Noise Exposure Alters Glutamatergic and GABAergic Synaptic Connectivity in the Hippocampus and Its Relevance to Tinnitus.

Accumulating evidence implicates a role for brain structures outside the ascending auditory pathway in tinnitus, the phantom perception of sound. In addition to other factors such as age-dependent hearing loss, high-level sound exposure is a prominent cause of tinnitus. Here, we examined how noise exposure altered the distribution of excitatory and inhibitory synaptic inputs in the guinea pig hippocampus and determined whether these changes were associated with tinnitus. In experiment one, guinea pigs were overexposed to unilateral narrow-band noise (98 dB SPL, 2 h). Two weeks later, the density of excitatory (VGLUT-1/2) and inhibitory (VGAT) synaptic terminals in CA1, CA3, and dentate gyrus hippocampal subregions was assessed by immunohistochemistry. Overall, VGLUT-1 density primarily increased, while VGAT density decreased significantly in many regions. Then, to assess whether the noise-induced alterations were persistent and related to tinnitus, experiment two utilized a noise-exposure paradigm shown to induce tinnitus and assessed tinnitus development which was assessed using gap-prepulse inhibition of the acoustic startle (GPIAS). Twelve weeks after sound overexposure, changes in excitatory synaptic terminal density had largely recovered regardless of tinnitus status, but the recovery of GABAergic terminal density was dramatically different in animals expressing tinnitus relative to animals resistant to tinnitus. In resistant animals, inhibitory synapse density recovered to preexposure levels, but in animals expressing tinnitus, inhibitory synapse density remained chronically diminished. Taken together, our results suggest that noise exposure induces striking changes in the balance of excitatory and inhibitory synaptic inputs throughout the hippocampus and reveal a potential role for rebounding inhibition in the hippocampus as a protective factor leading to tinnitus resilience.

Functional glutamate transporters are expressed in the carotid chemoreceptor.

BACKGROUND: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression. METHODS: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed. RESULTS: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB. CONCLUSIONS: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.

Neurochemical differences between target-specific populations of rat dorsal raphe projection neurons.

Serotonin (5-HT)-containing neurons in the dorsal raphe (DR) nucleus project throughout the forebrain and are implicated in many physiological processes and neuropsychiatric disorders. Diversity among these neurons has been characterized in terms of their neurochemistry and anatomical organization, but a clear sense of whether these attributes align with specific brain functions or terminal fields is lacking. DR 5-HT neurons can co-express additional neuroactive substances, increasing the potential for individualized regulation of target circuits. The goal of this study was to link DR neurons to a specific functional role by characterizing cells according to both their neurotransmitter expression and efferent connectivity; specifically, cells projecting to the medial prefrontal cortex (mPFC), a region implicated in cognition, emotion, and responses to stress. Following retrograde tracer injection, brainstem sections from Sprague-Dawley rats were immunohistochemically stained for markers of serotonin, glutamate, GABA, and nitric oxide (NO). 98% of the mPFC-projecting serotonergic neurons co-expressed the marker for glutamate, while the markers for NO and GABA were observed in 60% and less than 1% of those neurons, respectively. To identify potential target-specific differences in co-transmitter expression, we also characterized DR neurons projecting to a visual sensory structure, the lateral geniculate nucleus (LGN). The proportion of serotonergic neurons co-expressing NO was greater amongst cells targeting the mPFC vs LGN (60% vs 22%). The established role of 5-HT in affective disorders and the emerging role of NO in stress signaling suggest that the impact of 5-HT/NO co-localization in DR neurons that regulate mPFC circuit function may be clinically relevant.

Vesicular Glutamate Transporter Inhibitors: Structurally Modified Brilliant Yellow Analogs.

Glutamate uptake into synaptic vesicles in nerve terminals is a pivotal step in glutamate synaptic transmission. Glutamate is the major excitatory neurotransmitter and, as such, the vesicular glutamate transporter (VGLUT) responsible for this uptake is involved in a variety of nervous system functions and various types of pathophysiology. As yet, no VGLUT-specific, membrane-permeable agents have been developed to affect neuronal function in intact neurons, although two potent VGLUTspecific inhibitors are known. These compounds contain diazo and highly charged sulfonic acid groups, rendering them membrane-impermeable and potentially cytotoxic. In an effort to eliminate these undesirable properties, we have developed two novel agents, Brilliant Yellow analogs 1 and 2, which are free of these two groups. We show here that these agents retain highly VGLUT-selective inhibitory activity, despite their reduction in potency, and exhibit no significant cellular toxicity. Potential use of this molecular modification is discussed.

Functional Characterization of a Vesicular Glutamate Transporter in an Interneuron That Makes Excitatory and Inhibitory Synaptic Connections in a Molluscan Neural Circuit.

Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but was not directly confirmed. Here we study a suspected glutamatergic retraction interneuron, B64. We used the representational difference analysis (RDA) method to successfully clone an Aplysia vesicular glutamate transporter (ApVGLUT) from B64 and from a glutamatergic motor neuron B38. Previously, RDA was used to characterize novel neuropeptides. Here we demonstrate its utility for characterizing other types of molecules. Bioinformatics suggests that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and Caenorhabditis elegans VGLUTs. We expressed ApVGLUT in a cell line, and demonstrated that it indeed transports glutamate in an ATP and proton gradient-dependent manner. We mapped the ApVGLUT distribution in the CNS using in situ hybridization and immunocytochemistry. Further, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Although glutamate is primarily an excitatory transmitter in the mammalian CNS, B64 elicits inhibitory PSPs in protraction neurons to terminate protraction and excitatory PSPs in retraction neurons to maintain retraction. Pharmacological data indicated that both types of PSPs are mediated by glutamate. Thus, glutamate mediates the dual function of B64 in Aplysia. More generally, our systematic approaches based on RDA may facilitate analyses of transmitter actions in small circuits with identifiable neurons.

Expression of vesicular glutamate transporter 3 mRNA in the brain and retina of the pigeon.

Vesicular glutamate transporters (vGluTs), which accumulate glutamate into synaptic vesicles, are classified into three subtypes in mammalian brains: vGluT1, vGluT2, and vGluT3. VGluT3 is localized in non-glutamatergic neurons of the brain and retinal amacrine cells. In birds, the vGluT3 genome is found, but its distribution in the brain or retina is unknown. The present study was conducted to analyze vGluT3 cDNA sequence and elucidate its distribution in the pigeon brain and retina. The vGluT3 cDNA comprises 1761bp and showed 95% and 88% identity to the chicken and zebra finch vGluT3 cDNAs, respectively, and 74% identity to human vGluT3 cDNA. In situ hybridization revealed that the vGluT3 mRNA was expressed in neurons of the caudal linear nucleus (LC) of the brain and in amacrine cells of the inner nuclear layer of the retina. A combination of in situ hybridization and serotonin immunohistochemistry revealed three types of stained cells in LC and retina: vGluT3(+)/serotonin(+), vGluT3(+)/serotonin(-), and vGluT3(-)/serotonin(+). The vGluT3(+)/serotonin(+) cells were approximately 22% in LC and 16% in the retina. The present results suggest that the pigeon vGluT3 mRNA is comparable with the mammalian type.

Mechanisms of glutamate transport.

L-Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and plays important roles in a wide variety of brain functions, but it is also a key player in the pathogenesis of many neurological disorders. The control of glutamate concentrations is critical to the normal functioning of the central nervous system, and in this review we discuss how glutamate transporters regulate glutamate concentrations to maintain dynamic signaling mechanisms between neurons. In 2004, the crystal structure of a prokaryotic homolog of the mammalian glutamate transporter family of proteins was crystallized and its structure determined. This has paved the way for a better understanding of the structural basis for glutamate transporter function. In this review we provide a broad perspective of this field of research, but focus primarily on the more recent studies with a particular emphasis on how our understanding of the structure of glutamate transporters has generated new insights.

Transcript expression of vesicular glutamate transporters in lumbar dorsal root ganglia and the spinal cord of mice - effects of peripheral axotomy or hindpaw inflammation.

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.

VGLUT3-immunoreactive afferents of the lateral septum: ultrastructural evidence for a modulatory role of glutamate.

Through its extensive connections with various brain regions, the lateral septum (LS) participates in the processing of cognitive, emotional and autonomic information. It is decisively involved in the generation of behavioral responses according to environmental demands. Modulatory afferents reaching the LS from the brain stem (e.g. dopaminergic, serotonergic) play a role in the adjustment of these behavioral responses. Recently, a population of vesicular glutamate transporter 3-immunoreactive (VGLUT3-ir) fibers forming prominent pericellular basket-like structures (PBLS) was described in the rat LS. These VGLUT3-ir PBLS are distributed in a layer-like pattern, which is very typical for modulatory afferents of the LS. There is meanwhile broad evidence that glutamate can act as a modulatory or co-transmitter and that those neurons, which make use of this transmission mode, primarily express VGLUT3. Thus, the VGLUT3-ir fibers within the LS could also display features typical for non-canonical glutamatergic transmission. Employing pre-embedding electron microscopy for VGLUT3 in rats, we show now that the VGLUT3-ir fibers outlining LS neurons represent axonal terminals, which primarily form symmetric synapses with somata and proximal dendrites of their target neurons. Occasionally, we also found VGLUT3-ir terminals that make canonical asymmetric synapses on distal dendrites and spines. Thus, VGLUT3-ir boutons in the LS form two different, disproportionate, populations of synaptic contacts with their target neurons. The larger one of them is indicative of employing glutamate as a modulatory transmitter.

Vesicular glutamate transporters in axons that innervate the human dental pulp.

INTRODUCTION: Vesicular glutamate transporters (VGLUTs) are involved in the transport of transmitter glutamate into synaptic vesicles and are used as markers for glutamatergic neurons. METHODS: To assess which types of VGLUTs are involved in the glutamate signaling in pulpal axons and to investigate their distribution, we performed light microscopic immunohistochemistry by using antibodies against VGLUT1, VGLUT2, calcitonin gene-related peptide, and Western blot analysis in human dental pulp. RESULTS: VGLUT1 was expressed in a large number of pulpal axons, especially in the peripheral pulp where the axons branch extensively. The VGLUT1 immunopositive axons showed bead-like appearance, and the majority of these also expressed calcitonin gene-related peptide. VGLUT2 was expressed in few axons throughout the pulp. CONCLUSIONS: Our findings suggest that VGLUT1 is involved mainly in the glutamate-mediated signaling of pain, primarily at the level of the peripheral pulp.

Developmental expression of Synaptotagmin isoforms in single calyx of Held-generating neurons.

The large glutamatergic calyx of Held synapse in the auditory brainstem has become a powerful model for studying transmitter release mechanisms, but the molecular bases of presynaptic function at this synapse are not well known. Here, we have used single-cell quantitative PCR (qPCR) to study the developmental expression of all major Synaptotagmin (Syt) isoforms in putative calyx of Held-generating neurons (globular bushy cells) of the ventral cochlear nucleus. Using electrophysiological criteria and the expression of marker genes including VGluTs (vesicular glutamate transporters), Ca(2+) binding proteins, and the transcription factor Math5, we identified a subset of the recorded neurons as putative calyx of Held-generating bushy cells. At postnatal days 12-15 these neurons expressed Syt-2 and Syt-11, and also Syt-3, -4, -7 and -13 at lower levels, whereas Syt-1 and -9 were absent. Interestingly, early in development (at P3-P6), immature bushy cells expressed a larger number of Syt-isoforms, with Syt-1, Syt-5, Syt-9 and Syt-13 detected in a significantly higher percentage of neurons. Our study sheds light on the molecular properties of putative calyx of Held-generating neurons and shows the developmental regulation of the Syt-isoform expression profile in a single neuron type.

Nonserotonergic projection neurons in the midbrain raphe nuclei contain the vesicular glutamate transporter VGLUT3.

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network.

Glutamatergic afferents of the ventral tegmental area in the rat.

Glutamatergic inputs to the ventral tegmental area (VTA), thought crucial to the capacity of the VTA to detect and signal stimulus salience, have been reported to arise in but a few structures. However, the afferent system of the VTA comprises very abundant neurons within a large formation extending from the prefrontal cortex to the caudal brainstem. Neurons in nearly all parts of this continuum may be glutamatergic and equivalently important to VTA function. Thus, we sought to identify the full range of glutamatergic inputs to the VTA by combining retrograde transport of wheat germ agglutinin-bound gold after injections into the VTA with nonisotopic in situ hybridization of the vesicular glutamate transporters (VGLUTs) 1, 2, and 3. We found glutamatergic neurons innervating the VTA in almost all structures projecting there and that a majority of these are subcortical and VGLUT2 mRNA positive. The tremendous convergence of glutamatergic afferents from many brain areas in the VTA suggests that (1) the function of the VTA requires integration of manifold and diverse bits of information and (2) the activity of the VTA reflects the ongoing activities of various combinations of its afferents.