Markers Of GABAergic Interneurons Research Articles

Alterations of Perineuronal Net Expression and Abnormal Social Behavior and Whisker-dependent Texture Discrimination in Mice Lacking the Autism Candidate Gene Engrailed 2

GABAergic interneurons and perineuronal nets (PNNs) are important regulators of plasticity throughout life and their dysfunction has been implicated in the pathogenesis of several neuropsychiatric conditions, including autism spectrum disorders (ASD). PNNs are condensed portions of the extracellular matrix (ECM) that are crucial for neural development and proper formation of synaptic connections.We previously showed a reduced expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of adult mice lacking the Engrailed2 gene (En2-/- mice), a mouse model of ASD. Since alterations in PNNs have been proposed as a possible pathogenic mechanism in ASD, we hypothesized that the PNN dysfunction may contribute to the neural and behavioral abnormalities of En2-/- mice. Here, we show an increase in the PNN fluorescence intensity, evaluated by Wisteria floribunda agglutinin, in brain regions involved in social behavior and somatosensory processing. In addition, we found that En2-/- mice exhibit altered texture discrimination through whiskers and display a marked decrease in the preference for social novelty.Our results raise the possibility that altered expression of PNNs, together with defects of GABAergic interneurons, might contribute to the pathogenesis of social and sensory behavioral abnormalities.

LPS-induced inflammation reduces GABAergic interneuron markers and brain-derived neurotrophic factor in mouse prefrontal cortex and hippocampus

Inflammation, reduced gamma-aminobutyric acidergic (GABAergic) function and altered neuroplasticity are co-occurring pathophysiologies in major depressive disorder (MDD). However, the link between these biological changes remains unclear. We hypothesized that inflammation induces deficits in GABAergic interneuron markers and that this effect is mediated by brain-derived neurotrophic factor (BDNF). We report here that systemic inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) (0.125, 0.25, 0.5, 1, 2 mg/kg) in the first cohort of C57BL/6 mice (n = 72; 10–11 weeks; 50% female) resulted in increased interleukin 1-beta and interleukin-6 in prefrontal cortex (PFC) and hippocampus (HPC), as measured using enzyme-linked immunosorbent assay (ELISA). Quantitative real-time polymerase reaction (qPCR) was used to explore the effect of LPS on the expression of GABAergic interneuron markers. In the PFC of the second cohort (n = 39; 10–11 weeks; 50% female), 2 mg/kg of LPS decreased the expression of somatostatin (Sst) (p = 0.0014), parvalbumin (Pv) (p = 0.0257), cortistatin (Cort) (p = 0.0003), neuropeptide Y (Npy) (p = 0.0033) and cholecystokinin (Cck) (p = 0.0041), and did not affect corticotropin-releasing hormone (Crh) and vasoactive intestinal peptide (Vip) expression. In the HPC, 2 mg/kg of LPS decreased the expression of Sst (p = 0.0543), Cort (p = 0.0011), Npy (p = 0.0001), and Cck (p < 0.0001), and did not affect Crh, Pv, and Vip expression. LPS decreased the expression of Bdnf in the PFC (p < 0.0001) and HPC (p = 0.0003), which significantly correlated with affected markers (Sst, Pv, Cort, Cck, and Npy). Collectively, these results suggest that inflammation may causally contribute to cortical cell microcircuit GABAergic deficits observed in MDD.

A Novel Early Life Stress Model Affects Brain Development and Behavior in Mice

Early life stress (ELS) in developing children has been linked to physical and psychological sequelae in adulthood. In the present study, we investigated the effects of ELS on brain and behavioral development by establishing a novel ELS model that combined the maternal separation paradigm and mesh platform condition. We found that the novel ELS model caused anxiety- and depression-like behaviors and induced social deficits and memory impairment in the offspring of mice. In particular, the novel ELS model induced more enhanced depression-like behavior and memory impairment than the maternal separation model, which is the established ELS model. Furthermore, the novel ELS caused upregulation of arginine vasopressin expression and downregulation of GABAergic interneuron markers, such as parvalbumin (PV), vasoactive intestinal peptide, and calbindin-D28k (CaBP-28k), in the brains of the mice. Finally, the offspring in the novel ELS model showed a decreased number of cortical PV-, CaBP-28k-positive cells and an increased number of cortical ionized calcium-binding adaptors-positive cells in their brains compared to mice in the established ELS model. Collectively, these results indicated that the novel ELS model induced more negative effects on brain and behavioral development than the established ELS model.

Patterns of functional connectivity alterations induced by alcohol reflect somatostatin interneuron expression in the human cerebral cortex

Acute alcohol administration affects functional connectivity, yet the underlying mechanism is unknown. Previous work suggested that a moderate dose of alcohol reduces the activity of gamma-aminobutyric acidergic (GABAergic) interneurons, thereby leading to a state of pyramidal disinhibition and hyperexcitability. The present study aims to relate alcohol-induced changes in functional connectivity to regional genetic markers of GABAergic interneurons. Healthy young adults (N = 15, 5 males) underwent resting state functional MRI scanning prior to alcohol administration, immediately and 90 min after alcohol administration. Functional connectivity density mapping was performed to quantify alcohol-induced changes in resting brain activity between conditions. Patterns of differences between conditions were related to regional genetic markers that express the primary GABAergic cortical interneuron subtypes (parvalbumin, somatostatin, and 5-hydroxytryptamine receptor 3A) obtained from the Allen Human Brain Atlas. Acute alcohol administration increased local functional connectivity density within the visual cortex, sensorimotor cortex, thalamus, striatum, and cerebellum. Patterns of alcohol-induced changes in local functional connectivity density inversely correlated with somatostatin cortical gene expression. These findings suggest that somatostatin-expressing interneurons modulate alcohol-induced changes in functional connectivity in healthy individuals.

Neuroregenerative gene therapy to treat temporal lobe epilepsy in a rat model.

Temporal lobe epilepsy (TLE) is a common drug-resistant epilepsy associated with abundant cell death in the hippocampus. Here, we develop a novel gene therapy-mediated cell therapy that regenerates GABAergic neurons using internal hippocampal astrocytes to suppress seizure activity in a rat TLE model. We discovered that TLE-induced reactive astrocytes in the hippocampal CA1 region can be efficiently converted into GABAergic neurons after overexpressing a neural transcription factor NeuroD1. The astrocyte-converted neurons showed typical markers of GABAergic interneurons, fired action potentials, and formed functional synaptic connections with other neurons. Following NeuroD1-mediated astrocyte-to-neuron conversion, the number of hippocampal interneurons was significantly increased, and the spontaneous recurrent seizure (SRS) activity was significantly decreased. Moreover, NeuroD1 gene therapy treatment rescued total neuronal loss in the CA1 region and ameliorated the cognitive and mood dysfunctions in the TLE rat model. These results suggest that regeneration of GABAergic interneurons through gene therapy approach may provide a novel therapeutic intervention to treat drug-resistant TLE.

Mice deficient in the NAAG synthetase II gene Rimkla are impaired in a novel object recognition task.

N-acetylaspartylglutamate (NAAG) is an abundant neuropeptide in the mammalian nervous system, synthesized by two related NAAG synthetases I and II (NAAGS-I and -II) encoded by the genes Rimklb and Rimkla, respectively. NAAG plays a role in cognition and memory, according to studies using inhibitors of the NAAG hydrolase glutamate carboxypeptidase II that increase NAAG concentration. To examine consequences of reduced NAAG concentration, Rimkla-deficient (Rimkla-/- ) mice were generated. These mice exhibit normal NAAG level at birth, likely because of the intact Rimklb gene, but have significantly reduced NAAG levels in all brain regions in adulthood. In wild type mice NAAGS-II was most abundant in brainstem and spinal cord, as demonstrated using a new NAAGS-II antiserum. In the hippocampus, NAAGS-II was only detectable in neurons expressing parvalbumin, a marker of GABAergic interneurons. Apart from reduced open field activity, general behavior of adult (6months old) Rimkla-/- mice examined in different tests (dark-light transition, optokinetic behavior, rotarod, and alternating T-maze) was not significantly altered. However, Rimkla-/- mice were impaired in a short-term novel object recognition test. This was also the case for mice lacking NAA synthase Nat8l, which are devoid of NAAG. Together with results from previous studies showing that inhibition of the NAAG degrading enzyme glutamate carboxypeptidase II is associated with a significant improvement in object recognition, these results suggest a direct involvement of NAAG synthesized by NAAGS-II in the memory consolidation underlying the novel object recognition task.

Neonatal proinflammatory challenge evokes a microglial response and affects the ratio between subtypes of GABAergic interneurons in the hippocampus of juvenile rats: sex-dependent and sex-independent effects.

Neonatal proinflammatory challenge (NPC) may contribute to the development of psychiatric disorders in adults. A double exposure of neonatal rats to lipopolysaccharide, a component of cellular wall of gram-negative bacteria, on postnatal days 3 and 5 provokes the development of depressive- and anxiety-like behaviors. NPC impairs neuroplasticity and cognition in adult animals, significant modifications of neuroplasticity being evident even in adolescence. We studied effects of NPC on microglia and GABAergic neuronal population of the dorsal hippocampus in juvenile male and female rats using immunofluorescent histochemistry. The expression of glutamic acid decarboxylase-67 (GAD67) and calcium-binding proteins calretinin, calbindin, and parvalbumin were used as quantitative markers of GABAergic interneurons and their specific subpopulations, respectively. NPC induced changes of microglial morphology indicating inflammatory activation mostly expressed in CA3 field; the effect was similar in males and females. The number of GAD67 expressing neurons was similar in the dorsal hippocampus of females and males independently on the NPC. The portion of calbindin-immunoreactive GAD67-positive neurons significantly increased while the portion of calretinin-immunoreactive GAD67-positive neurons significantly decreased in the CA1 field of rats exposed to NPC independently on their sex. NPC did not affect the parvalbumin-positive subpopulation of GABAergic neurons in the hippocampus of rats of either sex. These data suggest that NPC-induced modification of GABAergic neuronal population composition under the proinflammatory conditions is involved in the maintenance of excitation/inhibition homeostatic balance in the hippocampus.

Altered Expression of GABAergic Markers in the Forebrain of Young and Adult Engrailed-2 Knockout Mice.

Impaired function of GABAergic interneurons, and the subsequent alteration of excitation/inhibition balance, is thought to contribute to autism spectrum disorders (ASD). Altered numbers of GABAergic interneurons and reduced expression of GABA receptors has been detected in the brain of ASD subjects and mouse models of ASD. We previously showed a reduced expression of GABAergic interneuron markers parvalbumin (PV) and somatostatin (SST) in the forebrain of adult mice lacking the Engrailed2 gene (En2-/- mice). Here, we extended this analysis to postnatal day (P) 30 by using in situ hybridization, immunohistochemistry, and quantitative RT-PCR to study the expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of En2-/- and wild type (WT) mice. In addition, GABA receptor subunit mRNA expression was investigated by quantitative RT-PCR in the same brain regions of P30 and adult En2-/- and WT mice. As observed in adult animals, PV and SST expression was decreased in En2-/- forebrain of P30 mice. The expression of GABA receptor subunits (including the ASD-relevant Gabrb3) was also altered in young and adult En2-/- forebrain. Our results suggest that GABAergic neurotransmission deficits are already evident at P30, confirming that neurodevelopmental defects of GABAergic interneurons occur in the En2 mouse model of ASD.

Cortical GABAergic Dysfunction in Stress and Depression: New Insights for Therapeutic Interventions.

Major depressive disorder (MDD) is a debilitating illness characterized by neuroanatomical and functional alterations in limbic structures, notably the prefrontal cortex (PFC), that can be precipitated by exposure to chronic stress. For decades, the monoaminergic deficit hypothesis of depression provided the conceptual framework to understand the pathophysiology of MDD. However, accumulating evidence suggests that MDD and chronic stress are associated with an imbalance of excitation–inhibition (E:I) within the PFC, generated by a deficit of inhibitory synaptic transmission onto principal glutamatergic neurons. MDD patients and chronically stressed animals show a reduction in GABA and GAD67 levels in the brain, decreased expression of GABAergic interneuron markers, and alterations in GABAA and GABAB receptor levels. Moreover, genetically modified animals with deletion of specific GABA receptors subunits or interneuron function show depressive-like behaviors. Here, we provide further evidence supporting the role of cortical GABAergic interneurons, mainly somatostatin- and parvalbumin-expressing cells, required for the optimal E:I balance in the PFC and discuss how the malfunction of these cells can result in depression-related behaviors. Finally, considering the relatively low efficacy of current available medications, we review new fast-acting pharmacological approaches that target the GABAergic system to treat MDD. We conclude that deficits in cortical inhibitory neurotransmission and interneuron function resulting from chronic stress exposure can compromise the integrity of neurocircuits and result in the development of MDD and other stress-related disorders. Drugs that can establish a new E:I balance in the PFC by targeting the glutamatergic and GABAergic systems show promising as fast-acting antidepressants and represent breakthrough strategies for the treatment of depression.

Abnormal development of the inferior temporal region in fetuses with Down syndrome.

Down syndrome (DS) is a genetic condition associated with impairment in several cognitive domains. Previous evidence showed a notable neurogenesis reduction in the hippocampal region of DS fetuses, which may account for the impairment of declarative memory that characterizes DS starting from early life stages. The fusiform gyrus (FG) and the inferior temporal gyrus (ITG) play a key role in visual recognition memory, a function that is impaired in children and adults with DS. The goal of the current study was to establish whether fetuses with DS (17-21 weeks of gestation) exhibit neuroanatomical alterations in the FG and ITG that may underlie recognition memory impairment. We found that the FG and ITG of fetuses with DS had a reduced thickness and fewer cells in comparison with euploid fetuses. Moreover, DS fetuses had fewer cells expressing the neuronal marker NeuN than euploid fetuses, but a similar number of cells expressing the astrocytic marker GFAP and, consequently, a higher percentage of astrocytes. Immunohistochemistry for calretinin (CR), a marker of GABAergic interneurons, showed that in DS fetuses the ratio of CR-positive vs. CR-negative cells was greater than in euploid fetuses, both in the FG (177%) and ITG (161%). An increased ratio of CR-positive vs. CR-negative cells was also found in the entorhinal cortex, hippocampus and dentate gyrus. Results provide novel evidence that the FG and ITG of DS fetuses exhibit numerous developmental defects. These defects may underlie the functional alterations in visual recognition memory observed in children with DS.

Chronic Stress Exposure Reduces Parvalbumin Expression in the Rat Hippocampus through an Imbalance of Redox Mechanisms: Restorative Effect of the Antipsychotic Lurasidone

BackgroundPsychiatric disorders are associated with altered function of inhibitory neurotransmission within the limbic system, which may be due to the vulnerability of selective neuronal subtypes to challenging environmental conditions, such as stress. In this context, parvalbumin-positive GABAergic interneurons, which are critically involved in processing complex cognitive tasks, are particularly vulnerable to stress exposure, an effect that may be the consequence of dysregulated redox mechanisms.MethodsAdult Male Wistar rats were subjected to the chronic mild stress procedure for 7 weeks. After 2 weeks, both control and stress groups were further divided into matched subgroups to receive chronic administration of vehicle or lurasidone (3 mg/kg/d) for the subsequent 5 weeks. Using real-time RT-PCR and western blot, we investigated the expression of GABAergic interneuron markers and the levels of key mediators of the oxidative balance in the dorsal and ventral hippocampus.ResultsChronic mild stress induced a specific decrease of parvalbumin expression in the dorsal hippocampus, an effect normalized by lurasidone treatment. Interestingly, the regulation of parvalbumin levels was correlated to the modulation of the antioxidant master regulator NRF2 and its chaperon protein KEAP1, which were also modulated by pharmacological intervention.ConclusionsOur findings suggest that the susceptibility of parvalbumin neurons to stress may represent a key mechanism contributing to functional and structural impairments in specific brain regions relevant for psychiatric disorders. Moreover, we provide new insights on the mechanism of action of lurasidone, demonstrating that its chronic treatment normalizes chronic mild stress-induced parvalbumin alterations, possibly by potentiating antioxidant mechanisms, which may ameliorate specific functions that are deteriorated in psychiatric patients.

Age-Related Susceptibility to Epileptogenesis and Neuronal Loss in Male Fischer Rats Exposed to Soman and Treated With Medical Countermeasures.

Elderly individuals compose a large percentage of the world population; however, few studies have addressed the efficacy of current medical countermeasures (MCMs) against the effects of chemical warfare nerve agent exposure in aged populations. We evaluated the efficacy of the anticonvulsant diazepam in an old adult rat model of soman (GD) poisoning and compared the toxic effects to those observed in young adult rats when anticonvulsant treatment is delayed. After determining their respective median lethal dose (LD50) of GD, we exposed young adult and old adult rats to an equitoxic 1.2 LD50 dose of GD followed by treatment with atropine sulfate and the oxime HI-6 at 1 min after exposure, and diazepam at 30 min after seizure onset. Old adult rats that presented with status epilepticus were more susceptible to developing spontaneous recurrent seizures (SRSs). Neuropathological analysis revealed that in rats of both age groups that developed SRS, there was a significant reduction in the density of mature neurons in the piriform cortex, thalamus, and amygdala, with more pronounced neuronal loss in the thalamus of old adult rats compared with young adult rats. Furthermore, old adult rats displayed a reduced density of cells expressing glutamic acid decarboxylase 67, a marker of GABAergic interneurons, in the basolateral amygdala and piriform cortex, and a reduction of astrocyte activation in the piriform cortex. Our observations demonstrate the reduced effectiveness of current MCM in an old adult animal model of GD exposure and strongly suggest the need for countermeasures that are more tailored to the vulnerabilities of an aging population.

Cell Type-Specific Expression of Corticotropin-Releasing Hormone-Binding Protein in GABAergic Interneurons in the Prefrontal Cortex.

Corticotropin-releasing hormone-binding protein (CRH-BP) is a secreted glycoprotein that binds CRH with very high affinity to modulate CRH receptor activity. CRH-BP is widely expressed throughout the brain, with particularly high expression in regions such as the amygdala, hippocampus, ventral tegmental area and prefrontal cortex (PFC). Recent studies suggest a role for CRH-BP in stress-related psychiatric disorders and addiction, with the PFC being a potential site of interest. However, the molecular phenotype of CRH-BP-expressing cells in this region has not been well-characterized. In the current study, we sought to determine the cell type-specific expression of CRH-BP in the PFC to begin to define the neural circuits in which this key regulator is acting. To characterize the expression of CRH-BP in excitatory and/or inhibitory neurons, we utilized dual in situ hybridization to examine the cellular colocalization of CRH-BP mRNA with vesicular glutamate transporter (VGLUT) or glutamic acid decarboxylase (GAD) mRNA in different subregions of the PFC. We show that CRH-BP is expressed predominantly in GABAergic interneurons of the PFC, as revealed by the high degree of colocalization (>85%) between CRH-BP and GAD. To further characterize the expression of CRH-BP in this heterogenous group of inhibitory neurons, we examined the colocalization of CRH-BP with various molecular markers of GABAergic interneurons, including parvalbumin (PV), somatostatin (SST), vasoactive intestinal peptide (VIP) and cholecystokinin (CCK). We demonstrate that CRH-BP is colocalized predominantly with SST in the PFC, with lower levels of colocalization in PV- and CCK-expressing neurons. Our results provide a more comprehensive characterization of the cell type-specific expression of CRH-BP and begin to define its potential role within circuits of the PFC. These results will serve as the basis for future in vivo studies to manipulate CRH-BP in a cell type-specific manner to better understand its role in stress-related psychiatric disorders, including anxiety, depression and addiction.

Analyzing the influence of BDNF heterozygosity on spatial memory response to 17β-estradiol

The recent use of estrogen-based therapies as adjunctive treatments for the cognitive impairments of schizophrenia has produced promising results; however the mechanism behind estrogen-based cognitive enhancement is relatively unknown. Brain-derived neurotrophic factor (BDNF) regulates learning and memory and its expression is highly responsive to estradiol. We recently found that estradiol modulates the expression of hippocampal parvalbumin-positive GABAergic interneurons, known to regulate neuronal synchrony and cognitive function. What is unknown is whether disruptions to the aforementioned estradiol–parvalbumin pathway alter learning and memory, and whether BDNF may mediate these events. Wild-type (WT) and BDNF heterozygous (+/−) mice were ovariectomized (OVX) at 5 weeks of age and simultaneously received empty, estradiol- or progesterone-filled implants for 7 weeks. At young adulthood, mice were tested for spatial and recognition memory in the Y-maze and novel-object recognition test, respectively. Hippocampal protein expression of BDNF and GABAergic interneuron markers, including parvalbumin, were assessed. WT OVX mice show impaired performance on Y-maze and novel-object recognition test. Estradiol replacement in OVX mice prevented the Y-maze impairment, a Behavioral abnormality of dorsal hippocampal origin. BDNF and parvalbumin protein expression in the dorsal hippocampus and parvalbumin-positive cell number in the dorsal CA1 were significantly reduced by OVX in WT mice, while E2 replacement prevented these deficits. In contrast, BDNF+/− mice showed either no response or an opposite response to hormone manipulation in both behavioral and molecular indices. Our data suggest that BDNF status is an important biomarker for predicting responsiveness to estrogenic compounds which have emerged as promising adjunctive therapeutics for schizophrenia patients.

Loss of dopamine D2 receptors increases parvalbumin-positive interneurons in the anterior cingulate cortex.

Disruption to dopamine homeostasis during brain development has been implicated in a variety of neuropsychiatric disorders, including depression and schizophrenia. Inappropriate expression or activity of GABAergic interneurons are common features of many of these disorders. We discovered a persistent upregulation of GAD67+ and parvalbumin+ neurons within the anterior cingulate cortex of dopamine D2 receptor knockout mice, while other GABAergic interneuron markers were unaffected. Interneuron distribution and number were not altered in the striatum or in the dopamine-poor somatosensory cortex. The changes were already present by postnatal day 14, indicating a developmental etiology. D2eGFP BAC transgenic mice demonstrated the presence of D2 receptor expression within a subset of parvalbumin-expressing cortical interneurons, suggesting the possibility of a direct cellular mechanism through which D2 receptor stimulation regulates interneuron differentiation or survival. D2 receptor knockout mice also exhibited decreased depressive-like behavior compared with wild-type controls in the tail suspension test. These data indicate that dopamine signaling modulates interneuron number and emotional behavior and that developmental D2 receptor loss or blockade could reveal a potential mechanism for the prodromal basis of neuropsychiatric disorders.

Trajectory of the main GABAergic interneuron populations from early development to old age in the rat primary auditory cortex.

In both humans and rodents, decline in cognitive function is a hallmark of the aging process; the basis for this decrease has yet to be fully characterized. However, using aged rodent models, deficits in auditory processing have been associated with significant decreases in inhibitory signaling attributed to a loss of GABAergic interneurons. Not only are these interneurons crucial for pattern detection and other large-scale population dynamics, but they have also been linked to mechanisms mediating plasticity and learning, making them a prime candidate for study and modeling of modifications to cortical communication pathways in neurodegenerative diseases. Using the rat primary auditory cortex (A1) as a model, we probed the known markers of GABAergic interneurons with immunohistological methods, using antibodies against gamma aminobutyric acid (GABA), parvalbumin (PV), somatostatin (SOM), calretinin (CR), vasoactive intestinal peptide (VIP), choline acetyltransferase (ChAT), neuropeptide Y (NPY), and cholecystokinin (CCK) to document the changes observed in interneuron populations across the rat's lifespan. This analysis provided strong evidence that several but not all GABAergic neurons were affected by the aging process, showing most dramatic changes in expression of parvalbumin (PV) and somatostatin (SOM) expression. With this evidence, we show how understanding these trajectories of cell counts may be factored into a simple model to quantify changes in inhibitory signaling across the course of life, which may be applied as a framework for creating more advanced simulations of interneuronal implication in normal cerebral processing, normal aging, or pathological processes.

Mutations in the BLOC-1 Subunits Dysbindin and Muted Generate Divergent and Dosage-dependent Phenotypes

Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5(mu/mu)) and dysbindin (Bloc1s8(sdy/sdy)). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5(mu/mu) and Bloc1s8(sdy/sdy) mice. Unlike Bloc1s8(sdy/sdy), elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders.

Traumatic Brain Injury Increases Cortical Glutamate Network Activity by Compromising GABAergic Control

Traumatic brain injury (TBI) is a major risk factor for developing pharmaco-resistant epilepsy. Although disruptions in brain circuitry are associated with TBI, the precise mechanisms by which brain injury leads to epileptiform network activity is unknown. Using controlled cortical impact (CCI) as a model of TBI, we examined how cortical excitability and glutamatergic signaling was altered following injury. We optically mapped cortical glutamate signaling using FRET-based glutamate biosensors, while simultaneously recording cortical field potentials in acute brain slices 2-4 weeks following CCI. Cortical electrical stimulation evoked polyphasic, epileptiform field potentials and disrupted the input-output relationship in deep layers of CCI-injured cortex. High-speed glutamate biosensor imaging showed that glutamate signaling was significantly increased in the injured cortex. Elevated glutamate responses correlated with epileptiform activity, were highest directly adjacent to the injury, and spread via deep cortical layers. Immunoreactivity for markers of GABAergic interneurons were significantly decreased throughout CCI cortex. Lastly, spontaneous inhibitory postsynaptic current frequency decreased and spontaneous excitatory postsynaptic current increased after CCI injury. Our results suggest that specific cortical neuronal microcircuits may initiate and facilitate the spread of epileptiform activity following TBI. Increased glutamatergic signaling due to loss of GABAergic control may provide a mechanism by which TBI can give rise to post-traumatic epilepsy.

Activity of dlx5a/ dlx6a regulatory elements during zebrafish GABAergic neuron development

During vertebrate forebrain formation, Dlx homeobox genes play essential roles in the differentiation, migration and survival of subpallial precursor cells that will later give rise to diverse subtypes of γ-aminobutyric acid (GABA)-expressing neurons, including inhibitory cortical interneurons in mammals. They also participate in the regulation of the Gad genes encoding the enzymes necessary for GABA synthesis. In mice, at least four cis-regulatory elements (CREs) control Dlx expression in the telencephalon and diencephalon: URE2 and I12b in the Dlx1/ Dlx2 bigene cluster, and I56i and I56ii in the Dlx5/ Dlx6 bigene cluster. However, little is known so far with respect to the function of orthologous dlx genes and their regulatory elements during zebrafish GABAergic neuron development. To investigate whether similar dlx-mediated pathways exist in the early developing zebrafish forebrain, we generated independent lines of transgenic zebrafish carrying two distinct GFP reporter constructs driven by a β-globin minimal promoter: one containing a ∼1.4 kb dlx5a/ dlx6a intergenic sequence (encompassing I56i and I56ii) and one with a ∼1.1 kb fragment containing only the I56i CRE, respectively. The expression patterns of these two transgenes were compared with that obtained with another construct containing the ∼1.4 kb dlx5a/ dlx6a intergenic sequence and driven by a ∼3.5 kb dlx6a 5′-flanking fragment. Our comparative analysis showed that GFP expression of the three transgene is largely overlapping throughout the ventral forebrain. Intriguingly, the dlx6a 5′-flanking fragment has a major impact on transgene expression in the mesencephalic tectum. Furthermore, comparison of transgene expression between the ∼1.4 kb and ∼1.1 kb intergenic fragments did not show any specific spatial expression conferred by I56ii. Almost all GFP-expressing cells in the transgenic zebrafish are GABA-positive and also express various GABAergic interneuron markers. Together, our data suggest that zebrafish dlx5a/ dlx6a intergenic CREs may be involved in a conserved genetic pathway necessary for proper dlx expression during zebrafish GABAergic neuron development.

The immunhistochemical examination of GABAergic interneuron markers in the dorsolateral prefrontal cortex of patients with late-life depression

The "vascular depression" hypothesis has sought to explain differences in etiology between early and late life depression, and has been reinforced by recent imaging and morphometric studies. Gamma-aminobutyric acid (GABA) is thought to play a major role in the neurobiology of depression. However, it is unclear whether there is an effect on GABA neuronal subpopulations in an elderly depressed cohort. This study therefore examined immunohistochemically two calcium-binding proteins, calretinin and parvalbumin, which have been demonstrated to bind to two distinct GABAergic interneuron subpopulations, within the dorsolateral prefrontal cortex (DLPFC) of elderly depressed patients, against age-matched controls. Post-mortem tissue was obtained from nine controls and 11 depressed patients for the parvalbumin study and seven controls and 14 depressed patients in the calretinin study, and the mean percentage per area of immunohistochemical staining of the two antibodies was measured in individual layers and across the whole of the DLPFC. The study found a reduction in parvalbumin immunostaining in layer 6 (p = 0.05) of the DLFPC in elderly depressed patients. However, no significant changes were found in parvalbumin or calretinin immunostaining in the any other layer of the DLPFC in elderly depressed patients. The study does not suggest any change in GABA interneuron subpopulations, though significant reductions in layer 6 may represent subtle disturbance in GABA parvalbumin-expressing interneuron and glumatatergic pyramidal projection neuron regulation in late-life depression.