have been linked to anxiety, depression, and post-traumatic stress disorder. We investigated role of DAT using the new DAT heterozygous (DAT-HET) and homozygous mutant (DAT-KO) rat models of hyperdopaminergia. We studied the impact of two breeding conditions in spontaneous locomotor behavior of female rats. The classical colony, through mating DAT-HET males × DAT-HET females (breeding HET-HET), was used. A second WT colony was derived and maintained (breeding WT-WT). Additionally, a subgroup of rats was bred through mating DAT-KO males × WT females (atypical HET, breeding KO-WT). We studied the effects of genotype and its interaction with maternal care (depending by breeding condition). HET-HET breeding led to reduced activity in HET females compared to WT rats (from WT-WT breeding). However, HET females from KO-WT breeding did not differ so much from WT rats (WT-WT breeding). The maternal-care impact was then confirmed: HET mothers (breeding HET-HET) showed reduced liking/grooming of pups and increased digging away from nest, compared to WT mothers (breeding WT-WT). In their female offspring (HET, breeding HET-HET vs. WT, breeding WT-WT), isolation plus wet bedding induced higher and more persistent impact on activity of HET rats, even when the stressor was removed. Our results highlight the importance of epigenetic factors (e.g., maternal care) in responses to stress expressed by offspring at adulthood, quite independently of genotype. DAT hypofunction could determinate vulnerability to stressful agents via altered maternal care.

Regionally restricted modulation of Sam68 expression and Arhgef9 alternative splicing in the hippocampus of a murine model of multiple sclerosis.

Multiple sclerosis (MS) and its preclinical models are characterized by marked changes in neuroplasticity, including excitatory/inhibitory imbalance and synaptic dysfunction that are believed to underlie the progressive cognitive impairment (CI), which represents a significant clinical hallmark of the disease. In this study, we investigated several parameters of neuroplasticity in the hippocampus of the experimental autoimmune encephalomyelitis (EAE) SJL/J mouse model, characterized by rostral inflammatory and demyelinating lesions similar to Relapsing-Remitting MS. By combining morphological and molecular analyses, we found that the hippocampus undergoes extensive inflammation in EAE-mice, more pronounced in the CA3 and dentate gyrus (DG) subfields than in the CA1, associated with changes in GABAergic circuitry, as indicated by the increased expression of the interneuron marker Parvalbumin selectively in CA3. By laser-microdissection, we investigated the impact of EAE on the alternative splicing of Arhgef9, a gene encoding a post-synaptic protein playing an essential role in GABAergic synapses and whose mutations have been related to CI and epilepsy. Our results indicate that EAE induces a specific increase in inclusion of the alternative exon 11a only in the CA3 and DG subfields, in line with the higher local levels of inflammation. Consistently, we found a region-specific downregulation of Sam68, a splicing-factor that represses this splicing event. Collectively, our findings confirm a regionalized distribution of inflammation in the hippocampus of EAE-mice. Moreover, since neuronal circuit rearrangement and dynamic remodeling of structural components of the synapse are key processes that contribute to neuroplasticity, our study suggests potential new molecular players involved in EAE-induced hippocampal dysfunction.

Alternative splicing of neurexins 1-3 is modulated by neuroinflammation in the prefrontal cortex of a murine model of multiple sclerosis.

Mounting evidence points to immune-mediated synaptopathy and impaired plasticity as early pathogenic events underlying cognitive decline (CD) in Multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) mouse model of the disease. However, knowledge of the neurobiology of synaptic dysfunction is still incomplete. Splicing regulation represents a flexible and powerful mechanism involved in dynamic remodeling of the synapse, which allows the expression of synaptic protein variants that dynamically control the specificity of contacts between neurons. The pre-synaptic adhesion molecules neurexins (NRXNs) 1-3 play a relevant role in cognition and are alternatively spliced to yield variants that differentially cluster specific ligands in the postsynaptic compartment and modulate functional properties of the synaptic contact. Notably, mutations in these genes or disruption of their splicing program are associated with neuropsychiatric disorders. Herein, we have investigated how inflammatory changes imposed by EAE impact on alternative splicing of the Nrxn 1-3 mouse genes in the acute phase of disease. Due to its relevance in cognition, we focused on the prefrontal cortex (PFC) of SJL/J mice, in which EAE-induced inflammatory lesions extend to the rostral forebrain. We found that inclusion of the Nrxn 1-3 AS4 exon is significantly increased in the PFC of EAE mice and that splicing changes are correlated with local Il1ß-expression levels. This correlation is sustained by the concomitant downregulation of SLM2, the main splicing factor involved in skipping of the AS4 exon, in EAE mice displaying high levels of Il1ß- expression. We also observed that Il1ß-expression levels correlate with changes in parvalbumin (PV)-positive interneuron connectivity. Moreover, exposure to environmental enrichment (EE), a condition known to stimulate neuronal connectivity and to improve cognitive functions in mice and humans, modified PFC phenotypes of EAE mice with respect to Il1ß-, Slm2-expression, Nrxn AS4 splicing and PV-expression, by limiting changes associated with high levels of inflammation. Our results reveal that local inflammation results in early splicing modulation of key synaptic proteins and in remodeling of GABAergic circuitry in the PFC of SJL/J mice. We also suggest EE as a tool to counteract these inflammation-associated events, thus highlighting potential therapeutic targets for limiting the progressive CD occurring in MS.

Motor Transitions' Peculiarity of Heterozygous DAT Rats When Offspring of an Unconventional KOxWT Mating.

Causal factors of psychiatric diseases are unclear, due to gene × environment interactions. Evaluation of consequences, after a dopamine-transporter (DAT) gene knock-out (DAT-KO), has enhanced our understanding into the pathological dynamics of several brain disorders, such as Attention-Deficit/Hyperactivity and Bipolar-Affective disorders. Recently, our attention has shifted to DAT hypo-functional (heterozygous, HET) rodents: HET dams display less maternal care and HET females display marked hypo-locomotion if cared by HET dams (Mariano et al., 2019). We assessed phenotypes of male DAT-heterozygous rats as a function of their parents: we compared "maternal" origin (MAT-HET, obtained by breeding KO-male rats with WT-female dams) to "mixed" origin (MIX-HET, obtained by classical breeding, both heterozygous parents) of the allele. MAT-HET subjects had significantly longer rhythms of daily locomotor activity than MIX-HET and WT-control subjects. Furthermore, acute methylphenidate (MPH: 0, 1, 2 mg/kg) revealed elevated threshold for locomotor stimulation in MAT-HETs, with no response to the lower dose. Finally, by Porsolt-Test, MAT-HETs showed enhanced escape-seeking (diving) with more transitions towards behavioral despair (floating). When comparing both MAT- and MIX-HET to WT-control rats, decreased levels of DAT and HDAC4 were evident in the ventral-striatum; moreover, with respect to MIX-HET subjects, MAT-HET ones displayed increased DAT density in dorsal-striatum. MAT-HET rats displayed region-specific changes in DAT expression, compared to "classical" MIX-HET subjects: greater DAT availability may elevate threshold for dopamine action. Further behavioral and epigenetic characterizations of MAT-HETs, together with deeper characterization of maternal roles, could help to explore parent-of-origin mechanisms for such a peculiar phenotype.

Behavioral characterization of DAT-KO rats and evidence of asocial-like phenotypes in DAT-HET rats: The potential involvement of norepinephrine system.

Dopamine (DA) is a key neurotransmitter of the central nervous system, whose availability is regulated by the dopamine transporter (DAT). Deletion of DAT gene leading to hyperdopaminergia was previously performed on mouse models. This enabled recapitulation of the core symptoms of Attention-Deficit / Hyper-activity Disorder (ADHD), which include hyperactivity, inattention and cognitive impairment. We used recently developed DAT knockout (DAT-KO) rats to carry out further behavioral profiling on this novel model of hyperdopaminergia. DAT-KO rats display elevated locomotor activity and restless environmental exploration, associated with a transient anxiety profile. Furthermore, these rats show pronounced stereotypy and compulsive-like behavior at the Marble-Burying test. Homozygous DAT-KO rats mantain intact social interaction when tested in a social-preference task, while heterozygous (HET) rats show high inactivity associated with close proximity to the social stimulus. Ex-vivo evaluation of brain catecholamines highlighted increased levels of norepinephrine in the hippocampus and hypothalamus exclusively of heterozygous rats. Taken together, our data present evidence of unexpected asocial tendencies in heterozygous (DAT-HET) rats associated with neurochemical alterations in norepinephrine neurotransmission. We shed light on the behavioral and neurochemical consequences of altered DAT function in a higher, more complex model of hyperdopaminergia. Unraveling the role of DA neurotransmission in DAT-KO rats has very important implications in the understanding of many psychiatric illnesses, including ADHD, where alterations in DA system have been demonstrated.

Activation of 5-HT7 receptor by administration of its selective agonist, LP-211, modifies explorative-curiosity behavior in rats in two paradigms which differ in visuospatial parameters.

AIMS: The serotonin 7 receptor (5-HT7R) subtype, coded by Htr7 gene, is broadly expressed in the central nervous system (CNS) with clear involvement in behavioral functions such as learning/memory, regulation of mood, and circadian rhythms. In this study, we assessed effects of 5-HT7R stimulation by administration of its selective agonist, LP-211 (0.25 mg/kg i.p.), in adult Wistar-Han rats. METHODS: We used two different explorative-curiosity tests. Drug was administered either before one side-chamber familiarization (CF/V group) or immediately after it, to act on consolidation of familiarization (V/CF group). RESULTS: Exp. 1 for novelty seeking in black/white boxes (BWB), with door opening after 5 minutes in the familiar chamber, showed that (i) time spent in the novel environment (significantly higher than in familiar chamber for controls) is enhanced in V/CF group (potentiated recognition for a "visual" consolidation) and not different in CF/V group; (ii) activity and chamber transitions, made by CF/V rats, are significantly higher than for other groups (interference on recognition for a "spatial" acquisition). Exp. 2 for novelty preference in D- vs L-shaped chambers (D/L), with start from neutral center, gave different results: (i) time spent in the novel environment by CF/V group is significantly higher than other groups (potentiated "cognitive" acquisition); (ii) chamber transitions made by V/CF group are significantly higher than other groups (potentiated "emotional" consolidation). CONCLUSION: These apparently conflicting results may reflect LP-211 effects on visual vs spatial memory (D/L apparatus has more pronounced hippocampal components than BWB). However, further experiments are needed to analyze more in depth the mechanisms involved.