Amygdalar neurotransmission alterations in the BTBR mice model of idiopathic autism.

Autism Spectrum Disorders (ASD) are principally diagnosed by three core behavioural symptoms, such as stereotyped repertoire, communication impairments and social dysfunctions. This complex pathology has been linked to abnormalities of corticostriatal and limbic circuits. Despite experimental efforts in elucidating the molecular mechanisms behind these abnormalities, a clear etiopathogenic hypothesis is still lacking. To this aim, preclinical studies can be really helpful to longitudinally study behavioural alterations resembling human symptoms and to investigate the underlying neurobiological correlates. In this regard, the BTBR T+ Itpr3tf/J (BTBR) mice are an inbred mouse strain that exhibits a pattern of behaviours well resembling human ASD-like behavioural features. In this study, the BTBR mice model was used to investigate neurochemical and biomolecular alterations, regarding Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF), together with GABAergic, glutamatergic, cholinergic, dopaminergic and noradrenergic neurotransmissions and their metabolites in four different brain areas, i.e. prefrontal cortex, hippocampus, amygdala and hypothalamus. In our results, BTBR strain reported decreased noradrenaline, acetylcholine and GABA levels in prefrontal cortex, while hippocampal measurements showed reduced NGF and BDNF expression levels, together with GABA levels. Concerning hypothalamus, no differences were retrieved. As regarding amygdala, we found reduced dopamine levels, accompanied by increased dopamine metabolites in BTBR mice, together with decreased acetylcholine, NGF and GABA levels and enhanced glutamate content. Taken together, our data showed that the BTBR ASD model, beyond its face validity, is a useful tool to untangle neurotransmission alterations that could be underpinned to the heterogeneous ASD-like behaviours, highlighting the crucial role played by amygdala.

Association between social dominance hierarchy and PACAP expression in the extended amygdala, corticosterone, and behavior in C57BL/6 male mice.

The natural alignment of animals into social dominance hierarchies produces adaptive, and potentially maladaptive, changes in the brain that influence health and behavior. Aggressive and submissive behaviors assumed by animals through dominance interactions engage stress-dependent neural and hormonal systems that have been shown to correspond with social rank. Here, we examined the association between social dominance hierarchy status established within cages of group-housed mice and the expression of the stress peptide PACAP in the bed nucleus of the stria terminalis (BNST) and central nucleus of the amygdala (CeA). We also examined the relationship between social dominance rank and blood corticosterone (CORT) levels, body weight, motor coordination (rotorod) and acoustic startle. Male C57BL/6 mice were ranked as either Dominant, Submissive, or Intermediate based on counts of aggressive/submissive encounters assessed at 12 weeks-old following a change in homecage conditions. PACAP expression was significantly higher in the BNST, but not the CeA, of Submissive mice compared to the other groups. CORT levels were lowest in Submissive mice and appeared to reflect a blunted response following events where dominance status is recapitulated. Together, these data reveal changes in specific neural/neuroendocrine systems that are predominant in animals of lowest social dominance rank, and implicate PACAP in brain adaptations that occur through the development of social dominance hierarchies.

Puberty Blocker, Leuprolide, Reduces Sex Differences in Rough-and-Tumble Play and Anxiety-like Behavior in Juvenile Rats.

We examined the effect of the puberty blocker, leuprolide acetate, on sex differences in juvenile rough-and-tumble play behavior and anxiety-like behavior in adolescent male and female rats. We also evaluated leuprolide treatment on gonadal and pituitary hormone levels and activity-regulated cytoskeleton-protein messenger RNA levels within the adolescent amygdala, a region important both for rough-and-tumble play and anxiety-like behavior. Our findings suggest that leuprolide treatment lowered anxiety-like behavior during adolescent development, suggesting that the maturation of gonadotropin-releasing hormone systems may be linked to increased anxiety. These data provide a potential new model to understand the emergence of increased anxiety triggered around puberty. Leuprolide also reduced masculinized levels of rough-and-tumble play behavior, lowered follicle-stimulating hormone, and produced a consistent pattern of reducing or halting sex differences of hormone levels, including testosterone, growth hormone, thyrotropin, and corticosterone levels. Therefore, leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior, allowing for better harmonization of these systems following gender-affirming hormone treatment. These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior.

Adolescent alcohol exposure modifies adult anxiety-like behavior and amygdala sensitivity to alcohol in rats: Increased c-Fos activity and sex-dependent microRNA-182 expression.

Adolescent binge alcohol drinking is a serious health concern contributing to adult alcohol abuse often associated with anxiety disorders. We have used adolescent intermittent ethanol (AIE) administration as a model of binge drinking in rats in order to explore its long-term effect on the basolateral amygdala (BLA) responsiveness to alcohol and anxiety-like behavior. AIE increased the number of BLA c-Fos positive cells in adult Wistar rats and anxiety-like behavior assessed by the open field test (OFT). Additionally, in adult female rats receiving AIE BLA over expression of miR-182 was found. Therefore, our results indicate that alcohol consumption during adolescence can lead to enduring changes in anxiety-like behavior and BLA susceptibility to alcohol that may be mediated by sex-dependent epigenetic changes. These results contribute to understanding the mechanisms involved in the development of alcohol use disorders (AUD) and anxiety-related disorders.

NRG1-ErbB4 signaling in the medial amygdala controls mating motivation in adult male mice.

Motivation-driven mating is a basic affair for the maintenance of species. However, the underlying molecular mechanisms that control mating motivation are not fully understood. Here, we report that NRG1-ErbB4 signaling in the medial amygdala (MeA) is pivotal in regulating mating motivation. NRG1 expression in the MeA negatively correlates with the mating motivation levels in adult male mice. Local injection and knockdown of MeA NRG1 reduce and promote mating motivation, respectively. Consistently, knockdown of MeA ErbB4, a major receptor for NRG1, and genetic inactivation of its kinase both promote mating motivation. ErbB4 deletion decreases neuronal excitability, whereas chemogenetic manipulations of ErbB4-positive neuronal activities bidirectionally modulate mating motivation. We also identify that the effects of NRG1-ErbB4 signaling on neuronal excitability and mating motivation rely on hyperpolarization-activated cyclic nucleotide-gated channel 3. This study reveals a critical molecular mechanism for regulating mating motivation in adult male mice.

Insula→Amygdala and Insula→Thalamus Pathways Are Involved in Comorbid Chronic Pain and Depression-Like Behavior in Mice.

The comorbidity of chronic pain and depression poses tremendous challenges for the treatment of either one because they exacerbate each other with unknown mechanisms. As the posterior insular cortex (PIC) integrates multiple somatosensory and emotional information and is implicated in either chronic pain or depression, we hypothesize that the PIC and its projections may contribute to the pathophysiology of comorbid chronic pain and depression. We show that PIC neurons were readily activated by mechanical, thermal, aversive, and stressful and appetitive stimulation in naive and neuropathic pain male mice subjected to spared nerve injury (SNI). Optogenetic activation of PIC neurons induced hyperalgesia and conditioned place aversion in naive mice, whereas inhibition of these neurons led to analgesia, conditioned place preference (CPP), and antidepressant effect in both naive and SNI mice. Combining neuronal tracing, optogenetics, and electrophysiological techniques, we found that the monosynaptic glutamatergic projections from the PIC to the basolateral amygdala (BLA) and the ventromedial nucleus (VM) of the thalamus mimicked PIC neurons in pain modulation in naive mice; in SNI mice, both projections were enhanced accompanied by hyperactivity of PIC, BLA, and VM neurons and inhibition of these projections led to analgesia, CPP, and antidepressant-like effect. The present study suggests that potentiation of the PIC→BLA and PIC→VM projections may be important pathophysiological bases for hyperalgesia and depression-like behavior in neuropathic pain and reversing the potentiation may be a promising therapeutic strategy for comorbid chronic pain and depression.

Adolescent social isolation disrupts developmental tuning of neuropeptide circuits in the hypothalamus to amygdala regulating social and defensive behavior.

Engaging in positive social (i.e., prosocial) interactions during adolescence acts to modulate neural circuits that determine adult adaptive behavior. While accumulating evidence indicates that a strong craving for prosocial behavior contributes to sustaining neural development, the consequences of social deprivation during adolescence on social neural circuits, including those involving oxytocin (OXT) and vasopressin (AVP), are poorly characterized. We evaluated adaptive behaviors in socially isolated mice, including anxiety-like, social, and defensive behaviors, along with OXT and AVP neural profiles in relevant brain regions. Social isolation from postnatal day (P-)22 to P-48 induced enhanced defensive and exploratory behaviors, in nonsocial and social contexts. Unlike OXT neurons, AVP+ cell density in the paraventricular nucleus of the hypothalamus increases with age in males. Social isolation also modulated gene expression in the medial amygdala (MeA), including the upregulation of OXT receptors in males and the downregulation of AVP1a receptors in both sexes. Socially isolated mice showed an enhanced defensive, anogenital approach toward a novel adult female during direct social interactions. Subsequent c-Fos mapping revealed diminished neural activity in restricted brain areas, including the MeA, lateral septum, and posterior intralaminar nucleus of the thalamus, in socially isolated mice. These data indicate that neural signals arising from daily social interactions invoke region-specific modification of neuropeptide expression that coordinates with altered defensiveness and neural responsivities, including OXT- and AVP-projecting regions. The present findings indicate an involvement of OXT and AVP circuits in adolescent neural and behavioral plasticity that is tuned by daily social interaction.

Selective alterations of endocannabinoid system genes expression in obsessive compulsive disorder.

Obsessive Compulsive Disorder (OCD) is listed as one of the top 10 most disabling neuropsychiatric conditions in the world. The neurobiology of OCD has not been completely understood and efforts are needed in order to develop new treatments. Beside the classical neurotransmitter systems and signalling pathways implicated in OCD, the possible involvement of the endocannabinoid system (ECS) has emerged in pathophysiology of OCD. We report here selective downregulation of the genes coding for enzymes allowing the synthesis of the endocannabinoids. We found reduced DAGLα and NAPE-PLD in blood samples of individuals with OCD (when compared to healthy controls) as well as in the amygdala complex and prefrontal cortex of dopamine transporter (DAT) heterozygous rats, manifesting compulsive behaviours. Also mRNA levels of the genes coding for cannabinoid receptors type 1 and type 2 resulted downregulated, respectively in the rat amygdala and in human blood. Moreover, NAPE-PLD changes in gene expression resulted to be associated with an increase in DNA methylation at gene promoter, and the modulation of this gene in OCD appears to be correlated to the progression of the disease. Finally, the alterations observed in ECS genes expression appears to be correlated with the modulation in oxytocin receptor gene expression, consistently with what recently reported. Overall, we confirm here a role for ECS in OCD at both preclinical and clinical level. Many potential biomarkers are suggested among its components, in particular NAPE-PLD, that might be of help for a prompt and clear diagnosis.

Effect of a single psilocybin treatment on Fos protein expression in male rat brain.

Psilocybin has received attention as a treatment for depression, stress disorders and drug and alcohol addiction. To help determine the mechanisms underlying its therapeutic effects, here we examined acute effects of a range of behaviourally relevant psilocybin doses (0.1-3 mg/kg SC) on regional expression of Fos, the protein product of the immediate early gene, c-fos in brain areas involved in stress, reward and motivation in male rats. We also determined the cellular phenotypes activated by psilocybin, in a co-labeling analysis with NeuN, a marker of mature neurons, or Olig1, a marker of oligodendrocytes. In adult male Sprague-Dawley rats, psilocybin increased Fos expression dose dependently in several brain regions, including the frontal cortex, nucleus accumbens, central and basolateral amygdala and locus coeruleus. These effects were most marked in the central amygdala. Double labeling experiments showed that Fos was expressed in both neurons and oligodendrocytes. These results extend previous research by determining Fos expression in multiple brain areas at a wider psilocybin dose range, and the cellular phenotypes expressing Fos. The data also highlight the amygdala, especially the central nucleus, a key brain region involved in emotional processing and learning and interconnected with other brain areas involved in stress, reward and addiction, as a potentially important locus for the therapeutic effects of psilocybin. Overall, the present findings suggest that the central amygdala may be an important site through which the initial brain activation induced by psilocybin is translated into neuroplastic changes, locally and in other regions that underlie its extended therapeutic effects.

Projections from infralimbic medial prefrontal cortex glutamatergic outputs to amygdala mediates opioid induced hyperalgesia in male rats.

Repeated use of opioid analgesics may cause a paradoxically exacerbated pain known as opioid-induced hyperalgesia (OIH), which hinders effective clinical intervention for severe pain. Currently, little is known about the neural circuits underlying OIH modulation. Previous studies suggest that laterocapsular division of the central nucleus of amygdala (CeLC) is critically involved in the regulation of OIH. Our purpose is to clarify the role of the projections from infralimbic medial prefrontal cortex (IL) to CeLC in OIH. We first produced an OIH model by repeated fentanyl subcutaneous injection in male rats. Immunofluorescence staining revealed that c-Fos-positive neurons were significantly increased in the right CeLC in OIH rats than the saline controls. Then, we used calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) labeling and the patch-clamp recordings with ex vivo optogenetics to detect the functional projections from glutamate pyramidal neurons in IL to the CeLC. The synaptic transmission from IL to CeLC, shown in the excitatory postsynaptic currents (eEPSCs), inhibitory postsynaptic currents (eIPSCs) and paired-pulse ratio (PPR), was observably enhanced after fentanyl administration. Moreover, optogenetic activation of this IL-CeLC pathway decreased c-Fos expression in CeLC and ameliorated mechanical and thermal pain in OIH. On the contrary, silencing this pathway by chemogenetics exacerbated OIH by activating the CeLC. Combined with the electrophysiology results, the enhanced synaptic transmission from IL to CeLC might be a cortical gain of IL to relieve OIH rather than a reason for OIH generation. Scaling up IL outputs to CeLC may be an effective neuromodulation strategy to treat OIH.

Ketamine alleviates fear memory and spatial cognition deficits in a PTSD rat model via the BDNF signaling pathway of the hippocampus and amygdala.

BACKGROUND: Post-traumatic stress disorder (PTSD) is associated with traumatic stress experiences. This condition can be accompanied by learning and cognitive deficits. Studies have demonstrated that ketamine can rapidly and significantly alleviate symptoms in patients with chronic PTSD. Nonetheless, the effects of ketamine on neurocognitive impairment and its mechanism of action in PTSD remain unclear. METHODS: In this study, different concentrations of ketamine (5, 10, 15, and 20 mg/kg, i.p.) were evaluated in rat models of single prolonged stress and electrophonic shock (SPS&S). Expression levels of brain-derived neurotrophic factor (BDNF) and post-synaptic density-95 (PSD-95) in the hippocampus (HIP) and amygdala (AMG) were determined by Western blot analysis and immunohistochemistry. RESULTS: The data showed that rats subjected to SPS&S exhibited significant PTSD-like cognitive impairment. The effect of ketamine on SPS&S-induced neurocognitive function showed a U-shaped dose effect in rats. A single administration of ketamine at a dosage of 10-15 mg/kg resulted in significant changes in behavioral outcomes. These manifestations of improvement in cognitive function and molecular changes were reversed at high doses (15-20 mg/kg). CONCLUSION: Overall, ketamine reversed SPS&S-induced fear and spatial memory impairment and the down-regulation of BDNF and BDNF-related PSD-95 signaling in the HIP and AMG. A dose equal to 15 mg/kg rapidly reversed the behavioral and molecular changes and promoted the amelioration of cognitive dysfunction. The enhanced association of BDNF signaling with PSD-95 effects could be involved in the therapeutic efficiency of ketamine for PTSD.

Chronic kidney disease may evoke anxiety by altering CRH expression in the amygdala and tryptophan metabolism in rats.

Chronic kidney disease (CKD) is associated with anxiety; however, its exact mechanism is not well understood. Therefore, the aim of the present study was to assess the effect of moderate CKD on anxiety in rats. 5/6 nephrectomy was performed in male Wistar rats. 7 weeks after, anxiety-like behavior was assessed by elevated plus maze (EPM), open field (OF), and marble burying (MB) tests. At weeks 8 and 9, urinalysis was performed, and blood and amygdala samples were collected, respectively. In the amygdala, the gene expression of Avp and the gene and protein expression of Crh, Crhr1, and Crhr2 were analyzed. Furthermore, the plasma concentration of corticosterone, uremic toxins, and tryptophan metabolites was measured by UHPLC-MS/MS. Laboratory tests confirmed the development of CKD. In the CKD group, the closed arm time increased; the central time and the total number of entries decreased in the EPM. There was a reduction in rearing, central distance and time in the OF, and fewer interactions with marbles were detected during MB. CKD evoked an upregulation of gene expression of Crh, Crhr1, and Crhr2, but not Avp, in the amygdala. However, there was no alteration in protein expression. In the CKD group, plasma concentrations of p-cresyl-sulfate, indoxyl-sulfate, kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid, xanthurenic acid, 5-hydroxyindoleacetic acid, picolinic acid, and quinolinic acid increased. However, the levels of tryptophan, tryptamine, 5-hydroxytryptophan, serotonin, and tyrosine decreased. In conclusion, moderate CKD evoked anxiety-like behavior that might be mediated by the accumulation of uremic toxins and metabolites of the kynurenine pathway, but the contribution of the amygdalar CRH system to the development of anxiety seems to be negligible at this stage.

Inhibition of fatty acid binding protein-5 in the basolateral amygdala induces anxiolytic effects and accelerates fear memory extinction.

RATIONALE: The endocannabinoid (eCB) system critically controls anxiety and fear-related behaviours. Anandamide (AEA), a prominent eCB ligand, is a hydrophobic lipid that requires chaperone proteins such as Fatty Acid Binding Proteins (FABPs) for intracellular transport. Intracellular AEA transport is necessary for degradation, so blocking FABP activity increases AEA neurotransmission. OBJECTIVE: To investigate the effects of a novel FABP5 inhibitor (SBFI-103) in the basolateral amygdala (BLA) on anxiety and fear memory. METHODS: We infused SBFI-103 (0.5 µg-5 µg) to the BLA of adult male Sprague Dawley rats and ran various anxiety and fear memory behavioural assays, neurophysiological recordings, and localized molecular signaling analyses. We also co-infused SBFI-103 with the AEA inhibitor, LEI-401 (3 µg and 10 µg) to investigate the potential role of AEA in these phenomena. RESULTS: Acute intra-BLA administration of SBFI-103 produced strong anxiolytic effects across multiple behavioural tests. Furthermore, animals exhibited acute and long-term accelerated associative fear memory extinction following intra-BLA FABP5 inhibition. In addition, BLA FABP5 inhibition induced strong modulatory effects on putative PFC pyramidal neurons along with significantly increased gamma oscillation power. Finally, we observed local BLA changes in the phosphorylation activity of various anxiety- and fear memory-related molecular biomarkers in the PI3K/Akt and MAPK/Erk signaling pathways. At all three levels of analyses, we found the functional effects of SBFI-103 depend on availability of the AEA ligand. CONCLUSIONS: These findings demonstrate a novel intra-BLA FABP5 signaling mechanism regulating anxiety and fear memory behaviours, neuronal activity states, local anxiety-related molecular pathways, and functional AEA modulation.

Clinical Correlation of Altered Molecular Signatures in Epileptic Human Hippocampus and Amygdala.

Widespread alterations in the expression of various genes could contribute to the pathogenesis of epilepsy. The expression levels of various genes, including major inhibitory and excitatory receptors, ion channels, cell type-specific markers, and excitatory amino acid transporters, were assessed and compared between the human epileptic hippocampus and amygdala, and findings from autopsy controls. Moreover, the potential correlation between molecular alterations in epileptic brain tissues and the clinical characteristics of patients undergoing epilepsy surgery was evaluated. Our findings revealed significant and complex changes in the expression of several key regulatory genes in both the hippocampus and amygdala of patients with intractable epilepsy. The expression changes in various genes differed considerably between the epileptic hippocampus and amygdala. Different correlation patterns were observed between changes in gene expression and clinical characteristics, depending on whether the patients were considered as a whole or were subdivided. Altered molecular signatures in different groups of epileptic patients, defined within a given category, could be viewed as diagnostic biomarkers. Distinct patterns of molecular changes that distinguish these groups from each other appear to be associated with epilepsy-specific functional consequences.

Synaptic Involvement of the Human Amygdala in Parkinson's Disease.

α-Synuclein, a protein mostly present in presynaptic terminals, accumulates neuropathologically in Parkinson's disease in a 6-stage sequence and propagates in the nervous system in a prion-like manner through neurons and glia. In stage 3, the substantia nigra are affected, provoking motor symptoms and the amygdaloid complex, leading to different nonmotor symptoms; from here, synucleinopathy spreads to the temporal cortex and beyond. The expected increase in Parkinson's disease incidence accelerates the need for detection biomarkers; however, the heterogeneity of this disease, including pathological aggregates and pathophysiological pathways, poses a challenge in the search for new therapeutic targets and biomarkers. Proteomic analyses are lacking, and the literature regarding synucleinopathy, neural and glial involvement, and volume of the human amygdaloid complex is controversial. Therefore, the present study combines both proteomic and stereological probes. Data-independent acquisition-parallel accumulation of serial fragmentation proteomic analysis revealed a remarkable proteomic impact, especially at the synaptic level in the human amygdaloid complex in Parkinson's disease. Among the 199 differentially expressed proteins, guanine nucleotide-binding protein G(i) subunit alpha-1 (GNAI1), elongation factor 1-alpha 1 (EEF1A1), myelin proteolipid protein (PLP1), neuroplastin (NPTN), 14-3-3 protein eta (YWHAH), gene associated with retinoic and interferon-induced mortality 19 protein (GRIM19), and orosomucoid-2 (ORM2) stand out as potential biomarkers in Parkinson's disease. Stereological analysis, however, did not reveal alterations regarding synucleinopathy, neural or glial populations, or volume changes. To our knowledge, this is the first proteomic study of the human amygdaloid complex in Parkinson's disease, and it identified possible biomarkers of the disease. Lewy pathology could not be sufficient to cause neurodegeneration or alteration of microglial and astroglial populations in the human amygdaloid complex in Parkinson's disease. Nevertheless, damage at the proteomic level is manifest, showing up significant synaptic involvement.

Delivery of graphene oxide nanosheets modulates glutamate release and normalizes amygdala synaptic plasticity to improve anxiety-related behavior.

Graphene oxide nanosheets (GO) were reported to alter neurobiological processes involving cell membrane dynamics. GO ability to reversibly downregulate specifically glutamatergic synapses underpins their potential in future neurotherapeutic developments. Aberrant glutamate plasticity contributes to stress-related psychopathology and drugs which target dysregulated glutamate represent promising treatments. We find that in a rat model of post-traumatic stress disorder (PTSD), a single injection of GO to the lateral amygdala following the stressful event induced PTSD-related behavior remission and reduced dendritic spine densities. We explored from a mechanistic perspective how GO could impair glutamate synaptic plasticity. By simultaneous patch clamp pair recordings of unitary synaptic currents, live-imaging of presynaptic vesicle release and confocal microscopy, we report that GO nanosheets altered the probability of release enhancing the extinction of synaptic plasticity in the amygdala. These findings show that the modulation of presynaptic glutamate release might represent an unexplored target for (nano)pharmacological interventions of stress-related disorders.

Sex-differences in proteasome-dependent K48-polyubiquitin signaling in the amygdala are developmentally regulated in rats.

BACKGROUND: Sex differences have been observed in several brain regions for the molecular mechanisms involved in baseline (resting) and memory-related processes. The ubiquitin proteasome system (UPS) is a major protein degradation pathway in cells. Sex differences have been observed in lysine-48 (K48)-polyubiquitination, the canonical degradation mark of the UPS, both at baseline and during fear memory formation within the amygdala. Here, we investigated when, how, and why these baseline sex differences arise and whether both sexes require the K48-polyubiquitin mark for memory formation in the amygdala. METHODS: We used a combination of molecular, biochemical and proteomic approaches to examine global and protein-specific K48-polyubiquitination and DNA methylation levels at a major ubiquitin coding gene (Uba52) at baseline in the amygdala of male and female rats before and after puberty to determine if sex differences were developmentally regulated. We then used behavioral and genetic approaches to test the necessity of K48-polyubiquitination in the amygdala for fear memory formation. RESULTS: We observed developmentally regulated baseline differences in Uba52 methylation and total K48-polyubiquitination, with sexual maturity altering levels specifically in female rats. K48-polyubiquitination at specific proteins changed across development in both male and female rats, but sex differences were present regardless of age. Lastly, we found that genetic inhibition of K48-polyubiquitination in the amygdala of female, but not male, rats impaired fear memory formation. CONCLUSIONS: These results suggest that K48-polyubiquitination differentially targets proteins in the amygdala in a sex-specific manner regardless of age. However, sexual maturity is important in the developmental regulation of K48-polyubiquitination levels in female rats. Consistent with these data, K48-polyubiquitin signaling in the amygdala is selectively required to form fear memories in female rats. Together, these data indicate that sex-differences in baseline K48-polyubiquitination within the amygdala are developmentally regulated, which could have important implications for better understanding sex-differences in molecular mechanisms involved in processes relevant to anxiety-related disorders such as post-traumatic stress disorder (PTSD).

Male and female brains have differences in size, development, and cellular processes. Further, males and females have differences in likelihood of developing certain anxiety-related disorders, such as post-traumatic stress disorder (PTSD). We previously observed sex differences in a cellular mechanism that controls the destruction of proteins via tagging by the protein modifier ubiquitin in resting and behaviorally trained animals. We found that adult female rats "ubiquitinated" different proteins during learning and had more ubiquitin than male rats at rest in the amygdala, the brain region that controls emotional regulation. This study investigated if the sex difference in ubiquitin at rest changed as animals age, including the proteins being ubiquitinated and how the amount of ubiquitin was controlled. We also investigated if male and female rats need ubiquitin for memory formation. We found that males and females ubiquitinate different proteins, but that aging also contributes to changes in this, suggesting that sexual maturity may be important for controlling the amount of ubiquitin in females. Lastly, we found that only female rats needed ubiquitin in the amygdala for forming a fear memory. These results are important for understanding the role of ubiquitin activity at different developmental stages and for forming fear-based memories in both sexes. Since females are more likely to develop PTSD than males, these data could help understand how different cellular processes work together in PTSD development to create better treatment options.

Interleukin-1ß and TNF-α are elevated in the amygdala of adult rats prenatally exposed to ethanol.

Affective disorders, including anxiety and depression, developed in adult offspring of the mothers who consumed alcohol during pregnancy could be associated with an imbalance in neuroimmune factors in the amygdala (corpus amygdaloideum) resulted in impaired emotional stimulus processing. The aim of this study was to compare the content of cytokines TNF-α, IL-1α, IL-1ß, IL-10, and IL-17 in the amygdala of adult female rats exposed to alcohol in utero and control rats. Cytokine levels were evaluated using a multiplex immunoassay system; mRNA expression was investigated using a real-time reverse transcription-polymerase chain reaction (RT-qPCR) assay. Prenatal alcohol exposure led to the increase in the content of TNF-α and IL-1ß without significant changes in the mRNA expression level. Our data suggest that ethanol exposure to the fetus during pregnancy can result in long-term alterations in the content of the key neuroinflammatory factors in the amygdala, which in turn can be a risk factor for affective disorders in the adulthood.

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.