c-Fos Immunoreactivity Research Articles

Mapping of c-Fos Expression in Rat Brain Sub/Regions Following Chronic Social Isolation: Effective Treatments of Olanzapine, Clozapine or Fluoxetine

The c-Fos as a marker of cell activation is used to identify brain regions involved in stimuli processing. This review summarizes a pattern of c-Fos immunoreactivity and the overlapping brain sub/regions which may provide hints for the identification of neural circuits that underlie depressive- and anxiety-like behaviors of adult male rats following three and six weeks of chronic social isolation (CSIS), relative to controls, as well as the antipsychotic-like effects of olanzapine (Olz), and clozapine (Clz), and the antidepressant-like effect of fluoxetine (Flx) in CSIS relative to CSIS alone. Additionally, drug-treated controls relative to control rats were also characterized. The overlapping rat brain sub/regions with increased expression of c-Fos immunoreactivity following three or six weeks of CSIS were the retrosplenial granular cortex, c subregion, retrosplenial dysgranular cortex, dorsal dentate gyrus, paraventricular nucleus of the thalamus (posterior part, PVP), lateral/basolateral (LA/BL) complex of the amygdala, caudate putamen, and nucleus accumbens shell. Increased activity of the nucleus accumbens core following exposure of CSIS rats either to Olz, Clz, and Flx treatments was found, whereas these treatments in controls activated the LA/BL complex of the amygdala and PVP. We also outline sub/regions that might represent potential neuroanatomical targets for the aforementioned antipsychotics or antidepressant treatments.

8631 Tanycytic TSHR Activation Regulates Blood-Hypothalamus Barrier Plasticity, Ingestive Behavior, Adipose Remodeling, and Body Weight

Abstract Disclosure: L. Cullen: None. H.S. Kannangara: None. R. Witztum: None. J. Burgess: None. S. Miyashita: None. A. Gumerova: None. F. Korkmaz: None. S.L. Sims: None. U. Cheliadinova: None. G. Pevnev: None. O. Moldavski: None. O. Barak: None. S. Rojekar: None. V. Laurencin: None. T. Frolinger: None. T. Yuen: None. K. Goosens: None. W. Zhou: None. V. Ryu: None. M. Zaidi: None. The notion that TSH acts solely on its primary endocrine target, the thyroid gland, has long been challenged (Abe et al., 2003; PMID: 14567913). More recently, however, we and others have used single transcript technology to document, for the first time, Tshr expression in over 173 brain regions and nuclei, particularly hypothalamic tanycytes (eLife, 2022, PMID: 36052994). The latter cells are known to convey metabolic information across the blood-hypothalamus barrier (BHB) and regulate energy homeostasis. To study whether tanycytic TSHRs have a role in the regulation of BHB permeability, ingestive behavior, adipose tissue remodeling and body weight, we first compared BHB vascular permeability of food-deprived and ad libitum-fed Tshr+/- mice and their wild type (WT) littermates. Transmission electron microscopy and immunostaining was used to study the expression of the constitutive tight junction protein claudin-1 and the glycoprotein MECA-32, which are expressed in tanycytes and endothelial cells, respectively. In loss-of-function studies, fasting of Tshr+/- mice and of WT mice significantly increased the organization of tanycytic honeycomb pattern of claudin-1 and MECA-32-associated fenestral diaphragms in the median eminence and arcuate nucleus, suggesting decreased BHB permeability. As a result, peanut butter consumption following fasting was decreased significantly compared to control mice. In gain-of-function studies, intracerebroventricular (ICV) infusions of a highly specific TSHR agonist, MS438, profoundly increased BHB permeability in fasted mice, as evidenced by decreased claudin-1 and MECA-32 immunostaining and significant increases in peanut butter consumption. This was associated with greater loss of fat mass and body weight following food deprivation, as well as increased influx of fatty acids across the BHB, increased NPY expression (as a measure of increased appetite), and increased secretion of ghrelin from the stomach fundus. We further performed c-Fos immunostaining in brain centers regulating ingestive behavior. ICV MS438 significantly decreased c-Fos immunoreactivity in the hypothalamic paraventricular nucleus, arcuate nucleus and the nucleus of the solitary tract. Early studies using CRSIPR/Cas9 to delete Tshrs in tanycytes via ICV injection resulted in reduced body weight following food deprivation. Finally, using PRV152 (injected into fat tissue) and PRV614 (injected into stomach fundus), we colocalized immunostaining in the hypothalamic paraventricular nucleus, and hindbrain raphe pallidus. Collectively, these data establish a new role for tanycytic TSHRs in regulating BHB plasticity, ingestive behavior, adipose tissue remodeling, and body weight through a novel fast-forward circuit involving the hypothalamus, stomach fundus and fat depots. Presentation: 6/3/2024

Prophylactic (R,S)-ketamine and (2S,6S)-HNK decrease fear expression by differentially modulating fear neural ensembles

We previously reported that a single injection of (R,S)-ketamine or its metabolite (2S,6S)-hydroxynorketamine (HNK) prior to stress attenuates learned fear. However, whether these drugs attenuate learned fear through divergent or convergent effects on neural activity remains to be determined. 129S6/SvEv male mice were injected with saline, (R,S)-ketamine, or (2S,6S)-HNK one week before a 3-shock contextual fear conditioning (CFC) paradigm. Five days later, mice were re-exposed to the aversive context, and euthanized one hour later to quantify active cells. Brains were processed for c-fos immunoreactivity, and neural networks were built with a novel, wide-scale imaging pipeline. We found that (R,S)-ketamine and (2S,6S)-HNK attenuate learned fear. Fear-related neural activity was altered in: dorsal CA3 following (2S,6S)-HNK; ventral CA3 and CA1, infralimbic (IL) and prelimbic (PL) regions, insular cortex (IC), retrosplenial cortex (RSP), piriform cortex (PIR), nucleus reuniens (RE), and periaqueductal grey (PAG) following both (R,S)-ketamine and (2S,6S)-HNK; and in the paraventricular nucleus of thalamus (PVT) following (R,S)-ketamine. Dorsal CA3 and ventral hippocampus activation correlated with freezing in the (R,S)-ketamine group, and RSP activation correlated with freezing in both (R,S)-ketamine and (2S,6S)-HNK groups. (R,S)-ketamine increased connectivity between cortical and subcortical regions while (2S,6S)-HNK increased connectivity within these regions. This work identifies novel nodes in fear networks, involving the RE, PIR, IC, PAG and RSP, that can be targeted with neuromodulatory strategies or pharmaceutical compounds to treat fear-induced disorders. This approach could be used to optimize target engagement and dosing strategies of existing medications.

Chronic unpredictable stress during adolescence exerts sex-specific effects on depressive-like behavior and neural activation triggered by tail suspension test

During adolescence, acute stress can modify neuronal excitability in various brain regions, leading to negative behavioral outcomes. However, the impact of chronic stress during adolescence on neuronal responses to acute stimuli remains unclear. To address this, we subjected adolescent mice to 12 days of chronic unpredictable stress (CUS). Anxiety and depressive behaviors were evaluated, along with changes in c-Fos expression, which is one of the most widely used markers of neuronal activation. By comparing c-Fos immunoreactivity between the CUS and control groups both before and after the tail suspension test (TST), we found that adolescent CUS induced depressive behaviors in male mice, but not in female mice. Adolescent CUS primarily affected the excitability of neurons in the infralimbic cortex (IL), the dorsomedial and dorsolateral area of the bed nucleus of the stria terminalis (BNST), and the ventral hippocampus CA3. TST exerted a significant main effect on the density of c-Fos+ neurons in the prelimbic cortex (PL), infralimbic cortex (IL), cingulate areas 1 and 2 (Cg1, Cg2), the lateral septum (LS), Bed nucleus terminalis (BNST), and lateral habenular (LHb). Furthermore, the excitability of neurons in the paraventricular thalamic nucleus (PVT) was impacted by sex. These data suggest that adolescent CUS elicits region- and sex-specific modifications in TST-induced c-Fos expression, establishing a theoretical basis for understanding the pathophysiological alterations in mood disorders following adolescent stress.

Immune-mediated impairment of tonic immobility defensive behavior in an experimental model of colonic inflammation.

Ulcerative colitis has been associated with psychological distress and an aberrant immune response. The immunomodulatory role of systemic cytokines produced during experimental intestinal inflammation in tonic immobility (TI) defensive behavior remains unknown. The present study characterized the TI defensive behavior of guinea pigs subjected to colitis induction at the acute stage and after recovery from intestinal mucosa injury. Moreover, we investigated whether inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, IL-10, and prostaglandins) act on the mesencephalic nucleus, periaqueductal gray matter (PAG). Colitis was induced in guinea pigs by intrarectal administration of acetic acid. The TI defensive behavior, histology, cytokine production, and expression of c-FOS, IBA-1, and cyclooxygenase (COX)-2 in PAG were evaluated. Colitis reduced the duration of TI episodes from the first day, persisting throughout the 7-day experimental period. Neuronal c-FOS immunoreactivity was augmented in both columns of the PAG (ventrolateral (vlPAG) and dorsal), but there were no changes in IBA-1 expression. Dexamethasone, infliximab, and parecoxib treatments increased the duration of TI episodes, suggesting a modulatory role of peripheral inflammatory mediators in this behavior. Immunoneutralization of TNF-α, IL-1β, and IL-8 in the vlPAG reversed all effects produced by colitis. In contrast, IL-10 neutralization further reduced the duration of TI episodes. Our results reveal that peripherally produced inflammatory mediators during colitis may modulate neuronal functioning in mesencephalic structures such as vlPAG.

Prefrontal cortex-nucleus reuniens-hippocampus network exhibits sex-differentiated responses to stress and antidepressant treatment in rats.

Depression is a serious psychiatric disease, which is diagnosed twice as frequently in women than men. We have recently shown that lesioning or inactivation of the nucleus reuniens (RE), which interconnects the prefrontal cortex (PFC) and hippocampus, promoted resilience to stress in males, exerts an antidepressant effect in the Forced Swim Test (FST) and prevents the development of behavioral and neurobiological alterations induced by the chronic mild stress model of depression. In this study, we expand our findings on the FST in female rats and we investigate whether RE lesion presents sex differences following treatment with two distinct antidepressants, a selective serotonin reuptake inhibitor, i.e. sertraline and a tricyclic antidepressant, i.e. clomipramine. Male and female rats received either a surgical lesion of the RE or sham operation, then treated with vehicle, sertraline (10mg/kg) or clomipramine (10mg/kg) and were subjected to the FST. Activation of key brain areas of interest (PFC, Hippocampus and RE) were measured by c-Fos immunoreactivity. RE lesion induced an antidepressant-like phenotype in both female and male rats, confirming its crucial role in the stress response. Similarly to RE lesion, sertraline treatment resulted in increased swimming and decreased immobility duration, as well as enhanced head shake frequency, in both sexes. Notably, climbing behavior was increased only following clomipramine treatment. RE area was less active in females compared to male rats and in clomipramine-treated males compared to their corresponding vehicle-group. Activation of the PFC and the CA1 hippocampal area was reduced in clomipramine-treated females, in comparison to vehicle-treated female rats. This effect was not evident in males, which exhibited less activation in the PFC and the hippocampus than females. Re lesion proves equally effective in female and male rats, but sex is highlighted as a pivotal factor in behavioral and treatment response in FST, as well as in related circuit connectivity and activation.

Palatable solution overconsumption in the Cntnap2-/- murine model of autism: a link with oxytocin.

Dysregulated appetite is common in autism spectrum disorder (ASD) and it includes excessive interest in tasty foods. Overconsumption of palatable fluids has been found in the valproic acid-induced ASD rat. Though ASD has a strong genetic component, the link between ASD-related genes and appetite for palatable foods remains elusive. We focused on the CNTNAP2 gene whose deletion in mice recapitulates human ASD symptoms. We investigated whether Cntnap2-/- male mice consume greater amounts of palatable 10% sucrose, 0.1% saccharin, and 4.1% intralipid solutions offered in episodic meals either in a no-choice paradigm or a two-bottle choice test. We examined how sucrose intake affects c-Fos immunoreactivity in feeding-related brain areas. Finally, we determined doses at which intraperitoneal oxytocin decreases sucrose intake in mutants. In the single-bottle tests, Cntnap2-/- mice drank more sucrose, saccharin, and intralipid compared to WTs. Given a choice between two tastants, Cntnap2-/- mice had a higher preference for sucrose than intralipid. While the standard 1 mg/kg oxytocin dose reduced sucrose intake in WTs, a low oxytocin dose (0.1 mg/kg) decreased sucrose intake in Cntnap2-/- mice. Sucrose intake induced a more robust c-Fos response in wild-type (WT) than Cntnap2-/- mice in the reward and hypothalamic sites and it increased the percentage of Fos-immunoreactivity oxytocin neurons in WTs, but not in mutants. We conclude that Cntnap2-/- mice overconsume palatable solutions, especially sucrose, beyond levels seen in WTs. This excessive consumption is associated with blunted c-Fos immunoreactivity in feeding-related brain sites, and it can be reversed by low-dose oxytocin.

Effects of acute inhibition of dopamine β-hydroxylase on neural responses to pups in adult virgin male California mice (Peromyscus californicus)

The neural mechanisms underlying paternal care in biparental mammals are not well understood. The California mouse (Peromyscus californicus) is a biparental rodent in which virtually all fathers are attracted to pups, while virgin males vary widely in their behavior toward unrelated infants, ranging from attacking to avoiding to huddling and grooming pups. We previously showed that pharmacologically inhibiting the synthesis of the neurotransmitter norepinephrine (NE) with the dopamine β-hydroxylase inhibitor nepicastat reduced the propensity of virgin male and female California mice to interact with pups. The current study tested the hypothesis that nepicastat would reduce pup-induced c-Fos immunoreactivity, a cellular marker of neural activity, in the medial preoptic area (MPOA), medial amygdala (MeA), basolateral amygdala (BLA), and bed nucleus of the stria terminalis (BNST), brain regions implicated in the control of parental behavior and/or anxiety. Virgin males were injected with nepicastat (75 mg/kg, i.p.) or vehicle 2 hours prior to exposure to either an unrelated pup or novel object for 60 minutes (n = 4–6 mice per group). Immediately following the 60-minute stimulus exposure, mice were euthanized and their brains were collected for c-Fos immunohistochemistry. Nepicastat reduced c-Fos expression in the MeA and MPOA of pup-exposed virgin males compared to vehicle-injected controls. In contrast, nepicastat did not alter c-Fos expression in any of the above brain regions following exposure to a novel object. Overall, these results suggest that the noradrenergic system might influence MeA and MPOA function to promote behavioral interactions with pups in virgin males.

Regulating the activity of GABAergic neurons in the ventral pallidum alters the general anesthesia effect of propofol

The full mechanism of action of propofol, a commonly administered intravenous anesthetic drug in clinical practice, remains elusive. The focus of this study was the role of GABAergic neurons which are the main neuron group in the ventral pallidum (VP) closely associated with anesthetic effects in propofol anesthesia. The activity of VP GABAergic neurons following propofol anesthesia in Vgat-Cre mice was observed via detecting c-Fos immunoreactivity by immunofluorescence and western blotting. Subsequently, chemogenetic techniques were employed in Vgat-Cre mice to regulate the activity of VP GABAergic neurons. The role of VP GABAergic neurons in generating the effects of general anesthesia induced by intravenous propofol was further explored through behavioral tests of the righting reflex. The results revealed that c-Fos expression in VP GABAergic neurons in Vgat-Cre mice dramatically decreased after propofol injection. Further studies demonstrated that chemogenetic activation of VP GABAergic neurons during propofol anesthesia shortened the duration of anesthesia and promoted wakefulness. Conversely, the inhibition of VP GABAergic neurons extended the duration of anesthesia and facilitated the effects of anesthesia. The results obtained in this study suggested that regulating the activity of GABAergic neurons in the ventral pallidum altered the effect of propofol on general anesthesia.

Pharmacological characterization of sex differences in the effects of dopaminergic drugs on effort-based decision making in rats.

Motivational dysfunctions related to effort exertion are common in psychiatric disorders. Dopamine systems regulate exertion of effort and effort-based choice in humans and rodents. Previous rodent studies mainly employed male rats, and it is imperative to conduct studies in male and female rats. The present studies compared the effort-related effects of IP injections of the dopamine antagonists ecopipam and haloperidol, and the vesicular monoamine transport-2 inhibitor tetrabenazine (TBZ), in male and female rats using the fixed ratio 5/chow feeding choice task. Ecopipam (0.05-0.2mg/kg) and haloperidol (0.05-0.15mg/kg) induced a low-effort bias, decreasing lever pressing and increasing chow intake in males and females in the same dose range. With lever pressing, there was a modest but significant dose x sex interaction after ecopipam injection, but there was no significant interaction after administration of haloperidol. In the first study with TBZ (0.25-1.0mg/kg), there was a robust sex difference. TBZ shifted choice from lever pressing to chow intake in male rats, but was ineffective in females. In a second experiment, 2.0mg/kg affected choice behavior in both males and females. TBZ increased accumbens c-Fos immunoreactivity in a sex-dependent manner, with males significantly increasing at 1.0mg/kg, while females showed augmented immunoreactivity at 2.0mg/kg. The neural and behavioral effects of TBZ differed across sexes, emphasizing the importance of conducting studies in male and female rats. This research has implications for understanding the effort-related motivational dysfunctions seen in psychopathology.

The Granular Retrosplenial Cortex Is Necessary in Male Rats for Object-Location Associative Learning and Memory, But Not Spatial Working Memory or Visual Discrimination and Reversal, in the Touchscreen Operant Chamber.

The retrosplenial cortex (RSC) is a hub of diverse afferent and efferent projections thought to be involved in associative learning. RSC shows early pathology in mild cognitive impairment and Alzheimer's disease (AD), which impairs associative learning. To understand and develop therapies for diseases such as AD, animal models are essential. Given the importance of human RSC in object-location associative learning and the success of object-location associative paradigms in human studies and in the clinic, it would be of considerable value to establish a translational model of object-location learning for the rodent. For this reason, we sought to test the role of RSC in object-location learning in male rats using the object-location paired-associates learning (PAL) touchscreen task. First, increased cFos immunoreactivity was observed in granular RSC following PAL training when compared with extended pretraining controls. Following this, RSC lesions following PAL acquisition were used to explore the necessity of the RSC in object-location associative learning and memory and two tasks involving only one modality: trial-unique nonmatching-to-location for spatial working memory and pairwise visual discrimination/reversal. RSC lesions impaired both memory for learned paired-associates and learning of new object-location associations but did not affect performance in either the spatial or visual single-modality tasks. These findings provide evidence that RSC is necessary for object-location learning and less so for learning and memory involving the individual modalities therein.

Combination administration of alprazolam and N-Ethylmaleimide synergistically enhances sleep behaviors in mice with no potential CNS side effects.

N-Ethylmaleimide (NEM), an agonist of the potassium chloride cotransporters 2 (KCC2) receptor, has been correlated with neurosuppressive outcomes, including decreased pain perception and the prevention of epileptic seizures. Nevertheless, its relationship with sleep-inducing effects remains unreported. The present study aimed to investigate the potential enhancement of NEM on the sleep-inducing properties of alprazolam (Alp). The test of the righting reflex was used to identify the appropriate concentrations of Alp and NEM for inducing sleep-promoting effects in mice. Total sleep duration and sleep quality were evaluated through EEG/EMG analysis. The neural mechanism underlying the sleep-promoting effect was examined through c-fos immunoreactivity in the brain using immunofluorescence. Furthermore, potential CNS-side effects of the combination Alp and NEM were assessed using LABORAS automated home-cage behavioral phenotyping. Combination administration of Alp (1.84 mg/kg) and NEM (1.0 mg/kg) significantly decreased sleep latency and increased sleep duration in comparison to administering 1.84 mg/kg Alp alone. This effect was characterized by a notable increase in REM duration. The findings from c-fos immunoreactivity indicated that NEM significantly suppressed neuron activation in brain regions associated with wakefulness. Additionally, combination administration of Alp and NEM showed no effects on mouse neural behaviors during automated home cage monitoring. This study is the first to propose and demonstrate a combination therapy involving Alp and NEM that not only enhances the hypnotic effect but also mitigates potential CNS side effects, suggesting its potential application in treating insomnia.

High-Fat Diet Alters Neuronal Activity in Cardiometabolic-Regulatory Brain Regions in a Sex-Specific Manner

Obesity is a major public health problem that is associated with dysregulation of central nervous system circuits controlling cardiovascular and metabolic functions. High-fat diet (HFD)-induced obesity in animal models is known to widely influence neuronal function in metabolic-regulatory brain regions; however, the impact of HFD, and its interactions with sex, on neuronal activity in cardiovascular-regulatory brain regions is not fully understood. Thus, the objective of this study was to investigate differences in c-fos expression, a marker of neuronal activity, in key brain regions involved in cardiometabolic regulation in HFD-induced obese mice. We hypothesized that HFD would alter the pattern of neuronal activity in hypothalamic and brainstem regions in a sex-specific manner. To test this, male and female C57Bl/6J mice (n=6/group) were placed on a 60% HFD or matched control diet for 14 weeks. At the end of the diet period, brains were collected to assess for c-fos immunoreactivity in the paraventricular nucleus (PVN) and arcuate nucleus (ARC) of the hypothalamus, as well as in the nucleus of the solitary tract (NTS) and rostral ventrolateral medulla (RVLM) of the brainstem. Brain slices were imaged using a Keyence Fluorescent Microscope and c-fos positive cells were counted using QuPath software in a blinded manner. There was a significant effect due to a sex-by-diet interaction in all brain regions, with female mice having a lower number of c-fos positive neurons compared to males overall, and even more so under HFD conditions (PVN: 132±21 male control vs. 183±28 male HFD vs. 136±21 female control vs. 89±10 female HFD, Pdiet=0.916, Psex=0.041, Pint=0.028 two-way ANOVA; ARC: 92±23 male control vs. 108±16 male HFD vs. 90±7 female control vs. 41±6 female HFD, Pdiet=0.272, Psex=0.024, Pint=0.039; NTS: 72±20 male control vs. 114±115 male HFD vs. 68±10 female control vs. 31±6 female HFD, Pdiet=0.885, Psex=0.004, Pint=0.009; RVLM: 59±12 male control vs. 105±11 male HFD vs. 59±12 female control vs. 28±5 female HFD, Pdiet=0.464, Psex=0.002, Pint=0.002). Overall, these data suggest there are sex-specific differences in neuronal activity within key brain regions integral to cardiometabolic control, which may be influenced by HFD in females. Future studies will need to assess the importance of these changes in neural activity to functional outcomes under normal conditions, and in the context of obesity. Funding: NIH R01 HL156986. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in ethanol effects in knock-in mice expressing ethanol insensitive alpha1 and alpha2 glycine receptor subunits

AimsGlycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol. Main methodsWe used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm. Key findingsEthanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice. SignificanceThe results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.

Hypothalamic GABAergic Neurons Expressing Cellular Retinoic Acid Binding Protein 1 (CRABP1) Are Sensitive to Metabolic Status and Liraglutide in Male Mice

Introduction: Owing to their privileged anatomical location, neurons of the arcuate nucleus of the hypothalamus (ARC) play critical roles in sensing and responding to metabolic signals such as leptin and glucagon-like peptide 1 (GLP-1). In addition to the well-known proopiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons, subpopulations of GABAergic neurons are emerging as key regulators of energy balance. However, the precise identity of these metabolic neurons is still elusive. Here, we identified and characterized the molecular signature of a novel population of GABAergic neurons of the ARC expressing Cellular retinoic acid binding protein 1 (Crabp1). Methods: Using a combination of immunohistochemistry and in situ hybridization techniques, we investigated the expression of Crabp1 across the mouse brain and characterized the molecular identity of Crabp1ARC neurons. We also determined whether Crabp1ARC neurons are sensitive to fasting, leptin, and GLP1R agonism by assessing cFOS immunoreactivity as a marker of neuronal activity. Results: Crabp1ARC neurons represent a novel GABAergic neuronal population robustly enriched in the ARC and are distinct from the prototypical melanocortin neurons. Crabp1ARC neurons overlap with three subpopulations of yet uncharacterized ARC neurons expressing Htr3b, Tbx19, and Tmem215. Notably, Crabp1ARC neurons express receptors for metabolic hormones and their activity is modulated by the nutritional state and GLP1R agonism. Conclusion: Crabp1ARC neurons represent a novel heterogeneous population of GABAergic neurons sensitive to metabolic status.

Novel rat model of gaming disorder: assessment of social reward and sex differences in behavior and c-Fos brain activity.

In 2018, the International Classification of Diseases (ICD-11) classified Gaming Disorder (GD) as a mental disorder. GD mainly occurs among adolescents, who, after developing addiction, show psychopathological traits, such as social anxiety, depression, social isolation, and attention deficit. However, the different studies conducted in humans so far show several limitations, such as the lack of demographic heterogeneity and equal representation of age, differences in the type of game and in the follow-up period. Furthermore, at present, no animal models specific to GD are available. To address the lack of an experimental model for GD, in the present work, we proposed a new GD rat model to investigate some peculiar tracts of the disorder. Two-month-old Wistar Kyoto rats, both males and females, were subject to a five-week training with a new innovative touch-screen platform. After five weeks of training, rats were assessed for: (a) their attachment to the play under several conditions, (b) their hyperactivity during gaming, and (c) the maintenance of these conditions after a period of game pause and reward interruption. After sacrifice, using immunohistochemistry techniques, the immunoreactivity of c-Fos (a marker of neuronal activity) was analyzed to study different neural areas. After the training, the rats subjected to GD protocol developed GD-related traits (e.g., hyperactivity, loss control), and the behavioral phenotype was maintained consistently over time. These aspects were completely absent in the control groups. Lastly, the analysis of c-Fos immunoreactivity in prelimbic cortex (PrL), orbitofrontal cortex (OFC), nucleus Accumbens, amygdala and bed nucleus of stria terminalis (BNST) highlighted significant alterations in the GD groups compared to controls, suggesting modifications in neural activity related to the development of the GD phenotype. The proposal of a new GD rat model could represent an innovative tool to investigate, in both sexes, the behavioral and neurobiological features of this disorder, the possible role of external factors in the predisposition and susceptibility and the development of new pharmacological therapies.

Sensitivity to the initial rewarding effects of alcohol: Influence of age, sex, and β-endorphin.

Alcohol use disorders (AUDs) are widespread, devastating and complex. About 20% of people who consume alcohol develop problem use, accounting for over 5% of worldwide deaths. While numerous animal models have facilitated understanding of the consequences of excessive drinking, translational models allow for experimental manipulation of factors thought to contribute to AUD liability. We employ a single-exposure conditioned place preference assay (SE-CPP) to investigate the influence of age, sex and the opioid peptide β-endorphin (bE) on the initial rewarding effects of ethanol, a strong predictor of AUDs. Adolescent (PND28-35) and adult (PND70-90) male and female, control C57BL/6J and bE-deficient mice were tested following a single injection of 1.5 g/kg of ethanol. Following the SE-CPP test, animals were deeply anesthetized, sacrificed, and perfused, and the brains were subsequently sectioned at 40 microns and processed for immunohistochemical localization of c-fos. One-sample, two-tailed t-tests were used to assess drug preference or aversion and the locomotor effects of alcohol. In general, adults were more sensitive to the effects of alcohol than adolescents, and outcomes depended on sex and bE. For example, among females, adolescents were stimulated by the drug, but insensitive to locomotor effects as adults, while among males, adolescents were insensitive and adults sedated. Wild-type adolescents of both sexes failed to evince initial subjective reward from the drug, but bE-deficient adolescents, and all adult subjects, preferred a context once associated with ethanol over one that had been paired with saline. c-fos immunoreactivity in multiple brain regions was attenuated in bE-deficient animals, though influences of both sex and bE grew stronger with age. This study demonstrates the utility of the SE-CPP paradigm for elucidating factors that contribute to the liability for AUDs, and supports the growing body of research that shows that sensitivity to the rewarding effects of alcohol changes during the course of development. Our results also suggest that developmental contributions are sex-dependent, and may also depend on the influence of endogenous opioid signaling.

Evidence for a Role of 5-HT-glutamate Co-releasing Neurons in Acute Stress Mechanisms.

A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.

Retraction Note: The pattern of c-Fos immunoreactivity in the hindbrain of the rat following stomach distension.

Retraction Note: The pattern of c-Fos immunoreactivity in the hindbrain of the rat following stomach distension.

Augmenting glutamatergic, but not dopaminergic, activity in the nucleus accumbens shell disrupts responding to a discrete alcohol cue in an alcohol context.

Discrete alcohol cues and contexts are relapse triggers for people with alcohol use disorder exerting particularly powerful control over behaviour when they co-occur. Here, we investigated the neural substrates subserving the capacity for alcohol-associated contexts to elevate responding to an alcohol-predictive conditioned stimulus (CS). Specifically, rats were trained in a distinct 'alcohol context' to respond by entering a fluid port during a discrete auditory CS that predicted the delivery of alcohol and were familiarized with a 'neutral context' wherein alcohol was never available. When conditioned CS responding was tested by presenting the CS without alcohol, we found that augmenting glutamatergic activity in the nucleus accumbens (NAc) shell by microinfusing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) reduced responding to an alcohol CS in an alcohol, but not neutral, context. Further, AMPA microinfusion robustly affected behaviour, attenuating the number, duration and latency of CS responses selectively in the alcohol context. Although dopaminergic inputs to the NAc shell were previously shown to be necessary for CS responding in an alcohol context, here, chemogenetic excitation of ventral tegmental area (VTA) dopamine neurons and their inputs to the NAc shell did not affect CS responding. Critically, chemogenetic excitation of VTA dopamine neurons affected feeding behaviour and elevated c-fos immunoreactivity in the VTA and NAc shell, validating the chemogenetic approach. These findings enrich our understanding of the substrates underlying Pavlovian responding for alcohol and reveal that the capacity for contexts to modulate responding to discrete alcohol cues is delicately underpinned by the NAc shell.