Intraganglionic laminar endings and their relationships with neuronal and glial structures of myenteric ganglia in the esophagus of rat and mouse

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

Vesicular glutamate transporter 2 immunoreactivity in putative vagal mechanosensor terminals of mouse and rat esophagus: indication of a local effector function?

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent structures throughout the gastrointestinal tract. Previous ultrastructural investigations have revealed synaptic contacts of IGLEs on myenteric neurons. Thus, in addtion to functioning probably as mechanosensors, IGLEs may also synaptically influence myenteric neurons. In search of clues for potential transmitters in IGLEs, we investigated, by combined neuronal tracing and immunocytochemistry in the esophagus, the correlation between IGLEs and vesicular glutamate transporter 2 (VGLUT2), which is considered a reliable marker for glutamatergic neurons. In rat esophagus, IGLEs were immunostained with calretinin. In the mouse, anterograde wheat germ agglutinin/horseradish peroxidase (WGA-HRP) tracing from nodose ganglion was used in order to label esophageal IGLEs. Confocal laser scanning microscopy demonstrated that VGLUT2 immunoreactivity was highly colocalized with synaptophysin and that both calretinin and tyramide amplified WGA-HRP in rat and mouse esophagus, respectively. No colocalization was found with calcitonin gene-related peptide, a marker for spinal primary afferents. Thus, VGLUT2 is found in vagal afferent endings in the esophagus, suggesting that glutamate is contained in, and probably released from, synaptic vesicles previously described in IGLEs. Functional evidence pending, this finding is in favor of a local effector function of IGLEs onto myenteric neurons.     

Number and distribution of intraganglionic laminar endings in the mouse esophagus as demonstrated with two different immunohistochemical markers.

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus. Two specific markers for IGLEs were recently described in mouse: the purinergic P2 x 2 receptor and the vesicular glutamate transporter 2 (VGLUT2). This study aimed at comparing both markers with respect to their suitability for quantitative analysis. We counted IGLEs immunostained for VGLUT2 and P2 x 2, respectively, and mapped their distribution in esophageal wholemounts of C57Bl/6 mice. Numbers and distribution of IGLEs were compared with those of myenteric ganglia as demonstrated by cuprolinic blue histochemistry. Whereas the distribution of VGLUT2-immunopositive IGLEs closely matched that of myenteric ganglia, P2 x 2-immunopositive IGLEs were rarely found in upper and middle esophagus but increasingly in its lower parts. P2 x 2 stained only half the number of IGLEs found with VGLUT2 immunostaining. We also investigated the correlation between anterograde tracing and immunohistochemistry for identifying IGLEs. Confocal microscopy revealed colocalization of all three markers in approximately 50% of IGLEs. The remaining IGLEs showed only tracer and VGLUT2 labeling but no P2 x 2 immunoreactivity. Thus, VGLUT2 and P2 x 2 represent two specific markers for qualitative demonstration of esophageal IGLEs. However, VGLUT2 may be superior to P2 x 2 as a quantitative marker for IGLEs in the esophagus of C57Bl/6 mice.     

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.     

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system. This study was supported by the Johannes und Frieda Marohn-Stiftung, Erlangen.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

Inputs from intrinsic primary afferent neurons to nitric oxide synthase-immunoreactive neurons in the myenteric plexus of guinea pig ileum

Nitric oxide synthase (NOS) immunoreactivity occurs in two groups of neurons in the guinea pig small intestine: descending interneurons that are also immunoreactive for choline acetyltransferase (ChAT), and inhibitory motor neurons that lack ChAT immunoreactivity. Interneurons that are involved in local reflexes would be expected to have inputs from intrinsic primary afferent (sensory) neurons, most of which are calbindin-immunoreactive. We examined this possibility using triple staining for NOS, ChAT and calbindin immunoreactivity and investigated the relationships between calbindin-immunoreactive varicosities and the cell bodies of NOS-immunoreactive neurons, using high-resolution confocal microscopy and electron microscopy. By confocal microscopy, we found that the cell bodies of ChAT/NOS interneurons received 84 +/- 23 (mean +/- SD) direct appositions from calbindin-immunoreactive varicosities and that the cell bodies of NOS-inhibitory motor neurons received 82 +/- 20 appositions. Electron-microscopic examination of the relations of 265-calbindin-immunoreactive varicosities, at distances within the resolution of the confocal microscope (300 nm), to 30 NOS-immunoreactive nerve cells indicated that 84% formed close contacts or synapses and 16% were separated from neurons by thin glial cell processes. Thus, each NOS-immunoreactive nerve cell receives about 70 synaptic inputs or close contacts from the calbindin-immunoreactive varicosities of intrinsic primary afferent neurons. It is concluded that there are monosynaptic reflex connections in which intrinsic primary afferent neurons synapse directly with motor neurons and di- or poly-synaptic reflexes in which ChAT- and NOS-immunoreactive neurons are interneurons, interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons.     

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters

Studies of synapsin-deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild-type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double-labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.     

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.     

Adrenergic differentiation and SSR2a receptor expression in CAD-cells cultured in serum-free medium

The CAD cell line originates from catecholaminergic neurons in the central nervous system (CNS) of a simian virus large T antigen transgenic mouse. In the present study, we have immunohistochemically characterized the cell line after differentiation in serum-free medium, using immunofluorescence in combination with confocal laser scanning microscopy (CLSM), immunoblot, and ribonuclease protection assay (RPA). Tyrosine hydroxylase (TH)-, phenylethanolamine-N-methyltransferase (PNMT)-, neuropeptide Y (NPY)-, vesicular monoamine transporter subtype 2-, vasoactive intestinal peptide (VIP)-, somatostatin (SS)-, synaptophysin-, synaptic vesicle protein 2 (SV2)-, and growth-associated protein of 43 (GAP-43)-immunoreactivities (IRs) were present in the cells but not choline acetyltransferase and vesicular acetylcholine transporter. The immunoreactive substances were present in cell bodies in serum-containing medium (SCM), but after serum withdrawal (protein-free medium, PFM) these proteins and peptides were partially shifted into the long process and their varicosities. A few cells cultured in PFM were occasionally found with extremely high TH-immunoreactivity (IR) in cell bodies and processes. Growth-associated protein of 43-immunoreactivity was weak in SCM but was up-regulated (verified with immunoblot) in PFM and concentrated in varicosities along the processes and the distal tips of neurites. The somatostatin receptor subtype 2a (SSR(2(a))) was found in the cytoplasm and the plasma membrane of the CAD-cells. After serum deprivation, all three methods showed that SSR(2(a)) was up-regulated in the cells. Thus, the CAD cell line after differentiation may be suitable for studying dynamics of SSR(2(a)).     

Molecular cloning and functional characterization of human vesicular glutamate transporter 3

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.     

β-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons

β-Amyloid (Aβ) peptides may cause malfunction and death of neurons in Alzheimer’s disease. We investigated the effect of Aβ on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with Aβ(25-35) at 3 and 10 μM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal–glial cell cultures (from P1 rats), Aβ reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), Aβ(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that Aβ may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake. Special issue article in honor of Dr. Frode Fonnum.     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

Developmental pattern of three vesicular glutamate transporters in the myenteric plexus of the human fetal small intestine

Three vesicular glutamate transporters (VGLUT1-3) have previously been identified in the central nervous system, where they define the glutamatergic phenotype, and their expression is tightly regulated during brain development. In the present study we applied immunocytochemistry to examine the distribution of the immunoreactivity of all three VGLUTs during prenatal development of the myenteric plexus in the human small intestine. We also investigated changes in their localization in the different segments of the small intestine and in the different compartments of the developing myenteric ganglia. Immunoreactivity against all three VGLUTs was found predominantly in the ganglionic neuropil, interganglionic varicose fibers and perisomatic puncta, but cytoplasmic labeling with different intensities also occurred. Each transporter displayed a characteristic spatiotemporal expression pattern, with the transient increase or decrease of immunoreactive cell bodies, varicosities or perisomatic puncta, depending on the fetal age, the gut segment or the ganglionic compartment. Throughout gestational weeks 14-23, VGLUT1 immunoreactivity always predominated over VGLUT2 immunoreactivity, though both peaked around week 20. VGLUT3 immunoreactivity was less abundant in the developing myenteric plexus than those of VGLUT1 and VGLUT2 immunoreactivity. It was mainly expressed in the ganglionic neuropil and in the perisomatic puncta throughout the examined gestational period. Neuronal perikarya immunoreactive for VGLUT3 were restricted to between weeks 18 and 20 of gestation and exclusively to the oral part of the small intestine.     

Distribution of P2X<Subscript>3</Subscript> receptor immunoreactivity in myenteric ganglia of the mouse esophagus

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent terminals throughout the gut. Electrophysiological experiments revealed a modulatory role of ATP in the IGLE-mechanotransduction process and the P2X₂-receptor has been described in IGLEs of mouse, rat and guinea pig. Another purinoceptor, the P2X₃-receptor, was found in IGLEs of the rat esophagus. These findings prompted us to investigate occurrence and distribution of the P2X₃-receptor in the mouse esophagus. Using multichannel immunofluorescence and confocal microscopy, P2X₃-immunoreactivity (-iry) was found colocalized with the vesicular glutamate transporter 2 (VGLUT2), a specific marker for IGLEs, on average in three-fourths of esophageal IGLEs. The distribution of P2X₃ immunoreactive (-ir) IGLEs was similar to that of P2X₂-iry and showed increasing numbers towards the abdominal esophagus. P2X₃/P2X₂-colocalization within IGLEs suggested the occurrence of heteromeric P2X_2/3 receptors. In contrast to the rat, where only a few P2X₃-ir perikarya were described, P2X₃ stained perikarya in ~80% of myenteric ganglia in the mouse. Detailed analysis revealed P2X₃-iry in subpopulations of nitrergic (nNOS) and cholinergic (ChAT) myenteric neurons and ganglionic neuropil of the mouse esophagus. We conclude that ATP might act as a neuromodulator in IGLEs via a (P2X₂)-P2X₃ receptor-mediated pathway especially in the abdominal portion of the mouse esophagus.     

Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.     

The expression of vesicular glutamate transporters defines two classes of excitatory synapse

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.     

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.     

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l-glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l-glutamate in synaptic-like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l-glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT-PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co-localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l-glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.     

Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine

Antibodies against choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) were used to determine whether neurons that have previously been identified as intrinsic primary afferent neurons in the guinea-pig small intestine have a cholinergic phenotype. Cell bodies of primary afferent neurons in the myenteric plexus were identified by their calbindin immunoreactivity and those in the submucous plexus by immunoreactivity for substance P. High proportions of both were immunoreactive for ChAT, viz. 98% of myenteric calbindin neurons and 99% of submucosal substance P neurons. ChAT immunoreactivity also occurred in all nerve cell bodies immunoreactive for calretinin and substance P in the myenteric plexus, but in only 16% of nerve cells immunoreactive for nitric oxide synthase. VAChT immunoreactivity was in the majority of calbindin-immunoreactive varicosities in the myenteric ganglia, submucous ganglia and mucosa and also in the majority of the varicosities of neurons that were immunoreactive for calretinin and somatostatin and that had been previously established as being cholinergic. We conclude that the intrinsic primary afferent neurons are cholinergic and that they may release transmitter from their sensory endings in the mucosa.