GABAergic cell types in the superficial layers of the mouse superior colliculus

To begin to unravel the complexities of GABAergic circuits in the superior colliculus (SC), we utilized mouse lines that express green fluorescent protein (GFP) in cells that contain the 67 kDa isoform of glutamic acid decarboxylase (GAD67-GFP), or Cre-recombinase in cells that contain glutamic acid decarboxylase (GAD; GAD2-cre). We used Cre-dependent virus injections in GAD2-Cre mice and tracer injections in GAD67-GFP mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells that project to the pretectum (PT), ventral lateral geniculate nucleus (vLGN) or parabigeminal nucleus (PBG), and interneurons in the stratum griseum superficiale (SGS) that do not project outside the SC. We found that approximately 30% of SGS neurons in the mouse are GABAergic. Of these GABAergic neurons, we identified three categories of potential interneurons in the GAD67-GFP line (GABA+GFP ~45%, GABA+GFP + PV ~15%, and GABA+PV ~10%). GABAergic cells that did not contain GFP or PV were identified as potential projection neurons (GABA only ~30%). We found that GABAergic neurons that project to the PBG are primarily located in the SGS and exhibit narrow field vertical, stellate, and horizontal dendritic morphologies, while GABAergic neurons that project to the PT and vLGN are primarily located in layers ventral to the SGS. In addition, we examined GABA and GAD67-containing elements of the mouse SGS using electron microscopy to further delineate the relationship between GABAergic circuits and retinotectal input. Approximately 30% of retinotectal synaptic targets are the presynaptic dendrites of GABAergic interneurons, and GAD67-GFP interneurons are a source of these presynaptic dendrites.     

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.     

The Ventral Tegmental Area has calbindin neurons with the capability to co‐release glutamate and dopamine into the nucleus accumbens

The ventral tegmental area (VTA) has three major classes of neurons: dopaminergic (expressing tyrosine hydroxylase; TH), GABAergic (expressing vesicular GABA transporter; VGaT) and glutamatergic (expressing vesicular glutamate transporter 2; VGluT2). While VTA dopaminergic and GABAergic neurons have been further characterized by expression of calcium‐binding proteins (calbindin, CB; calretinin, CR or parvalbumin, PV), it is unclear whether these proteins are expressed in rat VTA glutamatergic neurons. Here, by a combination of in situ hybridization (for VGluT2 mRNA detection) and immunohistochemistry (for CB‐, CR‐ or PV‐detection), we found that among the total population of VGluT2 neurons, 30% coexpressed CB, 3% coexpressed PV and <1% coexpressed CR. Given that some VGluT2 neurons coexpress TH or VGaT, we examined whether these neurons coexpress CB, and found that about 20% of VGluT2‐CB neurons coexpressed TH and about 13% coexpressed VGaT. Because VTA TH‐CB neurons are known to target the nucleus accumbens (nAcc), we determined whether VGluT2‐CB‐TH neurons innervate nAcc, and found that about 80% of VGluT2‐CB neurons innervating the nAcc shell coexpressed TH. In summary, (a) CB, PV and CR are detected in subpopulations of VTA‐VGluT2 neurons; (b) CB is the main calcium‐binding protein present in VTA‐VGluT2 neurons; (c) one‐third of VTA‐VGluT2 neurons coexpress CB; (d) some VTA‐VGluT2‐CB neurons have the capability to co‐release dopamine or GABA, and (e) a subpopulation of VTA glutamatergic‐dopaminergic neurons innervates nAcc shell. These findings further provide evidence for molecular diversity among VTA‐VGluT2 neurons, neurons that may play a role in specific circuitry and behaviours.     

Developmental expression of GABA and subunits of the GABAA receptor complex in an inhibitory synaptic circuit in the rat cerebellum

The temporal relationship between the expression of a transmitter and its corresponding receptor may provide important insights into the development of synaptic circuits in the central nervous system. Here we examined the emergence of the inhibitory transmitter GABA, and subunits of the GABAA/benzodiazepine receptor complex in a well-characterized cerebellar circuit formed by granule cells and the synapses they make with Golgi II neurons in the cerebella of rats ranging in age from birth to 21 days. The presence of GABA was determined immunocytochemically. The presence of the GABAA receptor was demonstrated by localizing the alpha 1 subunit of the receptor using in situ hybridization and immunochemical localization of a 50 kDa benzodiazepine-binding subunit using monoclonal antibodies. Germinal cells of the external granular layer which give rise to granule cells did not express the GABAA receptor at any age. Similarly, receptor labeling could not be detected in granule cells during their postmitotic migratory period. In the internal granular layer, immature postmigratory granule cells are unlabeled. The expression of GABAA receptor subunits was first observed on the fifth postnatal day (P5) and then only in the more mature granule cells which have well elaborated dendrites in contact with presynaptic elements. The number of labeled neurons increased over the subsequent ages examined. Presynaptic elements in association with the dendrites of labeled granule cells had ultrastructural features characteristic of Golgi II cell axon terminals. These elements demonstrate GABA transmitter as early as P3, preceded by 2-3 days receptor labeling in the granular layer. Therefore, granule cells express GABAA receptor subunits only after they have completed migration and their dendrites have become involved in specific synaptic circuits, including innervation by GABAergic afferents.     

Quantitative analysis of neuronal diversity in the mouse olfactory bulb

Olfactory sensory information is processed and integrated by circuits within the olfactory bulb. Golgi morphology suggests the olfactory bulb contains several major neuronal classes. However, an increasingly diverse collection of neurochemical markers have been localized in subpopulations of olfactory bulb neurons. While the mouse is becoming the animal model of choice for olfactory research, little is known about the proportions of neurons expressing and coexpressing different neurochemical markers in this species. Here we characterize neuronal populations in the mouse main olfactory bulb, focusing on glomerular populations. Immunofluorescent labeling for: 1) calretinin, 2) calbindin D-28K (CB), 3) parvalbumin, 4) neurocalcin, 5) tyrosine hydroxylase (TH), 6) the 67-kDa isoform of GAD (GAD67), and 7) the neuronal marker NeuN was performed in mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65kDa (GAD65) promoter. Using unbiased stereological cell counts we estimated the total numbers of cells and neurons in the bulb and the number and percentage of neurons expressing and coexpressing different neurochemical populations in each layer of the olfactory bulb. Use of a genetic label for GAD65 and immunohistochemistry for GAD67 identified a much larger percentage of GABAergic neurons in the glomerular layer (55% of all neurons) than previously recognized. Additionally, while many glomerular neurons expressing TH or CB coexpress GAD, the majority of these neurons preferentially express the GAD67 isoform. These data suggest that the chemospecific populations of neurons in glomeruli form distinct subpopulations and that GAD isoforms are preferentially regulated in different neurochemical cell types.     

Gamma-aminobutyric acid, glutamic acid decarboxylase and tyrosine hydroxylase in rat striatum demonstrated by single and dual immunocytochemistry.

By means of dual ultrastructural immunostaining the followings patterns are visualized: gamma-aminobutyric acid (GABA) immunoreactive neurons, dendrites, axons and axon terminals and tyrosine hydroxylase (TH) immunopositive fibers, varicosities and boutons in rat striatum. Additionally single glutamic acid decarboxylase (GAD) immunolabeling is carried out. Four subgroups of GABA and GAD immunoreactive striatal neurons are revealed. These neuronal types are identified on the basis of sectional diameters, nuclear form and nuclear envelope invaginations, quantity of cytoplasm and cell organelles. Plasmalemmal appositions between GABAergic and between GABAergic and immunonegative neurons are observed. All subgroups of striatal GABAergic neurons contact with GABA and GAD immunoreactive, TH immunoreactive and immunonegative boutons. In the striatal neuropil numerous GABAergic, dopaminergic and immunonegative axonal endings synapsed with dendrites and spines are found out. Massive dopaminergic striatal structures using dual immunostaining is evident. Some GABA and GAD immunoreactive dendrites are revealed in direct contact with capillary walls.     

Heterogeneity of gamma-aminobutyric acid type A receptors in mitral and tufted cells of the rat main olfactory bulb

Mitral and tufted cells of the olfactory bulb receive strong gamma-aminobutyric acid (GABA)-ergic input and express GABA(A) receptors containing the alpha1 or alpha3 subunit. The distribution of these subunits was investigated in rats via multiple immunofluorescence and confocal microscopy, by using gephyrin as a marker of GABAergic synapses. A prominent immunoreactivity was detected throughout the external plexiform layer (EPL) and glomerular layer (GL). However, although staining for the alpha1 subunit was uniform throughout the EPL, that of the alpha3 subunit was most intense in the outer one-third of this layer. All mitral cells were positive for the alpha1 subunit. In contrast, the alpha3 subunit was restricted to a subpopulation of mitral cells, many of which also expressed calretinin. Likewise, external tufted cells could be subdivided into distinct groups, either singly labeled for the alpha1 or alpha3 subunit or doubly labeled. At the subcellular level, staining for the alpha1 and alpha3 subunits was punctate, forming clusters partially colocalized with gephyrin. However, many alpha1- and alpha3-positive clusters lacked gephyrin, suggesting the existence of either nonsynaptic GABA(A) receptor clusters or synaptic receptors not associated with gephyrin. Quantitative analysis of colocalization among the three markers in the inner EPL, outer EPL, and GL revealed considerable heterogeneity, suggestive of a differential organization of GABA(A) receptor subtypes in the apical and basal dendrites of mitral and tufted cells. Together these results reveal a complex subunit organization of GABA(A) receptors in the olfactory bulb and suggest that mitral and tufted cells participate in different synaptic circuits controlled by distinct GABA(A) receptor subtypes.     

Presynaptic But Not Postsynaptic GABA Signaling at Unitary Mossy Fiber Synapses

Dentate gyrus granule cells have been suggested to corelease GABA and glutamate both in juvenile animals and under pathological conditions in adults. Although mossy fiber terminals (MFTs) are known to express glutamic acid decarboxylase (GAD) in early postnatal development, the functional role of GABA synthesis in MFTs remains controversial, and direct evidence for synaptic GABA release from MFTs is missing. Here, using GAD67-GFP transgenic mice, we show that GAD67 is expressed only in a population of immature granule cells in juvenile animals. We demonstrate that GABA can be released from these cells and modulate mossy fiber excitability through activation of GABAB autoreceptors. However, unitary postsynaptic currents generated by individual, GAD67-expressing granule cells are purely glutamatergic in all postsynaptic cell types tested. Thus GAD67 expression does not endow dentate gyrus granule cells with a full GABAergic phenotype and GABA primarily instructs the pre- rather than the postsynaptic element.     

Expression of alpha 5 GABAA receptor subunit in developing rat hippocampus

The GABAergic system plays an important role in the hippocampal development. Here we have studied the developmental expression of the alpha 5 subunit of the GABA(A) receptor (from rat hippocampus) by RT-competitive PCR, immunoblot and immunocytochemistry. Our results demonstrated an early induction of the alpha 5 subunit expression (at mRNA and protein levels) during the first postnatal week, peaking at P5 and decreasing after this age. The peak of alpha 5 subunit expression precedes the peak of expression for the synaptophysin, GAD65 and GAD67. Thus, the increase in the alpha 5 GABA(A) receptor subunit expression may precede the GABAergic synaptogenesis. Importantly, between P0 and P7, the expression of the alpha 5 subunit was concentrated at the cell somata of the pyramidal and granular cells. After P10, its localization shifted from the cell bodies to the dendritic layers. This developmental pattern is similar to that reported for the Na(+)-K(+)-2Cl(-) system and it might be correlated with the transition from excitatory to inhibitory GABAergic activity.     

Distribution of gamma-aminobutyric acid receptors in cultured adrenergic and noradrenergic bovine chromaffin cells

Fluorescence imaging techniques for recording cytosolic [Ca(2+)](i) from single chromaffin cells were used to characterize and discriminate between cell subpopulations containing gamma-aminobutyric acid (GABA)(A) and GABA(B) receptor subtypes. By combining this methodology with the immunoidentification of individual chromaffin cells using specific antibodies against tyrosine hydroxylase (TH), phenyl-etanolamine-N-methyl transferase (PNMT), and glutamic acid decarboxylase (GAD) linked to different fluorescent probes, we have been able to ascribe single-cell calcium responses to identified adrenergic and noradrenergic chromaffin cells. GAD enzyme is present in 30% of the chromaffin cell population, located primarily in adrenergic cells; 86% of GAD(+) cells were also PNMT(+). GAD expression was not correlated with the presence of GABA receptors. GABA-responsive cells were found with equal frequency in the GAD(+) and GAD(-) groups. However, the expression of GABA receptors was correlated with the adrenergic phenotype. [Ca(2+)](i) responses to GABA were found more frequently in adrenergic than in noradrenergic cells. GABA(A) receptors are more evenly distributed; about 90% of GABA-responsive cells have them. GABA(B) receptors have a more restricted distribution (present in 45% of responding cells). The coexpression of both GABA(A) and GABA(B) subtypes is the rule; only a minor subpopulation (about 12%) displays exclusively GABA(B) receptors. GABA receptor subtypes are distributed in a similar way when chromaffin cells are separated according to GAD(+)/GAD(-) or PNMT(+)/PNMT(-) classifications, with only minor differences. These data indicate that the intrinsic GABAergic system in the adrenal medulla is not designed as a paracrine model in which a group of cells specializes in transmitter synthesis and a different group serves as a specific target.     

Specific cell types in cat retina express different forms of glutamic acid decarboxylase

We studied the expression of glutamate decarboxylase (GAD), GAD65 and GAD67, in cat retina by immunocytochemistry. About 10% of GABAergic amacrime cells express only GAD65 and 30% express only GAD67. Rougly 60% contain both forms of the enzyme, but GAD67 is present only at low levels in the majority of these double-labeled amacrine cells. The staining pattern in the inner plexiform layer (IPL) for the two GAD forms was also different. GAD65 was restricted to strata 1–4, and GAD67 was apparent throughout the IPL but was strongest in strata 1 and 5. This indicates that soams, as well as their processes, are differentially stained for the two forms of GAD. Cell types expressing only GAD65 include interplexiform cells, one type of cone bipolar cell, and at least one type of serotonin-accumulating amacrine cell. Cell types expressing only GAD67 include amacrine cells synthesizing dopamine, amacrine cells synthesizing nitric oxide (NO), and amacrine cells accumulating serotonin. Cholinergic amacrine cells express a low level of both GAD forms. Our findings in the retina are consistent with previous observations in the brain that GAD65 expression is greater in terminals than in somas. In addition, in retina most neurons expressing GAD67 also contain a second neurotransmitter as well as GABA, and they tend to be larger than neurons expressing GAD65. We propose that large cells have a greater demand for GABA than small cells, and thus require the constant, relatively unmodulated level of GABA that is provided by GAD67. © 1995 Willy-Liss, Inc.     

Training-induced changes in the expression of GABAA-associated genes in the amygdala after the acquisition and extinction of Pavlovian fear

Previous work suggests the gamma-aminobutyric acid (GABA)ergic system may be dynamically regulated during emotional learning. In the current study we examined training-induced changes in the expression of GABA(A)-related genes and the binding of GABA receptor radioligands in the amygdala after the acquisition and extinction of Pavlovian fear. Using in situ hybridization, we examined the expression pattern changes of mRNAs for GABAergic markers in the lateral, basolateral and central subdivisions of the amygdala in C57Bl/6J mice. These markers included GABA-synthesizing enzymes (GAD67 and GAD65), major GABA(A) receptor subunits (alpha1, alpha2, alpha3, alpha5, beta2 and gamma2) and the expression of mRNAs that are involved in a variety of GABA-related intracellular processes, including GABA transporter-1 (GAT1), GABA(A) receptor-associated protein and the GABA(A) clustering protein, gephyrin. With fear conditioning, we found decreased mRNA levels of alpha1, alpha5 and GAD67, as well as deceased benzodiazepine binding in the amygdala. Fear extinction induced an increase in mRNA levels of alpha2, beta2, GAD67 and gephyrin, as well as a decrease in GAT1. Together, these findings indicate that the acquisition of fear induced a downregulation of mRNA markers related to a decrease in amygdala GABAergic function, whereas the acquisition of fear extinction produced an upregulation of GABAergic markers related to enhanced GABAergic transmission.     

Novel interneuronal network in the mouse posterior piriform cortex

The neural circuits of the piriform cortex mediate field potential oscillations and complex functions related to integrating odor cues with behavior, affective states, and multisensory processing. Previous anatomical studies have established major neural pathways linking the piriform cortex to other cortical and subcortical regions and major glutamatergic and GABAergic neuronal subtypes within the piriform circuits. However, the quantitative properties of diverse piriform interneurons are unknown. Using quantitative neural anatomical analysis and electrophysiological recording applied to a GAD65-EGFP transgenic mouse expressing GFP (green fluorescent protein) under the control of the GAD65 promoter, here we report a novel inhibitory network that is composed of neurons positive for GAD65-EGFP in the posterior piriform cortex (PPC). These interneurons had stereotyped dendritic and axonal properties that were distinct from basket cells or interneurons expressing various calcium-binding proteins (parvalbumin, calbindin, and calretinin) within the PPC. The GAD65-GFP neurons are GABAergic and outnumbered any other interneurons (expressing parvalbumin, calbindin, and calretinin) we studied. The firing pattern of these interneurons was highly homogenous and is similar to the regular-spiking nonpyramidal (RSNP) interneurons reported in primary sensory and other neocortical regions. Robust dye coupling among these interneurons and expression of connexin 36 suggested that they form electrically coupled networks. The predominant targets of descending axons of these interneurons were the dendrites of Layer III principal cells. Additionally, synapses were found on dendrites and somata of deep Layer II principal neurons and Layer III basket cells. A similar interneuronal subtype was also found in GAD65-EGFP-negative mouse. The extensive dendritic bifurcation at superficial lamina IA among horizontal afferent fibers and unique axonal targeting pattern suggests that these interneurons may play a role in direct feedforward inhibitory and disinhibitory olfactory processing. We conclude that the GAD65-GFP neurons may play distinct roles in regulating information flow and olfactory-related oscillation within the PPC in vivo.     

Spatial distribution of synapses on tyrosine hydroxylase–expressing juxtaglomerular cells in the mouse olfactory glomerulus

Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial‐short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)‐immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ‐aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH‐expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non‐ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH‐expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non‐ON profiles. Thus, fewer proximal non‐ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic‐GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059–1074, 2017. © 2017 Wiley Periodicals, Inc.     

GABAergic input to the synaptic terminals of mb1 bipolar cells in the goldfish retina

An EM-autoradiographical/immunocytochemical technique was used to study amacrine cell synapses onto mb1 bipolar cell terminals in goldfish retina. Tissue was double labeled for [3H]GABA uptake and glutamate decarboxylase (GAD) immunolocalization. Nearly 90% of the amacrine cell synaptic processes onto both proximal and distal halves of mb1 terminals were labeled with either [3H]GABA or GAD-immunoreactivity (IR). Proximal half: 73% of the amacrine synapses were labeled with [3H]GABA uptake and 82% with GAD-IR; 88% of [3H]GABA labeled contacts were double labeled. Distal half: 17% of the amacrine synapses were labeled with [3H]GABA uptake and 67% with GAD-IR; 63% of [3H]GABA labeled contacts were double labeled. After consideration of the possible sources of [3H]GABA labeled synapses onto mb1 terminals, we concluded that the synaptic terminals of pyriform Ab amacrine cells double label for [3H]GABA and GAD-IR despite our previous report that Ab cell bodies do not stain for anti-catfish brain GAD antiserum. We suggest that Ab cells contain isoenzymes of GAD which differ in subcellular distribution, thereby accounting for the differential staining of the cell bodies and dendrites obtained with the GAD antiserum we used.     

GABA(A) receptor beta2/beta3 subunit and GAD67 immunoreactivity in the trigeminal motor nucleus during early postnatal development

GABA neurotransmission plays a role in brainstem circuitry responsible for jaw movements. We investigated the developmental relationship between terminals expressing GAD67 and GABA(A) receptor beta(2)/beta(3) subunit expression within the trigeminal motor nucleus. GAD67 immunoreactivity was intense throughout development. Neuropilar beta(2)/beta(3) immunoreactivity emerged during the 2nd postnatal week. Our data provide anatomical evidence for a GABAergic innervation of neonatal trigeminal motoneurons and suggest that beta(2)/beta(3) subunit expression is developmentally regulated in trigeminal motoneurons.