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• VGLUT1 and VGLUT2 are generally preserved in AD across the medial temporal lobe. • We report a lower density of VGLUT1 in the dentate gyrus stratum moleculare in AD. • VGLUT2 expression is lower in the AD subiculum and superior temporal gyrus. • VGLUT1 and VGLUT2 expression show a brain region-specific vulnerability in AD. Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets. [ABSTRACT FROM AUTHOR]

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene cause CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental syndrome where patients exhibit early-onset seizures, intellectual disability, stereotypies, limited or absent speech, autism-like symptoms and sensory impairments. Mounting evidences indicate that disrupted sensory perception and processing represent core signs also in mouse models of CDD; however we have very limited knowledge on their underlying causes. In this study, we investigated how CDKL5 deficiency affects synaptic organization and experience-dependent plasticity in the thalamo-cortical (TC) pathway carrying whisker-related tactile information to the barrel cortex (BC). By using synapse-specific antibodies and confocal microscopy, we found that Cdkl5-KO mice display a lower density of TC synapses in the BC that was paralleled by a reduction of cortico-cortical (CC) connections compared to wild-type mice. These synaptic defects were accompanied by reduced BC activation, as shown by a robust decrease of c-fos immunostaining, and atypical behavioral responses to whisker-mediated tactile stimulation. Notably, a 2-day paradigm of enriched whisker stimulation rescued both number and configuration of excitatory synapses in Cdkl5-KO mice, restored cortical activity and normalized behavioral responses to wild-type mice levels. Our findings disclose a novel and unsuspected role of CDKL5 in controlling the organization and experience-induced modifications of excitatory connections in the BC and indicate how mutations of CDKL5 produce failures in higher-order processing of somatosensory stimuli. This article is part of a Special Issue entitled: Animal Models of Neurodevelopmental Disorders. Unlabelled Image • CDKL5 regulates the organization of synaptic connectivity in the barrel cortex. • The barrel cortex of Cdkl5 mutants is less activated. • Cdkl5-KO mice show atypical whisker-mediated behavioral responses. • Experience-dependent structural plasticity is not prevented by loss of CDKL5. • Enhanced sensory stimulation rescues cortical and behavioral defects of Cdkl5-KOs. [ABSTRACT FROM AUTHOR]

• Cleavage of SNAP-25 reduced VGluT2 expression in rats with neuropathic pain. • Overexpression of SNAP-25 increased VGluT2 expression and induced allodynia in rats. • BoNT/A inhibited astrocyte activation, upregulation of VGluT2 abolished this effect. • PKA/p-CREB pathway was involved in SNAP-25-mediated regulation of neuropathic pain. Synaptosomal-associated protein 25 (SNAP-25) plays an important role in neuropathic pain. However, the underlying mechanism is largely unknown. Vesicular glutamate transporter 2 (VGluT2) is an isoform of vesicular glutamate transporters that controls the storage and release of glutamate. In the present study, we found the expression levels of VGluT2 correlated with the upregulation of SNAP-25 in the spinal cord of rats following chronic constriction injury (CCI)-induced neuropathic pain. Cleavage of SNAP-25 by Botulinum toxin A (BoNT/A) attenuated mechanical allodynia, downregulated the expression of VGluT2 and reduced glutamate release. Overexpression of VGluT2 abolished the antinociceptive effect of BoNT/A. Upregulation of SNAP-25 in naive rats increased VGluT2 expression and induced pain-responsive behaviors. In pheochromocytoma (PC12) cells, the expression of VGluT2 was also depended on SNAP-25 dysregulation. Moreover, we found VGluT2 was involved in SNAP-25-mediated regulation of astrocyte expression and activation of the PKA/p-CREB pathway mediated the upregulation of SNAP-25 in neuropathic pain. The findings of our study indicate that VGluT2 contributes to the effect of SNAP-25 in maintaining the development of neuropathic pain and suggests a novel mechanism underlying SNAP-25 regulation of neuropathic pain. [ABSTRACT FROM AUTHOR]

Highlights • VGLUT2 puncta density is upregulated in the cochlear nucleus ipsilateral to acoustic trauma. • VGLUT1 puncta density is downregulated in the cochlear nucleus contralateral to acoustic trauma. • Tinnitus does not correlate with measures of cochlear dysfunction or histopathology. • VGLUT asymmetries are abolished upon auditory–somatosensory bimodal stimulation treatment that reversed behavioral tinnitus. • Tinnitus-associated glutamatergic redistribution is likely the result of maladaptive somatosensory compensation. Abstract Tinnitus alters auditory–somatosensory plasticity in the cochlear nucleus (CN). Correspondingly, bimodal auditory–somatosensory stimulation treatment attenuates tinnitus, both in animals and humans (Marks et al., 2018). Therefore, we hypothesized that tinnitus is associated with altered somatosensory innervation of the CN. Here, we studied the expression of vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) in the CN, which reveals glutamatergic projections from the cochlea as well as somatosensory systems to this brainstem auditory center. Guinea pigs were unilaterally exposed to narrowband noise and behaviorally tested for tinnitus using gap-prepulse inhibition of the acoustic startle. Following physiological and behavioral measures, brain sections were immunohistochemically stained for VGLUT1 or VGLUT2. Puncta density was determined for each region of the ipsilateral and contralateral CN. Tinnitus was associated with an ipsilateral upregulation of VGLUT2 puncta density in the granule cell domain (GCD) and anteroventral CN (AVCN). Furthermore, there was a tinnitus-associated interaural asymmetry for VGLUT1 expression in the AVCN and deep layer of the dorsal CN (DCN3), due to contralateral downregulation of VGLUT1 expression. These tinnitus-related glutamatergic imbalances were reversed upon bimodal stimulation treatment. Tinnitus-associated ipsilateral upregulation of VGLUT2-positive projections likely derives from somatosensory projections to the GCD and AVCN. This upregulation may underlie the neurophysiological hallmarks of tinnitus in the CN. Reversing the increased ipsilateral glutamatergic innervation in the CN is likely a key mechanism in treating tinnitus. [ABSTRACT FROM AUTHOR]

Despite recent progress on neural pathways underlying individual behaviors, how an animal balances and prioritizes behavioral outputs remains poorly understood. While studying the relationship between hunger-induced feeding and pup-induced maternal behaviors in virgin female mice, we made the unexpected discovery that presence of pups strongly delayed and decreased food consumption. Strikingly, presence of pups also suppressed feeding induced by optogenetic activation of Agrp neurons. Such a suppressive effect inversely correlated with the extents of maternal behaviors, but did not rely on the display of these behaviors, and was also present in virgin males. Furthermore, chemogenetic activation of Vglut2+ neurons in the medial preoptic area (mPOA), a region critical for maternal behaviors and motivation, was sufficient to suppress hunger-induced feeding. However, muscimol inhibition of the mPOA, while disrupting maternal behaviors, did not prevent pup suppression of feeding, indicating that neural pathways in other brain regions may also mediate such an effect. Together, these results provide novel insights into neural coordination of pup care and feeding in mice and organizations of animal behaviors in general. [ABSTRACT FROM AUTHOR]

The subiculum is the output component of the hippocampal formation and holds a key position in the neural circuitry of memory. Previous studies have demonstrated the subiculum’s connectivity to other brain areas in detail; however, little is known regarding its internal structure. We investigated the cytoarchitecture of the temporal and mid-septotemporal parts of the subiculum using immunohistochemistry. The border between the CA1 region and subiculum was determined by both cytoarchitecture and zinc transporter 3 (ZnT3)-immunoreactivity (IR), whereas the border between the subiculum and presubiculum (PreS) was partially indicated by glutamate receptor 1 (GluR1)-IR. The subiculum was divided into proximal and distal subfields based on cytoarchitecture and immunohistochemistry for calbindin (CB), nitric oxide synthase (NOS) and Purkinje cell protein 4 (PCP4). The proximal subiculum (defined here as subiculum 2) was composed of five layers: the molecular layer (layer 1), the medium-sized pyramidal cell layer (layer 2) that contained NOS- and PCP4-positive neurons, the large pyramidal cell layer (layer 3) characterized by the accumulation of ZnT3- (more proximally) and vesicular glutamate transporter 2-positive (more distally) boutons, layer 4 containing polymorphic cells, and the deepest layer 5 composed of PCP4-positive cells with long apical dendrites that reached layer 1. The distal subiculum (subiculum 1) consisting of smaller neurons did not show these features. Quantitative analyses of the size and numerical density of somata substantiated this delineation. Both the proximal–distal division and five-layered structure in the subiculum 2 were confirmed throughout the temporal two-thirds of the subiculum. These findings will provide a new structural basis for hippocampal investigations. [ABSTRACT FROM AUTHOR]

The tectofugal pathway in birds consists of four stations, the retina, optic tectum, rotundal nucleus, and entopallium, and it conveys visual information via three ascending pathways. These pathways consist of retino-tectal, tecto-rotundal and rotundo-entopallial cells, all of which are glutamatergic. The present study examined the localization of ionotropic glutamate receptors (iGluRs) to identify the target areas of glutamatergic projections in the tectofugal pathway in pigeons. Nine subunits of iGluRs were analyzed using in situ hybridization as follows: AMPA receptors (GluA1, GluA2, GluA3, and GluA4), kainate receptors (GluK1, GluK2, and GluK4), and NMDA receptors (GluN1 and GluN2A). Hybridization signals of subunits showed various intensities in different cells. In the optic tectum, a strong to moderate expression was observed in layer 10 (GluA2, GluA3, GluK4, and GluN1) and layer 13 (GluA2, GluK4, GluN1, and GluN2A). The rotundal nucleus intensely expressed GluA3, GluA4, GluK1, and GluK4. In the entopallium, an intense to moderate expression of GluK1 and GluK4, and a moderate to weak expression of AMPA and NMDA receptors were observed. Furthermore, the parvocellular and magnocellular parts of the isthmic nuclei showed a strong expression of GluA2, GluA3, GluK4, and GluN1. The present findings demonstrate the expression of iGluRs in glutamatergic projection targets of the tectofugal pathway in birds and suggest a diversity of iGluRs in the transmission of visual information. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) is a serine–threonine protein kinase that controls protein synthesis in the nervous system. Here, we characterized the role of protein synthesis regulation due to mTOR signaling in rat dorsal root ganglion (DRG) following plantar incision. The number of phosphorylated mTOR (p-mTOR)-positive neurons was increased 2–4 days after the incision. Rapamycin inhibited p-mTOR expression in the DRG and thermal hypersensitivity 3 days but not 1 day after the incision. Vesicular glutamate transporter 2 (VGLUT2) expression was increased after the plantar incision, which was inhibited by rapamycin. These results demonstrated that tissue injury induces phosphorylation of mTOR and increased protein level of VGLUT2 in the DRG neurons. mTOR phosphorylation involves in maintenance of injury-induced thermal hypersensitivity. [ABSTRACT FROM AUTHOR]

Midbrain dopamine systems play important roles in Parkinson’s disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular diversity and participation in circuits involved in addiction, we hypothesize that individual VGluT2 subpopulations of neurons play unique roles in addiction and other disorders. [ABSTRACT FROM AUTHOR]

Abstract: Cardiorespiratory control neurons in the brainstem nucleus tractus solitarius (NTS) undergo dramatic expansion of dendritic arbors during the early postnatal period, when functional remodeling takes place within the NTS circuitry. However, the underlying molecular mechanisms of morphological maturation of NTS neurons are largely unknown. Our previous studies point to the neurotrophin brain-derived neurotrophic factor (BDNF), which is abundantly expressed by NTS-projecting primary sensory neurons, as a candidate mediator of NTS dendritogenesis. In the current study, we used neonatal rat NTS neurons in vitro to examine the role of BDNF in the dendritic development of neurochemically identified subpopulations of NTS neurons. In the presence of abundant glia, BDNF promoted NTS dendritic outgrowth and complexity, with the magnitude of the BDNF effect dependent on neuronal phenotype. Surprisingly, BDNF switched from promoting to inhibiting NTS dendritogenesis upon glia depletion. Moreover, glia depletion alone led to a significant increase in NTS dendritic outgrowth. Consistent with this result, astrocyte-conditioned medium (ACM), which promoted hippocampal dendritogenesis, inhibited dendritic growth of NTS neurons. The latter effect was abolished by heat-inactivation of ACM, pointing to a diffusible astrocyte-derived negative regulator of NTS dendritic growth. Together, these data demonstrate a role for BDNF in the postnatal development of NTS neurons, and reveal novel effects of glia on this process. Moreover, previously documented dramatic increases in NTS glial proliferation in victims of sudden infant death syndrome (SIDS) underscore the importance of our findings and the need to better understand the role of glia and their interactions with BDNF during NTS circuit maturation. Furthermore, while it has previously been demonstrated that the specific effects of BDNF on dendritic growth are context-dependent, the role of glia in this process is unknown. Thus, our data carry important implications for mechanisms of dendritogenesis likely beyond the NTS. [Copyright &y& Elsevier]

Abstract: Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2–4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18–28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. [ABSTRACT FROM AUTHOR]

Abstract: Ethanol exposure during postnatal days (PN) 4–6 in rats alters cerebellar development resulting in significant loss of Purkinje cells. There is little knowledge, however, on what happens to the neurons that survive. In this study, rat pups were treated with a daily dose of ethanol (either 3.6 or 4.5 g/kg body weight) delivered by intragastric intubation on PN4, PN4–6, or PN7–9. Then the interactions between climbing fibers and Purkinje cells were examined on PN14 using confocal microscopy. Mid-vermal cerebellar sections were stained with antibodies to calbindin-D28k (to visualize Purkinje cells) and vesicular glutamate transporter 2 (VGluT2, to visualize climbing fibers). Confocal z-stack images were obtained from Lobule 1 and analyzed with Imaris software to quantify the staining of the two antibodies. The VGluT2 immunostaining was significantly reduced in the PN4 and PN4–6 ethanol groups for the 4.5 g/kg dose level, compared to controls, indicating that the cerebellar circuitry was significantly altered following developmental ethanol exposure. Not only were there fewer Purkinje cells following ethanol exposure, but the surviving neurons had significantly fewer VGluT2-labeled synapses. These alterations in the synaptic integrity were both dose dependent and temporally dependent. [ABSTRACT FROM AUTHOR]

Abstract: Lamina I of the spinal cord contains many projection neurons that express the neurokinin 1 receptor (NK1r). It has been reported that these cells can undergo long-term potentiation (LTP), which may result from insertion of AMPA-type glutamate receptors (AMPArs) containing GluA1 or GluA4 subunits. We therefore investigated synaptic AMPAr expression on these cells with immunocytochemistry following antigen-retrieval. We also examined their density of glutamatergic input (by analysing AMPAr synaptic puncta and contacts from glutamatergic boutons), and phosphorylation of extracellular signal-regulated kinases (pERKs) following noxious stimulation. Our results indicate that there are two populations of NK1r-expressing projection neurons: large GluA4+/GluA1− cells with a high density of glutamatergic input and small GluA1+/GluA4− cells with a much lower input density. Results from pERK experiments suggested that the two groups may not differ in the types of noxious stimulus that activate them. Glutamatergic synapses on distal dendrites of the large cells were significantly longer than those on proximal dendrites, which presumably compensates for the greater attenuation of distally-generated excitatory postsynaptic currents (EPSCs). Both types of cell received contacts from peptidergic primary afferents, however, on the large cells these appeared to constitute over half of the glutamatergic synapses, and were often associated with elongated AMPAr puncta. This suggests that these afferents, which probably contain substance P, provide a powerful, secure synaptic input to large NK1r-expressing projection neurons. These results demonstrate the importance of GluA4-containing AMPArs in nociceptive transmission and raise the possibility that different forms of LTP in lamina I projection neurons may be related to differential expression of GluA1/GluA4. [Copyright &y& Elsevier]

Abstract: To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles. [Copyright &y& Elsevier]

Abstract: Synaptosome-associated protein of 25 kDa (SNAP25) is a component of the fusion complex that mediates synaptic vesicle exocytosis, regulates calcium dynamics and neuronal plasticity. Despite its crucial role in vesicle release, SNAP25 is not distributed homogenously within the brain. It seems to be virtually absent in mature inhibitory terminals and is observed in a subtype of excitatory neurons defined by the expression of vesicular glutamate transporter 1 (VGluT1). Since a complementary distribution of VGluT1 and VGluT2 in excitatory synapses is correlated with different probabilities of release (Pr), we evaluated whether SNAP25 localization is associated with specific synaptic properties. In the cerebellum, climbing fiber (CF) and parallel fiber (PF) inputs, which impinge onto the same Purkinje cell (PC), have very different functional properties. In the cerebellum of adult rats, using confocal and electron microscopy, we observed that VGluT2-positive CFs, characterized by a high Pr, only weakly express SNAP25, while VGluT1-positive PFs that show a low Pr abundantly express SNAP25. Moreover, SNAP25 was less profuse in the VGluT2-positive rosettes of mossy fibers (MFs) and was almost absent in inhibitory terminals. We extended our analysis to the SNAP23 homolog; this is expressed at different levels in both γ-aminobutyric acid-containing terminals (GABAergic) and glutamatergic terminals of the cerebellar cortex. In conclusion, the preferential localization of SNAP25 in specific synaptic boutons suggests a correlation between SNAP25 and the Pr. This evidence supports the hypothesis that SNAP25 has a modulatory role in shaping synaptic responses. [Copyright &y& Elsevier]

Abstract: Mesoventromedial dopamine neurons projecting from the medial ventral tegmental area to the ventromedial shell of the nucleus accumbens play a role in attributing incentive salience to environmental stimuli that predict important events, and appear to be particularly sensitive to the effects of psychostimulant drugs. Despite the observation that these dopamine neurons make up almost the entire complement of neurons in the projection, stimulating their cell bodies evokes a fast glutamatergic response in accumbens neurons. This is apparently due to dopamine neuron glutamate cotransmission, suggested by the extensive coexpression of vesicular glutamate transporter 2 (VGLUT2) in the neurons. To examine the interplay between the dopamine and glutamate signals, we used acute quasi-horizontal brain slices made from DAT-YFP mice in which the intact mesoventromedial projection can be visualized. Under current clamp, when dopamine neurons were stimulated repeatedly, dopamine neuron glutamate transmission showed dopamine-mediated facilitation, solely at higher, burst-firing frequencies. Facilitation was diminished under voltage clamp and flipped to inhibition by intracellular Cs+ or GDPβS, indicating that it was mediated postsynaptically. Postsynaptic facilitation was D1 mediated, required activation of NMDA receptors and closure of voltage gated K+-channels. When postsynaptic facilitation was blocked, D2-mediated presynaptic inhibition became apparent. These counterbalanced pre- and postsynaptic actions determine the frequency dependence of dopamine modulation; at lower firing frequencies dopamine modulation is not apparent, while at burst firing frequency postsynaptic facilitation dominates and dopamine becomes facilitatory. Dopamine neuron glutamate cotransmission may play an important role in encoding the incentive salience value of conditioned stimuli that activate goal-directed behaviors, and may be an important subtract for enduring drug-seeking behaviors. [Copyright &y& Elsevier]

Abstract: Olfactory information is initially processed through intricate synaptic interactions between glutamatergic projection neurons and GABAergic interneurons in the olfactory bulb. Although bulbar neurons and networks have been reported to develop even postnatally, much is yet unknown about the glutamatergic neuron development. To address this issue, we studied the postnatal ontogeny of vesicular glutamate transporters (VGLUT1 and VGLUT2) in the main olfactory bulb of rats, using in situ hybridization, immunohistochemistry, and their combination. In situ hybridization data showed that VGLUT1 mRNA is intensely expressed in differentiating mitral cells and smaller cells of the mitral cell layer (MCL) on postnatal day 1 (P1), and also at lower levels in small- and medium-sized cells, presumably tufted cell populations, of the external plexiform layer (EPL) from P5 onward. VGLUT2 mRNA was expressed in many MCL cell populations on P1, also in small- and medium-sized cells of the EPL at almost the same level as MCL cells between P5 and P7, and became apparently less intense in the MCL than in the EPL from P10 onward. The expression, unlike VGLUT1 mRNA, was also found in small-sized cells of the interglomerular region. In partial agreement with these data, immunohistochemical analyses demonstrated that subsets of mitral and EPL cells are stained for VGLUT1 or VGLUT2, with the former cells coexpressing both subtypes until P5. Moreover, a combined fluorescence in situ hybridization–immunohistochemical dual labeling of the P10 bulb revealed that neither VGLUT1 nor VGLUT2 mRNA is expressed in GABAergic or dopaminergic periglomerular cells, implying their expression in other periglomerular cell subclasses, external tufted cells and/or short-axon cells. Thus, the present study suggests that early in the postnatal development distinct glutamatergic bulbar neurons of rats express spatiotemporally either or both of the two VGLUT subtypes as a specific vesicular transport system, specifically contributing to glutamate-mediated neurobiological events. [Copyright &y& Elsevier]

Abstract: There is increasing evidence that a subset of midbrain dopamine (DA) neurons uses glutamate as a co-transmitter and expresses vesicular glutamate transporter (VGLUT) 2, one of the three vesicular glutamate transporters. In the present study, double in situ hybridization was used to examine tyrosine hydroxylase (TH) and VGLUT2 mRNA expression during the embryonic development of these neurons, and postnatally, in normal rats and rats injected with 6-hydroxydopamine (6-OHDA) at P4 to destroy partially DA neurons. At embryonic days 15 and 16, there was a regional overlap in the labeling of TH and VGLUT2 mRNA in the ventral mesencephalon, which was no longer found at late embryonic stages (E18–E21) and postnatally. In normal pups from P5 to P15, only 1–2% of neurons containing TH mRNA in the ventral tegmental area (VTA) and substantia nigra, pars compacta, also displayed VGLUT2 mRNA. In contrast, after the cerebroventricular administration of 6-OHDA at P4, 26% of surviving DA neurons in the VTA of P15 rats expressed VGLUT2. To search for a colocalization of TH and VGLUT2 protein in axon terminals of these neurons, the nucleus accumbens of normal and 6-OHDA-lesioned P15 rats was examined by electron microscopy after dual immunocytochemical labeling. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in the core of nucleus accumbens. In 6-OHDA-lesioned rats, the total number of TH positive terminals was considerably reduced, and yet the proportion also displaying VGLUT2 immunoreactivity was modestly but significantly increased (37%). These results lead to the suggestion that the glutamatergic phenotype of a VTA DA neurons is highly plastic, repressed toward the end of normal embryonic development, and derepressed postnatally following injury. They also support the hypothesis of co-release of glutamate and DA by mesencephalic neurons in vivo, at least in the developing brain. [Copyright &y& Elsevier]

Abstract: Experiments were done in urethane-anesthetized, barodenervated, male Wistar rats. Chemical stimulation of the hypothalamic paraventricular nucleus (PVN) by unilateral microinjections of N-methyl-d-aspartic acid (NMDA) elicited increases in mean arterial pressure (MAP) and greater splanchnic nerve activity (GSNA). The increases in the MAP and GSNA induced by chemical stimulation of the PVN were significantly exaggerated by bilateral microinjections of d(−)-2-amino-7-phosphono-heptanoic acid (d-AP7) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) (ionotropic glutamate receptor antagonists) into the medial subnucleus of the nucleus tractus solitarius (mNTS). These results were confirmed by single unit recordings; i.e. excitation of mNTS barosensitive neurons caused by chemical stimulation of the ipsilateral PVN was blocked by application of d-AP7 and NBQX to these neurons. Bilateral microinjections of d-AP7 and NBQX into the mNTS elicited pressor responses which were significantly attenuated by inhibition of PVN neurons by bilateral microinjections of muscimol. Unilateral microinjections of fluorogold into the mNTS resulted in bilateral retrograde labeling of the PVN neurons. Unilateral microinjections of biotinylated dextran amine into the PVN resulted in anterograde labeling of axons and terminals in the mNTS bilaterally and the labeled terminals exhibited vesicular glutamate transporter-2 immunoreactivity. These results indicated that 1) a tonically active glutamatergic bilateral projection from the PVN to the mNTS exists; 2) bilateral blockade of ionotropic glutamate receptors in the mNTS exaggerates the increases in MAP and GSNA, but not heart rate, to the chemical stimulation of the PVN; and 3) this projection may serve as a restraint mechanism for excitatory cardiovascular effects of PVN stimulation. [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]

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: 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: 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]

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]

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]

The rat retrotrapezoid nucleus (RTN) contains neurons that have a well-defined phenotype characterized by the presence of vesicular glutamate transporter 2 (VGLUT2) mRNA and a paired-like homeobox 2b (Phox2b)-immunoreactive (ir) nucleus and the absence of tyrosine hydroxylase (TH). These neurons are important to chemoreception. In the present study, we tested the hypothesis that the chemically-coded RTN neurons (ccRTN) (Phox2b(+)/TH(-)) are activated during an acute episode of running exercise. Since most RTN neurons are excited by the activation of perifornical and lateral hypothalamus (PeF/LH), a region that regulates breathing during exercise, we also tested the hypothesis that PeF/LH projections to RTN neurons contribute to their activation during acute exercise. In adult male Wistar rats that underwent an acute episode of treadmill exercise, there was a significant increase in c-Fos immunoreactive (c-Fos-ir) in PeF/LH neurons and RTN neurons that were Phox2b(+)TH(-) (p<0.05) compared to rats that did not exercise. Also the retrograde tracer Fluoro-Gold that was injected into RTN was detected in c-Fos-ir PeF/LH (p<0.05). In summary, the ccRTN neurons (Phox2b(+)TH(-)) are excited by running exercise. Thus, ccRTN neurons may contribute to both the chemical drive to breath and the feed-forward control of breathing associated with exercise., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the striatum, the dendritic tree of the two main populations of projection neurons, called "medium spiny neurons (MSNs)", are covered with spines that receive glutamatergic inputs from the cerebral cortex and thalamus. In Parkinson's disease (PD), striatal MSNs undergo an important loss of dendritic spines, whereas aberrant overgrowth of striatal spines occurs following chronic cocaine exposure. This review examines the possibility that opposite dopamine dysregulation is one of the key factors that underlies these structural changes. In PD, nigrostriatal dopamine degeneration results in a significant loss of dendritic spines in the dorsal striatum, while rodents chronically exposed to cocaine and other psychostimulants, display an increase in the density of "thin and immature" spines in the nucleus accumbens (NAc). In rodent models of PD, there is evidence that D2 dopamine receptor-containing MSNs are preferentially affected, while D1-positive cells are the main targets of increased spine density in models of addiction. However, such specificity remains to be established in primates. Although the link between the extent of striatal spine changes and the behavioral deficits associated with these disorders remains controversial, there is unequivocal evidence that glutamatergic synaptic transmission is significantly altered in both diseased conditions. Recent studies have suggested that opposite calcium-mediated regulation of the transcription factor myocyte enhancer factor 2 (MEF2) function induces these structural defects. In conclusion, there is strong evidence that dopamine is a major, but not the sole, regulator of striatal spine pathology in PD and addiction to psychostimulants. Further studies of the role of glutamate and other genes associated with spine plasticity in mediating these effects are warranted., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

While considerable effort has been made to investigate the neural mechanisms of pain, much less effort has been devoted to itch, at least until recently. However, itch is now gaining increasing recognition as a widespread and costly medical and socioeconomic issue. This is accompanied by increasing interest in the underlying neural mechanisms of itch, which has become a vibrant and rapidly-advancing field of research. The goal of the present forefront review is to describe the recent progress that has been made in our understanding of itch mechanisms., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)