Neuron-specific expression of reporter gene in transgenic mice carrying the 5′-upstream region of mouse P/Q-type Ca channel α1A subunit gene fused to E. coli lacZ reporter gene

To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel α_1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5′-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced. In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5′-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5′-upstream region failed to show reporter expression. Histological examinations showed that the three 5′-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla. The 1.5-kb 5′-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5′-upstream sequence between 1.5 kb and 6.3 kb. In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells. The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5′-upstream region. These results suggest that the expression of the mouse P/Q-type Ca²⁺ channel α_1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.     

Neuronal circuit‐dependent alterations in expression of two isoforms of glutamic acid decarboxylase in the hippocampus following electroconvulsive shock: A stereology‐based study

There is an increasing body of evidence suggesting that GABAergic dysfunction is involved in various psychiatric disorders. The goal of our study was to investigate the influences of electroconvulsive therapy (ECT), one of the most effective treatments for depression, on the GABAergic system in the hippocampus. In this stereology‐based study, we identified GABAergic neurons by immunostaining for two isoforms of glutamic acid decarboxylase (GAD), GAD65, and GAD67 and estimated the expression changes induced by single or repeated electroconvulsive shock (ECS; an animal model of ECT). The numerical density (ND) of entire population of GABAergic neurons (expressing GAD65 and/or GAD67) was seldom altered by the administration of ECS. GAD67‐positive (GAD67⁺) neurons were also rarely affected by ECS. On the other hand, the ND of GAD65⁺ neurons was changed in a layer‐specific manner. In the CA1 region, the ND of GAD65⁺ neurons was increased in the strata radiatum/lacunosum‐moleculare (SR/SLM) by repeated ECS. In the CA3 region, the ND of GAD65⁺ neurons was decreased in the stratum oriens and SR/SLM after single ECS. The expression ratio of GAD65 in GABAergic neurons was increased specifically in layers receiving afferents from the entorhinal cortex (EC), i.e., SR/SLM of the CA1 region and molecular layer of the dentate gyrus (DG), after repeated ECS administration, whereas the expression ratio of GAD67 in GABAergic neurons was decreased in several layers by the same treatment. These results indicate that the ECS‐induced changes in ND of GAD65⁺ or GAD67⁺ neurons were most likely due to alterations in GAD expression rather than actual increases or decreases in cell numbers. Altogether, the neuronal circuit‐dependent alterations in GABA‐mediated signaling may play a contributory role in the depression treatment process introduced by ECT. © 2009 Wiley‐Liss, Inc.     

The distribution of GAD67‐mRNA in the adult zebrafish (teleost) forebrain reveals a prosomeric pattern and suggests previously unidentified homologies to tetrapods

We used in situ hybridization on sections to examine the distribution of GAD67‐expressing cell populations in the entire forebrain of the adult zebrafish. GAD67 is predominantly expressed in the olfactory bulb (OB), all regions of the subpallium (including the dorsal, ventral, central, and lateral nucleus of the area ventralis [Vd, Vv, Vc, and Vl, respectively]), as well as preoptic (PPa, PPp, and PM), pretectal (PPd, PPv, PCN, PSp, and PSm), ventral (= pre‐) thalamic (I, VM, and VL), hypothalamic (Hr, Hi, and Hc), preglomerular (P, PGa, PGl, PGm, and RT), and posterior tubercular (TPp and TPm) nuclei. Only scattered GAD67‐expressing cells are seen in all pallial zones (Dm, Dd, Dc, Dl, and Dp) and in the previously unidentified bed nucleus of the stria medullaris (BNSM). The BNSM appears to be the adult teleostean derivative of the larval eminentia thalami (EmT). We identify the GAD67‐positive entopeduncular nucleus proper (EN) as being homologous to the entopeduncular nucleus of nonprimate mammals. GAD67 is strongly expressed in the anterior thalamic nucleus (A). The anterior thalamic nucleus is laterally bordered by a distinct GAD67‐expressing cell population, which we interpret as the previously unidentified reticular thalamic nucleus (RTN) of teleosts. Furthermore, we identified a GAD67‐positive thalamic nucleus, the intercalated nucleus (IC), which is sandwiched between the GAD67‐negative dorsal (DP) and central posterior (CP) thalamic nuclei. Overall, the distribution of GAD67‐expressing cells highly resembles the distribution of γ‐aminobutyric acid (GABA)/GAD67‐expressing cells found in the early zebrafish (teleost) forebrain and thus allows us to propose a prosomeric fate map of GABAergic cell populations. J. Comp. Neurol. 516:553–568, 2009. © 2009 Wiley‐Liss, Inc.     

Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse

Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.     

Selective loss of parvalbumin-positive GABAergic interneurons in the cerebral cortex of maternally stressed Gad1-heterozygous mouse offspring

Exposure to maternal stress (MS) and mutations in GAD1, which encodes the γ-aminobutyric acid (GABA) synthesizing enzyme glutamate decarboxylase (GAD) 67, are both risk factors for psychiatric disorders. However, the relationship between these risk factors remains unclear. Interestingly, the critical period of MS for psychiatric disorders in offspring corresponds to the period of GABAergic neuron neurogenesis and migration in the fetal brain, that is, in the late stage of gestation. Indeed, decrement of parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC) and hippocampus (HIP) has often been observed in schizophrenia patients. In the present study, we used GAD67-green fluorescent protein (GFP) knock-in mice (that is, mice in which the Gad1 gene is heterozygously deleted; GAD67^+/GFP) that underwent prenatal stress from embryonic day 15.0 to 17.5 and monitored PV-positive GABAergic neurons to address the interaction between Gad1 disruption and stress. Administration of 5-bromo-2-deoxyuridine revealed that neurogenesis of GFP-positive GABAergic neurons, but not cortical plate cells, was significantly diminished in fetal brains during MS. Differential expression of glucocorticoid receptors by different progenitor cell types may underlie this differential outcome. Postnatally, the density of PV-positive, but not PV-negative, GABAergic neurons was significantly decreased in the mPFC, HIP and somatosensory cortex but not in the motor cortex of GAD67^+/GFP mice. By contrast, these findings were not observed in wild-type (GAD67^+/+) offspring. These results suggest that prenatal stress, in addition to heterozygous deletion of Gad1, could specifically disturb the proliferation of neurons destined to be PV-positive GABAergic interneurons.     

Abnormalities in cortical interneuron subtypes in ephrin‐B mutant mice

To explore roles for ephrin‐B/EphB signaling in cortical interneurons, we previously generated ephrin‐B (Efnb1/b2/b3) conditional triple mutant (TM^lz) mice using a Dlx1/2.Cre inhibitory neuron driver and green fluorescent protein (GFP) reporters for the two main inhibitory interneuron groups distinguished by expression of either glutamic acid decarboxylase 1 (GAD1; GAD67‐GFP) or 2 (GAD2; GAD65‐GFP). This work showed a general involvement of ephrin‐B in migration and population of interneurons into the embryonic neocortex. We now determined whether specific interneurons are selectively affected in the adult brains of TM^lz.Cre mice by immunostaining with antibodies that identify the different subtypes. The results indicate that GAD67‐GFP‐expressing interneurons that also express parvalbumin (PV), calretinin (CR) and, to a lesser extent, somatostatin (SST) and Reelin (Rln) were significantly reduced in the cortex and hippocampal CA1 region in TM^lz.Cre mutant mice. Neuropeptide Y (NPY) interneurons that also express GAD67‐GFP were reduced in the hippocampal CA1 region, but much less so in the cortex, although these cells exhibited abnormal cortical layering. In GAD65‐GFP‐expressing interneurons, CR subtypes were reduced in both cortex and hippocampal CA1 region, whereas Rln interneurons were reduced exclusively in hippocampus, and the numbers of NPY and vasoactive intestinal polypeptide (VIP) subtypes appeared normal. PV and CR subtype interneurons in TM^lz.Cre mice also exhibited reductions in their perisomatic area, suggesting abnormalities in dendritic/axonal complexity. Altogether, our data indicate that ephrin‐B expression within forebrain interneurons is required in specific subtypes for their normal population, cortical layering and elaboration of cell processes.     

In vivo knockdown of GAD67 in the amygdala disrupts fear extinction and the anxiolytic-like effect of diazepam in mice

In mammals, γ-aminobutyric acid (GABA) transmission in the amygdala is particularly important for controlling levels of fear and anxiety. Most GABA synthesis in the brain is catalyzed in inhibitory neurons from ℒ-glutamic acid by the enzyme glutamic acid decarboxylase 67 (GAD67). In the current study, we sought to examine the acquisition and extinction of conditioned fear in mice with knocked down expression of the GABA synthesizing enzyme GAD67 in the amygdala using a lentiviral-based (LV) RNA interference strategy to locally induce loss-of-function. In vitro experiments revealed that our LV-siRNA-GAD67 construct diminished the expression of GAD67 as determined with western blot and fluorescent immunocytochemical analyses. In vivo experiments, in which male C57BL/6J mice received bilateral amygdala microinjections, revealed that LV-siRNA-GAD67 injections produce significant inhibition of endogenous GAD67 when compared with control injections. In contrast, no significant changes in GAD65 expression were detected in the amygdala, validating the specificity of LV knockdown. Behavioral experiments showed that LV knockdown of GAD67 results in a deficit in the extinction, but not the acquisition or retention, of fear as measured by conditioned freezing. GAD67 knockdown did not affect baseline locomotion or basal measures of anxiety as measured in open field apparatus. However, diminished GAD67 in the amygdala blunted the anxiolytic-like effect of diazepam (1.5 mg kg^–1) as measured in the elevated plus maze. Together, these studies suggest that of GABAergic transmission in amygdala mediates the inhibition of conditioned fear and the anxiolytic-like effect of diazepam in adult mice.     

Both upstream and intragenic sequences of the human neurofilament light gene direct expression oflacZ in neurons of transgenic mouse embryos

Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to thelacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression oflacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promotelacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression oflacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences containcis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directslacZ expression in embryonic neurons.     

Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study

The central nucleus of the amygdala is interconnected with a variety of visceral and autonomic nuclei of the brainstem. These include the parabrachial nucleus, the nucleus of the solitary tract, the nucleus ambiguus and the dorsal motor nucleus of the vagus. Despite repeated attempts, neurochemical characterization of the major subcortical connections of the central nucleus has not yet been accomplished. Based on earlier immunohistochemical and in situ hybridization evidence indicating the presence of numerous GABAergic neurons in the macaque monkey central nucleus, we predicted that a sizeable portion of the descending projections may be GABAergic. We tested this hypothesis using a novel double labelling method with gold conjugated WGA-apoHRP as a retrograde tracer and in situ hybridization for detecting the mRNA that encodes the enzyme glutamic acid decarboxylase (GAD67) as a marker for GABAergic cells. Following WGA-apoHRP-gold injections into the brainstem, a large number of retrogradely labelled cells was observed in the medial and lateral divisions of the central nucleus. Of the retrogradely labelled cells observed in the medial division of the central nucleus, approximately half were double-labelled for GAD67 mRNA; about 30% double labelling was observed in the lateral division. These data support the view that a sizeable component of the central nucleus projection to the brainstem is GABAergic.