Glutamate neurons in the substantia nigra compacta and retrorubral field

Dopaminergic neurons of the substantia nigra compacta (SNC), ventral tegmental area (VTA) and retrorubral field (RRF) play a role in reward, motivation, learning, memory, and movement. These neurons are intermingled with GABAergic neurons. Recent evidence shows that the VTA contains glutamatergic neurons expressing vesicular glutamate transporter type 2 (VGluT2); some of them co‐express tyrosine hydroxylase (TH). Here, we used a combination of radioactive in situ hybridisation and immunohistochemistry to explore whether any of the vesicular glutamate transporters [vesicular glutamate transporter type 1 (VGluT1), VGluT2, or vesicular glutamate transporter type 3 (VGluT3)] were encoded by neurons in the SNC or RRF. We found expression of VGluT2 mRNA, but not of VGluT1 or VGluT3, in the SNC and RRF. These VGluT2 neurons rarely showed TH immunoreactivity. Within the SNC, the VGluT2 neurons were infrequently found at the rostral level, but were often seen at the medial and caudal levels intercalated in the mediolateral portion of the dorsal tier, at a ratio of one VGluT2 neuron per 4.4 TH neurons. At this level, VGluT2 neurons were also found in the adjacent substantia nigra reticulata and substantia nigra pars lateralis. Within the RRF, the VGluT2 neurons showed an increasing rostrocaudal gradient of distribution. The RRF proportion of VGluT2 neurons in relation to TH neurons was constant throughout the rostrocaudal levels, showing an average ratio of one VGluT2 neuron per 1.7 TH neurons. In summary, we provide evidence indicating that the SNC and RRF, which are traditionally considered to be dopaminergic areas, have neurons with the ability to participate in glutamate signaling.     

GABAergic and glutamatergic efferents of the mouse ventral tegmental area

The role of dopaminergic (DA) projections from the ventral tegmental area (VTA) in appetitive and rewarding behavior has been widely studied, but the VTA also has documented DA‐independent functions. Several drugs of abuse, act on VTA GABAergic neurons, and most studies have focused on local inhibitory connections. Relatively little is known about VTA GABA projection neurons and their connections to brain sites outside the VTA. This study employed viral‐vector‐mediated cell‐type‐specific anterograde tracing, classical retrograde tracing, and immunohistochemistry to characterize VTA GABA efferents throughout the brain. We found that VTA GABA neurons project widely to forebrain and brainstem targets, including the ventral pallidum, lateral and magnocellular preoptic nuclei, lateral hypothalamus, and lateral habenula. Minor projections also go to central amygdala, mediodorsal thalamus, dorsal raphe, and deep mesencephalic nuclei, and sparse projections go to prefrontal cortical regions and to nucleus accumbens shell and core. These projections differ from the major VTA DA target regions. Retrograde tracing studies confirmed results from the anterograde experiments and differences in projections from VTA subnuclei. Retrogradely labeled GABA neurons were not numerous, and most non‐tyrosine hydroxylase/retrogradely labeled cells lacked GABAergic markers. Many non‐TH/retrogradely labeled cells projecting to several areas expressed VGluT2. VTA GABA and glutamate neurons project throughout the brain, most prominently to regions with reciprocal connections to the VTA. These data indicate that VTA GABA and glutamate neurons may have more DA‐independent functions than previously recognized. J. Comp. Neurol. 522:3308–3334, 2014. © 2014 Wiley Periodicals, Inc.     

GFRalpha-1 mRNA in dopaminergic and nondopaminergic neurons in the substantia nigra and ventral tegmental area.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for several types of neurons, including dopaminergic (DAergic) neurons. GDNF binds with high affinity to the GDNF family receptor alpha-1 (GFRalpha-1), which is highly expressed in the midbrain. Using anatomical and lesion techniques, we demonstrated that GFRalpha-1 was expressed in DAergic and non-DAergic neurons in the rat midbrain. Immunohistochemical characterization of GFRalpha-1-expressing neurons indicated that most of the neurons that were immunopositive for the DAergic marker tyrosine hydroxylase (TH) expressed GFRalpha-1 in the substantia nigra pars compacta (SNC). In contrast, fewer TH-containing neurons expressed GFRalpha-1 in the substantia nigra pars reticulata (SNR) and the ventral tegmental area (VTA). Depletion of GFRalpha-1/TH neurons was observed in the SNC following treatment with the neurotoxin 6-hydroxydopamine (6-OHDA); however, GFRalpha-1 expression remained in some neurons located in the SNR. The gamma-aminobutyric acid (GABA)ergic nature of GFRalpha-1-expressing neurons located in the SNR, which were resistant to (6-hydroxydopamine) 6-OHDA, was established by their expression of glutamic acid decarboxylase (GAD; the synthesizing enzyme for GABA). Further analysis indicated that coexpression of GFRalpha-1 and GAD varied in a rostrocaudal gradient in the SNR, substantia nigra pars lateralis (SNL), and VTA. Midbrain DAergic and GABAergic neurons have been previously classified according to their Ca(2+) binding protein (CaBP) content; thus, we also sought to investigate the proportion of midbrain GFRalpha-1-expressing neurons containing parvalbumin (PV), calbindin (CB), and calretinin (CR) in the midbrain. Although GFRalpha-1 expression was found mainly in CB- and CR-immunoreactive neurons, it was rarely observed in PV-immunolabeled neurons. Analysis of the proportion of GFRalpha-1-expressing neurons for each CaBP subpopulation indicated the coexistence of GFRalpha-1 with CR in the VTA and all subdivisions of the SN; double-labeled GFRalpha-1/CR neurons were distributed in the SNC, SNR, SNL, and VTA. GFRalpha-1/CB neurons were also detected in the SNC, SNL, and VTA. Expression of GFRalpha-1 in DAergic and non-DAergic neurons in the rat SN and VTA suggests that GDNF, via GFRalpha-1, might modulate DAergic and GABAergic functions in the nigrostriatal, mesolimbic, and nigrothalamic circuits of the adult rat.     

Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic

Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). We first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. We established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). We found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system.     

Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat

The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.     

GABAergic phenotype of periglomerular cells in the rodent olfactory bulb

Periglomerular (PG) cells in the rodent olfactory bulb are heterogeneous anatomically and neurochemically. Here we investigated whether major classes of PG cells use gamma-aminobutyric acid (GABA) as a neurotransmitter. In addition to three known subtypes of PG cells expressing tyrosine hydroxylase (TH), calbindin D-28k (CB), and calretinin (CR), we identified a novel PG cell population containing the GABAA receptor alpha5 subunit. Consistent with previous studies in the rat, we found that TH-positive cells were also labeled with antibodies against GABA, whereas PG cells expressing CB or the alpha5 subunit were GABA-negative. Using GAD67-GFP knockin mice, we found that all PG cell subtypes expressed GAD67-GFP. Calretinin labeled the major fraction (44%) of green fluorescent protein (GFP)-positive cells, followed by TH (16%), CB (14%), and the alpha5 subunit (13%). There was no overlap between these neuronal populations, which accounted for approximately 85% of GAD67-GFP-positive cells. We then demonstrated that PG cells labeled for TH, CB, or CR established dendrodendritic synapses expressing glutamic acid decarboxylase (GAD) or the vesicular inhibitory amino acid transporter, VGAT, irrespective of their immunoreactivity for GABA. In addition, CB-, CR-, and TH-positive dendrites were apposed to GABAA receptor clusters containing the alpha1 or alpha3 subunits, which are found in mitral and tufted cells, and the alpha2 subunit, which is expressed by PG cells. Together, these findings indicate that all major subtypes of PG cells are GABAergic. In addition, they show that PG cells provide GABAergic input to the dendrites of principal neurons and are interconnected with other GABAergic interneurons, which most likely are other PG cells.     

Parvalbumin-producing cortical interneurons receive inhibitory inputs on proximal portions and cortical excitatory inputs on distal dendrites

To examine inputs to parvalbumin (PV)-producing interneurons, we generated transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein specifically in the interneurons, and completely visualized their dendrites and somata. Using immunolabeling for vesicular glutamate transporter (VGluT)1, VGluT2, and vesicular GABA transporter, we found that VGluT1-positive terminals made contacts 4- and 3.1-fold more frequently with PV-producing interneurons than VGluT2-positive and GABAergic terminals, respectively, in the primary somatosensory cortex. Even in layer 4, where VGluT2-positive terminals were most densely distributed, VGluT1-positive inputs to PV-producing interneurons were 2.4-fold more frequent than VGluT2-positive inputs. Furthermore, although GABAergic inputs to PV-producing interneurons were as numerous as VGluT2-positive inputs in most cortical layers, GABAergic inputs clearly preferred the proximal dendrites and somata of the interneurons, indicating that the sites of GABAergic inputs were more optimized than those of VGluT2-positive inputs. Simulation analysis with a PV-producing interneuron model compatible with the present morphological data revealed a plausible reason for this observation, by showing that GABAergic and glutamatergic postsynaptic potentials evoked by inputs to distal dendrites were attenuated to 60 and 87%, respectively, of those evoked by somatic inputs. As VGluT1-positive and VGluT2-positive axon terminals were presumed to be cortical and thalamic glutamatergic inputs, respectively, cortical excitatory inputs to PV-producing interneurons outnumbered the thalamic excitatory and intrinsic inhibitory inputs more than two-fold in any cortical layer. Although thalamic inputs are known to evoke about two-fold larger unitary excitatory postsynaptic potentials than cortical ones, the present results suggest that cortical inputs control PV-producing interneurons at least as strongly as thalamic inputs.     

Relative contributions and mapping of ventral tegmental area dopamine and GABA neurons by projection target in the rat

The ventral tegmental area (VTA) is a heterogeneous midbrain structure that contains dopamine (DA), GABA, and glutamate neurons that project to many different brain regions. Here, we combined retrograde tracing with immunocytochemistry against tyrosine hydroxylase (TH) or glutamate decarboxylase (GAD) to systematically compare the proportion of dopaminergic and GABAergic VTA projections to 10 target nuclei: anterior cingulate, prelimbic, and infralimbic cortex; nucleus accumbens core, medial shell, and lateral shell; anterior and posterior basolateral amygdala; ventral pallidum; and periaqueductal gray. Overall, the non-dopaminergic component predominated VTA efferents, accounting for more than 50% of all projecting neurons to each region except the nucleus accumbens core. In addition, GABA neurons contributed no more than 20% to each projection, with the exception of the projection to the ventrolateral periaqueductal gray, where the GABAergic contribution approached 50%. Therefore, there is likely a significant glutamatergic component to many of the VTA's projections. We also found that VTA cell bodies retrogradely labeled from the various target brain regions had distinct distribution patterns within the VTA, including in the locations of DA and GABA neurons. Despite this patterned organization, VTA neurons comprising these different projections were intermingled and never limited to any one subregion. These anatomical results are consistent with the idea that VTA neurons participate in multiple distinct, parallel circuits that differentially contribute to motivation and reward. While attention has largely focused on VTA DA neurons, a better understanding of VTA subpopulations, especially the contribution of non-DA neurons to projections, will be critical for future work.     

Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area

Ventral tegmental area (VTA) dopamine (DA) neurons and their forebrain projections are critically involved in reward processing and cognitive functions. Descending projections from the lateral habenula (LHb) play a central role in inhibiting DA cell activity in response to the absence of expected rewards. As LHb efferents are reportedly glutamatergic, their ability to inhibit DA cells would theoretically require a disynaptic connection involving VTA GABA neurons and their local collateral inputs to DA cells. We therefore used anterograde tract-tracing from the LHb to investigate the relative selectivity of LHb synapses onto GABA versus DA VTA neurons. LHb axons were visualized using immunoperoxidase, and DA and GABA cells were marked by immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA, respectively. By ultrastructural analysis, 16% of LHb axons were observed to form synaptic contacts in the VTA, and most of these were of an intermediate morphological type that did not exhibit definitive asymmetric or symmetric character. LHb axons synaptically targeted TH- and GABA-labeled dendrites to a comparable extent (45 and 52% observed incidence, respectively). Pre-embedding immunogold labeling for the vesicular glutamate transporter type 2 and post-embedding immunogold staining for GABA confirmed that approximately 85% of LHb terminals were glutamatergic and not GABAergic. These results suggest that the robust inhibition of DA cells evoked by the LHb is unlikely to arise from a selective innervation of VTA GABA neurons. Moreover, the LHb may mediate a direct excitation of DA cells that is over-ridden by indirect inhibition originating from an extrinsic source.     

Vesicular glutamate transporter 2 protein and mRNA containing neurons in the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus is the key structure of the control of circadian rhythms and has a rich glutamatergic innervation. Besides the presence of glutamatergic afferents, several findings also suggest the existence of glutamatergic efferents from the suprachiasmatic nucleus to its target neurons in various prominent hypothalamic cell groups. However, there is no direct neuromorphological evidence for the presence of glutamatergic neurons in the suprachiasmatic nucleus. Therefore, the purpose of the present investigations was to try to clarify this question. Immunocytochemistry was used at the light and electron microscopy level to identify vesicular glutamate transporter type 2 (VGluT2) immunopositive (presumed glutamatergic) neurons in the rat suprachiasmatic nucleus. In addition VGluT2 mRNA expression in neurons of the nucleus was also addressed with radioisotopic in situ hybridization. Both at the light and electron microscopy level we detected VGluT2 positive neurons, which did not contain GABA, vasoactive intestinal polypeptide or vasopressin. Further, we demonstrated the expression of VGluT2 mRNA in a few cells within the suprachiasmatic nucleus; these glutamatergic cells were distinct from somatostatin mRNA expressing neurons. As VGluT2 is a selective marker of glutamatergic neuronal elements, the present observations provide direct neuromorphological evidence for the presence of glutamatergic neurons in the cell group.     

Chemoarchitecture of GABAergic neurons in the ferret superior colliculus

gamma-Aminobutyric acid (GABA)ergic neurons are thought to play a key role both in visual processing and in the complex sensory-motor transformations that take place in the mammalian superior colliculus. To understand the organization of GABAergic neurons in the ferret superior colliculus, we applied antisera to several markers of GABAergic function, including GABA, two isoforms of its synthetic enzyme glutamic acid decarboxylase (GAD-65 and GAD-67), and the GABA transporter, GAT-1. We also applied antisera to several calcium binding proteins (calbindin [CB], calretinin [CR], and parvalbumin [PV]) and neuronal nitric oxide synthase (NOS), chemical markers that colocalize with GABA in some areas of the central nervous system. The distribution of GABAergic neurons in the ferret is similar to that of other mammalian species. GABAergic neurons in the ferret superior colliculus were small, morphologically diverse, and widely distributed throughout all layers of the colliculus. As has been shown in other mammalian species, neurons expressing PV, CB, CR, and NOS were differentially distributed in layers and patches throughout the ferret colliculus. None of these markers, however, showed a distribution that mirrored that of GABAergic neurons. Furthermore, few GABAergic neurons colocalized these neurochemical markers. Only 14% of GABAergic neurons in the superficial layers and 18% of neurons in the deeper layers colocalized PV, 14% of GABAergic neurons in the superficial layers and 10% in the deeper layers colocalized CB, and only 1% of GABAergic neurons in both the superficial and deep layers colocalized nitric oxide synthase. Thus, the arrangement of GABAergic neurons in the ferret superior colliculus is broadly distributed and is distinct from other recognized organizational patterns in the superior colliculus.     

Calcium binding protein markers of GABA deficits in schizophrenia — post mortem studies and animal models

As quantitative neuroimaging continues to elucidate the gross neuropathology of schizophrenia, neurochemical and histological studies have contributed to defining this pathology in terms of neurotransmitter dysfunction. Increasingly, there is evidence implicating neurons containing the major inhibitory neurotransmitter of the brain--gamma-aminobutyric acid (GABA). Benes was the first to demonstrate deficits in some morphological subtypes of interneurons in the frontal cortex in schizophrenia. We identified that this was specific to a subgroup of GABAergic interneurons containing parvalbumin (PV), which is found in the fast-firing cells providing inhibitory control of the cortico-fugal pyramidal cells. PV is notable in being expressed late in development; the late expression of this protective calcium binding protein (CBP) may impart an early vulnerability to these neurons, indicating a possible mechanism for the developmental origins of schizophrenia. Cortical GABAergic neurons expressing the CBP calretinin (CR) are unaffected in schizophrenia, although those containing calbindin (CB) are also diminished in number. These deficits in PV and CB are notable in also being observed in bipolar disorder, indicating how the close aetiological relationship of these two psychiatric disorders is reflected in their pathology. One of the most substantial abnormalities seen in post-mortem brain tissue is the hippocampal deficit of PV-containing neurons, again in the absence of effects on CR-positive cells. This deficit occurring in a structure implicated in cognitive symptomatology may well have functional relevance, and we find it can be induced by a model of the disease, sub-chronic phencyclidine (PCP) administration, that can also produce cognitive disturbances. This PCP model, like schizophrenia, demonstrates other neurochemical changes which include indicators of glutamatergic dysfunction. The temporal and aetiological relationships between glutamatergic and GABAergic deficits remains unclear, but may well relate to an initial loss/dysfunction of GABA/PV neurons that subsequently gives rise to a glutamatergic pathology.     

Optogenetic excitation in the ventral tegmental area of glutamatergic or cholinergic inputs from the laterodorsal tegmental area drives reward

Converging evidence shows that ventral tegmental area (VTA) dopamine neurons receive laterodorsal tegmental nucleus (LDTg) cholinergic and glutamatergic inputs. To test the behavioral consequences of selectively driving the two sources of excitatory LDTg input to the VTA, channelrhodopsin‐2 (ChR2) was expressed in LDTg cholinergic neurons of ChAT::Cre mice (ChAT‐ChR2 mice) or in LDTg glutamatergic neurons of VGluT2::Cre mice (VGluT2‐ChR2 mice). Mice were tested in a 3‐chamber place preference apparatus where entry into a light‐paired chamber resulted in VTA light stimulation of LDTg‐cholinergic or LDTg‐glutamatergic axons for the duration of a chamber stay. ChAT‐ChR2 mice spent more time in the light‐paired chamber and subsequently showed conditioned place preference for the light‐paired chamber in the absence of light. VGluT2‐ChR2 mice, entered the light‐paired chamber significantly more times than a light‐unpaired chamber, but remained in the light‐paired chamber for short time periods and did not show a conditioned place preference. When each entry into the light‐paired chamber resulted in a single train of VTA light stimulation, VGluT2‐ChR2 mice entered the light‐paired chamber significantly more times than the light‐unpaired chamber, but spent approximately equal amounts of time in the two chambers. VTA excitation of LDTg‐glutamatergic inputs may be more important for reinforcement of initial chamber entry while VTA excitation of LDTg‐cholinergic inputs may be more important for the rewarding effects of chamber stays. We suggest that LDTg‐cholinergic and LDTg‐glutamatergic inputs to the VTA each contribute to the net rewarding effects of exciting LDTg axons in the VTA.     

Inhibition of GABAergic neurotransmission in the ventral tegmental area by cannabinoids

It was shown recently that Delta9-tetrahydrocannabinol, like several other drugs eliciting euphoria, stimulates dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens. The aim of the present work was to clarify the mechanism of this stimulatory effect. Our hypothesis was that cannabinoids depress the GABAergic inhibition of dopaminergic neurons in the VTA. Electrophysiological properties of VTA neurons in rat coronal midbrain slices were studied with the patch-clamp technique. GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked by electrical stimulation in the vicinity of the recorded neurons. The amplitude of IPSCs was depressed by the synthetic mixed CB1/CB2 cannabinoid receptor agonist WIN55212-2 (10(-6) and 10(-5) m). The CB1 cannabinoid receptor antagonist SR141716A (10(-6) m) prevented the inhibition produced by WIN55212-2 (10(-5) m). Two observations showed that IPSCs were depressed with a presynaptic mechanism. WIN55212-2 (10(-5) m) did not change the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. Currents evoked by pressure ejection of muscimol from a pipette were also not changed by WIN55212-2 (10(-5) m). The results indicate that activation of CB1 cannabinoid receptors inhibits GABAergic neurotransmission in the VTA with a presynaptic mechanism. Depression of the GABAergic inhibitory input of dopaminergic neurons would increase their firing rate in vivo. Accordingly, dopamine release in the projection region of VTA neurons, the nucleus accumbens, would also increase.     

Dopaminergic axons in different divisions of the adult rat striatal complex do not express vesicular glutamate transporters

Midbrain dopamine neurons signal rapid information about rewards and reward-related events. It has been suggested that this fast signal may, in fact, be conveyed by co-released glutamate. Evidence that dopamine neurons co-release glutamate comes largely from studies involving cultured neurons or tissue from young animals. Recently, however, it has been shown that this dual glutamatergic/dopaminergic phenotype declines with age, and can be induced by injury, suggesting that it is not a key feature of adult dopamine neurons. Here, we provide further support for this view by showing that dopaminergic axons and terminals in subregions of the adult striatum do not express vesicular glutamate transporters (VGluT1, VGluT2 or VGluT3). Striatal tissue from the adult rat was immunolabelled to reveal tyrosine hydroxylase (TH; biosynthetic enzyme of dopamine) and one of the three known VGluTs. Importantly, we compared the immunogold labelling for each of the VGluTs associated with TH-positive structures with background labelling at the electron microscopic level. In addition, we carried out a subregional analysis of the core and shell of the nucleus accumbens. We found that dopaminergic axons and terminals in the dorsolateral striatum and ventral striatum (nucleus accumbens core and shell) do not express VGluT1, VGluT2 or VGluT3. We conclude, therefore, that in the normal, adult rat striatum, dopaminergic axons do not co-release glutamate.     

Changes in density of calcium‐binding‐protein‐immunoreactive GABAergic neurons in prefrontal cortex in schizophrenia and bipolar disorder

There is evidence that GABAergic neurotransmission is altered in mental disorders such as schizophrenia (SCZ) and bipolar disorder (BPD). The calcium‐binding proteins (CBPs) calbindin (CB), calretinin (CR), and parvalbumin (PV) are used as markers of specific subpopulations of cortical GABAergic interneurons. We examined the postmortem prefrontal cortical region (Brodmann's area 9) of patients with SCZ and BPD, and of age‐matched control subjects, excluding suicide cases. The laminar density of neurons immunoreactive (IR) for three CBPs, namely CB, CR, and PV, was quantified. The densities of CB‐IR neurons in layer 2 and PV‐IR neurons in layer 4 in the SCZ subjects decreased compared with those in the control subjects. When CBP‐IR neurons were classified according to their size, a reduction in the density of medium CB‐IR neurons in layer 2 in SCZ subjects and an increase in the density of large CR‐IR neurons in layer 2 in BPD subjects were observed. These results suggest that alterations in specific GABAergic neurons are present in mental disorders, and that such alterations may reflect the vulnerability toward the disorders.