Vesicular glutamate transporter 1 immunoreactivity in extrinsic and intrinsic innervation of the rat esophagus

Encouraged by the recent finding of vesicular glutamate transporter 2 (VGLUT2) immunoreactivity (-ir) in intraganglionic laminar endings (IGLEs) of the rat esophagus, we investigated also the distribution and co-localization patterns of VGLUT1. Confocal imaging revealed substantial co-localization of VGLUT1-ir with selective markers of IGLEs, i.e., calretinin and VGLUT2, indicating that IGLEs contain both VGLUT1 and VGLUT2 within their synaptic vesicles. Besides IGLEs, we found VGLUT1-ir in both cholinergic and nitrergic myenteric neuronal cell bodies, in fibers of the muscularis mucosae, and in esophageal motor endplates. Skeletal neuromuscular junctions, in contrast, showed no VGLUT1-ir. We also tested for probable co-localization of VGLUT1-ir with markers of extrinsic and intrinsic esophageal innervation and glia. Within the myenteric neuropil we found, besides co-localization of VGLUT1 and substance P, no further co-localization of VGLUT1-ir with any of these markers. In the muscularis mucosae some VGLUT1-ir fibers were shown to contain neuronal nitric oxide synthase (nNOS)-ir. VGLUT1-ir in esophageal motor endplates was partly co-localized with vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT)-ir, but VGLUT1-ir was also demonstrated in separately terminating fibers at motor endplates co-localized neither with ChAT/VAChT-ir nor with nNOS-ir, suggesting a hitherto unknown glutamatergic enteric co-innervation. Thus, VGLUT1-ir was found in extrinsic as well as intrinsic innervation of the rat esophagus.     

Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells

The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing (n = 3), eye enucleation (n = 3), and untreated (n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells.     

Vesicular neurotransmitter transporter: bioenergetics and regulation of glutamate transport

Glutamate plays essential roles in chemical transmission as a major excitatory neurotransmitter. The accumulation of glutamate in secretory vesicles is mediated by vesicular glutamate transporters (VGLUTs) that together with the driving electrochemical gradient of proteins influence the subsequent quantum release of glutamate and the function of higher-order neurons. The vesicular content of glutamate is well correlated with membrane potential (Δψ), which suggests that Δψ determines the vesicular glutamate concentration. The transport of glutamate into secretory vesicles is highly dependent on Cl(-). This anion stimulates glutamate transport but is inhibitory at higher concentrations. Accumulating evidence indicates that Cl(-) regulates glutamate transport through control of VGLUT activity and the H(+) electrochemical gradient. Recently, a comprehensive study demonstrated that Cl(-) regulation of VGLUT is competitively inhibited by metabolic intermediates such as ketone bodies. It also showed that ketone bodies are effective in controlling epilepsy. These results suggest a correlation between metabolic state and higher-order brain function. We propose a novel function for Cl(-) as a fundamental regulator for signal transmission.     

Methyl-beta-cyclodextrin influences glutamate transport in the rat brain nerve terminals by depletion of membrane cholesterol

Role of membrane cholesterol in direct and reversed function of Na+ -dependent glutamate transporters and exocytosis was investigated. The depletion of membrane cholesterol by methyl-beta-cyclodextrin (MebetaCD) resulted in a dose-dependent significant reduction of the L-[14C]glutamate uptake by synaptosomes. Treatment of synaptosomes with 15 mM MebetaCD caused a decrease in the velocity of L-[14C]glutamate uptake by 49 +/- 4% (P < or = 0.05). The depolarization stimulated Ca2+ -dependent glutamate release that occurred via reverse functioning of glutamate transporters decreased insignificantly for 1 min from 8.0 +/- 0.4% to 6.7 +/- 0.4% of total accumulated synaptosomal label after MebetaCD treatment. The depletion of membrane cholesterol resulted in a reduction of the depolarization evoked exocytotic release from 8.0 +/- 1.0% to 4.2 +/- 1.0% of total synaptosomal label. Thus, cholesterol depletion was found to decrease significantly the Na+ -dependent uptake and exocytotic release of glutamate.     

Modulating effect of glutamate transporter inhibitors on accumulation and release of the neuromediator by the brain nerve terminals in rats

L-[14C]glutamate uptake and release processes in nerve terminals has been investigated using the nontransportable and transportable competitive inhibitors of glutamate transport as tools. The effects of DL-threo-beta-benzyloxyaspartate (DL-TBOA) and DL-threo-beta-hydroxyaspartate (DL-THA) on the accumulation of L-[14C]glutamate have been evaluated after the exposure of rats to centrifuge-induced hypergravity. Both analogs potently inhibited the L-[14C]glutamate uptake in a dose-dependent manner (100 microM glutamate, 30 s incubation period). The IC50 values for DL-TBOA calculated on the basis of curves of non-linear regression kinetic analysis was 18 +/- 2 microM and 11 +/- 2 microM (p < or = 0.05) before and after the exposure to artificial gravity, respectively. L-THA, transportable analog, exhibited similar inhibitory characteristics (18 +/- 2 and 12 +/- 2 microM, respectively). We have also demonstrated that DL-TBOA exerted slighter effect on depolarization-evoked carrier-mediated L-[14C]glutamate release in control rats in comparison with gravity-loaded ones. Thus, DL-TBOA had complex effect on glutamatergic transmission, inhibited uptake and release of L-glutamate, and perhaps, became more potent under centrifuge-induced hypergravity.     

Topographical organization of TRPV1-immunoreactive epithelium and CGRP-immunoreactive nerve terminals in rodent tongue

Transient receptor potential vanilloid subfamily member 1 (TRPV1) is activated by capsaicin, acid, and heat and mediates pain through peripheral nerves. In the tongue, TRPV1 expression has been reported also in the epithelium. This indicates a possibility that sensation is first received by the epithelium. However, how nerves receive sensations from the epithelium remains unclear. To clarify the anatomical basis of this interaction, we performed immunohistochemical studies in the rodent tongue to detect TRPV1 and calcitonin gene-related peptide (CGRP), a neural marker. Strong expression of TRPV1 in the epithelium was observed and was restricted to the apex of the tongue. Double immunohistochemical staining revealed that CGRP-expressing nerve terminals were in close apposition to the strongly TRPV1-expressing epithelium of fungiform papilla in the apex of rodent tongues. These results suggest that the TRPV1-expressing epithelium monitors the oral environment and acquired information may then be conducted to the adjacent CGRP-expressing terminals.     

Presynaptic mechanisms underlying the alpha-lipoic acid facilitation of glutamate exocytosis in rat cerebral cortex nerve terminals

The antioxidant alpha-lipoic acid has been reported to prevent and reverse age-related impairments in learning and memory. However, it is unclear how alpha-lipoic acid improves cognitive function. In this study, the effect of alpha-lipoic acid on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes) was examined. We found that alpha-lipoic acid potently facilitated 4-aminopyridine (4AP)-evoked glutamate release, and this release facilitation results from an enhancement of vesicular exocytosis and not from an increase of non-vesicular release. Examination of the effect of alpha-lipoic acid on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release was associated with an increase in voltage-dependent Ca(2+) influx. Consistent with this, alpha-lipoic acid-mediated facilitation of glutamate release was completely prevented in synaptosomes pretreated with a wide spectrum blocker of the N- and P/Q-type Ca(2+) channels, omega-conotoxin MVIIC. The facilitatory effect of alpha-lipoic acid on Ca(2+) influx was not due to an increase of synaptosomal excitability because alpha-lipoic acid did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. In addition, both ionomycin and hypertonic sucrose-induced glutamate release were enhanced by alpha-lipoic acid. Furthermore, disruption of cytoskeleton organization with cytochalasin D occluded the facilitatory effect of alpha-lipoic acid on 4AP or ionomycin-evoked glutamate release. These results suggest that the antioxidant alpha-lipoic acid enhances the Ca(2+) entry through presynaptic N- and P/Q-type Ca(2+) channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes. Also, activation of PKA and PKC may underlie, at least in part, the alpha-lipoic acid-mediated facilitation of glutamate release observed here as alpha-lipoic acid-enhanced 4AP and ionomycin-evoked glutamate release were significantly attenuated by PKA and PKC inhibitors. This finding may provide some information regarding the mechanism of action of alpha-lipoic acid in the central nervous system (CNS).     

Fangchinoline inhibits glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

Fangchinoline, an active component of radix stephaniae tetrandrinea, has been shown to possess neuroprotective properties. It has been reported that excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. The primary purpose of the present study was to investigate the effect of fangchinoline on glutamate release in rat cerebral cortex nerve terminals and to explore the possible mechanism. Fangchinoline inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) in a concentration-dependent manner, and this phenomenon resulted from a reduction of vesicular exocytosis but not from an inhibition of Ca²⁺-independent efflux via glutamate transporter. Fangchinoline did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. Fangchinoline-mediated inhibition of glutamate release was significantly prevented by the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, and by the PKC inhibitors, GF109203X and Ro318220. In addition, the glutamate release mediated by direct Ca²⁺ entry with Ca²⁺ ionophore (ionomycin) was unaffected by fangchinoline, which suggests that the inhibitory effect of fangchinoline is not due to directly interfering with the release process at some point subsequent to Ca²⁺ influx. These results suggest that fangchinoline inhibits glutamate release from the rat cortical synaptosomes through the suppression of voltage-dependent Ca²⁺ channel activity and subsequent reduces Ca²⁺ entry into nerve terminals, rather than any upstream effect on nerve terminal excitability. This inhibition appears to involve the suppression of PKC signal transduction pathway. This finding may explain the neuroprotective effects of fangchinoline against neurotoxicity.     

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X₇ receptors, it is still unclear whether neuronal functions can be attributed to P2X₇ receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X₇ receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of 'nude' release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X₇ receptors. Together, our findings suggest that (i) P2X₇ receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X₇ receptors are localized on glutamatergic nerve terminals; (iii) P2X₇ receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca²⁺-dependent vesicular release; and (iv) the P2X₇ receptor itself constitutes a significant Ca²⁺-independent mode of exit for glutamate.     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Carbonic anhydrase in the carotid body and the carotid sinus nerve

It is well known that carbonic anhydrase plays an important role in the physiological responses of carotid-body chemoreceptors to hypercapnia. Nevertheless the precise location of the enzyme within the carotid body has been a matter of controversy for many years. Using the Hansson method we found histochemical evidence that this enzyme is localized in type I cells. Type II cells and nerve terminals did not show enzymatic activity. These results allow us to define the carotid body as a secondary receptor in the context of the "acidic hypothesis" of transduction in the carotid body.     

Quantitative studies of rat carotid body type I cell nerve endings

The results of a stereological and morphometric analysis of rat carotid body type I cell nerve endings are described. 66.9% of endings possessed symmetrical junctions. Of the remaining endings, 3.6% were presynaptic and 26% were postsynaptic to type I cells; 3.6% of endings had a reciprocal configuration. Apart from membrane specialisations, no other ultrastructural criteria were found to distinguish the different types of endings. Ventilation with 100% and 10% oxygen showed that the hypoxic mixture reduced synaptic vesicle concentration in the nerve endings; this effect was independent of the innervation to the carotid body.     

Carbonic anhydrase in the carotid body and the carotid sinus nerve

Summary It is well known that carbonic anhydrase plays an important role in the physiological responses of carotidbody chemoreceptors to hypercapnia. Nevertheless the precise location of the enzyme within the carotid body has been a matter of controversy for many years. Using the Hansson method we found histochemical evidence that this enzyme is localized in type I cells. Type II cells and nerve terminals did not show enzymatic activity. These results allow us to define the carotid body as a secondary receptor in the context of the acidic hypothesis of transduction in the carotid body.     

Vesicular monoamine transporter 2 mediates fear behavior in mice

A subset of people exposed to a traumatic event develops post‐traumatic stress disorder (PTSD), which is associated with dysregulated fear behavior. Genetic variation in SLC18A2, the gene that encodes vesicular monoamine transporter 2 (VMAT2), has been reported to affect risk for the development of PTSD in humans. Here, we use transgenic mice that express either 5% (VMAT2‐LO mice) or 200% (VMAT2‐HI mice) of wild‐type levels of VMAT2 protein. We report that VMAT2‐LO mice have reduced VMAT2 protein in the hippocampus and amygdala, impaired monoaminergic vesicular storage capacity in both the striatum and frontal cortex, decreased monoamine metabolite abundance and a greatly reduced capacity to release dopamine upon stimulation. Furthermore, VMAT2‐LO mice showed exaggerated cued and contextual fear expression, altered fear habituation, inability to discriminate threat from safety cues, altered startle response compared with wild‐type mice and an anxiogenic‐like phenotype, but displayed no deficits in social function. By contrast, VMAT2‐HI mice exhibited increased VMAT2 protein throughout the brain, higher vesicular storage capacity and greater dopamine release upon stimulation compared with wild‐type controls. Behaviorally, VMAT2‐HI mice were similar to wild‐type mice in most assays, with some evidence of a reduced anxiety‐like responses. Together, these data show that presynaptic monoamine function mediates PTSD‐like outcomes in our mouse model, and suggest a causal link between reduced VMAT2 expression and fear behavior, consistent with the correlational relationship between VMAT2 genotype and PTSD risk in humans. Targeting this system is a potential strategy for the development of pharmacotherapies for disorders like PTSD.     

Corticotropin-releasing factor (CRF)-immunoreactive neurons in the mammillary body of the rat

Summary The presence and distribution of CRF-immunoreactive cells and nerve fibers were studied in the mammillary body of the rat, 12 days after placing various types of lesions within the hypothalamus. Anterior and anteriolateral cuts, placed in the midhypothalamus immediately behind the paraventricular nuclei resulted in an almost complete disappearance of CRF-immunoreactive fibers from the median eminence and simultaneous appearance of CRF-containing neurons in the mammillary body. Posterior or postero-lateral hypothalamic cuts carried out in front of the mammillary body caused the accumulation of CRF-immunoreactive material in neurons and neural processes located behind the cut-line. This type of intervention had no effect on the quantity of CRF fibers in the median eminence. A cut running through the central part of the mammillary body in the frontal plane resulted in appearance of CRF neurons only in the posterior half of the mammillary region. Placing a cut behind and over the mammillary body, CRF-immunoreactive neurons became detectable below the superior cut-line. No immunoreactive neurons were observed in the mammillary body when the frontal cut reached the base of the brain at the posterior border of the nucleus, leaving intact its anterior and superior connections. In all these cases when the mammillo-thalamic tract was transected, CRF neurons became detectable in the mammillary body.