An immunohistochemical and radioimmunological study of the distribution of [met5]- and [leu5]-enkephalin in the gastrointestinal tract.

The identification of opiate-like substances in extracts of the gastrointestinal tract and nervous system of vertebrates suggests that the known endogenous opiate-like peptides [Met⁵]- and [Leu⁵]-enkephalin might have a role in neurotransmission. In this study the gastrointestinal tract of guinea-pigs, rats and hamsters was examined by the immunoperoxidase-bridge method using specific antisera raised against [Met⁵]- and [Leu⁵]-enkephalin. Immunostained nerve fibers were most numerous in Meissner's plexus of the duodenum and in the circular muscle layer of the stomach and rectum of the guinea-pig. Nerve fibers in the guinea-pig esophagus and cardia of the stomach stained with [Met⁵]- but not with [Leu⁵]-enkephalin antiserum. Staining was not observed in any epithelial cells. The regional distribution of these peptides was also examined by radioimmunoassay of extracts of the gut of guinea-pigs and rats. The highest concentrations of [Met⁵]- and [Leu⁵]-enkephalin were found in extracts of guinea-pig duodenum at a ratio of 11:1, respectively.These findings provide evidence for an enkephalinergic nervous system in the gastrointestinal tract.     

Distribution of [Met5]- and [Leu5]-enkephalin-, vasoactive intestinal polypeptide- and substance P-like immunoreactivities in human adrenal glands

There is recent evidence that the amine storing cells of mammalian adrenal medulla also contain bioactive peptides. In the present study we examined human adrenal glands with the immunoperoxidase-bridge method using specific antisera raised against [Met⁵]- and [Leu⁵]-enkephalin, vasoactive intestinal polypeptide hormone (VIP), and substance P. Approximately one-third of the cells in the adrenal medulla demonstrated enkephalin-like immunoreactivity. The intensity of the immunostain varied among individual cells but did not appear to correlate with amine content, as determined by the formaldehyde-induced fluorescence of catecholamines. An abundant network of varicose fibre-like structures and dots, representing preterminal and terminal nerves, demonstrated vasoactive intestinal polypeptide-like immunoreactivity and were found in close proximity to medullary gland cells. Substance P-like immunoreactivity was observed in a few fibres in the medulla and cortex. However, we could not detect cells containing vasoactive intestinal polypeptide- or substance P-like immunoreactivity in adrenal glands. p ]The present findings suggest that human adrenal medullary cells contain both [Met⁵]- and [Leu⁵]-enkephalin and are richly innervated by peptidergic nerves containing vasoactive intestinal polypeptide. These peptides may modulate the release and effects of catecholamines in the adrenal medulla. The nerves with substance P-like immunoreactivity may represent a separate peptidergic neuronal system.     

[Met5]enkephalin-Arg6-Phe7- and [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibres and neurons in the superior cervical ganglion of the rat

[Met⁵]enkephalin-Arg⁶-Phe⁷- (MEAP-) and [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸- (MEAGL-) immunoreactivity was studied by indirect immunohistochemistry in the superior cervical ganglion of the rat with specific antisera produced in rabbits against the corresponding synthetic opioid peptides. Several MEAP- and a few MEAGL-immunoreactive principal nerve cells were observed in the ganglion, while the small intensely fluorescent cells appeared as non-reactive. The superior cervical ganglion also contained dense networks of MEAP- and MEAGL-immunoreactive nerve fibres, which often formed basket-like structures around the principal nerve cells and small intensely fluorescent cells. After ligation of the preganglionic nerve trunk with simultaneous transection of the main postganglionic trunks, a distinct accumulation of both MEAP- and MEAGL- immunoreactivity was observed on both sides of the ligature. Ligation of the preganglionic nerve trunk caused a marked decrease in the number of both MEAP- and MEAGL-immunoreactive nerve fibres in the ganglion. Ligation of the main postganglionic nerve trunks with simultaneous preganglionic nerve division resulted in accumulation of MEAP- and MEAGL-immunoreactive material on the ganglionic side of the ligature in both the external and internal carotid nerve. After division of both the pre- and postganglionic nerve trunks, some immunoreactive nerve fibres and principal nerve cells were still observed in the ganglion. A few immunoreactive neurons and nerve fibres were also observed in the ganglion stellatum. A large number of MEAP- and MEAGL-immunoreactive nerve fibres was detected in the spinal cord at the levels C6-Th6. A few neurons in the intermediolateral cell column of the spinal cord at levels C8-Thl showed MEAP- but not MEAGL-immunoreactivity. The cultured superior cervical ganglion contained a few MEAP-immunoreactive neurons, and the fibre outgrowth showed immunoreactivity both to MEAP and MEAGL. In electron microscopy, MEAGL-immunoreactivity in the superior cervical ganglion was localized in nerve fibres containing neurotubules and in principal nerve cells.The present results demonstrate that the rat superior cervical ganglion contains both extrinsic and intrinsic MEAP- and MEAGL-immunoreactive nerve fibres. Most of these fibres are of preganglionic origin. Both the principal nerve and small intensely fluorescent cells are often surrounded by MEAP- or MEAGL-immunoreactive nerve fibres and may receive innervation by these fibres. Several ganglionic neurons projecting to the sympathetic target tissues show MEAP- and/or MEAGL-immunoreactivity. Since MEAP- and MEAGL sequences are included in the proenkephalin A molecule, but not in the other opioid precursor molecules, it is evident that a neuronal system containing proenkephalin-A-derived enkephalins, is present in the rat superior cervical ganglion.     

Extracellular K+ concentration during electrical stimulation of rat isolated sympathetic ganglia,vagus and optic nerves

Recordings of extracellular potassium concentration ([K⁺]_e) were made in rat isolated sympathetic ganglia, vagus and optic nerves using ion sensitive microelectrodes. Repetitive orthodromic stimulation of ganglia resulted in [K⁺]_e increases of up to 7 mmol/1 above resting level (6 mmol/1), which were followed by post-stimulus undershoots. Activation of vagal A and B fibres did not significantly alter [K⁺]_e but C-fibre activity induced rises of up to 5 mmol/1. Repetitive stimulation of the predominantly mylinated optic nerve resulted in [K⁺]_e rises of up to 2.5 mmol/1. In the ganglion and vagus nerve, application of ouabain (30–1000 μmol/1) led to a raised baseline [K⁺]_e concentration, an increase in the peak achieved during stimulation and a reduced undershoot amplitude. The amplitude of the undershoot in normal solution was shown to be dependent on the duration of the preceding stimulation period as well as the amplitude of the preceding [K⁺]_e rise. In ganglia and vagus nerves, bath application of γ-aminobutyric acid (10–100μmol/1) and carbachol (10–1000 μmol/1) also elevated [K⁺]_e.It is concluded that repetitive activity in rat peripheral and central nerve fibres leads to significant changes in extracellular K⁺ ion-concentration and that the restoration of these levels is strongly dependent on the intact activity of the membrane Na^+/K⁺ pump.     

Location of substance P-, bombesin-gastrin-releasing peptide, [Met5]enkephalin- and [Met5]enkephalin-Arg6-Phe7-like immunoreactivities in adult human sympathetic ganglia

Indirect immunofluorescent methods were used to study peptides in human paravertebral sympathetic ganglia. [Met5]enkephalin, [Met5]enkephalin-Arg6-Phe7 and bombesin-gastrin-releasing peptide-like immunoreactivities were localized in varicose nerve fibers, which often formed basket-like networks around principal ganglion cells. Substance P-like immunoreactivity appeared frequently as solitary varicose nerve fibers and occasionally as networks. No immunolabeled cell bodies were discovered with any of the antisera used, including antibodies raised against molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH2. The results demonstrate the presence of peptides within nerve fibers and terminals but not cell bodies of human paravertebral sympathetic ganglia. The localization suggests that the peptides have neurotransmitter or neuromodulator roles in the ganglia.     

Regional distribution of endorphin,met5-enkephalin and leu5-enkephalin in the pigeon brain

The distribution of β-endorphin and enkephalin in the pigeon forebrain by immunohistochemistry and radioimmunoassay is essentially analogous to mammals. Both endorphin- and enkephalin-reactive fibers have a similar periventricular distribution, but the enkephalin fibers are more extensive and are also found in the paleostriatum, limbic regions and brain stem, pituitary stalk and notably, penetrating the organum vasculosum hypothalami. There was poor correlation between endorphin and enkephalin regional contents by radioimmunoassay. In contrast, a highly significant correlation was observed between Met⁵-enkephalin and Leu⁵-enkephalin regional distribution. These data support the view that enkephalin neurons and endorphin neurons are independent central neuronal systems.     

Two Classes of Antagonist Interact with Receptors for the Mitogenic Neuropeptides Bombesin,Bradykinin, and Vasopressin

Abstract

While screening neuropeptides for activity as growth factors we have found that bradykinin is a mitogen for Swiss 3T3 cells. It acts synergistically with insulin, and maximal effect is obtained at 10 nM. It acts through a distinct receptor, characterized as a B, subtype using bradykinin analogues. The neuropeptides bombesin and vasopressin are also potent mitogens for Swiss 3T3 cells. The substance P antagonists [DArg¹, DPro², DTrp^7,9, Leu¹¹] substance P and (DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹] substance P are inhibitors of DNA synthesis stimulated by both bombesin and vasopressin. In the present study they were found also to inhibit bradykinin-induced mitogenesis. In contrast, the ligand-specific antagonists [Leu¹³-Ψ(CH₂NH)Leu¹⁴]bombesin, [Pmp¹, OMeTyr², Arg⁸]vasopressin and [DArg¹¹, Hyp³, Thi^3,8, DPhe⁷]bradykinin showed no cross-inhibition with each others receptors. We propose therefore that the receptors for the mitogenic neuropeptides bombesin, vasopressin, and bradykinin can interact with two classes of antagonist, one recognizing the ligand binding site (e.g., [Leu¹³-Ψ(CH₂ NH)Leu¹⁴]bombesin) and the other recognizing a common domain shared by the three receptors (e.g., [DArg¹, DPhe⁵, DTrP^7,9, Leu¹¹] substance P).     

High-resolution radioautographic study of the serotonin innervation of the rat corpus striatum after intraventricular administration of [3H]5-hydroxytryptamine

In the rat striatum, identification of the serotonin-containing nerve endings at the ultrastructural level has been done by radioautography after ventriculodsternal perfusion of [³H]serotonin. Only the ventricular border of the middle part of the nucleus caudatus-putamen (neostriatum) was studied and, in addition, a small dorsal part of the globus pallidus.For every 100 μm²of thin sections of the neostriatum, 0.5 labelled structures were found, which were either axons (14%), preterminal enlargements (21.5%) or nerve terminals (64.5%). Three kinds of labelled nerve endings could be recognized according to the size and shape of their synaptic vesicles, although all contained occasional large granular vesicles. The first type (36%) was characterized by small and often granular, ovoid vesicles mixed with tubular structures of the same diameter (20 nm). The second type (28%) contained crowded spherical vesicles of 45 nm in diameter which were mostly clear. The third type (36%) contained a mixture of both types of synaptic vesicles. The labelled nerve terminals in the neostriatum showed a few synaptic contacts (mean of 14%) which were, however, more numerous in the second type of nerve endings (25–30%) and mainly of the asymmetric type. In the globus pallidus the serotonin-labelled nerve terminals resembled those in the neostriatum but one additional type was observed, which was characterized by only a few typical synaptic vesicles and by numerous large granular vesicles.The observations made in this study indicate that the serotonin-containing varicosities of the striatum are very similar to those described in some other nuclei. They exhibit characteristic large granular vesicles and the synaptic vesicles often have an eccentric tiny granule after administration of exogenous serotonin and/or inhibition of monoamine oxidase. The peculiar type of nerve endings with tubular vesicles described by others in the cerebellum and in the locus coeruleus was also present in the neostriatum. The morphological heterogeneity of serotonin-containing nerve endings within a single nucleus or from one nucleus to another, is discussed in relation to the amine content, the stage of formation of the synaptic vesicles and their origin in different raphe nuclei.     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

Incorporation of [3H]2-deoxyglucose into glycogen in nervous tissues

Mice were injected with [³H]2-deoxyglucose and after 1 h high molecular weight glycogen was extracted from brain, liver and muscle tissues. 1–2% of the total radioactivity in each tissue was recovered in the glycogen fraction. Isolated buccal ganglia of the pond snail,Planorbis, and isolated abdominal ganglia of the horse leechHaemopis, were exposedin vitro to [³H]2-deoxyglucose for 1 h. 1–10% of the total radioactivity in these tissues was located in the high molecular weight glycogen fraction. Treatment of the extracted labelled glycogen fractions with amyloglucosidase caused release of the label in a manner consistent with the breakdown of labelled glycogen. Ganglia of snail and leech were exposed to [³H]2-deoxyglucose, fixed in glutaraldehyde and osmium tetroxide solutions, and prepared for autoradiography using aqueous histological processing. Light and electron microscope autoradiography showed that over 90% of the label was positively associated with glycogen particles (α- and β-particles). Certain previously published reports on the incorporation of 2-deoxyglucose into glycogen are discussed in relation to these findings.It is concluded that [³H]2-deoxyglucose is partially incorporated into glycogen in nervous tissue; the labelled 2-deoxyglycogen withstands aqueous histological processing and can be visualized directly by autoradiography.     

A novel, [tyrosyl-3,5-3H]oxytocin binding,uterine cell population in the rat

The cellular localization of uterine oxytocin binding sites in the rat was studied by means of in vitro receptor autoradiography. Using [tyrosyl-3,5-³H]oxytocin as ligand, binding sites were localized in tissue sections from uteri of estrous, mated, and artificially cervically stimulated rats (n = 4 per group), and specificity of binding was investigated by means of simultaneous incubations with oxytocin, [Gly⁴, Thr⁷]oxytocin and [Arg]vasopressin. A previously unidentified type of cell was densely labelled by tritiated oxytocin. The labelled cells were preferentially localized near the endomyometrial border and at the interface of the circular and longitudinal muscle layers. In addition, these cells were found in the muscle layers. The dense labelling of these cells, which did not constitute part of the endometrial epithelium or blood vessels, was abolished when oxytocin or [Arg]vasopressin, but not [Gly⁴, Thr⁷]oxytocin, was added to the incubation medium. Binding of the radioligand was also found on muscle cells of the circular and longitudinal layers of the myometrium and cells of the endometrial luminal and glandular epithelium. Whereas incubation with oxytocin and [Gly⁴, Thr⁷]oxytocin diminished the labelling in both myometrium and endometrium, incubation with [Arg]vasopressin reduced labelling only in the myometrium. Similar results were obtained in tissues from rats in different reproductive states. This study demonstrates the presence of oxytocin binding sites in three different types of cell in the uterus of the rat. While the sites in the myometrium may be associated with the contractile response of this type of tissue to oxytocin, the functional significance of oxytocin binding sites on the endometrial epithelium and in the densely labelled, scattered cell remains to be elucidated. © 1992 Wiley-Liss, Inc.     

[3H]haloperidol and [3H]spiroperidol receptor binding after striatal injection of kainic acid

[³]Haloperidol and [³H]spiroperidol binding studies after kainate injection into the striatum indicate the presence of dopamine receptive sites not located on post-synaptic membranes. However, the physiological meaning of these “presynaptic” sites remains to be established.     

Clusters of axonal Na+ channels adjacent to remyelinating Schwann cells

Summary Rat sciatic nerve fibres were demyelinated by injection of lysolecithin and examined at several stages as Schwann cells proliferated, adhered, and initiated remyelination. Immunoperoxidase EM has been used to follow the clustering of Na⁺ channels that represents an early step in the formation of new nodes of Ranvier. At the peak of demyelination, 1 week postinjection, only isolated sites, suggestive of the original nodes, were labelled. As Schwann cells adhered and extended processes along the axons, regions of axonal Na⁺ channel immunoreactivity were often found just beyond their leading edges. These channel aggregates were associated only with the axolemma and Na⁺ channels were not detected on glial membranes. Sites with more than one cluster in close proximity and broadly labelled aggregates between Schwann cells suggested that new nodes of Ranvier formed as neighbouring Na⁺ channel groups merged. Schwann cells thus seem to play a major role in ion channel distributions in the axolemma. In all of these stages Na⁺ channel label was found primarily just outside the region of close contact between axon and Schwann cell. This suggests that Schwann cell adherence acts in part to exclude Na⁺ channels, or that diffusible substances are involved and can act some distance from regions of direct contact.     

Effect of Concanavalin A on Intracellular K+ and Na+ Concentration and K+ Transport of Human Lymphocytes

Incubation of human lymphocytes with ConA causes an increase in [Na⁺]; and a decrease in [K⁺];. This effect is not due to the experimental washing procedure, but is due to the ConAinduced increase in permeability which is not fully compensated by the increase in active transport.The ConA-induced increase in ⁴²K⁺ uptake consists of an increase in leak flux which is independent of [Na⁺]_o, and of an increase in pump flux which is dependent on [Na⁺]₀.The increase in leak flux may be caused by increased membrane fluidity. The increase in pump flux may be produced by the increased [Na⁺]. and by a stimulation of Na⁺, K⁺ ATPase.     

Serotonergic synaptic input to facial motoneurons: localization by electronmicroscopic autoradiography

Serotonergic nerve terminals in the facial motor nucleus were labelled with [³H]5-hydroxy-tryptamine. When serotonergic nerve terminals were destroyed (by the selective neurotoxin 5,7-dihydroxytryptamine) the labelling was lost. By electron-microscopic autoradiography, labelled serotonergic terminals were found to make axo-dendritic or axo-somatic junctions with facial motor neurons. No axo-axonic junctions were observed.These morphological findings are consistent with physiological studies which indicate that 5-hydroxy-tryptamine facilitates the excitation of facial motoneurons through a direct postsynaptic action.     

Synthesis,storage and release of [14C]acetylcholine in isolated rat diaphragm muscles

1. Segments of rat diaphragms were kept in choline-free media for 4 hr and were then exposed to a physiological concentration of [¹⁴C]-choline (30 μM) at 37° C. The synthesis, storage and subsequent release of [¹⁴C]acetylcholine by the muscles was assessed by isotopic- and bio-assays after isolation of the transmitter by paper electrophoresis.2. Replacement of endogenous acetylcholine (0·92 μ-mole/kg) with labelled acetylcholine proceeded slowly at rest, but rapidly during nerve stimulation. [¹⁴C]Acetylcholine accumulated most rapidly when hydrolysis of the released transmitter, and thus the re-use of endogenous choline, was prevented by an esterase inhibitor. Fully replaced stores were maintained during nerve stimulation by synthesis rates sufficient to replenish at least 35% of the store size in 5 min.3 In the presence of hemicholinium-3, which inhibits choline uptake, acetylcholine stores declined rapidly during stimulation, and residual synthesis was slight, indicating little intraneural choline. Net choline uptake into nerve terminals was estimated from the highest observed synthesis rate and from previous measurements of the number and size of terminals, as 3-6 p-mole/cm² sec.4. Transmitter synthesis was localized in the region of end-plates, and was reduced to a few per cent of normal 6 weeks after phrenic nerve section. Release experiments suggested that at least half of the acetylcholine in phrenic nerves is in their terminals; from this content and the morphology of the terminals, the average concentration of transmitter in the whole endings would appear to be about 50 m-mole/l. Homogenization of the muscles freed choline acetyltransferase into solution, but left some [¹⁴C]acetylcholine associated with small particles, presumably synaptic vesicles.5. Resting transmitter release was about 0·013% of stores/sec. With 360 nerve impulses at 1-20/sec, release increased up to 0·43% of stores/sec, and amounted to 3·5-7 × 10^-18 moles per end-plate per impulse. The release rate was unaffected by the doubling of store size which occurred with eserine, but the extra transmitter did help to maintain releasable stores during prolonged stimulation. Experiments with fractional store labelling indicated that newly synthesized acetylcholine was preferentially released.6. Preformed [³H]acetylcholine was not taken up and retained by muscle or nerve cells in the absence of an esterase inhibitor. With eserine present, labelled acetylcholine was taken up uniformly by muscle segments; when eserine was then removed, radioactive acetylcholine remained only near neuromuscular junctions.     

Distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in rat hippocampal long-term slice cultures isolated from external afferents

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [³H]MK-801 and [³H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [³H]MK-801 and [³H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [³H]Kainate in the cultures exceeded that in the hippocampi in situ 3–5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the ”superficiomembranal’’ axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [³H]MK-801 and [³H]Kainate binding sites, but not of [³H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures. Accepted: 6 February 2001     

Potassium-evoked release of [14C]GABA and [3H]taurine from rat olfactory bulb slices

Rat olfactory bulb slices were preloaded with [³H] taurine or with [¹⁴C] GABA. Upon stimulation of the slices with increasing concentrations of KCl, we observed release of [³H] taurine or [¹⁴C] GABA. Superfusion of the slices with high concentrations of K⁺ in the absence of Ca²⁺ in the perfusion medium, led to a marked decrease in the stimulated release of both [³H] taurine and [¹⁴C] GABA.     

A radioautographic study of [3H]L-glutamate and [3H]L-glutamine uptake in the guinea-pig cochlea

Since glutamate has been recently proposed as a possible transmitter of the sensory hair cells in the cochlea, a radioautographic study was performed to look for the in vitro uptake of [³H] l-glutamate and [³H]l-glutamine. Several experimental conditions were applied. The control experimental procedures consisted in an incubation with one of the labelled tracers (10 min), followed by a post-incubation (3 × 10 min) without tracer. In these experiments, either with [³H]l-glutamate or [³H]l-glutamine, the following structures were labelled: inner hair cells, glial cells of the osseous spiral lamina and areas of the inner spiral and tunnel spiral bundles. When these experiments were carried out in absence of Na⁺, these labellings were strongly decreased. When the incubation was not followed by a post-incubation, the results differed depending on the tracer: with [³H]l-glutamate, the glial cells and the areas of inner spiral and tunnel spiral bundles were labelled, whereas with [³H]l-glutamine, mainly the inner hair cells were labelled. An addition of l-methionine-dl-sulfoximine, a glutamine synthetase inhibitor, into the incubation and post-incubation media, produced a decrease of the labelling of the inner hair cells and of the glial cells. An addition of unlabelled glutamine to the post-incubation media decreased the inner hair cell labelling, while a similar addition of unlabelled glutamate did not. In either case, neither the outer hair cells, the second type of sensory cells, nor the spiral ganglion neurons were labelled.These results suggest that in the cochlea, glutamate and glutamine have their metabolisms linked together, as in some parts of the central nervous system. Correlated to biochemical and electro physiological data these results support the hypothesis that glutamate could be the neurotransmitter of the inner hair cells.     

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.