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.     

Forebrain GABAergic projections from the dorsal raphe nucleus identified by using GAD67–GFP knock‐in mice

The dorsal raphe nucleus (DR) contains serotonergic (5‐HT) neurons that project widely throughout the forebrain. These forebrain regions also receive innervation from non–5‐HT neurons in the DR. One of the main groups of non–5‐HT neurons in the DR is γ‐aminobutyric acid (GABA)ergic, but their projections are poorly understood due to the difficulty of labeling these neurons immunohistochemically. To identify GABAergic projection neurons within the DR in the current study, we used a knock‐in mouse line in which expression of green fluorescent protein (GFP) is controlled by the glutamic acid decarboxylase (GAD)67 promotor. Projections of GAD67–GFP neurons to the prefrontal cortex (PFC), nucleus accumbens (NAC), and lateral hypothalamus (LH) were evaluated by using retrograde tract tracing. The location of GAD67–GFP neurons projecting to each of these areas was mapped by rostrocaudal and dorsoventral location within the DR. Overall, 16% of DR neurons projecting to either the PFC or NAC were identified as GAD67–GFP neurons. GAD67–GFP neurons projecting to the PFC were most commonly found ventrally, in the rostral two‐thirds of the DR. NAC‐projecting GAD67–GFP neurons had an overlapping distribution that extended dorsally. GAD67–GFP neurons made a larger contribution to the projection of the DR to the LH, accounting for 36% of retrogradely labeled neurons, and were widespread throughout the DR. The current data indicate that DR GABAergic neurons not only may have the capacity to influence local network activity, but also make a notable contribution to DR output to multiple forebrain targets. J. Comp. Neurol. 520:4157–4167, 2012. © 2012 Wiley Periodicals, Inc.     

Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex

Gamma-aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.     

Long-range GABAergic projection in a circuit essential for vocal learning

The anterior forebrain pathway (AFP) in the passerine song system is essential for song learning but not for song production. Several lines of evidence suggest that area X, a major nucleus in the AFP, forms part of the avian striatum. A key feature of striatal projection neurons is that they use the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Some area X neurons express GABA-like immunoreactivity, but the neurotransmitter phenotype of the projection neurons is largely unknown. To determine whether area X projection neurons are GABAergic, we used immunocytochemistry and confocal microscopy to examine whether these neurons in adult male zebra finches express the GABA synthetic enzyme glutamic acid decarboxylase (GAD). We observed numerous large and small GAD+ somata in area X, and dense GAD+ terminals, but no GAD+ somata in the target of area X, the medial nucleus of the dorsolateral thalamus (DLM). The density of GAD+ terminals in DLM was strongly reduced by ibotenic acid lesions of area X. After tracer injection into the DLM, all of the retrogradely labeled neurons in area X were GAD+. After tracer injection into area X, the vast majority of anterogradely labeled terminals in DLM were GAD+. We conclude that area X neurons projecting to DLM express GAD and are thus likely GABAergic. If this projection is indeed inhibitory, information processing in the AFP is substantially more complicated than previously realized. Moreover, because a GABAergic projection to a thalamic target is reminiscent of pallidal rather than of striatal circuitry, area X may contain both striatal and pallidal components.     

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.     

Inhibitory neurons in the anterior piriform cortex of the mouse: Classification using molecular markers

The primary olfactory cortex (or piriform cortex, PC) is attracting increasing attention as a model system for the study of cortical sensory processing, yet little is known about inhibitory neurons in the PC. Here we provide the first systematic classification of GABA‐releasing interneurons in the anterior PC of mice, based on the expression of molecular markers. Our experiments used GAD67‐GFP transgenic mice, in which gamma‐aminobutyric acid (GABA)‐containing cells are labeled with green fluorescent protein (GFP). We first confirmed, using paired whole‐cell recordings, that GFP⁺ neurons in the anterior PC of GAD67‐GFP mice are functionally GABAergic. Next, we performed immunolabeling of GFP⁺ cells to quantify their expression of every possible pairwise combination of seven molecular markers: calbindin, calretinin, parvalbumin, cholecystokinin, neuropeptide Y, somatostatin, and vasoactive intestinal peptide. We found that six main categories of interneurons could be clearly distinguished in the anterior PC, based on the size and laminar location of their somata, intensity of GFP fluorescence, patterns of axonal projections, and expression of one or more of the seven markers. A number of rarer categories of interneurons could also be identified. These data provide a road map for further work that examines the functional properties of the six main classes of interneurons. Together, this information elucidates the cellular architecture of the PC and provides clues about the roles of GABAergic interneurons in olfactory processing. J. Comp. Neurol. 518:1670–1687, 2010. © 2009 Wiley‐Liss, Inc.     

A subpopulation of gonadotropin‐releasing hormone neurons in the adult mouse forebrain is γ‐Aminobutyric acidergic

Gonadotropin‐releasing hormone (GnRH) neurons play a pivotal role in reproductive function. GnRH is released in distinct pulses that are regulated by neurotransmitters or neuromodulators. With immunohistochemistry and GAD67‐GFP knockin mice, this study shows for the first time that a subset of GnRH neurons in the forebrain of adult mouse is γ‐aminobutyric acid (GABA)‐ergic. There is a gender difference in the percentage of GnRH neurons expressing GAD67‐GFP in female vs. male mice. The percentage of GnRH neurons expressing GAD67‐GFP decreased after castration of female mice and increased to the normal female level after estradiol treatment. The percentage of GnRH neurons expressing GAD67‐GFP did not change significantly in intact, castrated, or castration + testosterone propionate‐treated male mice. During the female estrous cycle, the percentage of GnRH neurons expressing GAD67‐GFP was higher during the estrous stage than during the diestrous stage. During sexual maturation of postnatal development, GnRH neurons did not express GAD67‐GFP until postnatal day (P) 15, and the gender differences were first detected at P30, which corresponds to the maturation stage. In conclusion, our data suggest that 1) a subset of GnRH neurons in mouse forebrain is GABA‐ergic, 2) expression of GAD67‐GFP in GnRH neurons is at least in part regulated by estrogen, and 3) GnRH neurons secrete GABA to regulate themselves. © 2015 Wiley Periodicals, Inc.     

The distribution of GABA-containing perikarya,fibers, and terminals in the forebrain and midbrain of pigeons,with particular reference to the basal ganglia and its projection targets

Immunohistochemical techniques were used to study the distributions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid (GABA) in pigeon forebrain and midbrain to determine the organization of GABAergic systems in these brain areas in birds. In the basal ganglia, numerous medium-sized neurons throughout the striatum were labeled for GABA, while pallidal neurons, as well as a small population of large, aspiny striatal neurons, labeled for GAD and GABA. GAD+ and GABA+ fibers and terminals were abundant throughout the basal ganglia, and GABAergic fibers were found in all extratelencephalic targets of the basal ganglia. Most of these targets also contained numerous GABAergic neurons. In pallial regions, approximately 10-12% of the neurons were GABAergic. The outer rind of the pallium was more intensely labeled for GABAergic fibers than the core. The olfactory tubercle region, the ventral pallidum, and the hypothalamus were extremely densely labeled for GABAergic fibers, while GABAergic neurons were unevenly distributed in the hypothalamus. GABAergic neurons and fibers were abundant in the dorsalmost part of thalamus and the dorsal geniculate region, while GABAergic neurons and fibers were sparse (or lightly labeled) in the thalamic nuclei rotundus, triangularis, and ovoidalis. Further, GABAergic neurons were abundant in the superficial tectal layers, the magnocellular isthmic nucleus, the inferior colliculus, the intercollicular region, the central gray, and the reticular formation. GABAergic fibers were particularly abundant in the superficial tectal layers, the parvocellular isthmic nucleus, the inferior colliculus, the intercol-licular region, the central gray, and the interpeduncular nucleus. These results suggest that GABA plays a role as a neurotransmitter in nearly all fore- and midbrain regions of birds, and in many instances the observed distributions of GABAergic neurons and fibers closely resemble the patterns seen in mammals, as well as in other vertebrates. © 1994 Wiley-Liss, Inc.     

Characterization of GABAergic neurons in rapid-eye-movement sleep controlling regions of the brainstem reticular formation in GAD67-green fluorescent protein knock-in mice

Recent experiments suggest that brainstem GABAergic neurons may control rapid-eye-movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67-GFP knock-in mice) exhibit numerous GFP-positive neurons in the central gray and reticular formation, allowing on-line identification in vitro . Small (10–15 µm) or medium-sized (15–25 µm) GFP-positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double-labeled for GFP and GABA, confirming that GFP-positive neurons are GABAergic. Whole-cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP-positive neurons were spontaneously active at 3–12 Hz, fired tonically, and possessed a medium-sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low-threshold calcium spike. GFP-positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP-positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP-positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol-inhibited GABAergic PnO neurons project to, and inhibit, REM-on SubC reticular neurons during waking, whereas carbachol-excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine-inhibited GABAergic neurons.     

Electrophysiological and morphological characteristics of GABAergic respiratory neurons in the mouse pre-Bötzinger complex

The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre‐Bötzinger complex. We used 67‐kDa isoform of glutamic acid decarboxylase‐green fluorescence protein (GAD67‐GFP) (Δneo) knock‐in (GAD67^GFP/+) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre‐Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre‐Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP‐positive neurons in the pre‐Bötzinger complex were recorded in a perforated whole‐cell patch‐clamp mode. Six of 32 GFP‐positive neurons were respiratory and the remaining 26 neurons were non‐respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post‐synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP‐negative neurons were recorded in the pre‐Bötzinger complex. GFP‐positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP‐positive inspiratory neurons had bipolar, triangular or crescent‐shaped somata and GFP‐negative inspiratory neurons had multipolar‐shaped somata. The somata of GFP‐positive inspiratory neurons were smaller than those of GFP‐negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre‐Bötzinger complex.     

Low voltage-activated calcium and fast tetrodotoxin-resistant sodium currents define subtypes of cholinergic and noncholinergic neurons in rat basal forebrain

Neurons of the basal forebrain (BF) possess unique combinations of voltage-gated membrane currents. Here, we describe subtypes of rat basal forebrain neurons based on patch-clamp analysis of low-voltage activated (LVA) calcium and tetrodotoxin-resistant (TTX-R) sodium currents combined with single-cell RT-PCR analysis. Neurons were identified by mRNA expression of choline acetyltransferase (ChAT+, cholinergic) and glutamate decarboxylase (GAD67, GABAergic). Four cell types were encountered: ChAT+, GAD+, ChAT+/GAD+ and ChAT-/GAD- cells. Both ChAT+ and ChAT+/GAD+ cells (71/75) displayed LVA currents and most (34/39) expressed mRNA for LVA Ca(2+) channel subunits. Ca(v)3.2 was detected in 31/34 cholinergic neurons and Ca(v)3.1 was expressed in 6/34 cells. Three cells expressed both subunits. No single neurons showed Ca(v)3.3 mRNA expression, although BF tissue expression was observed. In young rats (2-4 mo), ChAT+/GAD+ cells displayed larger LVA current densities compared to ChAT+ neurons, while these latter neurons displayed an age-related increase in current densities. Most (29/38) noncholinergic neurons (GAD+ and ChAT-/GAD-) possessed fast TTX-R sodium currents resembling those mediated by Na(+) channel subunit Na(v)1.5. This subunit was expressed predominately in noncholinergic neurons. No cholinergic cells (0/75) displayed fast TTX-R currents. The TTX-R currents were faster and larger in GAD+ neurons compared to ChAT-/GAD- neurons. The properties of ChAT+/GAD+ neurons resemble those of ChAT+ neurons, rather than of GAD+ neurons. These results suggest novel features of subtypes of cholinergic and noncholinergic neurons within the BF that may provide new insights for understanding normal BF function.     

Cholinergic responses in GABAergic and non-GABAergic neurons in the intermediate gray layer of mouse superior colliculus

Neurons in the intermediate gray layer (SGI) of the mammalian superior colliculus (SC) receive dense cholinergic innervations from the brainstem parabrachial region. Such cholinergic inputs may influence execution of orienting behaviors. To obtain deeper insights into how the cholinergic inputs modulate the SC local circuits, we analysed the cholinergic responses in identified γ‐aminobutyric acid (GABA)ergic and non‐GABAergic neurons using SC slices obtained from GAD67‐GFP knock‐in mice. The responses of SGI neurons to cholinergic agonists were various combinations of fast inward currents mediated mainly via α4β2 and partly by α7 nicotinic receptors (nIN), slow inward currents caused by activation of M1 plus M3 muscarinic receptors (mIN), and slow outward currents caused by activation of M2 muscarinic receptors (mOUT). The most common cholinergic responses in non‐GABAergic neurons was nIN + mIN + mOUT (38/68), followed by nIN + mIN (16/68), nIN + mOUT (11/68), nIN only (2/68), and no response (1/68). On the other hand, the major response pattern in GABAergic neurons was either nIN only (26/54) or nIN + mIN (21/54), followed by nIN + mOUT (4/54), mOUT only (2/54), and no response (1/54). Thus, major effects of cholinergic inputs to both SGI GABAergic and non‐GABAergic neurons are excitatory, but the response patterns in these two types of SGI neurons are different. Thus, actions of the cholinergic inputs to non‐GABAergic and GABAergic SGI neurons are not simple push–pull mechanisms, like excitation vs inhibition, but might cooperate to balance the level of excitation and inhibition for setting the state of the response property of the local circuit.     

Response of the GABAergic and dopaminergic mesostriatal projections to the lesion of the contralateral dopaminergic mesostriatal pathway in the rat.

Although dopamine is the main neurotransmitter in the mesostriatal system, recent studies indicate the existence of two nigrostriatal GABAergic projections: one arising from neurons immunoreactive for GABA, glutamic acid decarboxylase (GAD67), and parvalbumin (PV) lying in the substantia nigra pars reticulata (nigrostriatal GABA cells) and the other arising from a subpopulation of dopaminergic neurons lying in the substantia nigra pars compacta and ventral tegmental area, which under normal conditions, contains mRNA for GAD65 (one of the two isoforms of glutamic acid decarboxylase), but which is not immunoreactive for GABA and GAD65 (nigrostriatal dopaminergic [DA]/GABA cells). With the aim of improving our knowledge about the interaction between the nigrostriatal system of both brain hemispheres, we have studied the response of these three components of the mesostriatal system (GABA, DA/GABA, and DA) to the lesion of the contralateral mesostriatal DA pathway, by using morphological and neurophysiological techniques. Our findings show that, in the side contralateral to the lesion, (1) the number of nigrostriatal GABA cells increases from 6% to 17% with respect to the total number of nigrostriatal cells, (2) the soma of DA/GABA cells becomes immunoreactive for GABA and GAD65, and (3) there is an increase in the firing rate and burst activity of DA-neurons, except in those projecting to the striatum, which may be under the action of the GABA hyperactivity. Taken together, our results suggest that the GABAergic components of the mesostriatal projection play a regulatory role on the DA component, activated or upregulated after contralateral DA lesion and are probably addressed to restoring the functional symmetry in basal ganglia and to slowing down the contralateral progression of DA-cell degeneration in Parkinson's disease.     

Alpha 2 adrenergic receptors on GABAergic,putative sleep-promoting basal forebrain neurons

The basal forebrain plays an important role in the modulation of cortical activity and sleep-wake states. Yet its role must be multivalent as lesions reportedly diminish cortical fast activity and also cortical slow activity along with slow wave sleep (SWS). Basal forebrain cholinergic vs. GABAergic cell groups could differentially influence these processes. By labelling recorded neurons with Neurobiotin (Nb) using the juxtacellular technique and identifying them by immunostaining, we previously found that whereas all cholinergic cells increased their firing, the majority of GABAergic neurons decreased their firing in association with evoked cortical activation in urethane-anaesthetized rats. Here, we examined the possibility that such GABAergic, cortical activation 'off' cells might bear alpha 2 adrenergic receptors (alpha2AR) through which noradrenaline (NA) could inhibit them during cortical activation. First using simple dual-immunostaining for glutamic acid decarboxylase (GAD) and the alpha2AAR, we found that the majority (approximately 60%) of GAD-immunopositive (GAD+) neurons through the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) were labelled for the alpha2AAR. Second, in urethane-anaesthetized rats, we examined whether Nb-labelled, GAD+ cortical activation 'off' neurons that discharged maximally in association with cortical slow wave activity, were immunopositive for alpha2AAR. We found that all the Nb+/GAD+'off' cells were labelled for the alpha2AAR. Such cells could be inhibited in association with cortical activation and waking when noradrenergic locus coeruleus (LC) neurons discharge and be disinhibited with cortical slow waves and SWS when these neurons become inactive. We thus propose that alpha2AR-bearing GABAergic basal forebrain neurons constitute sleep-active and sleep-promoting neurons.     

The effect of prefrontal stimulation on the firing of basal forebrain neurons in urethane anesthetized rat

The basal forebrain (BF) contains a heterogeneous population of cholinergic and non-cholinergic corticopetal neurons and interneurons. Neurons firing at a higher rate during fast cortical EEG activity (f>16Hz) were called F cells, while neurons that increase their firing rate during high-amplitude slow-cortical waves (f<4Hz) were categorized as S-cells. The prefrontal cortex (PFC) projects heavily to the BF, although little is known how it affects the firing of BF units. In this study, we investigated the effect of stimulation of the medial PFC on the firing rate of BF neurons (n=57) that were subsequently labeled by biocytin using juxtacellular filling (n=22). BF units were categorized in relation to tail-pinch induced EEG changes. Electrical stimulation of the medial PFC led to responses in 28 out of 41 F cells and in 8 out of 9 S cells. Within the sample of responsive F cells, 57% showed excitation (n=8) or excitation followed by inhibitory period (n=8). The remaining F cells expressed a short (n=6) or long inhibitory (n=6) response. In contrast, 6 out of the 8 responsive S cells reduced their firing after prefrontal stimulation. Among the F cells, we recovered one cholinergic neuron and one parvalbumin-containing (PV) neuron using juxtacellular filling and subsequent immunocytochemistry. While the PV cell displayed short latency facilitation, the cholinergic cell showed significant inhibition with much longer latency in response to the prefrontal stimulus. This is in agreement with previous anatomical data showing that prefrontal projections directly target mostly non-cholinergic cells, including GABAergic neurons.     

Distinct glutamatergic and GABAergic subsets of hypothalamic pro‐opiomelanocortin neurons revealed by in situ hybridization in male rats and mice

Pro‐opiomelanocortin (POMC) and agouti‐related protein (AGRP) neurons in the hypothalamus regulate various aspects of energy homeostasis and metabolism. POMC and AGRP neurons, respectively, agonize and antagonize melanocortin receptors on their common downstream neurons. However, it is unknown whether they also reciprocally stimulate and inhibit the same neurons by amino acid transmitters. Whereas AGRP neurons are mostly GABAergic, surprisingly, only a small population of POMC neurons has been found to be glutamatergic, and a significantly larger subpopulation to be GABAergic. To further examine amino acid phenotypes of POMC neurons, we studied mRNA expression for the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), and the GABA synthetic enzyme, glutamic acid decarboxylase 67 (GAD67), in POMC neurons of both rats and mice by using in situ hybridization techniques. In rats, approximately 58% of POMC neurons were labeled for VGLUT2 and 37% for GAD67 mRNA. In mice, approximately 43% of POMC neurons contained VGLUT2, and 54% contained GAD67 mRNA. In both species, a prominent mediolateral distribution pattern was observed at rostral and mid levels of the POMC cell group with VGLUT2–POMC neurons dominating in lateral portions and GAD67–POMC neurons in medial portions. These data demonstrate that both glutamatergic and GABAergic cells are present in comparably significant numbers among POMC neurons. Their glutamatergic or GABAergic phenotype may represent a major functional division within the POMC cell group. J. Comp. Neurol. 521:3287–3302, 2013. © 2013 Wiley Periodicals, Inc.     

Molecular heterogeneity of aggrecan‐based perineuronal nets around five subclasses of parvalbumin‐expressing neurons in the mouse hippocampus

Subsets of GABAergic neurons are surrounded by perineuronal nets (PNNs), which play a critical role in the regulation of neural plasticity and neuroprotection. Although the plant lectin Wisteria floribunda agglutinin (WFA) has been commonly used to label PNNs, WFA only detects N‐acetyl‐d ‐galactosamine on aggrecan, a member of the lectican family. In this study, we used WFA and the antibody against the core protein of aggrecan (ACAN) to investigate the molecular heterogeneity of aggrecan‐based PNNs around five subclasses of parvalbumin‐expressing (PV+) γ‐aminobutyric acid (GABA)ergic neurons in the CA1 and CA3 regions of the mouse hippocampus. The vast majority of ACAN+ PNNs were colocalized with WFA in the stratum pyramidale, whereas a substantial population of ACAN+ PNNs lacked WFA labeling in the stratum oriens. We then defined the subclasses of PV+ neurons based on their cellular locations, molecular expression, and septal projection. Like the WFA+ PNNs, ACAN+ PNNs surrounded PV+ basket cells and bistratified cells but not axo‐axonic cells. Unlike the WFA+ PNNs, ACAN+ PNNs frequently surrounded PV+ oriens‐lacunosum moleculare cells and hippocampo‐septal cells. Interestingly, the relative densities of GABAergic synapses were higher around PV+ neurons with ACAN+ PNNs than around those without ACAN+ PNNs. Degradation of WFA+ PNNs by chondroitinase ABC did not affect the GABAergic synaptic densities around PV+ neurons. Our findings suggest that the molecular composition of aggrecan‐based PNNs around PV+ neurons may differ in a subclass‐specific manner, and also might help determine the functional involvement of PNNs in the regulation of GABAergic synapses around PV+ neurons in the hippocampus. J. Comp. Neurol. 525:1234–1249, 2017. © 2016 Wiley Periodicals, Inc.     

Parvalbumin,calbindin, or calretinin in cortically projecting and GABAergic,cholinergic, or glutamatergic basal forebrain neurons of the rat

The basal forebrain (BF) plays an important role in modulating cortical activity and facilitating processes of attention, learning, and memory. This role is subserved by cholinergic neurons but also requires the participation of other noncholinergic neurons. Noncholinergic neurons include gamma-amino butyric acidergic (GABAergic) neurons, some of which project in parallel with the cholinergic cells to the cerebral cortex, others of which project caudally or locally. With the original aim of distinguishing different subgroups of GABAergic neurons, we examined immunostaining for the calcium binding proteins (CBPs) parvalbumin (Parv), calbindin (Calb), and calretinin (Calret) in the rat. Although the CBP(+) cell groups were distributed in a coextensive manner with the GABAergic cells, they were collectively more numerous. Of cells retrogradely labeled with cholera toxin (CT) from the prefrontal or parietal cortex, Parv(+) and Calb(+) cells, but not Calret(+) cells, represented substantial proportions ( approximately 35-45% each) that collectively were greater than that of GABAergic projection neurons. From dual immunostaining for the CBPs and glutamic acid decarboxylase (GAD), it appeared that the vast majority (>90%) of the Parv(+) group was GAD(+), whereas only a small minority (<10%) of the Calb(+) or Calret(+) group was GAD(+). Significant proportions of Calb(+) (>40%) and Calret(+) (>80%) neurons were immunopositive for phosphate-activated glutaminase, the synthetic enzyme for transmitter glutamate. The results suggested that, whereas Calret(+) cells predominantly comprise caudally or locally projecting, possibly glutamatergic BF neurons, Parv(+) cells likely comprise the cortically projecting GABAergic BF neurons and Calb(+) cells the cortically projecting, possibly glutamatergic BF neurons that would collectively participate with the cholinergic cells in the modulation of cortical activity.     

A dense cluster of D1+ cells in the mouse nucleus accumbens

The striatum is known to be largely composed of intermingled medium‐sized projection neurons expressing either the D₁ or the D₂ dopamine receptors. In the present study, we took advantage of the double BAC Drd1a‐TdTomato/Drd2‐GFP (D₁/D₂) transgenic mice to reveal the presence of a peculiar cluster of densely‐packed D₁+ cells located in the shell compartment of the nucleus accumbens. This spherical cluster has a diameter of 110 µm and is exclusively composed by D₁+ cells, which are all immunoreactive for the neuronal nuclear marker (NeuN). However, in contrast to other D₁+ or D₂+ striatal cells, those that form the accumbens cluster are devoid of calbindin (CB) and DARPP‐32, two faithful markers for striatal projection neurons. Using GAD‐GFP transgenic mice, we confirm the GABAergic nature of the D₁+ clustered neurons. Intracellular injections from fixed brain slices indicate that these neurons are endowed with distinctive morphological features, including a small (5–6 µm), round cell body giving rise to a single primary dendrite that branches into two secondary processes. Single‐neuronal injections combined to electron microscopy reveal the existence of GAP junctions linking these D₁+ cells. Based on their location, morphological characteristics and neurochemical phenotype, we conclude that the D₁+ accumbens cluster form a highly compact group of small neurons distinct from the larger and more diffusely distributed D₁+ or D₂+ striatal projection neurons that surround it. This remarkable nucleus might play a crucial role in the limbic function of the murine striatum.     

GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep

The lateral hypothalamus (LH), where wake‐active orexin (Orx)‐containing neurons are located, has been considered a waking center. Yet, melanin‐concentrating hormone (MCH)‐containing neurons are codistributed therein with Orx neurons and, in contrast to them, are active during sleep, not waking. In the present study employing juxtacellular recording and labeling of neurons with Neurobiotin (Nb) in naturally sleeping–waking head‐fixed rats, we identified another population of intermingled sleep‐active cells, which do not contain MCH (or Orx), but utilize γ‐aminobutyric acid (GABA) as a neurotransmitter. The ‘sleep‐max’ active neurons represented 53% of Nb‐labeled MCH‐(and Orx) immunonegative (?) cells recorded in the LH. For identification of their neurotransmitter, Nb‐labeled varicosities of the Nb‐labeled/MCH? neurons were sought within sections adjacent to the Nb‐labeled soma and immunostained for the vesicular transporter for GABA (VGAT) or for glutamate. A small proportion of sleep‐max Nb+/MCH? neurons (19%) discharged maximally during slow‐wave sleep (called ‘S‐max’) in positive correlation with delta electroencephalogram activity, and from VGAT staining of Nb‐labeled varicosities appeared to be GABAergic. The vast proportion of sleep‐max Nb+/MCH? neurons (81%) discharged maximally during paradoxical sleep (PS, called ‘P‐max’) in negative correlation with electromyogram amplitude, and from Nb‐labeled varicosities also appeared to be predominantly GABAergic. Given their discharge profiles across the sleep–wake cycle, P‐max together with S‐max GABAergic neurons could thus serve to inhibit other neurons of the arousal systems, including local Orx neurons in the LH. They could accordingly dampen arousal with muscle tone and promote sleep, including PS with muscle atonia.