GABAergic Markers Research Articles

Dichotomous Distribution of Putative Cholinergic Interneurons in Mouse Accessory Olfactory Bulb.

Sensory information processing in the olfactory bulb (OB) relies on diverse populations of bulbar interneurons. In rodents, the accessory OB (AOB) is divided into two bulbar regions, the anterior (aAOB) and posterior (pAOB), which differ substantially in their circuitry connections and associated behaviors. We previously identified and characterized a large number of morphologically diverse cholinergic interneurons in the main OB (MOB) using transgenic mice to visualize the cell bodies of choline acetyltransferase (ChAT-expressing neurons and immunolabeling (Krosnowski et al., 2012)). However, whether there are cholinergic neurons in the AOB is controversial and there is no detailed characterization of such neurons. Using the same line of ChAT(bacterial artificial chromosome, BAC)-enhanced green fluorescent protein (eGFP) transgenic mice, we investigated cholinergic neurons in the AOB. We found significant differences in the number and location of GFP-expressing (GFP+), putative cholinergic interneurons between the aAOB and pAOB. The highest numbers of GFP+ interneurons were found in the aAOB glomerular layer (aGL) and pAOB mitral/tufted cell layer (pMCL). We also noted a high density of GFP+ interneurons encircling the border region of the pMCL. Interestingly, a small subset of glomeruli in the middle of the GL receives strong MCL GFP+ nerve processes. These local putative cholinergic-innervated glomeruli are situated just outside the aGL, setting the boundary between the pGL and aGL. Many but not all GFP+ neurons in the AOB were weakly labeled with antibodies against ChAT and vesicular acetylcholine transporter (VAChT). We further determined if these GFP+ interneurons differ from other previously characterized interneuron populations in the AOB and found that AOB GFP+ interneurons express neither GABAergic nor dopaminergic markers and most also do not express the glutamatergic marker. Similar to the cholinergic interneurons of the MOB, some AOB GFP+ interneurons express the calcium binding protein, calbindin-D28K. Moreover, exposure to either a male intruder or soiled bedding from a mating cage leads to an increase in the number of c-Fos-expressing MCL GFP+ neurons. Taken together, our data reveal a population of largely unidentified putative cholinergic neurons in the AOB. Their dichotomous distribution in the aAOB and pAOB suggests region-specific cholinergic involvement in olfactory information processing.

Trans-generation enrichment of clozapine-responsiveness trait in mice using a subchronic hypo-glutamatergic model of schizophrenia:A preliminary study

BackgroundSchizophrenia patients who do not respond to clozapine treatment represent the most debilitating type of schizophrenia with unmet needs for novel interventions. To date there is no validated animal model for clozapine-refractory schizophrenia. MethodsWe used poor performance in the social preference (SP) test of C57/BL mice exposed to subchronic phencyclidine (PCP) as a correlate of negative signs of schizophrenia. Subsequently the mice were treated with clozapine and according to their SP they were defined as responding (i.e. clozapine/PCP ratio>1.5 SD) or non-responsive to clozapine. In each generation the responding mice were mated to produce the next generation. Unfortunately, the clozapine- non-responsive mice failed to proliferate and were thus excluded from the analyses. This forward genetic paradigm was used to produce the next generation of clozapine-responding mice. We assessed brain glutamic acid decarboxylase-67 (GAD67) protein levels, as a GABA-ergic marker, in the F2 and F3 generations. ResultsAlready in the F1 generation of male mice, but not females, it was possible to discriminate between clozapine-responders and non-responders. The rate of responders within each consecutive generation, increased. The increase was more pronounced in females. Up-regulation of GAD67 levels was detected between F2 and F3 only in male clozapine-responder mice, but not in females. ConclusionsThis preliminary proof-of-concept study succeeded in producing a trans-generation enrichment of clozapine-responsiveness trait in a hypo-glutamatergic animal model of negative signs of schizophrenia. This model may serve as a platform to better characterize the clozapine responsiveness trait and offer a model for clozapine-responsive schizophrenia.

Characterization of GABAergic Marker Expression in the Chronic Unpredictable Stress Model of Depression

Background Evidence continues to build suggesting that the GABAergic neurotransmitter system is altered in brains of patients with major depressive disorder. However, there is little information available related to the extent of these changes or the potential mechanisms associated with these alterations. As stress is a well-established precipitant to depressive episodes, we sought to explore the impact of chronic stress on GABAergic interneurons. Methods Using western blot analyses and quantitative real-time polymerase chain reaction, we assessed the effects of five-weeks of chronic unpredictable stress exposure on the expression of GABA-synthesizing enzymes (GAD65 and GAD67), calcium-binding proteins (calbindin, parvalbumin, and calretinin), and neuropeptides co-expressed in GABAergic neurons (somatostatin, neuropeptide Y, vasoactive intestinal peptide, and cholecystokinin) in the prefrontal cortex and hippocampus of rats. We also investigated the effects of corticosterone and dexamethasone exposure on these markers in vitro in primary cortical and hippocampal cultures. Results We found that chronic unpredictable stress induced significant reductions of GAD67 protein levels in both the prefrontal cortex and hippocampus of chronic unpredictable stress-exposed rats but did not detect changes in GAD65 protein expression. Similar protein expression changes were found in vitro in cortical neurons. In addition, our results provide clear evidence of reduced markers of interneuron population(s), namely somatostatin and neuropeptide Y, in the prefrontal cortex, suggesting these cell types may be selectively vulnerable to chronic stress. Conclusion Together, this work highlights that chronic stress induces regional and cell type-selective effects on GABAergic interneurons in rats. These findings provide additional supporting evidence that stress-induced GABA neuron dysfunction and cell vulnerability play critical roles in the pathophysiology of stress-related illnesses, including major depressive disorder.

청소년기 동물모델에서 구속 스트레스로 유발된 불안, 우울행동에 미치는 영향

Purpose: Some of the adolescent drinks more sugar-sweetened beverages. However, there is little evidence on the effect of eating behavior on emotional state and neurochemical changes under stress, especially on the levels of typical inhibitory neurotransmitters and gamma-aminobutyric acid. This article demonstrates that sucrose or saccharin drink reduces stress-related behavior responses and GABAergic deficits in adolescent rats. Methods: We randomly assigned 7-weeks-old Sprague-Dawley male rats to three groups: control group (Control), restraint stress only group (Stress), and restraint stress with unrestricted access to saccharin solution (Saccharin) and sucrose solution (Sucrose) as a positive control. We evaluated both anxious and depressive moods using an open field test and forced swim test, respectively. Using western blot analyses, the expression of a GABA-synthesizing enzyme, glutamate decarboxylase-67 (GAD67) and GABAergic markers, including calbindin and parvalbumin was assessed in the prefrontal cortex and the amygdala. Results: We found that both the drinks alleviated anxiety and depressive moods, induced significant attenuation in GAD67 level, and reduced calbindin level under stress in the prefrontal cortex and the amygdala. Conclusion: The results provide an understanding of the effect of sucrose or saccharin drink on stress-related responses. We propose the consumption of sweet drinks as a plausible strategy to alleviate stress-related alterations in adolescents. Key Words: Sugar-sweetened beverages; Artificially sweetened beverage; Gamma-aminobutyric acid; Adolescent; Emotions Key Words: 가당 음료; ì²­ì†Œë „; ìŠ¤íŠ¸ë ˆìŠ¤; ê°ì •; GABA성 ì‹ ê²½ì„¸í¬

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes.

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

The transcription factor Npas4 contributes to adolescent development of prefrontal inhibitory circuits, and to cognitive and emotional functions: Implications for neuropsychiatric disorders

The adolescent brain is marked by functional and structural modifications, particularly within the inhibitory system of the prefrontal cortex (PFC). These changes are necessary for the acquisition of adult cognitive functions and emotion regulation, and impairments in these processes are associated with neuropathologies such as schizophrenia and affective disorders. The molecular mechanisms regulating this adolescent refinement of prefrontal inhibitory circuits remain largely unknown. Here we demonstrate that the transcription factor Npas4 plays a major role in this process. Using a series of behavioral, molecular, pharmacological and genetic approaches in mice, we demonstrate that deficiency in Npas4 affects adolescent expression of multiple markers of GABAergic transmission in the PFC, including parvalbumin and GAD67, in a sex-specific manner. This abnormal pattern of expression of GABAergic markers is associated with sex-specific cognitive and emotional impairments that occur only when Npas4 deficiency begins at adolescence but not post-adolescence. Finally, we show that chronic treatment with the GABA enhancing drug sodium valproate during adolescence is sufficient to induce long-lasting recovery of the molecular and behavioral abnormalities observed in Npas4 deficient mice. Altogether, we provide evidence for the involvement of the transcription factor Npas4 to the structural changes that affect prefrontal inhibitory circuits during adolescence. Further investigations of Npas4 role in the adolescent brain might provide new insights on the molecular mechanisms underlying neuropsychiatric disorders that emerge during adolescence.

Chronic lurasidone treatment normalizes GABAergic marker alterations in the dorsal hippocampus of mice exposed to prenatal immune activation

Prenatal maternal infection represents a risk factor for the development of psychopathologic conditions later in life. Clinical evidence is also supported by animal models in which the vulnerability to develop a schizophrenic-like phenotype likely originates from inflammatory processes as early as in the womb. Prenatal immune challenge, for example, induces a variety of long-term behavioral alterations in mice, such as deficits in recognition and spatial working memory, perseverative behaviors and social impairments, which are relevant to different symptom clusters of schizophrenia.Here, we investigated the modulation of GABAergic markers in the dorsal and ventral hippocampus of adult mice exposed to late prenatal immune challenge with the viral mimetic Poly(I:C) (polyriboinosinic-polyribocytidilic-acid) at gestational day 17, and we evaluated the ability of chronic treatment with the multi-receptor antipsychotic lurasidone to modulate the alterations produced by maternal infection. Poly(I:C) mice show a significant reduction of key GABAergic markers, such as GAD67 and parvalbumin, specifically in the dorsal hippocampus, which were normalized by chronic lurasidone administration. Moreover, chronic drug administration increases the expression of the pool of brain derived neurotrophic factor (BDNF) transcripts with the long 3’-UTR as well as the levels of mature BDNF protein in the synaptosomal compartment, selectively in dorsal hippocampus.All in all, our findings demonstrate that lurasidone is effective in ameliorating molecular abnormalities observed in Poly(I:C) mice, providing further support to the neuroplastic properties of this multi-receptor antipsychotic drug.

Reducing GABAA-mediated inhibition improves forelimb motor function after focal cortical stroke in mice.

A deeper understanding of post-stroke plasticity is critical to devise more effective pharmacological and rehabilitative treatments. The GABAergic system is one of the key modulators of neuronal plasticity, and plays an important role in the control of “critical periods” during brain development. Here, we report a key role for GABAergic inhibition in functional restoration following ischemia in the adult mouse forelimb motor cortex. After stroke, the majority of cortical sites in peri-infarct areas evoked simultaneous movements of forelimb, hindlimb and tail, consistent with a loss of inhibitory signalling. Accordingly, we found a delayed decrease in several GABAergic markers that accompanied cortical reorganization. To test whether reductions in GABAergic signalling were causally involved in motor improvements, we treated animals during an early post-stroke period with a benzodiazepine inverse agonist, which impairs GABAA receptor function. We found that hampering GABAA signalling led to significant restoration of function in general motor tests (i.e., gridwalk and pellet reaching tasks), with no significant impact on the kinematics of reaching movements. Improvements were persistent as they remained detectable about three weeks after treatment. These data demonstrate a key role for GABAergic inhibition in limiting motor improvements after cortical stroke.

Sustained synchronized neuronal network activity in a human astrocyte co-culture system.

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

Transient Developmental Purkinje Cell Axonal Torpedoes in Healthy and Ataxic Mouse Cerebellum.

Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells – known as torpedoes – have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently during development in rat cerebellum. The function of Purkinje cell axonal torpedoes in health as well as in disease is poorly understood. We investigated the properties of developmental torpedoes in the postnatal mouse cerebellum of wild-type and transgenic mice. We found that Purkinje cell axonal torpedoes transiently appeared on axons of Purkinje neurons, with the largest number of torpedoes observed at postnatal day 11 (P11). This was after peak developmental apoptosis had occurred, when Purkinje cell counts in a lobule were static, suggesting that most developmental torpedoes appear on axons of neurons that persist into adulthood. We found that developmental torpedoes were not associated with a presynaptic GABAergic marker, indicating that they are not synapses. They were seldom found at axonal collateral branch points, and lacked microglia enrichment, suggesting that they are unlikely to be involved in axonal refinement. Interestingly, we found several differences between developmental torpedoes and disease-related torpedoes: developmental torpedoes occurred largely on myelinated axons, and were not associated with changes in basket cell innervation on their parent soma. Disease-related torpedoes are typically reported to contain neurofilament; while the majority of developmental torpedoes did as well, a fraction of smaller developmental torpedoes did not. These differences indicate that developmental torpedoes may not be functionally identical to disease-related torpedoes. To study this further, we used a mouse model of spinocerebellar ataxia type 6 (SCA6), and found elevated disease-related torpedo number at 2 years. However, we found normal levels of developmental torpedoes in these mice. Our findings suggest that the transient emergence of Purkinje cell axonal torpedoes during the second postnatal week in mice represents a normal morphological feature in the developing cerebellar microcircuit.

Influence of tetramethylenedisulfotetramine on synchronous calcium oscillations at distinct developmental stages of hippocampal neuronal cultures

The spatial and temporal patterns of spontaneous synchronous Ca2+ oscillations (SCOs) regulate physiological pathways that influence neuronal development, excitability, and health. Hippocampal neuronal cultures (HN) and neuron/glia co-cultures (HNG) produced from neonatal mice were loaded with Fluo-4/AM and SCOs recorded in real-time using a Fluorescence Imaging Plate Reader at different developmental stages in vitro. HNG showed an earlier onset of SCOs, with low amplitude and low frequency SCOs at 4days in vitro (DIV), whereas HN were quiescent at this point. SCO amplitude peaked at 9 DIV for both cultures. SCO network frequency peaked at 12 DIV in HN, whereas in HNG the frequency peaked at 6 DIV. SCO patterns were associated with the temporal development of neuronal networks and their ratio of glutamatergic to GABAergic markers of excitatory/inhibitory balance. HN and HNG exhibited differential responses to the convulsant tetramethylenedisulfotetramine (TETS) and were highly dependent on DIV. In HN, TETS triggered an acute rise of intracellular Ca2+ (Phase I response) only in 14 DIV and a sustained decrease of SCO frequency with increased amplitude (Phase II response) at all developmental stages. In HNG, TETS decreased the SCO frequency and increased the amplitude at 6 and 14 but not 9 DIV. There was no acute Ca2+ rise (Phase I response) in any age of HNG tested with TETS. These data demonstrated the importance of glia and developmental stage in modulating neuronal responses to TETS. Our results illustrate the applicability of the model for investigating how caged convulsants elicit abnormal network activity during the development of HN and HNG cultures in vitro.

Development of putative inhibitory neurons in the embryonic and postnatal mouse superficial spinal dorsal horn.

The superficial spinal dorsal horn is the first relay station of pain processing. It is also widely accepted that spinal synaptic processing to control the modality and intensity of pain signals transmitted to higher brain centers is primarily defined by inhibitory neurons in the superficial spinal dorsal horn. Earlier studies suggest that the construction of pain processing spinal neural circuits including the GABAergic components should be completed by birth, although major chemical refinements may occur postnatally. Because of their utmost importance in pain processing, we intended to provide a detailed knowledge concerning the development of GABAergic neurons in the superficial spinal dorsal horn, which is now missing from the literature. Thus, we studied the developmental changes in the distribution of neurons expressing GABAergic markers like Pax2, GAD65 and GAD67 in the superficial spinal dorsal horn of wild type as well as GAD65-GFP and GAD67-GFP transgenic mice from embryonic day 11.5 (E11.5) till postnatal day 14 (P14). We found that GABAergic neurons populate the superficial spinal dorsal horn from the beginning of its delineation at E14.5. We also showed that the numbers of GABAergic neurons in the superficial spinal dorsal horn continuously increase till E17.5, but there is a prominent decline in their numbers during the first two postnatal weeks. Our results indicate that the developmental process leading to the delineation of the inhibitory and excitatory cellular assemblies of pain processing neural circuits in the superficial spinal dorsal horn of mice is not completed by birth, but it continues postnatally.

Altered cortical processing of motor inhibition in schizophrenia

Inhibition is considered a key mechanism in schizophrenia. Short-latency intracortical inhibition (SICI) in the motor cortex is reduced in schizophrenia and is considered to reflect locally deficient γ-aminobutyric acid (GABA)-ergic modulation. However, it remains unclear how SICI is modulated during motor inhibition and how it relates to neural processing in other cortical areas. Here we studied motor inhibition Stop signal task (SST) in stabilized patients with schizophrenia (N = 28), healthy siblings (N = 21) and healthy controls (n = 31) matched in general cognitive status and educational level. Transcranial magnetic stimulation (TMS) and functional magnetic resonance imaging (fMRI) were used to investigate neural correlates of motor inhibition.SST performance was similar in patients and controls. SICI was modulated by the task as expected in healthy controls and siblings but was reduced in patients with schizophrenia during inhibition despite equivalent motor inhibition performance. fMRI showed greater prefrontal and premotor activation during motor inhibition in schizophrenia. Task-related modulation of SICI was higher in subjects who showed less inhibition-related activity in pre-supplementary motor area (SMA) and cingulate motor area. An exploratory genetic analysis of selected markers of inhibition (GABRB2, GAD1, GRM1, and GRM3) did not explain task-related differences in SICI or cortical activation. In conclusion, this multimodal study provides direct evidence of a task-related deficiency in SICI modulation in schizophrenia likely reflecting deficient GABA-A related processing in motor cortex. Compensatory activation of premotor areas may explain similar motor inhibition in patients despite local deficits in intracortical processing. Task-related modulation of SICI may serve as a useful non-invasive GABAergic marker in development of therapeutic strategies in schizophrenia.

Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons.

The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non-GABAergic phenotype, while they express well-established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (-) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV+ /NOS- /GAD67+ , PV+ /NOS+ /GAD67+ , PV+ /NOS- /GAD67- , and PV- /NOS+ /GAD67- . Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67- IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67+ IC neurons remained unchanged in all subdivisions. The NDs of PV+ /NOS- /GAD67+ neurons and PV- /NOS+ /GAD67- neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV+ /NOS+ /GAD67+ neurons and PV+ /NOS- /GAD67- neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67+ IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868-884, 2017. © 2016 Wiley Periodicals, Inc.

Overexpression of Glutamate Decarboxylase in Mesenchymal Stem Cells Enhances Their Immunosuppressive Properties and Increases GABA and Nitric Oxide Levels.

The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers in vitro, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders.

Striosome–dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons

The dopamine systems of the brain powerfully influence movement and motivation. We demonstrate that striatonigral fibers originating in striosomes form highly unusual bouquet-like arborizations that target bundles of ventrally extending dopamine-containing dendrites and clusters of their parent nigral cell bodies. Retrograde tracing showed that these clustered cell bodies in turn project to the striatum as part of the classic nigrostriatal pathway. Thus, these striosome-dendron formations, here termed "striosome-dendron bouquets," likely represent subsystems with the nigro-striato-nigral loop that are affected in human disorders including Parkinson's disease. Within the bouquets, expansion microscopy resolved many individual striosomal fibers tightly intertwined with the dopamine-containing dendrites and also with afferents labeled by glutamatergic, GABAergic, and cholinergic markers and markers for astrocytic cells and fibers and connexin 43 puncta. We suggest that the striosome-dendron bouquets form specialized integrative units within the dopamine-containing nigral system. Given evidence that striosomes receive input from cortical regions related to the control of mood and motivation and that they link functionally to reinforcement and decision-making, the striosome-dendron bouquets could be critical to dopamine-related function in health and disease.

GABAergic Synapses at the Axon Initial Segment of Basolateral Amygdala Projection Neurons Modulate Fear Extinction

Inhibitory synaptic transmission in the amygdala has a pivotal role in fear learning and its extinction. However, the local circuits formed by GABAergic inhibitory interneurons within the amygdala and their detailed function in shaping these behaviors are not well understood. Here we used lentiviral-mediated knockdown of the cell adhesion molecule neurofascin in the basolateral amygdala (BLA) to specifically remove inhibitory synapses at the axon initial segment (AIS) of BLA projection neurons. Quantitative analysis of GABAergic synapse markers and measurement of miniature inhibitory postsynaptic currents in BLA projection neurons after neurofascin knockdown ex vivo confirmed the loss of GABAergic input. We then studied the impact of this manipulation on anxiety-like behavior and auditory cued fear conditioning and its extinction as BLA related behavioral paradigms, as well as on long-term potentiation (LTP) in the ventral subiculum–BLA pathway in vivo. BLA knockdown of neurofascin impaired ventral subiculum–BLA–LTP. While this manipulation did not affect anxiety-like behavior and fear memory acquisition and consolidation, it specifically impaired extinction. Our findings indicate that modification of inhibitory synapses at the AIS of BLA projection neurons is sufficient to selectively impair extinction behavior. A better understanding of the role of distinct GABAergic synapses may provide novel and more specific targets for therapeutic interventions in extinction-based therapies.

Dopamine D1-like Receptors Regulate Constitutive, μ-Opioid Receptor-Mediated Repression of Use-Dependent Synaptic Plasticity in Dorsal Horn Neurons: More Harm than Good?

The current study reports on a synaptic mechanism through which D1-like receptors (D1LRs) modulate spinal nociception and plasticity by regulating activation of the μ-opioid receptor (MOR).D1LR stimulation with agonist SKF 38393 concentration-dependently depressed C-fiber-evoked potentials in rats receiving spinal nerve ligation (SNL), but not in uninjured rats. Depression was prevented by MOR- but not GABA-receptor blockade. Neurons expressing the D1 subtype were immunopositive for met-enkephalin and vesicular glutamate transporter VGLUT2, but not for GABAergic marker vGAT.Nerve ligation was followed by increased immunoreactivity for D1 in synaptic compartment (P3) in dorsal horn homogenates and presynaptic met-enkephalin-containing boutons. SNL led to increased immunoreactivity for met-enkephalin in dorsal horn homogenates, which was dose-dependently attenuated by selective D1LR antagonist SCH 23390. During blockade of either D1R or MOR, low-frequency (0.2 or 3 Hz) stimulation (LFS) to the sciatic nerve induced long-term potentiation (LTP) of C-fiber-evoked potentials, revealing a constituent role of both receptors in repressing afferent-induced synaptic plasticity. LFS consistently induced NMDA receptor-dependent LTP in nerve-injured rats. The ability of MOR both to prevent LTP and to modulate mechanical and thermal pain thresholds in behavioral tests was preserved in nerve-ligated rats that were postoperatively treated with SCH 23390. D1LR priming for 30 min sufficed to disrupt MOR function in otherwise naive rats via a mechanism involving receptor overuse.The current data support that, whereas D1LR-modulated MOR activation is instrumental in antinociception and endogenous repression of synaptic plasticity, this mechanism deteriorates rapidly by sustained use, generating increased vulnerability to afferent input. The current study shows that dopamine D1-like receptors (D1LRs) and μ-opioid receptors (MOR) in the spinal dorsal horn constitutively repress the expression of synaptic long-term potentiation (LTP) of C-fiber-evoked potentials. Anatomical data are provided supporting that the D1 subtype regulates MOR function by modulating met-enkephalin release. Sustained neuropathic pain induced by spinal nerve ligation is accompanied by D1R and met-enkephalin upregulation, acquired D1LR-mediated antinociception, and a loss of endogenous repression of further synaptic plasticity. We show that the ability of MOR to oppose LTP is rapidly impaired by sustained D1LR activation via a mechanism involving sustained MOR activation.

Effect of Methamphetamine Exposure on Expression of Calcium Binding Proteins in Rat Frontal Cortex and Hippocampus.

Methamphetamine (METH) is a psychostimulant drug with potent effects on the central nervous system that can cause psychotic symptoms similar to those of schizophrenia. Specific alterations in GABAergic neuronal markers have been reported in schizophrenia and animal models of psychotic illness. The aim of this study was to determine whether there are changes in subpopulations of GABAergic neurons, defined by the presence of calcium binding proteins (CBPs), in animal models of METH abuse. Rats received acute (Binge) doses of 4×6mg/kg, a chronic escalating dose regime (0.1-4mg/kg over 14days) or a combination of the two and were compared with a vehicle-administered control group. Brains were taken and sections of frontal cortex (Cg1) and hippocampus (dentate gyrus and CA1-3 regions) underwent immunostaining for three CBPs [parvalbumin (PV), calbindin (CB), and calretinin (CR)]. Significant decreases in PV-immunoreactive (IR) neurons in each METH group and all regions were observed. Smaller METH-induced deficits in CB-IR cells were observed, reaching significance primarily following chronic METH regimes, while CR-IR was significantly reduced only in frontal cortex following chronic administration. These results suggest that METH regimes in rats can induce selective deficits in GABAergic neuronal subtypes similar to those seen in schizophrenia and may underlie the psychosis and/or cognitive impairment that can occur in METH abuse and dependence.

Anti-NMDA receptor autoantibodies and associated neurobehavioral pathology in mice are dependent on age of first exposure to Toxoplasma gondii

BackgroundToxoplasma gondii is a pathogen implicated in psychiatric disorders. As elevated antibodies to T. gondii are also present in non-symptomatic individuals, we hypothesized that the age during first exposure to the pathogen may affect symptom manifestation. We tested this hypothesis by evaluating neurobehavioral abnormalities and the immune response in mice following adolescent or adult T. gondii infection. MethodsMice were infected with T. gondii at postnatal day 33 (adolescent/juvenile) or 61 (adult). At 8weeks post-infection (wpi), pre-pulse inhibition of the acoustic startle (PPI) in mice administered MK-801 (0.1 and 0.3mg/kg) and amphetamine (5 and 10mg/kg) was assessed. Peripheral (anti-T. gondii, C1q-associated IgG and anti-GLUN2 antibodies) and central (C1q and Iba1) markers of the immune response were also evaluated. In addition, regional brain expression of N-methyl-d-aspartate receptor (NMDAR) subunits (GLUN1 and GLUN2A), glutamatergic (vGLUT1, PSD95) and GABAergic (GAD67) markers, and monoamines (DA, NE, 5-HT) and their metabolites were measured. ResultsJuvenile and adult infected mice exhibited opposite effects of MK-801 on PPI, with decreased PPI in juveniles and increased PPI in adults. There was a significantly greater elevation of GLUN2 autoantibodies in juvenile-compared to adult-infected mice. In addition, age-dependent differences were found in regional expression of NMDAR subunits and markers of glutamatergic, GABAergic, and monoaminergic systems. Activated microglia and C1q elevations were found in both juvenile- and adult-T. gondii infected mice. ConclusionsOur study demonstrates that the age at first exposure to T. gondii is an important factor in shaping distinct behavioral and neurobiological abnormalities. Elevation in GLUN2 autoantibodies or complement protein C1q may be a potential underlying mechanism. A better understanding of these age-related differences may lead to more efficient treatments of behavioral disorders associated with T. gondii infection.