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Background: Schizophrenia is a chronic, severe mental illness with profound emotional and economic burdens for those afflicted and their families. An increasing number of studies have found that schizophrenia is marked by dysregulation of glutamatergic neurotransmission. While numerous studies have found alterations of postsynaptic molecules in schizophrenia, a growing body of evidence implicates presynaptic factors. Vesicular glutamate transporters (VGLUTs) have been identified and are known to package glutamate into vesicles in the presynaptic terminal for subsequent release into the synaptic cleft. Recent studies have shown that VGLUTs regulate synaptic activity via the amount of glutamate released. Accordingly, we hypothesized that VGLUTs are altered in schizophrenia, contributing to dysfunction of presynaptic activity. Methods: Using in situ hybridization and Western blot analysis, we investigated alterations in VGLUT1 and VGLUT2 transcript and protein expression in the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and a comparison group. Results: We found increased VGLUT1 transcript and reduced VGLUT1 protein expression in the ACC, but not DLPFC, in schizophrenia. Vesicular glutamate transporter 2 was unchanged at both levels of gene expression. We did not find changes in VGLUT1 messenger RNA (mRNA) or protein levels following 28-day treatment of rats with haloperidol (2 mg/kg/day), suggesting that our findings in schizophrenia are not due to an effect of antipsychotic treatment. Conclusions: Overall, our data suggest decreased glutamate release in the ACC, as well as discordant regulation of VGLUT1 expression at different levels of gene expression. [Copyright &y& Elsevier]

Abstract: Midbrain dopamine neurons in the ventral tegmental area, substantia nigra and retrorubral field play key roles in reward processing, learning and memory, and movement. Within these midbrain regions and admixed with the dopamine neurons, are also substantial populations of GABAergic neurons that regulate dopamine neuron activity and have projection targets similar to those of dopamine neurons. Additionally, there is a small group of putative glutamatergic neurons within the ventral tegmental area whose function remains unclear. Although dopamine neurons have been intensively studied and quantified, there is little quantitative information regarding the GABAergic and glutamatergic neurons. We therefore used unbiased stereological methods to estimate the number of dopaminergic, GABAergic and glutamatergic cells in these regions in the rat. Neurons were identified using a combination of immunohistochemistry (tyrosine hydroxylase) and in situ hybridization (glutamic acid decarboxylase mRNA and vesicular glutamate transporter 2 mRNA). In substantia nigra pars compacta 29% of cells were glutamic acid decarboxylase mRNA-positive, 58% in the retrorubral field and 35% in the ventral tegmental area. There were further differences in the relative sizes of the GABAergic populations in subnuclei of the ventral tegmental area. Thus, glutamic acid decarboxylase mRNA-positive neurons represented 12% of cells in the interfascicular nucleus, 30% in the parabrachial nucleus, and 45% in the parainterfascicular nucleus. Vesicular glutamate transporter 2 mRNA-positive neurons were present in the ventral tegmental area, but not substantia nigra or retrorubral field. They were mainly confined to the rostro-medial region of the ventral tegmental area, and represented approximately 2–3% of the total neurons counted (∼1600 cells). These results demonstrate that GABAergic and glutamatergic neurons represent large proportions of the neurons in what are traditionally considered as dopamine nuclei and that there are considerable heterogeneities in the proportions of cell types in the different dopaminergic midbrain regions. [Copyright &y& Elsevier]

Two cardinal features of Parkinson's disease (PD) pathophysiology are a loss of glutamatergic synapses paradoxically accompanied by an increased glutamatergic transmission to the striatum. The exact substrate of this increased glutamatergic drive remains unclear. The striatum receives glutamatergic inputs from the thalamus and the cerebral cortex. Using vesicular glutamate transporters (vGluTs) 1 and 2 as markers of the corticostriatal and thalamostriatal afferents, respectively, we examined changes in the synaptology and relative prevalence of striatal glutamatergic inputs in methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys using electron microscopic immunoperoxidase and confocal immunofluorescence methods. Our findings demonstrate that the prevalence of vGluT1-containing terminals is significantly increased in the striatum of MPTP-treated monkeys (51.9 ± 3.5% to 66.5 ± 3.4% total glutamatergic boutons), without any significant change in the pattern of synaptic connectivity; more than 95% of vGluT1-immunolabeled terminals formed axo-spinous synapses in both conditions. In contrast, the prevalence of vGluT2-immunoreactive terminals did not change after MPTP treatment (21.7 ± 1.3% vs. 21.6 ± 1.2% total glutamatergic boutons). However, a substantial increase in the ratio of axo-spinous to axo-dendritic synapses formed by vGluT2-immunoreactive terminals was found in the pre-caudate and post-putamen striatal regions of MPTP-treated monkeys, suggesting a certain degree of synaptic reorganization of the thalamostriatal system in parkinsonism. About 20% of putative glutamatergic terminals did not show immunoreactivity in striatal tissue immunostained for both vGluT1 and vGluT2, suggesting the expression of another vGluT in these boutons. These findings provide striking evidence that suggests a differential degree of plasticity of the corticostriatal and thalamostriatal system in PD. [ABSTRACT FROM AUTHOR]

Abstract: Cocaine- and amphetamine-regulated transcript (CART) peptides have a wide CNS distribution and appear to play an important role in a number of physiological processes including reward and reinforcement, feeding, locomotion, stress responses and perception of pain. We have further investigated the expression of CART peptides in rat cerebellum, using multiple fluorescent antibodies. Dense fibre-like immunofluorescence was observed in the molecular layer of the vestibular cerebellum (paraflocculus and lobes IX and X of the vermis). There was little or no immunofluorescence in any other region of the cerebellum (vermis or hemispheres). The immunofluorescence in lobes IX and X of the vermis showed a parasagittal banding pattern, with one medial and two lateral bands. We have provided several lines of evidence that CART peptides are expressed by climbing fibres: (1) There is CART peptide immunofluorescence in the inferior olivary complex, the source of climbing fibres. (2) No cerebellar cell bodies were labelled by the CART peptide antibody. (3) The developmental profile of CART peptide expression was consistent with climbing fibre development and finally (4) triple antibody labelling revealed co-expression of CART peptides with VGluT2 (the glutamate transporter at climbing fibre synapses) at boutons innervating calbindin D-28K labelled proximal Purkinje cell dendrites. Analysis of VGluT2 and CART peptide labelling in more detail showed that not all climbing fibres in the vestibular cerebellum expressed CART peptides but those fibres which did express CART peptides were immunofluorescent at all Purkinje cell synapses. [Copyright &y& Elsevier]

The ventral spinal cord consists of interneuron groups arising from distinct, genetically defined, progenitor domains along the dorsoventral axis. Many of these interneuron groups settle in the ventral spinal cord which, in mammals, contains the central pattern generator for locomotion. In order to better understand the locomotor networks, we have used different transgenic mice for anatomical characterization of one of these interneuron groups, called V2 interneurons. Neurons in this group are either V2a interneurons marked by the postmitotic expression of the transcription factor Chx10, or V2b interneurons which express the transcription factors Gata2 and Gata3. We found that all V2a and most V2b interneurons were ipsilaterally projecting in embryos as well as in newborns. V2a interneurons were for the most part glutamatergic while V2b interneurons were mainly GABAergic or glycinergic. Furthermore, we demonstrated that a large proportion of V2 interneurons expressed the axon guidance molecule EphA4, a molecule previously shown to be important for correct organization of locomotor networks. We also showed that V2 interneurons and motor neurons alone did not account for all EphA4-expressing neurons in the spinal cord. Together, these findings enable a better interpretation of neural networks underlying locomotion, and open up the search for as yet unknown components of the mammalian central pattern generator. [ABSTRACT FROM AUTHOR]

Abstract: During the last two decades attention has been focussed on the role of different neuropeptides in hypothalamic control of feeding behavior. Several hypothalamic peptides that participate in the control of ingestive behavior are produced in neuronal cell bodies of the arcuate nucleus and/or the lateral hypothalamic area. Apart from producing orexigenic or anorexigenic compounds of peptidergic nature, these neurons also produce excitatory and inhibitory amino acid neurotransmitters. The role of GABA and glutamate in regulating energy balance has received less attention in comparison to neuropeptides. The arcuate nucleus–median eminence area, a region with a weak blood–brain barrier, contains at least two neuronal cell populations that exert opposing actions on energy balance. The majority of the neurons located in the ventromedial aspect of the arcuate nucleus, which produce the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP), contain in addition the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter (VGAT), thereby supporting their GABAergic nature. Some neurons producing pro-opiomelanocortin (POMC)- and cocaine- and amphetamine-regulated transcript (CART), located in the ventrolateral division of the arcuate nucleus have recently been reported to contain the vesicular glutamate transporter 2 (VGLUT2), a marker for glutamatergic neurons, and the acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) as well as the vesicular ACh transporter (VAChT), supporting also a cholinergic phenotype. In the lateral hypothalamic area, hypocretin/orexin neurons express VGLUT1 or VGLUT2, but not GAD, whereas some melanin-concentrating hormone (MCH) cells contain GAD. These observations support the view that several classical transmitters, relatively neglected feeding transmitters candidates, are present in key neurons that regulate body weight and consequently may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in regulation of energy balance. [Copyright &y& Elsevier]

Abstract: We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the proteins they express, by studying the degree of co-localization of synapsin (SYN) I and II, synaptophysin (SYP) I and II, synaptosomal-associated protein (SNAP)-25 and SNAP-23 in vesicular glutamate transporter (VGLUT) 1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta in the rat cerebral cortex. Co-localization studies showed that SYNI and II were expressed in ∼90% of VGLUT1+, ∼30% of VGLUT2+ and 30–50% of VGAT+ puncta; SYPI was expressed in ∼95% of VGLUT1+, 30% of VGLUT2+, and 45% of VGAT+ puncta; SYPII in ∼7% of VGLUT1+, 3% of VGLUT2+, and 20% of VGAT+ puncta; SNAP-25 in ∼94% of VGLUT1+, 5% of VGLUT2+, and 1% of VGAT+ puncta, and SNAP-23 in ∼3% of VGLUT1+, 86% of VGLUT2+, and 22% of VGAT+ puncta. Since SYPI, which is considered ubiquitous, was expressed in about half of GABAergic axon terminals, we studied its localization electron microscopically and in immunoisolated synaptic vesicles: these studies showed that ∼30% of axon terminals forming symmetric synapses were SYPI-negative, and that immunoisolated VGAT-positive synaptic vesicles were relatively depleted of SYPI as compared with VGLUT1+ vesicles. Overall, the present investigation shows that in the cerebral cortex of rats distinct presynaptic proteins involved in neurotransmitter release are differentially expressed in GABAergic and in the two major types of glutamatergic axon terminals in the cerebral cortex of rats. [Copyright &y& Elsevier]

Abstract: The immunocytochemical localisation of vesicular glutamate transporters, VGLUT1 and VGLUT2, was employed to identify putative glutamatergic axon terminals innervating pelvic motoneurons. VGLUT1 terminals were sparsely distributed within lumbosacral spinal motoneuron pools, including the dorsolateral nucleus, retrodorsolateral nucleus and spinal nucleus of the bulbospongiosus. This was in marked contrast to VGLUT2 which was expressed in a robust innervation of these areas. Retrograde tracing was used to reveal motoneurons innervating the levator ani (LA) muscle. On these neurons, associations with VGLUT2 immunoreactive terminals were abundant while those with VGLUT1 were rare. Ultrastructural investigations revealed that VGLUT2 immunoreactive terminals made asymmetric synaptic contacts with dendrites of retrogradely labelled LA motoneurons. Quantification of VGLUT2 immunoreactive boutons in close association with these dendrites was carried out in young and aged animals using light microscopy. This revealed a significant decline in the numbers of VGLUT2 immunoreactive boutons on the more distal dendrites of motoneurons in aged rats. VGLUT2 boutons were reduced by approximately 21% from 11.25±0.5 per 35-μm length of dendrite in young rats to 8.89±0.5 in aged animals. This decline in glutamatergic input may reduce the excitability of LA motoneurons and consequently decrease the capacity of the rat to induce reflexive erections. [Copyright &y& Elsevier]

Abstract: Dopamine and GABA neurons in the ventral tegmental area project to the nucleus accumbens and prefrontal cortex and modulate locomotor and reward behaviors as well as cognitive and affective processes. Both midbrain cell types receive synapses from glutamate afferents that provide an essential control of behaviorally-linked activity patterns, although the sources of glutamate inputs have not yet been completely characterized. We used antibodies against the vesicular glutamate transporter subtypes 1 and 2 (VGlut1 and VGlut2) to investigate the morphology and synaptic organization of axons containing these proteins as putative markers of glutamate afferents from cortical versus subcortical sites, respectively, in rats. We also characterized the ventral tegmental area cell populations receiving VGlut1+ or VGlut2+ synapses according to their transmitter phenotype (dopamine or GABA) and major projection target (nucleus accumbens or prefrontal cortex). By light and electron microscopic examination, VGlut2+ as opposed to VGlut1+ axon terminals were more numerous, had a larger average size, synapsed more proximally, and were more likely to form convergent synapses onto the same target. Both axon types formed predominantly asymmetric synapses, although VGlut2+ terminals more often formed synapses with symmetric morphology. No absolute selectivity was observed for VGlut1+ or VGlut2+ axons to target any particular cell population. However, the synapses onto mesoaccumbens neurons more often involved VGlut2+ terminals, whereas mesoprefrontal neurons received relatively equal synaptic inputs from VGlut1+ and VGlut2+ profiles. The distinct morphological features of VGlut1 and VGlut2 positive axons suggest that glutamate inputs from presumed cortical and subcortical sources, respectively, differ in the nature and intensity of their physiological actions on midbrain neurons. More specifically, our findings imply that subcortical glutamate inputs to the ventral tegmental area expressing VGlut2 predominate over cortical sources of excitation expressing VGlut1 and are more likely to drive the behaviorally-linked bursts in dopamine cells that signal future expectancy or attentional shifting. [Copyright &y& Elsevier]

Abstract: Glutamatergic pathways play a key role in the functional organization of neuronal circuits involved in Parkinson disease (PD). Recently, three vesicular glutamate transporters (VGLUT1–3) were identified. VGLUT1 and VGLUT2 are responsible for the uploading of glutamate into synaptic vesicles and are the first specific markers of glutamatergic neurons available. Here, we analyzed the expression of VGLUT1 and VGLUT2 in autopsy tissues of PD patients and matched controls using Western blot and immunoautoradiography. VGLUT1 and VGLUT2 expression was increased in the Parkinsonian putamen by 24% and 29%, respectively (p <0.01). In contrast, only VGLUT1 was dramatically decreased in the prefrontal and temporal cortex of PD patients (∼50%, p <0.01 and p <0.001, respectively). These findings demonstrate the existence of profound alterations of glutamatergic transmission in PD, which are likely to contribute to the motor and cognitive impairments associated with the disease, and should thus be taken into account in the treatment of PD. [Copyright &y& Elsevier]

Abstract: Three vesicular glutamate transporters have been identified in mammals. Two of them, VGLUT1 and VGLUT2, define the glutamatergic phenotype and their distribution in the brain is almost complementary. In the present study we examined the distribution and expression levels of these two VGLUTs during postnatal development of the mouse barrel cortex. We also investigated changes in the localization of VGLUT1 and VGLUT2 within particular compartments of the barrel field (barrels/septa) during its development. We found differences in the time course of developmental expression, with VGLUT1 peaking around P14, while VGLUT2 increased gradually until adulthood. Over the examined period (P3 – adult) both transporters had stronger expression in the barrel interiors, and in this compartment VGLUT2 dominated, whereas in the inter-barrel septa VGLUT1 dominated over VGLUT2. Furthermore, we found that some nerve terminals in the barrel cortex coexpressed both transporters until adulthood. Colocalization was observed within the barrels, but not within the septa. [Copyright &y& Elsevier]

Abstract: Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated. [Copyright &y& Elsevier]

The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or γ-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co–expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic. [ABSTRACT FROM AUTHOR]

Abstract: Vesicular glutamate transporters (VGLUTs) are involved in storing glutamate for secretion at the level of glutamatergic axon terminals, and for this reason they have been extensively used as markers to identify glutamate-releasing cells. Platelets have been considered as a suitable model for studying glutamatergic dysfunction because they perform glutamate uptake and express both external transporters, and NMDA-like receptors. Here, we show that platelets express the pre-synaptic markers VGLUT1 and VGLUT2 and release glutamate following aggregation, implying a possible contributory role in the pathophysiology of stroke, migraine, and other excitotoxic disorders. [Copyright &y& Elsevier]

Abstract: The aortic depressor nerve (ADN) primarily transmits baroreceptor signals from the aortic arch to the nucleus tractus solitarii. Cell bodies of neurons that send peripheral fibers to form the ADN are located in the nodose ganglion (NG). Studies have implicated glutamate and nitric oxide in transmission of baroreflex signals; therefore, we tested the hypothesis that ADN neurons contain either vesicular glutamate transporters (VGLUTs) or neuronal nitric oxide synthase (nNOS) or both. We applied a fluorescent tracer, tetramethyl rhodamine dextran (TRD), to rat ADN to identify ADN neurons and then performed immunofluorescent labeling for nNOS and VGLUTs 1, 2, and 3 in NG sections. We found that VGLUT2-immunoreactivity (IR) and VGLUT3-IR was present in a significantly higher proportion of TRD positive neurons than in TRD negative neurons. In contrast, the percentage of TRD positive neurons containing VGLUT1-IR or nNOS-IR did not differ from that of TRD negative neurons. We also observed that the percentage of TRD positive neurons containing both VGLUT2-IR and nNOS-IR and the percentage of TRD positive neurons containing both VGLUT3-IR and nNOS-IR were significantly higher than that of TRD negative neurons. On the other hand, colocalization of VGLUT1-IR and nNOS-IR in TRD positive neurons did not differ from that of TRD negative neurons. These results support our hypothesis and suggest prominent roles of VGLUT2-IR containing neurons and VGLUT3-IR containing neurons in transmitting cardiovascular signals via the ADN to the brain stem. [Copyright &y& Elsevier]

Abstract: In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions. [Copyright &y& Elsevier]

Abstract: The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements. [Copyright &y& Elsevier]

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6-OHDA in the medial forebrain bundle. Thirty days post-lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro-Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal-projecting neurons (FG-labeled) by in situ hybridization and the results were confirmed by laser-guided capture microdissection microscopy followed by real-time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine-depleted striatum. Moreover, PF neurons innervating the dopamine-depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation. [ABSTRACT FROM AUTHOR]

Abstract: Although multiple effects of GABAB receptor activation on synaptic transmission in the striatum have been described, the precise locations of the receptors mediating these effects have not been determined. To address this issue, we carried out pre-embedding immunogold electron microscopy in the rat using antibodies against the GABAB receptor subunits, GABAB1 and GABAB2. In addition, to investigate the relationship between GABAB receptors and glutamatergic striatal afferents, we used antibodies against the vesicular glutamate transporters, vesicular glutamate transporter 1 and vesicular glutamate transporter 2, as markers for glutamatergic terminals. Immunolabeling for GABAB1 and GABAB2 was widely and similarly distributed in the striatum, with immunogold particles localized at both presynaptic and postsynaptic sites. The most commonly labeled structures were dendritic shafts and spines, as well as terminals forming asymmetric and symmetric synapses. In postsynaptic structures, the majority of labeling associated with the plasma membrane was localized at extrasynaptic sites, although immunogold particles were also found at the postsynaptic specialization of some symmetric, putative GABAergic synapses. Labeling in axon terminals was located within, or at the edge of, the presynaptic active zone, as well as at extrasynaptic sites. Double labeling for GABAB receptor subunits and vesicular glutamate transporters revealed that labeling for both GABAB1 and GABAB2 was localized on glutamatergic axon terminals that expressed either vesicular glutamate transporter 1 or vesicular glutamate transporter 2. The patterns of innervation of striatal neurons by the vesicular glutamate transporter 1- and vesicular glutamate transporter 2-positive terminals suggest that they are selective markers of corticostriatal and thalamostriatal afferents, respectively. These results thus provide evidence that presynaptic GABAB heteroreceptors are in a position to modulate the two major excitatory inputs to striatal spiny projection neurons arising in the cortex and thalamus. In addition, presynaptic GABAB autoreceptors are present on the terminals of spiny projection neurons and/or striatal GABAergic interneurons. Furthermore, the data indicate that GABA may also affect the excitability of striatal neurons via postsynaptic GABAB receptors. [Copyright &y& Elsevier]

Abstract: The electrical stimulation of a specific hypothalamic area rapidly evokes attacks in rats. Noteworthy, attack-related hypothalamic structures were identified in all species studied so far. The area has been extensively mapped in rats, and its anatomical connections have been studied in detail. However, technical difficulties precluded earlier the precise identification of the neural elements mediating the aggressive effects of stimulation. It now appears that a dense and distinct group of glutamatergic cells expressing vesicular glutamate transporter 2 mRNA extends over the entire hypothalamic attack area. Rostral parts overwhelmingly contained glutamatergic neurons. In more caudal parts, glutamatergic and fewer GABAergic neurons were found. The remarkable similarity in the distribution of hypothalamic attack area and glutamatergic cell groups suggests that these cells mediate the aggressive effects of stimulation. Surprisingly, thyrotropin releasing hormone mRNA was co-localized in a subset of glutamatergic neurons. Such neurons were present at all rostro-caudal levels of the hypothalamic attack area, except for that part of the hypothalamic attack area extending into the ventro-lateral part of the ventromedial hypothalamic nucleus. Earlier data on the projections of hypothalamic thyrotropin releasing hormone neurons suggest that this subpopulation plays a specific role in attack behavior. Thus, we identified three neuronal phenotypes in the hypothalamic structure that is involved in the induction of attacks: glutamatergic neurons co-expressing thyrotropin releasing hormone, glutamatergic neurons without thyrotropin releasing hormone, and GABAergic neurons dispersed among the glutamatergic cells. Assessing the specific roles and connections of these neuron subpopulations would contribute to our understanding of the mechanisms underlying attack behavior and aggression. [Copyright &y& Elsevier]

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/ synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons. [ABSTRACT FROM AUTHOR]

Abstract: Glutamate plays a role in the central regulation of the hypothalamic–pituitary–adrenal (HPA) and thyroid (HPT) axes. Until the recent discovery of vesicular glutamate transporters (VGLUT1–3), there was no specific tool for the examination of the putative morphological relationship between the glutamatergic and the hypophysiotropic systems. Using antisera against VGLUT2, corticotropin-releasing hormone (CRH), and prothyrotropin-releasing hormone (proTRH) (178–199), we performed double-labeling immunocytochemistry at light and electron microscopic levels in order to study the glutamatergic innervation of the CRH- and TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN). Fine VGLUT2-immunoreactive (IR) axons very densely innervated the parvocellular subdivisions of the PVN. VGLUT2-IR axons established juxtapositions with all parvocellular CRH- and TRH-synthesizing neurons. The innervation was similarly intense in all parvocellular subdivisions of the PVN. At ultrastructural level, VGLUT2-IR terminals frequently established synapses with perikarya and dendrites of the CRH- and proTRH-IR neurons. These findings demonstrate that glutamatergic neurons directly innervate hypophysiotropic CRH and TRH neurons in the PVN and, therefore, support the hypothesis that the glutamate-induced activation of the HPA and HPT axes may be accomplished by a direct action of glutamate on hypophysiotropic CRH and TRH systems. [Copyright &y& Elsevier]

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus and may be involved in local reflex control. We recently detected extensive though not complete colocalization of the vesicular glutamate transporter 2 (VGLUT2) with markers for IGLEs. To elucidate this colocalization mismatch, this study aimed at identifying markers for nitrergic, cholinergic, peptidergic, and adrenergic neurons and glial cells, which may colocalize with VGLUT2 outside of IGLEs. Confocal imaging revealed, besides substantial colocalization of VGLUT2 and substance P (SP), no other significant colocalizations of VGLUT2 and immunoreactivity for any of these markers within the same varicosities. However, we found close contacts of VGLUT2-positive structures to vesicular acetylcholine transporter, choline acetyltransferase, neuronal nitric oxide synthase, galanin, neuropeptide Y, and vasoactive intestinal peptide immunoreactive cell bodies and varicosities, as well as to glial cells. Neuronal perikarya were never positive for VGLUT2. Thus, VGLUT2 was almost exclusively found in IGLEs and may serve as a specific marker for them. In addition, many IGLEs also contained SP. The close contacts established by IGLEs to myenteric cell bodies, dendrites, and varicose fibers suggest that IGLEs modulate various types of enteric neurons and vice versa. [ABSTRACT FROM AUTHOR]

Glutamate, the principal excitatory neurotransmitter in the spinal cord, acts primarily through AMPA receptors. Although all four AMPA subunits are expressed by spinal neurons, we know little about their distribution at glutamatergic synapses. We used an antigen-unmasking technique to reveal the synaptic distribution of glutamate receptor (GluR) 1-4 subunits with confocal microscopy. After pepsin treatment, punctate staining was seen with antibodies against each subunit: GluR2-immunoreactive puncta were distributed throughout the gray matter, whereas GluR1-immunoreactive puncta were restricted to the dorsal horn and were most numerous in laminas I-II. Punctate staining for GluR3 and GluR4 was found in all laminas but was weak in superficial dorsal horn. Colocalization studies showed that GluR2 was present at virtually all (98%) puncta that were GluR1, GluR3, or GluR4 immunoreactive and that most (>90%) immunoreactive puncta in laminas IV, V, and IX showed GluR2, GluR3, and GluR4 immunoreactivity. Evidence that these puncta represented synaptic receptors was obtained with electron microscopy and by examining the association of GluR2- and GluR 1-immunoreactive puncta with glutamatergic boutons (identified with vesicular glutamate transporters or markers for unmyelinated afferents). The great majority (96%) of these boutons were associated with GluR2-immunoreactive puncta. Our findings suggest that GluR2 is almost universally present at AMPA-containing synapses, whereas GluR1 is preferentially associated with primary afferent terminals. We also found a substantial, rapid increase in staining for synaptic GluR1 subunits phosphorylated on the $845 residue in the ipsilateral dorsal horn after peripheral noxious stimulation. This finding demonstrates plastic changes, presumably contributing to central sensitization, at the synaptic level. [ABSTRACT FROM AUTHOR]

Glutamate, the major excitatory transmitter in the central nervous system, has been speculated for years to influence mammalian motor endplates but trials to identify glutamatergic motor terminals failed because specific markers were not available. Recently, antibodies to vesicular glutamate transporters (VGLUTs) opened new possibilities for further morphological investigations. We detected VGLUT1 immunoreactivity (-ir), but not VGLUT2-ir and VGLUT3-ir, respectively, in many motor nerve terminals in motor endplates of the mouse esophagus as identified by α-bungarotoxin or colocalization of VGLUT1 with choline acetyltransferase. These findings suggest that glutamate is co-stored with acetylcholine in esophageal neuromuscular junctions. Surprisingly, we found neither VGLUT1-ir nor VGLUT2-ir or VGLUT3-ir in neuromuscular junctions of somitic and branchiogenic skeletal muscles. This may reflect differences in functional properties and the embryonic origin between skeletal and esophageal striated muscle fibers. [Copyright &y& Elsevier]

Previously we reported that glutamate and neuronal nitric oxide synthase (nNOS) colocalize in neurons of the nucleus tractus solitarii (NTS). That finding provided anatomical support for the suggestion that nitric oxide and glutamate interact in cardiovascular regulation by the NTS. Here we test the hypothesis that nNOS colocalizes with vesicular glutamate transporters (VGluT1 and VGluT2) in the NTS. Immunoreactivity (IR) for VGluT better identifies glutamatergic terminals than does glutamate-IR, which may label metabolic as well as transmitter stores of the amino acid. We used fluorescent immunohistochemistry combined with confocal laser scanning microscopy to study IR for VGluT1, VGluT2 and nNOS in rat NTS. A high density of VGluT1-IR positive fibers was present in the gracilis and cuneatus nuclei while in the NTS we found a moderate density in the lateral and interstitial subnuclei and a low density in the dorsolateral, ventral and intermediate subnuclei. The medial, central, commissural and gelatinosus subnuclei contained few VGluT1-IR containing fibers. Thus, VGluT1 containing fibers are not prominent in portions of the NTS where cardiovascular afferent fibers terminate. In contrast, we found a high density of VGluT2-IR containing fibers in the gelatinosus subnucleus and subpostremal area and a moderate density in cardiovascular regions such as the dorsolateral and medial subnuclei as well as in the central and lateral subnuclei. We found a low density in the ventral, intermediate, interstitial and commissural subnuclei. VGluT1-IR and VGluT2-IR rarely colocalized in fibers within the NTS. VGluT1-IR did not colocalize with nNOS, but VGluT2-IR and nNOS-IR colocalized in fibers in all NTS subnuclei. When compared with the other NTS subnuclei, the dorsolateral, gelatinosus and subpostremal subnuclei had higher frequencies of colocalization of VGluT2-IR and nNOS-IR. VGluT2-IR positive fibers were also apposed to nNOS-IR positive fibers throughout the NTS. These data support our hypothesis and confirm that glutamatergic fibers in the NTS contain nNOS. [Copyright &y& Elsevier]

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)-α and ER-β. Evidence suggests that oestrogen-receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin-releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor-expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter-2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERα mRNA-(35S-labelling) and VGLUT2 mRNA-(digoxigenin-labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERα mRNA are consistent with previous data in the literature. Dual-labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 ± 3.4% of the ERα mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERα expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen-receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones. [ABSTRACT FROM AUTHOR]

Parasympathetic preganglionic neurons of the superior salivatory nucleus (SSN), which projects to the pterygopalatine ganglion (PPG), modulate salivation, lacrimation, and cerebrovascular tone. Our previous studies suggest that excitatory projections from the nucleus tractus solitarii modulate cerebrovascular tone by actions on SSN neurons. In this study we sought to test the hypothesis that N-methyl-d-aspartate (NMDA) type glutamate receptors and vesicular glutamate transporters (VGLUT) are present in the SSN and that SSN neurons receive glutamatergic input. In six rats we injected tetramethylrhodamine dextran (TRD), a fluorescent tracer, unilaterally into the PPG to label SSN neurons. Four days later, rats were perfused and brain stem sections containing the SSN were processed for fluorescent immunohistochemistry for N-methyl-d-aspartate receptor subunit 1 (NMDAR1) and vesicular glutamate transporters (VGLUT1 and VGLUT2). Confocal laser scanning microscopy showed that 88±3% of TRD-labeled SSN neurons contained NMDAR1-immunoreactivity (IR). The surrounding neuropil contained numerous fibers labeled for VGLUT2-IR, but not VGLUT1-IR. Double fluorescent immunohistochemistry for NMDAR1 and VGLUT2 revealed that fibers containing VGLUT2-IR were often in close proximity to cell bodies or proximal dendrites of TRD-labeled SSN neurons that were positive for NMDAR1-IR. These studies support our hypothesis that NMDA receptors and VGLUT are present in the SSN. They further provide support for the suggestion that there are glutamatergic inputs to SSN neurons and would be consistent with an excitatory input that could regulate cerebrovascular tone. [Copyright &y& Elsevier]

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh, and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite its evident importance to learning theory and models, the manner in which the lateral perforant path (LPP) transforms signals from entorhinal cortex to hippocampus is not well understood. The present studies measured synaptic responses in the dentate gyrus (DG) of adult mouse hippocampal slices during different patterns of LPP stimulation. Theta (5 Hz) stimulation produced a modest within-train facilitation that was markedly enhanced at the level of DG output. Gamma (50 Hz) activation resulted in a singular pattern with initial synaptic facilitation being followed by a progressively greater depression. DG output was absent after only two pulses. Reducing release probability with low extracellular calcium instated frequency facilitation to gamma stimulation while long-term potentiation, which increases release by LPP terminals, enhanced within-train depression. Relatedly, per terminal concentrations of VGLUT2, a vesicular glutamate transporter associated with high release probability, were much greater in the LPP than in CA3-CA1 connections. Attempts to circumvent the potent gamma filter using a series of short (three-pulse) 50 Hz trains spaced by 200 ms were only partially successful: composite responses were substantially reduced after the first burst, an effect opposite to that recorded in field CA1. The interaction between bursts was surprisingly persistent (>1.0 s). Low calcium improved throughput during theta/gamma activation but buffering of postsynaptic calcium did not. In all, presynaptic specializations relating to release probability produce an unusual but potent type of frequency filtering in the LPP. Patterned burst input engages a different type of filter with substrates that are also likely to be located presynaptically. KEY POINTS: The lateral perforant path (LPP)-dentate gyrus (DG) synapse operates as a low-pass filter, where responses to a train of 50 Hz, γ frequency activation are greatly suppressed. Activation with brief bursts of γ frequency information engages a secondary filter that persists for prolonged periods (lasting seconds). Both forms of LPP frequency filtering are influenced by presynaptic, as opposed to postsynaptic, processes; this contrasts with other hippocampal synapses. LPP frequency filtering is modified by the unique presynaptic long-term potentiation at this synapse. Computational simulations indicate that presynaptic factors associated with release probability and vesicle recycling may underlie the potent LPP-DG frequency filtering., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Surfactant protein C (SP-C) modulates cerebrospinal fluid (CSF) rheology. During ageing, its declining levels are accompanied by an increased burden of white matter lesions. Pulmonary SP-C intermediates harbouring the BRICHOS-domain prevent protein misfolding in the lungs. Thus, cerebral SP-C intermediates may counteract cerebral β-amyloidosis, a hallmark of Alzheimer's disease (AD). However, data on the molecular neuroanatomy of SP-C and its alterations in wildtype and triple transgenic (3xTg) mice, featuring essential elements of AD-neuropathology, are lacking. Therefore, this study investigated SP-C-containing structures in murine forebrains and their spatial relationships with vascular, glial and neuronal components of the neurovascular unit. Fluorescence labelling demonstrated neuronal SP-C in the medial habenula, the indusium griseum and the hippocampus. Glial counterstaining elucidated astrocytes in the corpus callosum co-expressing SP-C and S100β. Notably, perineuronal nets were associated with SP-C in the nucleus reticularis thalami, the lateral hypothalamus and the retrosplenial cortex. In the hippocampus of aged 3xTg mice, an increased number of dot-like depositions containing SP-C and Reelin, but devoid of BRICHOS-immunoreactivity were observed apart from AD-like lesions. Wildtype and 3xTg mice revealed an age-dependent increase of such deposits markedly pronounced in about 24-month-old 3xTg mice. SP-C levels of the intracellular and extracellular compartments in each group revealed an inverse correlation of SP-C and Reelin, with reduced SP-C and increased Reelin in an age-dependent fashion especially in 3xTg mice. Taken together, extracellular SP-C, as modulator of glymphatic clearance and potential ligand of PNs, declines in 3xTg mice, which show an accumulation of extracellular Reelin depositions during ageing. • Surfactant protein-C (SP-C) in mouse forebrain is of neuronal and astroglial origin. • The majority of cerebral surfactant protein-C is located in the brain interstitium. • SP-C-immunoreactivity is spatially linked to lectin-stained perineuronal nets. • Changes of SP-C and Reelin levels are age-dependent and inversely correlated. • Different amount of hippocampal SP-C deposits in wildtype and 3xTg mice. [ABSTRACT FROM AUTHOR]

The ventral tegmental area (VTA) is well known for regulating reward consumption, learning, memory, and addiction behaviors through mediating dopamine (DA) release in downstream regions. Other than DA neurons, the VTA is known to be heterogeneous and contains other types of neurons, including glutamate neurons. In contrast to the well-studied and established functions of DA neurons, the role of VTA glutamate neurons is understudied, presumably due to their relatively small quantity and a lack of effective means to study them. Yet, emerging studies have begun to reveal the importance of glutamate release from VTA neurons in regulating diverse behavioral repertoire through a complex intra-VTA and long-range neuronal network. In this review, we summarize the features of VTA glutamate neurons from three perspectives, namely, cellular properties, neural connections, and behavioral functions. Delineation of VTA glutamatergic pathways and their interactions with VTA DA neurons in regulating behaviors may reveal previously unappreciated functions of the VTA in other physiological processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cai and Tong.)

Glutamate is packaged in vesicles via two main vesicular transporter (VGLUT) proteins, VGLUT1 and VGLUT2, which regulate its storage and release from synapses of excitatory neurons. Studies in rodents, primates, ferrets, and tree shrews suggest that these transporters may identify distinct subsets of excitatory projections in visual structures, particularly in thalamocortical pathways where they tend to correlate with modulatory and driver projections, respectively. Despite being a well-studied model of thalamocortical connectivity, little is known about their expression pattern in the cat visual system. To expand current knowledge on their distribution and how they correlated with known driver and modulator projecting sites, we examined the protein expression patterns of VGLUT1 and VGLUT2 in the visual thalamus of the cat (lateral geniculate nucleus and the pulvinar complex). We also studied their expression pattern in relevant visual structures projecting to or receiving significant thalamic projections, such as the primary visual cortex and the superior colliculus. Our results indicate that both VGLUTs are consistently present throughout the cat visual system and show laminar or nuclei specificity in their distribution, which suggests, as in other species, that VGLUT1 and VGLUT2 represent distinct populations of glutamatergic projections., (© 2021 Wiley Periodicals LLC.)

Morphine is widely used in the treatment of moderate to severe pain. Long-term use of morphine leads to various adverse effects, such as tolerance and hyperalgesia. Vesicular glutamate transporter 2 (VGluT2) accumulates glutamate into synaptic vesicles and plays multiple roles in the central nervous system. However, the specific role of VGluT2 in morphine tolerance has not been fully elucidated. Here, we investigated the regulatory role of VGluT2 in morphine tolerance and assessed the potential role of the brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrkB) pathway in VGluT2 mediated morphine antinociceptive tolerance in mice. In the present study, we found that VGluT2 is upregulated in the spinal cord after the development of morphine tolerance. Furthermore, inhibition of VGluT2 with its antagonist (Chicago sky blue 6 B, CSB6B) or knockdown of VGluT2 by lentivirus restored the analgesic effect of morphine, suppressed the activation of astrocytes and microglia, and decreased glial-derived pro-inflammatory cytokines. Overexpression of VGluT2 by lentivirus facilitated morphine tolerance and mechanical hyperalgesia. In addition, we found the expression of BDNF is correlated with VGluT2 expression in the spinal cord after chronic morphine administration. Intrathecal injection of the BDNF/TrkB pathway antagonist K252a attenuated the development of morphine tolerance and decreased the expression of VGluT2 in the spinal cord, which suggested the BDNF/TrkB pathway participates in the regulation of VGluT2 in morphine tolerance. This study elucidates the functional capability of VGluT2 in modulating morphine tolerance and identifies a novel mechanism and promising therapeutic target for morphine tolerance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 He, Xu, Zhang, Ding, Guo, Zou and Wang.)

• Four subtypes of GABAergic cells can be distinguished in the mouse inferior colliculus. • This may indicate conservation of these GABAergic subtypes across rodent species. • Cholinergic boutons form close associations with GABAergic and glutamatergic neurons in the inferior colliculus. • All four subtypes of GABAergic cells have close associations with cholinergic boutons. • This indicates widespread effects of acetylcholine on excitatory and inhibitory cells across the mouse inferior colliculus. Acetylcholine (ACh) is a neuromodulator that has been implicated in multiple roles across the brain, including the central auditory system, where it sets neuronal excitability and gain and affects plasticity. In the cerebral cortex, subtypes of GABAergic interneurons are modulated by ACh in a subtype-specific manner. Subtypes of GABAergic neurons have also begun to be described in the inferior colliculus (IC), a midbrain hub of the auditory system. Here, we used male and female mice (Mus musculus) that express fluorescent protein in cholinergic cells, axons, and boutons to look at the association between ACh and four subtypes of GABAergic IC cells that differ in their associations with extracellular markers, their soma sizes, and their distribution within the IC. We found that most IC cells, including excitatory and inhibitory cells, have cholinergic boutons closely associated with their somas and proximal dendrites. We also found that similar proportions of each of four subtypes of GABAergic cells are closely associated with cholinergic boutons. Whether the different types of GABAergic cells in the IC are differentially regulated remains unclear, as the response of cells to ACh is dependent on which types of ACh receptors are present. Additionally, this study confirms the presence of these four subtypes of GABAergic cells in the mouse IC, as they had previously been identified only in guinea pigs. These results suggest that cholinergic projections to the IC modulate auditory processing via direct effects on a multitude of inhibitory circuits. [ABSTRACT FROM AUTHOR]