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The diffusible intercellular messenger nitric oxide may have a modulatory role in the thalamus and this action may be mediated via activation of soluble guanylate cyclase. In order to investigate this possibility, we applied the cyclic-GMP analogue 8-Bromo-cyclic-GMP (8-Br-cGMP) onto neurones in the ventrobasal and lateral geniculate nuclei of the thalamus in anaesthetised rats, and compared its effects with those of a nitric oxide donor. 8-Br-cGMP enhanced the responses of neurones to iontophoretically applied NMDA and AMPA. Furthermore, somatosensory and visual responses of ventrobasal and lateral geniculate neurones were enhanced to 274+/-76% and 217+/-69% of control values, respectively. These effects were similar to those seen with nitric oxide donors in this study and previous work from this laboratory. When applied to thalamic neurones in an in vitro slice preparation, 8-Br-cGMP caused a membrane depolarisation associated with a decrease in input resistance. These findings indicate that activation of guanylate cyclase can cause a membrane depolarisation of thalamic neurones in vitro, and that this effect is sufficient to enhance action responses to ionotropic glutamate receptor stimulation via either exogenous agonists or sensory stimulation., (Copyright 1999 Elsevier Science B.V.)

Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. One important mediator of sensitization is bradykinin (BK), a peptide released as a consequence of tissue damage. BK enhances the membrane ionic current activated by heat in nociceptive neurons, using a pathway that involves activation of protein kinase C (PKC). We find that five PKC isoforms are present in sensory neurons but that only PKC-epsilon is translocated to the cell membrane by BK. The heat response is sensitized when constitutively active PKC-epsilon is incorporated into nociceptive neurons. Conversely, BK-induced sensitization is suppressed by a specific peptide inhibitor of PKC-epsilon. We conclude that PKC-epsilon is principally responsible for sensitization of the heat response in nociceptors by bradykinin.

Latexin, a carboxypeptidase A inhibitor, is expressed in a cell type-specific manner in both central and peripheral nervous systems in the rat. In the neocortex, a specific subpopulation of neurons in layers V and VI expresses latexin. In the primary sensory ganglia, the expression is restricted to smaller diameter neurons. As a first step to clarify regulatory mechanisms underlying cell type-specific expression of latexin, we have determined the organization of the rat latexin gene and analyzed its regulatory elements. The latexin gene spans approximately 5.8 kb, and consists of six exons and five introns. Three transcription initiation sites were mapped. The upstream region lacks typical TATA or CAAT boxes but has several GC-rich sites. To assess promoter activity, the luciferase reporter gene fused to the 5'-flanking region (6.4 kb) of the latexin gene was transiently transfected into several cell lines. Luciferase activity was 2-8 times higher in latexin-expressing cells (PC12) than non-expressing cells (NS20 and L6). Deletion analysis with PC12 cells revealed that a core promoter is located between nucleotide positions -261 and -201 relative to the A of the initiation codon. Nerve growth factor (NGF)-responsive element(s) is located between positions -518 and -262, in which AP-1, AP-2 and NF-kappaB binding sites are found. Furthermore, we demonstrate that a 1.3 kb genomic fragment containing the first intron has transcriptional enhancing activity in PC12 cells. These results suggest that up and downstream regulatory elements are involved in the control of cell type-specific expression of latexin., (Copyright 1999 Elsevier Science B.V.)

The purpose of this study was to determine whether there is a specific organization of the primary sensory innervation on to identified vascular neurons in the inferior mesenteric ganglion (IMG) in guinea-pig. Retrograde tracers were placed intraluminally in inferior mesenteric artery (IMA) or inferior mesenteric vein (IMV) in vitro to identify ganglionic neurons as arterial, venous or unlabeled neurons. The distribution of primary sensory nerve fibers containing calcitonin gene-related peptide (CGRP), neuronal nitric oxide synthase (NOS) and substance P immunoreactivity (SP-IR) was compared before and after treatment with capsaicin. In control animals the density of immunoreactivity varied both with the transmitter and the type of neuron innervated. The density of immunoreactivity for all the three substances was reduced by capsaicin treatment. The degree of reduction of immunoreactivity in the fibers varied with the transmitter and the type of neuron. The density of CGRP and SP immunoreactive fibers was greatest around unlabeled neurons; 78% of the CGRP fibers were of primary sensory origin and all of the SP fibers were primary sensory. Around arterial neurons 44% of the CGRP fibers were of primary sensory origin and around venous 68% were primary sensory. NOS positive innervation around venous neurons was denser than around arterial neurons and all of it was completely (97%) eliminated by capsaicin, indicating that it was solely of primary sensory origin. We conclude that the primary sensory fibers innervating the IMG are differentially distributed to arterial and venous neurons and that the pattern of distribution is characteristic for each sensory neurotransmitter., (Copyright 1999 Elsevier Science B.V.)

The aims of this study were to improve insight into cAMP signaling in myenteric neurons and glia and identify the adenylyl cyclase (AC) isoforms expressed in myenteric ganglia of the guinea-pig small intestine. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520 nm/580 nm fluorescence emission ratio of the protein kinase A fluorosensor FlCRhR. Forskolin or pituitary adenylyl cyclase activating peptide caused an increase in cAMP levels in cell somas and neurites and elicited a slow EPSP-like response in myenteric AH/Type 2 neurons, whereas the inactive form of forskolin was without these effects. Glia displayed similar cAMP responses. Immunoblot analysis showed that AC I, III and IV were present in myenteric ganglia, with AC I being detected as two bands of 160 kDa and 185 kDa, AC III as two bands near 220 kDa, and AC IV as two bands of greater than 220 kDa. Pretreatment with N-ethylmaleimide and N-glycosidase F revealed an AC IV band at 115 kDa. Preabsorption with specific blocking peptides prevented detection of AC I or AC IV immunoreactive proteins. In ganglia which expressed strong AC IV immunoreactivity, no immunoreactive bands were detected for AC II, AC V/VI, AC VII or AC VIII. The amount of AC isoforms expressed in myenteric ganglia followed the order of AC IV&z.Gt;III>I. Immunofluorescent labeling studies revealed that AC I, AC III and AC IV were variably expressed in myenteric neurons and glia of the duodenum, jejunum and ileum. In the guinea-pig ileum, AC I, III and IV immunoreactivities were respectively present in 26%, 58% and 89% of calbindin-D28-colabeled myenteric neurons. These findings suggest that (1) AC I, AC III and AC IV variably contribute to cAMP signaling in myenteric ganglia, (2) AC I, AC III and AC IV may be differentially expressed in distinct subsets of calbindin-D28 neurons which may represent intrinsic primary afferent myenteric neurons. Our study also provides direct evidence for activation of cAMP-dependent protein kinase., (Copyright 1999 Published by Elsevier Science B.V.)

FRAP (fluoride-resistant acid phosphatase)-reactivity in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis (STNC) was examined by light and electron microscopy. Degenerated figures of terminals caused by capsaicin were compared with the FRAP-positive terminals. Scalloped (fan-like) or indented, sinuous, slender, and cap-like figures with closely packed agranular synaptic vesicles of various sizes were common to both FRAP-positive and capsaicin-sensitive terminals. These terminals had glomerular or nonglomerular endings. Sometimes FRAP-positive and capsaicin-sensitive glomerular terminals made presynapses with surrounding dendrites. Frequently, both nonglomerular terminals were in direct contact with the neuronal soma. The terminal features of FRAP-positive and capsaicin-sensitive ones in the mouse STNC are the same as those seen in the superficial dorsal horn of the spinal cord. These findings suggest that some of the FRAP-positive terminals are capsaicin-sensitive, thereby indicating their nociceptive primary afferent.

We investigated roles of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 microM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 microM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 microM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth., (Copyright 1999 Elsevier Science B.V.)

Histochemical detection of NADPH-d activity in rat barrel-field cortex reveals four types of distributions. (i) A transient, diffuse neuropil staining is visible in the cortical plate and in deeper layers until postnatal day (P) 4. Thereafter, until P15, it is segregated in whisker-specific patches in layer IV, then the pattern gradually disappears, becoming virtually indistinct by P21. This transient patterning of diffuse NADPH-d activity in layer IV disappears after cortical injections of kainic acid and is affected by neonatal damage to the contralateral snout. An intense labeling (ii) of scattered cells and (iii) of a plexus of fibers is present. With maturation, the cells become localized mostly in layers II/III, in the lower part of layer V, and in layer VI. They are sparse in layer I, in upper layer V, and in layer IV where their somata are located primarily in the interbarrel septa. (iv) Light staining of cortical neurons is detected mostly in layers II-IV but occasionally also in layers V-VI. Cytochrome c oxidase (CO)-positive patches associated with barrels are first detected in layer IV around P4-P5; their staining density increases with development, then stays high. In the adult, CO activity is moderate in supragranular layers, highest in the barrels in layer IV, low in upper layer V, medium dense in the deeper half of layer V, and low in lamina VI. Thus, NADPH-d and CO activities are not necessarily colocalized in the rodent barrel-field cortex. The varied (transient and long-lasting) distributions of NADPH-d activity indicate that the enzyme and its associated production of NO serve multiple roles in developing and adult barrel-field cortex., (Copyright 1999 Academic Press.)

Phosphatidylinositol 3-kinase (PI3-kinase) enzymes are key signalling molecules in the PC12 and neuronal cell survival pathway and are also involved in the regulation of retrograde axonal transport of nerve growth factor (NGF), with sympathetic neurons more sensitive to the effects of wortmannin/LY294002 than sensory neurons (Bartlett et al. [1997]; Brain Res. 761:257-262; Reynolds et al. [1998] Brain Res. 798:67-74). In this article, we characterized the mRNA expression of PI3-kinase isoforms in mouse sympathetic superior cervical ganglia (SCG) and sensory trigeminal ganglia (TGG) and examined the subcellular locations of immunoreactivity of the PI3-kinase isoforms in mouse cultured SCG and dorsal root ganglion (DRG) neurons. Both the SCG and the TGG express mRNA for the p110alpha, beta, gamma, delta, and vps34p PI3-kinase isoforms, but the TGG and not the SCG express mRNA for the p170 PI3-kinase isoform. In cultured SCG and DRG neurons, p110alpha, beta, and gamma immunoreactivity is in the SCG and DRG growth cones, and predominantly in puncta throughout the growth cone varicosity. However, in the cell bodies immunoreactivity varied, p110alpha is localized predominantly at the plasma membrane, while p110beta and gamma is localized in the perinuclear region of the cells. In addition, unlike other cell types, wortmannin has little effect on actin filament polymerization in either mouse cultured SCG or DRG neurons.

The effect of noradrenaline on the glycine response was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus using nystatin perforated patch recording configuration under voltage-clamp conditions. Noradrenaline reversibly potentiated the 10(-5)M glycine-induced Cl- current in a concentration-dependent manner. Single channel recordings in a cell-attached mode revealed that noradrenaline decreased the closing time of the glycine-activated channel activity. Noradrenaline neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. Clonidine mimicked and yohimbine blocked the noradrenaline action on glycine response. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride, protein kinase A inhibitor, mimicked the effect of noradrenaline on glycine response. Noradrenaline failed to affect the glycine response in the presence of these intracellular cyclic AMP and protein kinase A modulators. However, noradrenaline further enhanced the glycine response even in the presence of phorbol-12-myristate-13-acetate and chelerythrine, a protein kinase C inhibitor. Pertussis toxin treatment for 6-8 h blocked the noradrenaline facilitatory effect on the glycine response. In addition, noradrenaline potentiated the strychnine-sensitive postsynaptic currents evoked in a slice preparation of sacral dorsal commissural nucleus. These results suggest that the activation of alpha2-adrenoceptor by noradrenaline coupled with pertussis toxin-sensitive G-proteins reduces intracellular cyclic AMP formation through the inhibition of adenyl cyclase. The reduction of cyclic AMP decreases the protein kinase A activity, thus resulting in the potentiation of the glycinergic inputs to the sacral dorsal commissural neurons. It is thus feasible that the noradrenergic input to the sacral dorsal commissural nucleus modulates such nociceptive signals as pain by intracellular enhancing the glycine response.

Much attention has focused on the important role played by phosphatases in the control of gene transcription, cell differentiation and memory regulation. It is also clear that phosphatases may regulate a number of biochemical pathways which can modulate cellular function. Of particular interest is the role of phosphatases in the control of neuronal function. Alterations in neuronal function may contributed to the heightened airways responsiveness observed in asthma to a number of physiological stimuli including distilled water, sulfur dioxide, metabisulfite, hypertonic saline, exercise, allergens, viruses and cold air. An understanding of the mechanisms which regulate the function of sensory nerves could have important clinical implications. In this review we will highlight a number of studies that have investigated the role of phosphatases in the regulation of airway nerve function.

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.

Sex differences in the rat brain are dependent, in part, on oestrogen exposure during specific developmental perinatal periods. The availability of oestrogen requires precursor androgen and the presence of intraneuronal aromatase. To examine sites of oestrogen formation and action in the brain, immunocytochemical and biochemical localization of aromatase in the rat brain were determined between embryonic day 14 and postnatal day 20. Aromatase-immunolabelled neuronal profiles were present in hypothalamic, cortical and limbic regions. Surprisingly, aromatase immunoreactivity was also observed in non-limbic regions of the immature brain where it was previously unsuspected. Among these regions, aromatase staining was robust in developing sensory systems, including primary afferents of the olfactory, trigeminal, vestibulocochlear, and visual systems. To determine whether this aromatase is functional in these systems, i.e. converts testosterone to estradiol, the trigeminal nerve was dissected from the hindbrain of perinatal animals and studied for enzyme activity by the tritium release method. The dpm/mg protein/h tritium release in these tissues equalled that of hypothalamic or limbic controls, indicating that these sensory areas are sites of in-situ estradiol synthesis. Our data suggests that aromatase (estradiol)-dependent mechanisms may play a role in the differentiation and maturation of sensory pathways, which, in turn, may contribute to sex differences in the activity of these systems.

Complete information concerning possible alterations in nitric oxide production in the rat brainstem trigeminal system after peripheral nerve lesion is still lacking. This being the case, density of NADPH-diaphorase positive neurons in the trigeminal mesencephalic nucleus after experimental infraorbital nerve transection was studied. In the lesioned side, an ipsilateral increase in NADPH-d positive neurons was found at postoperative days 4 and 6 with respect to contralateral, without changes after a 2 months period. These data suggest that nitric oxide could be involved in regeneration of afferent fibers concerned with the periodontal receptors of maxillary teeth.

The inferior colliculus is a central auditory structure which serves as a site for the integration of ascending and descending auditory information. Changes in central auditory structures may occur with acoustic exposure, which cannot be explained by alterations in cochlear function alone. Rats were exposed to a 10-kHz tone at 100 dB SPL for 9 h. Auditory brainstem response measures showed an initial 25-30-dB threshold shift across all tested frequencies. By 30 days post-exposure, thresholds for clicks and most frequencies returned to near control levels; however, thresholds remained elevated at 10 and 20 kHz. Inner hair cell loss was confined to apical and basal ends of the cochlea, and did not exceed 20%. Inferior colliculus levels of the two isoforms of the GABA synthetic enzyme glutamate decarboxylase (65,000 and 67,000 mol. wt forms) were measured immediately post-exposure (0 h) and at two and 30 days post-exposure using quantitative immunocytochemical and western blotting techniques. Zero-hour measures revealed a significant increase in the level of glutamate decarboxylase (mol. wt 67,000) protein (118%), as well as in the optical density (35%) of immunolabeled cells. By 30 days post-exposure, inferior colliculus protein levels of both glutamate decarboxylase isoforms were significantly below unexposed controls (39% and 21% for the 65,000 and 67,000 mol. wt forms, respectively). These studies describe increased markers for GABA immediately following acoustic exposure, followed by a decline to below control levels from two to 30 days post-exposure. It remains to be determined whether noise trauma-induced changes in glutamate decarboxylase levels in the inferior colliculus reflect protective up-regulation in response to intense stimulation, followed by the establishment of new neurotransmitter equilibrium levels.

The cAMP signaling cascade has been implicated in several stages of memory formation. We have examined activation of this cascade by serotonin (5-HT) in the sensory neurons of Aplysia. We find that different patterns of 5-HT exposure induce three distinct modes of PKA activation. First, a single 5 min pulse induces transient (5 min) PKA activation that requires neither transcription nor translation. Second, 4-5 pulses induce intermediate-term persistent activation (3 hr duration) that requires translation but not transcription. Third, 5 pulses of 5-HT, as well as continuous (90 min) exposure, induce long-term persistent activation 20 hr later, which requires both transcription and translation. Thus, in the sensory neurons, different patterns of 5-HT give rise to three independent phases of PKA activation that differ in their induction requirements, their temporal profiles, and their molecular mechanisms.

The neuropeptide calcitonin gene-related peptide (CGRP) is expressed by one-third of adult rat lumbar dorsal root ganglion (DRG) neurons, many of which mediate pain sensation or cause vasodilation. The factors that regulate the developmental expression of CGRP are poorly understood. Embryonic DRG neurons initially lack CGRP. When these neurons were stimulated in culture by serum or persistent 50 mM KCl application, the same percentage of CGRP-immunoreactive (CGRP-IR) neurons developed in vitro as was seen in the adult DRG in vivo. The addition of the L-type calcium channel blockers, 5 microM nifedipine or 10 microM verapamil, dramatically decreased the proportion of CGRP-IR neurons that developed, although the N-type calcium channel blocker, 2.5 microM omega-conotoxin, was less effective. By contrast, the sodium channel blocker 1 microM tetrodotoxin had no effect on CGRP expression after depolarization. Fura-2 ratiometric imaging demonstrated that mean intracellular free calcium levels increased from 70 to 135 nM with chronic depolarization, and the addition of nifedipine inhibited that increase. Only a subpopulation of neurons had elevated calcium concentrations during chronic depolarization, and they were correlated with CGRP expression. Key signal transduction pathways were tested pharmacologically for their role in CGRP expression after depolarization; the addition of the CaM kinase inhibitor KN-62 reduced the proportion of CGRP-IR neurons to basal levels. By contrast, protein kinase A and protein kinase C were not implicated in the depolarization-induced CGRP increases. These data suggest that depolarization and the subsequent Ca2+-based signal transduction mechanisms play important roles in the de novo expression of CGRP by specific embryonic DRG neurons.

The distribution and morphology of nitric oxide-synthesizing elements in the human olfactory bulb were studied using NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry. NADPH-diaphorase was detected in all olfactory fibers and groups of superficial short-axon cells, deep short-axon cells, stellate cells and abundant centrifugal fibers. Similar cell types were nitric oxide synthase immunoreactive but olfactory fibers were immunonegative. The distribution patterns of nitric oxide-synthesizing elements showed significant differences from what has been reported in the olfactory bulb of macrosmatic mammals including rodents and insectivores. These differences are likely to correlate with interspecies differences in the processing of olfactory information.

Retrograde labeling of duodenum-sphincter of Oddi (SO) preparations in vitro with the carbocyanine dye DiI revealed that duodenal neurons project to the SO. The duodenum-SO-projecting neurons were immunoreactive (IR) for choline acetyltransferase but not nitric oxide synthase or calretinin, indicating that this is a cholinergic projection and that this pathway is distinct from the circuitry involved in the ascending limb of the peristaltic reflex. Approximately 20% of the duodenum-SO projection neurons were IR for calbindin. Calbindin-IR nerves within SO ganglia degenerated when the SO was maintained in organ culture alone, but persisted when the SO was cultured with the duodenum intact. Therefore, SO ganglia are a target of the calbindin-positive duodenum-SO projection. Because calbindin is a marker of intrinsic sensory neurons that have processes that pass to the mucosa, these neurons are in position to detect the release of a compound from the mucosa and signal its release to SO ganglia. When applied to retrogradely labeled neurons, cholecystokinin (CCK) elicited a prolonged depolarization, indicating that duodenum-SO-projecting neurons could be capable of detecting CCK released from the mucosa. It is proposed that the role of the intrinsic sensory neurons that project to the SO may be to signal the postprandial release of CCK, thus providing an instruction to decrease SO resistance and facilitate the flow of bile into the duodenum.

In order to identify cytochemical traits relevant to understanding excitatory neurotransmission in brainstem auditory nuclei, we have analyzed in the dorsal cochlear nucleus the synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc, three molecules probably involved in different steps of excitatory glutamatergic signaling. High levels of glutamate immunolabeling were found in three classes of synaptic endings in the dorsal cochlear nucleus, as determined by quantitation of immunogold labeling. The first type included auditory nerve endings, the second were granule cell endings in the molecular layer, and the third very large endings, better described as "mossy." This finding points to a neurotransmitter role for glutamate in at least three synaptic populations in the dorsal cochlear nucleus. The same three types of endings enriched in glutamate immunoreactivity also contained histochemically detectable levels of aspartate aminotransferase activity, suggesting that this enzyme may be involved in the synaptic handling of glutamate in excitatory endings in the dorsal cochlear nucleus. There was also extrasynaptic localization of the enzyme. Zinc ions were localized exclusively in granule cell endings, as determined by a Danscher-selenite method, suggesting that this ion is involved in the operation of granule cell synapses in the dorsal cochlear nucleus.

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.

In the present study, histochemical techniques combined with more conventional anatomical methods were used to refine the identification of the nucleus of the optic tract and the nuclei of the accessory optic system in the opossum. The distribution of the enzyme cytochrome oxidase (CO) was examined in the cells and the neuropil of the opossum's mesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract (NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that had received bilateral injections of horseradish peroxidase centered in the region of the inferior olive, were subjected to assays for CO and horseradish peroxidase. The region occupied by CO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeled cells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivary and CO-positive cells were confined within similar boundaries, the latter are up to twofold more numerous than the former. As revealed by cytochrome oxidase histochemistry, the outlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessory optic system coincided with those revealed by the histochemistry for nicotinamide dinucleotide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin beta subunit and alternate sections processing for NADPH-d and CO, the distribution of labeled retinal terminal fields in the mesodiencephalic region was shown to be coincident with regions of high levels of histochemical labeling. These results are discussed in the light of previous anatomofunctional assessments of the pretectum and accessory optic system.

Subplate neurons, the first neurons of the cerebral cortex to differentiate and mature, are thought to be essential for the formation of connections between thalamus and cortex, such as the system of ocular dominance columns within layer 4 of visual cortex. To learn more about the requirement for subplate neurons in the formation of thalamocortical connections, we have sought to identify the neurotransmitters and peptides expressed by the specific class of subplate neurons that sends axonal projections into the overlying visual cortex. To label retrogradely subplate neurons, fluorescent latex microspheres were injected into primary visual cortex of postnatal day 28 ferrets, just prior to the onset of ocular dominance column formation. Subsequently, neurons were immunostained with antibodies against glutamate, glutamic acid decarboxylase (GAD-67), parvalbumin, neuropeptide Y (NPY), somatostatin (SRIF), or nitric oxide synthase (NOS). Retrograde labeling results indicate that the majority of subplate neurons projecting into the cortical plate reside in the upper half of the subplate. Combined immunostaining and microsphere labeling reveal that about half of cortically projecting subplate neurons are glutamatergic; most microsphere-labeled subplate neurons do not stain for GAD-67, parvalbumin, NPY, SRIF, or NOS. These observations suggest that subplate neurons can provide a significant glutamatergic synaptic input to the cortical plate, including the neurons of layer 4. If so, excitation from the axons of subplate neurons may be required in addition to that from lateral geniculate nucleus neurons for the activity-dependent synaptic interactions that lead to the formation of ocular dominance columns during development.

The coexistence of S100beta with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100beta immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100beta-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100beta-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100beta-ir. Many neurons in the nodose ganglion colocalized S100beta-ir and NADPH-d activity, whereas S100beta-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100beta- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100beta and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

The structure of the nervous network and the distribution of tyrosine hydroxylase (TH)- and various neuropeptide-containing nerves were immunohistochemically studied in the glottis of the dog. The nervous network in the glottis revealed apparent regional differences in morphology. The nervous network in the cartilaginous vocal fold of the posterior glottis consisted of nerve bundles running parallel to the edge of the vocal fold. Only a small number of nerve bundles were observed in the anterior glottis, specifically in membranous vocal fold. In the subepithelial layer of the posterior glottis, a moderate number of galanin (GAL)-immunoreactive nerve fibers were observed, while only a few fibers were present in the anterior glottis. Numerous vasoactive intestinal peptide (VIP)-, GAL-, methionine-enkephalin (ENK)- and TH-immunoreactive nerve fibers were observed within and around the laryngeal submucosal seromucous gland. Many TH- and neuropeptide Y (NPY)-immunoreactive fibers were arranged around the blood vessels. In the epithelia, free nerve endings with immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) was observed. Furthermore, nerve cell bodies with SP-, VIP-, GAL-, ENK-, and NPY-immunoreactivity were observed in the deep region of the submucosal layer. The results from the present study suggest that there is autonomic regulation of the glottis. Regional structural differences in the nervous network of the glottis may reflect functional differences.

Enteric nerves can function normally without connections with the central nervous system. A contributing component of the functional autonomy exhibited by enteric nerves is their plasticity. In the present study, the number of nitric oxide synthase-immunoreactive (NOS-ir) myenteric neurons and inhibitory neuromuscular transmission were studied in extrinsically denervated ileal segments. Segments of ileum were extrinsically denervated by crushing the mesenteric blood vessels supplying a loop of ileum in anesthetized guinea pigs. Some unoperated animals were treated with capsaicin or 6-hydroxydopamine (6-OHDA) to disrupt primary afferent and sympathetic nerves, respectively. NOS-ir was localized using indirect immunofluorescence. Nerve-mediated relaxations of longitudinal muscle were studied in vitro using standard methods. At 7 weeks after extrinsic denervation there was a 93% increase in the number of NOS-ir myenteric neurons. The number of neurons containing detectable vasoactive intestinal peptide-ir neurons was not changed after extrinsic denervation. Neurogenic relaxations caused by 10, 20 and 50 Hz transmural stimulation were larger in extrinsically-denervated tissues compared to control tissues. The NOS antagonist, nitro-L-arginine (300 microM) inhibited neurogenic relaxations in control and extrinsically-denervated tissues. Capsaicin- but not 6-OHDA-treatment mimicked the effects of extrinsic denervation on NOS-ir and neurogenic relaxations of the longitudinal muscle. Active or passive properties of the longitudinal muscle were unaffected by extrinsic denervation. These data indicate that extrinsic denervation is associated with an increase in the number of myenteric neurons expressing detectable NOS-ir and potentiation of inhibitory transmission to longitudinal muscle. This effect is due to loss of extrinsic sensory nerves.

Afferent projections to the mediodorsal and anterior thalamic nuclei in the cat were studied by means of stereotaxic injection of neuronal tracers (horseradish peroxidase and fluorochromes). Acetylcholinesterase reaction was studied, as well as horseradish peroxidase and NADPH-diaphorase colocation in neuronal bodies which send and receive projections to and from the mediodorsal thalamic nucleus. Based on the connectivity and histochemistry findings, the possibility that prion agents responsible for fatal familial insomnia spread from the mediodorsal and anterior thalamic nuclei through a retrograde pathway is discussed. The possible pathophysiological implication of nitrergic systems in fatal familial insomnia is also considered.

Co-localization of mu-opioid receptor (MOR)-like immunoreactivity (-LI) with substance P (SP)-LI, calcitonin gene-related peptide (CGRP)-LI and nitric oxide synthase (NOS)-LI in the nodose, petrosal and jugular ganglia was examined in the rat by a double immunofluorescence histochemical method. About 0.6%, 41% and 95% of neurons with MOR-LI, respectively, in the nodose, petrosal and jugular ganglia showed SP-LI; about 2%, 51% and 66% of MOR-like immunoreactive neurons displayed CGRP-LI in the nodose, petrosal and jugular ganglia, respectively. In addition, about 59% of MOR-like immunoreactive neurons in the nodose ganglia displayed NOS-LI, whereas no NOS-LI was detected in the petrosal or jugular ganglion. These data provide evidence for co-localization of MOR-LI with SP-LI, CGRP-LI and NOS-LI in the vagal and glossopharyngeal afferent neurons, and suggest that MOR may regulate the release of SP, CGRP and nitric oxide from the visceral primary afferent terminals in the nucleus of the solitary tract of the rat., (Copyright 1998 Elsevier Science B.V.)

Acetylcholine (ACh) has long been considered a neurotransmitter candidate in the efferent vestibular system of mammals. Recently, choline acetyltransferase (ChAT), the synthesizing enzyme for ACh, was immunocytochemically localized in all five end-organs of the rat vestibule (Kong et al. (1994) Hear. Res. 75, 192-200). However, there is little information in the literature concerning the cholinergic innervation in the vestibular periphery of man. In the present study the ultrastructural localization of the ChAT-like immunoreactivity in the human vestibular periphery was investigated in order to reveal the cholinergic innervation in the human vestibular end-organs. A modified method of pre-embedding immunoelectron microscopy was applied. It was found that the ChAT-like immunoreactivity was located in the bouton-type vesiculated nerve terminals in the vestibular neurosensory epithelia of man. These ChAT-like immunostained nerve terminals make synaptic contacts either with afferent chalices surrounding type I vestibular sensory hair cells, or with type II vestibular sensory hair cells. These results show that the ChAT-like immunoreactivity in the human vestibular periphery is confined to the efferent vestibular system. The ChAT-containing efferents innervate both type I hair cells and type II hair cells, making postsynaptic and presynaptic contacts, respectively. This study presents evidence that ACh is a neurotransmitter candidate in the efferent vestibular system of man.

In this report, we have tested the hypothesis that brain-derived neurotrophic factor (BDNF) is an anterograde neurotrophic factor in the CNS and have focused on central noradrenergic neurons that synthesize BDNF. Double-label immunocytochemistry for BDNF and dopamine-beta-hydroxylase (DBH), a marker for noradrenergic neurons, demonstrated that BDNF is partially localized to noradrenergic nerve fibers and terminals in the adult rat brain. To test the functional importance of this anterograde BDNF, we analyzed transgenic mice carrying a DBH-BDNF minigene. Increased synthesis of BDNF in noradrenergic neurons of DBH-BDNF mice caused elevated TrkB tyrosine kinase activation throughout postnatal life in the neocortex, a noradrenergic target region. This afferently regulated increase in TrkB receptor activity led to long-lasting alterations in cortical morphology. To determine whether noradrenergic neuron-expressed BDNF also anterogradely regulated neuronal survival, we examined a second noradrenergic target, neonatal facial motoneurons. One week after axotomy, 72% of facial motoneurons were lost in control animals, whereas only 30-35% were lost in DBH-BDNF transgenic mice. Altogether, these results indicate that BDNF is anterogradely transported to fibers and terminals of noradrenergic neurons, that anterogradely secreted BDNF causes activation of TrkB in target regions, and that this secretion has functional consequences for target neuron survival and differentiation. This presynaptic secretion of BDNF may provide a cellular mechanism for modulating neural circuitry, in either the developing or mature nervous system.

Several lines of evidence suggest that neurotrophin administration may be of some therapeutic benefit in the treatment of peripheral neuropathy. However, a third of sensory neurons do not express receptors for the neurotrophins. These neurons are of small diameter and can be identified by the binding of the lectin IB4 and the expression of the enzyme thiamine monophosphatase (TMP). Here we show that these neurons express the receptor components for glial-derived neurotrophic factor (GDNF) signaling (RET, GFRalpha-1, and GFRalpha-2). In lumbar dorsal root ganglia, virtually all IB4-labeled cells express RET mRNA, and the majority of these cells (79%) also express GFRalpha-1, GFRalpha-2, or GFRalpha-1 plus GFRalpha-2. GDNF, but not nerve growth factor (NGF), can prevent several axotomy-induced changes in these neurons, including the downregulation of IB4 binding, TMP activity, and somatostatin expression. GDNF also prevents the slowing of conduction velocity that normally occurs after axotomy in a population of small diameter DRG cells and the A-fiber sprouting into lamina II of the dorsal horn. GDNF therefore may be useful in the treatment of peripheral neuropathies and may protect peripheral neurons that are refractory to neurotrophin treatment.

Neonatal destruction of the nigrostrial dopaminergic (DA) system with 6-hydroxydopamine leads to serotonergic (5-HT) hyperinnervation of the striatum. However, it is not clear whether this occurs in adult animals. We investigated whether serotonergic sprouting occurs in adult mice subjected to bilateral lesion of the DA system by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The effects of the lesion were evaluated using a new rotarod test and immunohistochemistry. One hundred days after the last MPTP lesion, there was a clear bilateral serotonergic hyperinnervation throughout the striatum. Additionally, those mice showing the highest reductions in striatal tyrosine hydroxylase (TH) immunoreactivity and in rotarod performance showed the highest density of serotonergic innervation (116% increase). The functional consequences of this process in Parkinson's disease and secondary parkinsonism remain to be clarified.

The lectin soybean agglutinin (SBA) from Glycine max binds to small-sized dorsal root ganglion cells and their central terminals in the superficial dorsal horn of the spinal cord. Here we investigated the ability of SBA and SBA conjugated to horseradish peroxidase (SBA-HRP) to trace thin calibre afferents into the spinal cord from a peripheral nerve. Following injection into the sciatic nerve, labelled cells in the dorsal root ganglion were predominantly small-sized but some medium-sized cells were also labelled. Colocalization studies of transported SBA with various neuronal markers showed that all neurons that transported SBA-HRP showed SBA binding, indicating high uptake specificity for the conjugate. 15% were immunoreactive for RT97 indicating that some axons were myelinated, and 54% also expressed binding sites for isolectin B4 from Griffonia simplicifolia, a selective marker for a subpopulation of unmyelinated afferents. With regard to neurotransmitter content, 43% of the SBA cells contained calcitonin gene-related peptide, 33% contained substance P and 2.5% somatostatin. In addition, 3% contained carbonic anhydrase. Centrally, injection of SBA in the sciatic nerve resulted in labelled terminals in somatotopically appropriate regions of laminae I-II of the dorsal horn, and in the gracile nucleus. A few neurons in the dorsal horn were labelled indicating that some transneuronal transport of SBA had occurred. The results show that SBA can be used as a transganglionic tracer to label fine calibre primary afferents that project to laminae I-II of the spinal cord and the gracile nucleus. It appears to label more afferents than isolectin B4, including also a subpopulation of myelinated afferents.

In the substantia nigra pars compacta, many phenylethanolamine-N-methyltransferase immunoreactive (PNMT-ir) terminals as well as serotonin-ir terminals were observed for the first time to be very closely situated to the tyrosine hydroxylase (TH)-ir, aromatic L-amino acid decarboxylase-ir, and GTP cyclohydrolase I (GCH)-ir dopaminergic cells [Nagatsu, I., Arai, R., Sakai, M., Yamawaki, Y., Takeuchi, T., Karasawa, N. and Nagatsu, T., Neurosci. Lett., 224 (1997) 185-188]. Immunohistochemical colocalization of TH with GCH or PNMT in the somata and dendrites of TH-positive neurons in the rostral ventrolateral reticular formation of the medulla oblongata (C1 region, [Hokfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Brain Res., 66 (1974) 235-251]) was proved by a double-labeling immunofluorescence method with a confocal laser-scanning microscope, indicating that the neurons are adrenergic. These results suggest that dopaminergic neurons in the substantia nigra receive PNMT-ir, adrenergic afferents from the C1 region of the medulla oblongata.

In normotensive rats, increased renal pelvic pressure stimulates the release of prostaglandin E and substance P, which in turn leads to an increase in afferent renal nerve activity (ARNA) and a contralateral natriuresis, a contralateral inhibitory renorenal reflex. In spontaneously hypertensive rats (SHR), increasing renal pelvic pressure failed to increase afferent renal nerve activity. The inhibitory nature of renorenal reflexes indicates that impaired renorenal reflexes could contribute to increased sodium retention in SHR. Phorbol esters, known to activate protein kinase C, increase afferent renal nerve activity in Wistar-Kyoto rats (WKY) but not in SHR. We examined the mechanisms involved in the impaired responses to renal sensory receptor activation in SHR. The phorbol ester 4beta-phorbol 12,13-dibutyrate increased renal pelvic protein kinase C activity similarly in SHR and WKY. Increasing renal pelvic pressure increased afferent renal nerve activity in WKY (27+/-2%) but not in SHR. Renal pelvic release of prostaglandin E increased similarly in WKY and SHR, from 0.8+/-0.1 to 2.0+/-0.4 ng/min and 0.7+/-0.1 to 1.4+/-0.2 ng/min. Renal pelvic release of substance P was greater (P<.01) in WKY, from 16.3+/-3.8 to 41.8+/-7.4 pg/min, than in SHR, from 9.9+/-1.7 to 17.0+/-3.2 pg/min. In WKY, renal pelvic administration of substance P at 0.8, 4, and 20 microg/mL increased ARNA 382+/-69, 750+/-233, and 783+/-124% second (area under the curve of afferent renal nerve activity versus time). In SHR, substance P at 0.8 to 20 microg/mL failed to increase ARNA. These findings demonstrate that the impaired afferent renal nerve activity response to increased renal pelvic pressure is related to decreased release of substance P and/or impaired activation of substance P receptors.

Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. Here we report that the transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated in vitro by Aplysia mitogen-activated protein kinase (apMAPK). We have cloned the cDNA encoding apMAPK and show that apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength.

The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model for neurotoxicological studies, including further studies of neurotoxic mechanisms of TMT. Four-week-old cultures, derived from 7-day-old donor rats and grown in serum-free medium, were exposed to TMT (0.5-100 microM) for 24 h followed by 24 h in normal medium. TMT-induced neurodegeneration was then monitored by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux into the culture medium, (c) cellular cobalt uptake as an index of calcium influx, (d) ordinary Nissl cell staining, and (e) immunohistochemical staining for microtubule-associated protein 2 (MAP-2). Cellular degeneration as assessed by densitometric measurements of PI uptake displayed a dose and time-dependent increase, with the following ranking of vulnerability of the hippocampal subfields: FD>CA4>/=CA3c>CA1>CA3ab. This differential neuronal vulnerability observed by PI uptake was confirmed by MAP-2 immunostaining and corresponded to in vivo cell stain observations of rats acutely exposed to TMT. The mean PI uptake of the cultures and the LDH efflux into the medium were highly correlated. The combined results obtained by the different markers indicate that the hippocampal slice culture method is a feasible model for further studies of TMT neurotoxicity.

We have isolated and characterized mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development. Analysis of both loss and gain of gene function conditions during embryonic and postembryonic development revealed specific requirements for neurotactin during axon outgrowth, fasciculation, and guidance. Furthermore, embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, displayed phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes.

To examine the influence of activity-dependent cues on differentiation of primary afferent neurons, we investigated the short- and long-term effects of depolarization and calcium influx on expression of transmitter traits in sensory ganglion cell cultures. We focused on expression of tyrosine hydroxylase (TH), a marker for dopaminergic neurons, in developing petrosal ganglion (PG), nodose ganglion, and dorsal root ganglion neurons grown in the presence or absence of depolarizing concentrations of KCl. Exposure to 40 mM KCl increased the proportion of TH-immunoreactive neurons in all three ganglia in a developmentally regulated manner that corresponded to the temporal pattern of dopaminergic expression in vivo. PG neurons, for example, were most responsive to elevated KCl on embryonic day 16.5 (E16.5), the age at which the dopaminergic phenotype is first detectable in vivo. However, KCl was relatively ineffective at increasing TH expression in neonatal PG, indicating a critical period for induction of this phenotype by depolarization. Detailed analysis of TH induction in PG neurons demonstrated that, although N-type calcium channels carried the majority of the high voltage-activated calcium current, only L-type calcium channel blockade inhibited the effect of elevated KCl. Further studies revealed that after removal of high KCl, neurons remained sensitized to subsequent stimulation for >1 week. Specifically, cultures exposed to KCl beginning on E16.5 (the conditioning stimulus), then returned to control medium, and subsequently re-exposed to elevated KCl after 9 d (the test stimulus) contained fourfold more TH-positive neurons than did cultures exposed to the test stimulus alone. Moreover, blockade of L-type calcium channels during the conditioning stimulus completely abolished long-term potentiation of the TH response to elevated KCl. These findings demonstrate a novel role for L-type calcium channels in activity-dependent plasticity of transmitter expression in sensory neurons and indicate that exposure to depolarizing stimuli during early development may alter neuronal response properties at later ages.

Adrenal chromaffin cells produce analgesic substances, such as catecholamines and enkephalins, and intrathecal (i.t.) implantation of either allografted adrenal tissue or xenogenic chromaffin cells produce antinociception in animals. We evaluated the analgesic effect of bovine chromaffin cells in a model of central pain in which rats exhibit chronic allodynia-like behavior after photochemically induced ischemic spinal cord injury. Bovine chromaffin cells or endothelial cells were injected i.t. onto the lumbar spinal cord and their effects on mechanical and cold allodynia-like behaviors were studied for up to 8 weeks. The chronic allodynia-like behavior was stable for months without signs of remission and i.t. implantation of human endothelial cells did not alleviate the chronic allodynia-like behavior for the entire observation period. In contrast, 2 weeks after i.t. implantation of bovine chromaffin cells, the mechanical allodynia was abolished in the spinally injured rats, and the enhanced response to cold stimuli was significantly reduced. The overall effects were significant up to 8 weeks after i.t. implantation, although the anti-allodynic effect decreased towards the end of the observation period. No signs of side-effects were noted after i.t. implantation. The allodynia-like state was temporarily restored by naloxone (0.5 mg/kg) or phentolamine (0.3 mg/kg) injected intraperitoneally. Immunohistochemical examination revealed that tyrosine hydroxylase (TH)-positive chromaffin cells could be identified adjacent to the spinal cord up to 4 weeks after i.t. implantation, whereas at 8 weeks the TH-positive cells were sparse. It is concluded that bovine chromaffin cells stay viable in rat spinal cord for a considerable period of time after i.t. administration and alleviate chronic allodynia-like behavior in spinally injured rats, possibly through activation of opioid and alpha-adrenoceptors. The present results further document a new therapeutic approach for the treatment of chronic neuropathic pain.

The aim of this study was to reveal mucosal projections of myenteric neurones in the stomach by using the neuronal tracer DiI (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorat) in combination with immunohistochemical detection of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS). The mucosal application of one DiI coated glass bead (diameter 50-100 microm) labelled on average 167 +/- 58 neurones in the myenteric plexus (n = 9 preparations). Most labelled cells were ChAT-positive (74%), the remaining cells were NOS-positive (n = 6). The vast majority of ascending DiI-labelled neurones were ChAT-positive (94%), whereas most descending neurones were NOS-positive (75%). ChAT- and NOS-positive fibers were demonstrated in the mucosa. Results suggest that ascending and descending myenteric neuronal pathways releasing acetylcholine and nitric oxide, respectively, are involved in control of mucosal functions in the stomach.

The role of cAMP and protein kinase A (PKA) in nociceptor sensitisation has been studied in frog skin in vitro. Multifibre nerve responses during noxious heating were enhanced by adding forskolin, an agent that elevates cAMP. H-89, a PKA antagonist, blocked the increased responses due to forskolin, but did not affect baseline responses. Thus, cAMP appears to act via PKA when sensitising nociceptors in frog skin.

Metabotropic glutamate receptors (mGluRs), which couple glutamate to second messengers, have important roles in the regulation of movement by the basal ganglia. We used two polyclonal antisera to mGluR1a and mGluR2/3 and confocal laser microscopy to investigate the localization of these receptors in the basal ganglia of the rat. The mGluRs were visualized in combination with an antibody to tyrosine hydroxylase (TH), an antibody to microtubule-associated protein 2 (MAP2, a dendritic marker), or SV2 (an antibody to a protein associated with presynaptic terminals). In the neostriatum, punctate mGluR1a immunoreactivity (ir) was present in the neuropil. This staining did not colocalize with MAP2-ir or SV2-ir and was not altered by decortication or unilateral 6-hydroxydopamine (6-OHDA) lesions. In the globus pallidus and substantia nigra pars reticulata, however, mGluR1a-ir was tightly clustered along large MAP2-ir dendrites. In contrast to the variations in mGluR1a-ir staining, similar punctate neuropil mGluR2/3-ir staining was observed within all basal ganglia structures. In the neostriatum, these puncta were abundant; unlike mGluR1a, many mGluR2/3-ir puncta colocalized with SV2-ir, and striatal mGluR2/3-ir puncta were markedly reduced in number after decortication. Neither mGluR1a-ir nor mGluR2/3-ir could be detected in TH-ir soma within substantia nigra pars compacta, or in TH-ir striatal terminals. Overall, our observations suggest that mGluR1a and mGluR2/3 receptors have distinct cellular localizations in different components of the basal ganglia circuitry and are likely to subserve distinct functions. Our data support the presence of mGluR2/3 on the terminals of corticostriatal afferents, where they may regulate glutamate release. In contrast, mGluR1a appears to be a postsynaptic receptor of neurons in the neostriatum, globus pallidus, and substantia nigra pars reticulata.

The trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN) neurons are involved in the transmission of orofacial sensory information. The presence of nitric oxide (NO), a putative neurotransmitter substance in the nervous system, was examined in the cat TrG and MTN using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry. In the TrG, where the majority of the trigeminal primary afferent perikarya are located, most of the intensely NADPH-d/ NOS-stained cells were small in size and distributed randomly throughout the ganglion. The medium-sized neurons were moderately stained. A plexus of pericellular varicose arborizations around large unstained ganglion cells and densely stained fibers in-between could also be observed. In the caudal part of the MTN, both NADPH-d activity and NOS immunoreactivity was present in MTN neurons. In addition, a few scattered NADPH-d/NOS-containing neurons were found in the mesencephalic-pontine junction part of the nucleus. In contrast, only nerve fibers and their terminals were present at a more rostral level in the mid- and rostral MTN. MTN neuronal perikarya were enveloped in fine basket-like NADPH-d/ NOS-positive networks. Differential expression patterns of NOS and its marker NADPH-d suggest that trigeminal sensory information processing in the cat MTN is controlled by nitrergic input through different mechanisms. We introduce the concept that NO can act as a neurotransmitter in mediating nociceptive and proprioceptive information from periodontal mechanoreceptors but may also participate in modulating the activity of jaw-closing muscle afferent MTN neurons.

In situ expression of c-fos observed in response to phenylephrine (PE)-induced hypertension provided a basis for characterizing the organization and neurotransmitter specificity of neurons at nodal points of medullary baroreflex circuitry. Sustained hypertension induced by a moderate dose of PE provoked patterns of c-fos mRNA and protein expression that conformed in the nucleus of the solitary tract (NTS) to the termination patterns of primary baroreceptor afferents and in the caudal ventrolateral medulla (CVLM) to a physiologically defined depressor region. A majority of barosensitive CVLM neurons concurrently displayed markers for the GABAergic phenotype; few were glycinergic. Phenylephrine-sensitive GABAergic neurons that were retrogradely labeled after tracer deposits in pressor sites of the rostral ventrolateral medulla (RVLM) occupied a zone extending approximately 1.4 mm rostrally from the level of the calamus scriptorius, intermingled partly with catecholaminergic neurons of the A1 and C1 cell groups. By contrast, barosensitive neurons of the NTS were found to be phenotypically complex, with very few projecting directly to the RVLM. Extensive colocalization of PE-induced Fos-IR and markers for the nitric oxide phenotype were seen in a circumscribed, rostral, portion of the baroreceptor afferent zone of the NTS, whereas only a small proportion of PE-sensitive neurons in the NTS were found to be GABAergic. PE treatment parameters have been identified that provide a basis for defining and characterizing populations of neurons at the first station in the central processing of primary baroreceptor input and at a key inhibitory relay in the CVLM.

As part of a recent program on the evolution of somatosensory systems in vertebrates, the cytoarchitecture, chemoarchitecture, and fiber connections of the caudal rhombencephalic alar plate were studied in the ribbed newt, Pleurodeles waltl. This part of the brain stem includes ill-defined dorsal column and lateral cervical nuclei. A cytoarchitectonic analysis revealed that the caudal medullary alar plate consists of an inner and an outer cell layer. The dorsomedial part of the outer cell layer at the obex level contains the dorsal column nucleus (DCN), whereas its ventrolateral part constitutes the lateral cervical nucleus (LCN). NADPH-diaphorase histochemistry and calbindin D-28k immunohistochemistry clearly delineate the main components of the compact inner cell layer, i.e. the nucleus of the solitary tract dorsally and the nucleus of the descending trigeminal tract ventrally. Neither NADPH-diaphorase-labeled nor calbindin D-28k positive neurons were observed in the DCN and LCN. With anterograde and retrograde tracing, the DCN and LCN were further delineated. Labeling of ascending dorsal root projections showed that the dorsal column and the DCN are somatotopically arranged: lumbar primary afferent fibers terminate on medial DCN neurons, whereas cervical primary afferent fibers terminate on lateral DCN neurons. The LCN is densely innervated by the dorsolateral funiculus. Retrograde tracing showed extensive, predominantly contralateral projections of both the DCN and LCN to the torus semicircularis and the ventral thalamus. These data show that even in the poorly segregated caudal rhombencephalic alar plate of urodeles a DCN and LCN can be distinguished with afferent and efferent projections comparable to those in anurans and other terrestrial vertebrates.

Previous studies have shown that the guinea pig inferior mesenteric artery receives spinal sensory vasodilatory innervation, which can be activated by colon distention and electrical nerve stimulation. In the present study, we investigated the hypotheses that nitric oxide synthase (NOS) is present in guinea pig primary sensory neurons in the dorsal root ganglion (DRG) and in nerve fibers surrounding the mesenteric arteries, and that nitric oxide (NO) is a sensory neurotransmitter in the inferior mesenteric artery in vitro. Double-labeling immunohistochemistry showed that neuronal NOS-IR was found in 12% of cells of guinea pig thoracic and lumbar DRGs; in 95.1% of these cells it was colocalized with substance P (SP), and SP immunoreactivity (SP-IR) was present in 23% of cells of the same DRGs. Neuronal NOS-like immunoreactivity was localized in nerve fibers surrounding guinea pig mesenteric artery and 25% of them were double stained with SP-IR. Endothelium-denuded inferior mesenteric artery preparations in vitro were incubated with guanethidine (30 microns, 30 min) and pre-contracted with methoxamine (30 microns). The NO donors, sodium nitroprusside (1 micron) and L-nitrosocysteine (300 microns), produced 91.0 +/- 5.5 and 90.4 +/- 9.6% vasodilatation of total vasodilatation in the vessel segments, respectively, which was capsaicin- or tetrodotoxin-insensitive. Repetitive electrical field stimulation of the preparations produced a frequency-dependent vasodilatation which was reduced by pretreatment with capsaicin or by tetrodotoxin (10 microns). The NOS inhibitor N omega-nitro-L-arginine (L-NNA) (100 microns, 30 min) diminished the nerve-evoked vasodilatation from 41.8 +/- 8.4 to 21.4 +/- 9.7% at 2 Hz and from 50.8 +/- 5.6 to 19.0 +/- 7.3% at 15 Hz (P < 0.05), whereas NG-nitro-L-arginine methyl ester (L-NAME, 100 microns-1 mM) did not significantly inhibit the relaxation. The stereo isomer nitro-D-arginine (D-NNA, 100 microns, 30 min) was ineffective. These findings suggest that NO is a neurotransmitter released from primary sensory nerves which mediates vasodilation in vitro.

Steroids which are synthesized within the nervous system, such as progesterone, have been termed 'neurosteroids'. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3beta-hydroxysteroid dehydrogenase and can convert [3H]pregnenolone to [3H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [3H]pregnenolone, they produce 5-10 times more [3H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5alpha-dihydroprogesterone, but unlike Schwann cells, they do not produce 3alpha,5alpha-tetrahydroprogesterone, a potent positive allosteric modulator of gamma-aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [3H]pregnenolone to progesterone and its 5alpha-reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.

The cross-sectional areas and succinate dehydrogenase activities of L5 dorsal root ganglion neurons in rats were determined after 14 days of spaceflight and after nine days of recovery. The mean and distribution of the cross-sectional areas were similar to age-matched, ground-based controls for both the spaceflight and for the spaceflight plus recovery groups. The mean succinate dehydrogenase activity was significantly lower in spaceflight compared to aged-matched control rats, whereas the mean succinate dehydrogenase activity was similar in age-matched control and spaceflight plus recovery rats. The mean succinate dehydrogenase activity of neurons with cross-sectional areas between 1000 and 2000 microns2 was lower (between 7 and 10%) in both the spaceflight and the spaceflight plus recovery groups compared to the appropriate control groups. The reduction in the oxidative capacity of a subpopulation of sensory neurons having relatively large cross-sectional areas immediately following spaceflight and the sustained depression for nine days after returning to 1 g suggest that the 0 g environment induced significant alterations in proprioceptive function.

Intraperitoneal (i.p.) administration of sulfated CCK octapeptide (CCK-8S) has been shown to induce changes in neuronal activity in the nucleus of the solitary tract (NTS) and area postrema (AP), sensory parts of the dorsal vagal complex (DVC), and in the paraventricular nucleus of the hypothalamus (PVN), as determined by activation of c-fos expression. Whether peripheral CCK influences neuronal activity in the locus coeruleus (LC)/subcoeruleus nucleus (SC) was investigated in awake rats at intraperitoneal (i.p.) injection of CCK-8S by c-Fos immunohistochemistry. CCK-8S i.p. (25, 50, and 100 micrograms/kg, respectively) dose-dependently increased the average number of c-Fos-LI-positive cells/section in the LC/SC by the factor 5.9, 8.2, and 11.7, respectively. Pretreatment with the CCK-A receptor antagonist MK-329 (devazepide; 1 mg/kg and 2 mg/kg i.p.) reduced the CCK-induced increase in c-fos expression in the LC/SC by 54% and 75%, respectively; the CCK-B receptor antagonist L-365,260 had no effect. Perivagal capsaicin pretreatment diminished the CCK-induced increase in the number of c-Fos-LI-positive cells in the LC/SC by 65%. In comparison, the CCK-A antagonist devazepide (1 mg/kg and 2 mg/kg i.p.) reduced the increase in c-fos expression by 76% and 88% in the PVN, 69% and 88% in the NTS, 86% and 83%, respectively, in the AP. Capsaicin diminished the CCK-induced increase in c-Fos-LI-positive cells in the PVN by 64%, in the NTS by 60%, but in the AP only by 25%. Immunostaining against the nuclear antigen c-Fos and the cytoplasmatic antigen tyrosine hydroxylase (TH) showed that 40% of all c-Fos-LI-positive cells in the LC/SC were TH-LI positive at 25 micrograms CCK/kg. The data indicate that CCK-8S i.p. induces modulation of neuronal activity in the LC/SC, DVC and PVN predominantly by peripheral action of CCK-A receptors and capsaicin-sensitive vagal afferents. These findings suggest that the LC/SC is involved in CNS-mediated regulatory influences of peripheral CCK.