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The peptidergic neurons play important roles such as neuromodulatory and neuroendocrine functions in the central nervous system. However, our knowledge about the organization and the function of the peptidergic neuromodulator systems is still very poor. The terminal nerve GnRH peptidergic neurons of a teleost, the dwarf gourami (Colisa lalia), serve as an excellent model system for such study. The cell bodies are large and make up a tight cell cluster, and the easy access to the cell bodies on the ventral surface of the brain makes the electrophysiological measurements in a precisely controlled manner. Here we show direct evidence to demonstrate the electrical coupling and the synchronization of the neural firing activity among the terminal nerve GnRH neurons by using the double patch-clamp recording technique. The electrical coupling coefficient was strong enough (ranged from 0.083 to 0.370) to synchronize spontaneous firings of GnRH neurons in the cluster. A model, in which the firings in the cluster occur within a small time window (dozens of milliseconds), was verified by using the serial loose-seal extracellular patch-clamp recordings and the cross-correlogram analysis. The present findings provide several insights for understanding the physiological mechanisms and functional significance of synchronized activities in the peptidergic and/or aminergic neuromodulator system as well as in the peptidergic neuroendocrine cells.

Purpose: To determine the effect of aging on corneal epithelial nerve density in an animal model., Methods: Corneal whole mounts from rats aged 6, 12, 18, and 24 months were stained immunohistochemically with antisera against the pan-neuronal marker neurotubulin. Epithelial nerve terminals and subbasal nerves in standardized 1-mm(2) central and peripheral zones from each cornea were drawn using a drawing tube attached to a light microscope. Images were scanned, and nerve densities were calculated as the percentage of each 1-mm(2) area occupied by nerves. The diameters of subbasal nerves in 6- and 24-month old animals were measured. Subbasal nerve vortices were analyzed qualitatively with reference to location, morphologic appearance, and directionality., Results: Epithelial nerve terminal density decreased by approximately 50% between 6 and 24 months. The rate of decline was roughly linear and similar in both central and peripheral cornea. In contrast, subbasal nerve density increased by more than 50% between 6 and 24 months in both central and peripheral cornea. The mean diameter of corneal subbasal nerves decreased approximately 30% (0.384 microm vs. 0.271 microm) between 6 and 24 months. The morphologic appearance and directionality of the subbasal nerve vortex demonstrated considerable interanimal variability and did not correlate with age., Conclusions: Rat corneal nerve terminal density decreases, but corneal subbasal nerve density increases, as a function of age. The age-related loss of nerve terminal density seen in the rat cornea is in keeping with the decreased corneal sensitivity reported in elderly humans and may contribute to the pathogenesis of dry eye disease in aged persons.

Despite intensive study, our understanding of the neuronal structures responsible for transducing the broad spectrum of environmental energies that impinge upon the skin has rested on inference and conjecture. This major shortcoming motivated the development of ex vivo somatosensory system preparations in neonatal mice in the hope that their small size might allow the peripheral terminals of physiologically identified sensory neurons to be labeled intracellularly for direct study. The present report describes the first such study of the peripheral terminals of four slowly adapting type I low-threshold mechanoreceptors (SAIs) that innervated the back skin of neonatal mice. In addition, this report includes information on the central anatomy of the same SAI afferents that were identified peripherally with both physiological and anatomical means, providing an essentially complete view of the central and peripheral morphology of individual SAI afferents in situ. Our findings reveal that SAIs in neonates are strikingly adult-like in all major respects. Afferents were exquisitely sensitive to mechanical stimuli and exhibited a distinctly irregular, slowly adapting discharge to stimulation of 1-4 punctate receptive fields in the skin. Their central collaterals formed transversely oriented and largely nonoverlapping arborizations limited to regions of the dorsal horn corresponding to laminae III-V. Their peripheral arborizations were restricted entirely within miniaturized touch domes, where they gave rise to expanded disc-like endings in close apposition to putative Merkel cells in basal epidermis. These findings therefore provide the first direct confirmation of the functional morphology of this physiologically unique afferent class., ((c) 2007 Wiley-Liss, Inc.)

Talpid moles are small insectivores that live in dark underground tunnels. They depend heavily on touch to navigate and find food. Most species have an array of complex epidermal sensory structures called Eimer's organs that cover the tip of the nose. In this study, the anatomy of Eimer's organ was examined in the coast mole and star-nosed mole by using the fluorescent styryl pyridinium dye AM1-43 and immunocytochemical staining for neurofilament 200 and substance P. In addition, DiI was used to label neural components of Eimer's organ. AM1-43 labeled all of the Eimer's organ receptors after systemic injection, suggesting a role in mechanotransduction. Immunostaining with neurofilament 200 and substance P labeled distinct subtypes of sensory fibers. Substance P labeled a group of free nerve endings along the outer edge of Eimer's organ, indicating a nociceptive role for these fibers. In contrast, neurofilament 200 labeled a more central set of nerve endings, suggesting that these fibers function as low-threshold mechanoreceptors. By labeling subsets of trigeminal afferents distant from the receptor array with DiI, we revealed innervation patterns indicating that one afferent supplies the outer, substance P-positive set of free nerve endings, whereas several afferents differentially innervate the central free nerve endings. Our results suggest that the free nerve endings innervating Eimer's organ are largely mechanosensitive and may play an important role in the rapid sensory discrimination observed in these species.

Descriptions of morphologically well-defined sensory airway receptors are sparse, in contrast to the multiplicity of airway receptors that have been identified electrophysiologically. The present study aimed at further determining the location, morphology and neurochemical coding of subepithelial receptor-like structures that have been sporadically reported in the wall of large diameter airways. The results were compared with those obtained for pulmonary neuroepithelial bodies (NEBs), which are complex intraepithelial sensory airway receptors. Multiple immunocytochemical staining showed branching laminar subepithelial receptor-like endings, which were found to intercalate in the smooth muscle layer of intrapulmonary conducting airways in rats. Because of the consistent intimate association with the airway smooth muscle, the laminar terminals will further be referred to as 'smooth muscle-associated airway receptors (SMARs)'. SMARs were characterised by their Na(+)/K(+)-ATPase alpha3, vesicular glutamate transporter 1 (VGLUT1) and VGLUT2-immunoreactivity, expression of the ATP receptor P2X(3), and the presence of calcium-binding proteins. Nerve fibres giving rise to SMARs were shown to be myelinated and to have a vagal origin. Interestingly, the neurochemical coding and receptor-like appearance of SMARs appeared to be almost identical to at least part of the complex vagal sensory terminals in NEBs. Intraepithelial nerve endings in pulmonary NEBs were indeed also shown to originate from myelinated vagal afferent nerve fibres, and to express Na(+)/K(+)-ATPase alpha3, VGLUT1, VGLUT2, P2X(3) and calcium-binding proteins. Since several of the latter proteins have been reported as markers for mechanoreceptor terminals in other organs, both SMARs and the vagal nodose nerve terminals in NEBs seem good candidates to represent the morphological counterparts of at least subsets of the extensive population of physiologically characterised myelinated vagal airway mechanoreceptors. The observation that SMARs and NEBs are regularly found in each other's immediate neighbourhood, and the very similar characteristics of their nerve terminals, point out that the interpretation of electrophysiological data based on 'local' stimuli should be made with great caution.

During the last decade a wealth of new information about the properties of the exocytotic fusion pore is changing our current view of exocytosis. The exocytotic fusion pore, a necessary stage before the full merging of the vesicle membrane with the plasma membrane, is becoming a key cellular structure that might critically control the amount of neurotransmitter released into the synaptic cleft and that can be subjected to control by second messengers and phosphorylated proteins. Fusion pores form, expand to fully merge membranes, or can close leaving an intact and identical synaptic vesicle in place for a new round of exocytosis. Transient formation of fusion pores is the mechanistic representation of the "kiss-and-run" hypothesis of transmitter release and offers new alternatives for synaptic vesicle recycling besides to the classical mechanism mediated by clathrin coat endocytosis. For vesicle recycling transient fusion pores ensures a fast mechanism for maintaining an active pool of synaptic vesicles. The size reached by transient fusion pores and the time spent on the open state can determine the release of subquantal synaptic transmission, which could be a mechanism of synaptic potentiation. In this review we will described the electrophysiological and fluorescence methods that contribute to further explore the biophysical properties of the exocytotic fusion pore and the relevant experiments obtained by these methods.

Purpose: To measure subbasal nerve density and orientation in normal human corneas across a broad age range., Methods: Sixty-five normal corneas of 65 subjects were examined by using tandem scanning confocal microscopy. Ages of subjects ranged from 15 to 79 years (mean 46 +/- 19 years), with 5 subjects from each hemidecade. Subbasal nerve fiber bundles appeared as bright, well-defined linear structures in confocal images of the central cornea. Images from 3 to 8 scans per eye (mean 4.6 +/- 1.8 scans) were randomly presented to a masked observer for analysis. The mean subbasal nerve density (total nerve length [microm] within a confocal image [area = 0.166 mm]), the mean nerve number per confocal scan, and the mean nerve orientation were determined by using a custom software program. Correlations between age and nerve density and age and nerve orientation were assessed by using Pearson correlation coefficients., Results: The subbasal nerve plexus was visible in the central cornea of all subjects. The mean subbasal nerve density was 8404 +/- 2012 microm/mm (range 4735 to 14,018 microm/mm). The mean subbasal nerve number was 4.6 +/- 1.6 nerves (range 1 to 8 nerves). The mean subbasal nerve orientation was 94 +/- 16 degrees (range 58 to 146 degrees). There was no correlation between age and subbasal nerve density (r = 0.21, P = 0.09) or between age and subbasal nerve orientation (r = -0.19, P = 0.12)., Conclusion: The density and orientation of the subbasal nerve plexus in the central human cornea does not change with age.

Palisade endings form a cuff of nerve terminals around the tip of muscle fibres. They are found only in extraocular muscles, but no definite evidence for their role in eye movements has been established. Palisade endings have been reported in all species so far investigated except the rat. In this study we demonstrate that antibodies against SNAP-25, the synaptosomal associated protein of 25 kDa, reliably visualize the complete motor, sensory and autonomic innervation of the extraocular muscles in human, monkey and rat. The SNAP-25 antibody can be combined with other immunofluorescence procedures, and is used here to study properties of palisade endings. With SNAP-25 immunolabelling putative palisade endings are identified in the rat for the first time. They are not well branched, but fulfil several criteria of palisade endings, being associated with non-twitch fibres as shown by double labelling with 'myosin heavy chain slow-twitch' antibodies. The putative palisade endings of the rat lack alpha-bungarotoxin binding, which implies that these synapses are sensory. If palisade endings are sensory then they could function as an eye muscle proprioceptor. They seem to be a general feature of all vertebrate eye muscles, unlike the other two extraocular proprioceptors, muscle spindles and Golgi tendon organs, the presence of which varies widely between species.

Purpose: To analyze palisade endings in cat extraocular muscles (EOMs) and to clarify whether these EOM-specific organs are sensory or motor., Methods: Twelve cats aged between 1 and 16 years were analyzed. Whole EOM tendons were immunostained using four different combinations of triple fluorescence labeling. Triple labeling included antibodies against choline acetyltransferase (ChAT), neurofilament, synaptophysin, and alpha-bungarotoxin. Preparations were examined by confocal laser scanning microscopy. ChAT-labeled EOMs were also analyzed by immunoelectron microscopy. Three-dimensional reconstructions were made of palisade endings., Results: Palisade endings were found in the distal and proximal myotendinous regions of cat EOMs. These endings arose from thin nerve fibers coming from the muscle and extending into the tendon. There, the nerve fibers turned back 180 degrees to divide into terminal branches around the muscle fiber tips. Terminal branches established numerous contacts with the tendon attached to the muscle fiber tip and only a few contacts with the muscle fiber. Often, nerve fibers forming palisade endings on muscle fiber tips were observed to establish multiple motor contacts on muscle fibers outside palisade endings. Three-dimensional reconstructions depicted the complex morphology of the palisade endings. All nerve fibers supplying palisade endings stained positively for ChAT and neurofilament. All nerve terminals in palisade endings were ChAT and synaptophysin positive. Only neuromuscular contacts in palisade endings were positive for alpha-bungarotoxin, as well., Conclusions: This study provides evidence that palisade endings in cat EOMs have effector function. The findings may be of significance for strabismus surgery because palisade endings are also found in human EOMs.

In the livers of humans, cats, guinea pigs, and tupaia, nerve endings are distributed all over the hepatic lobules. Nerve endings in the intralobular spaces are localized mainly in the Disse spaces and are oriented toward the hepatic stellate cells (HSCs), sinusoidal endothelial cells, and hepatocytes. They are especially closely related to HSCs. Various neurotransmitters such as substance P exist in the nerve endings. In addition, HSCs possess endothelin (ET) and adrenergic receptors and contract in response to the corresponding agonists. In contrast, nitric oxide (NO) inhibits the contraction of HSCs. HSCs thus appear to be involved in the regulation of hepatic sinusoidal microcirculation by contraction and relaxation. In the cirrhotic liver, intralobular innervation is decreased, but ET, ET receptors, and NO are overexpressed in the HSCs. These findings indicate that HSCs in cirrhotic liver may play an important role in the sinusoidal microcirculation through agents such as ET or NO rather than through intralobular innervation., (Copyright 2004 Wiley-Liss, Inc.)

Vagal mechanoreceptors to the guinea-pig oesophagus, recorded extracellularly, in vitro, fired spontaneously at 3.3 +/- 0.2 Hz, (n = 75, from 57 animals), and had low thresholds to circumferential stretch. In this study, we have investigated whether mechanotransduction by intraganglionic laminar endings (IGLEs) directly relies on mechano-gated ion channels, or whether it is due to chemical activation by neurotransmitters (glutamate or ATP) released from other cells during mechanical distortion. Rapid distortion of focal transduction sites (IGLEs) evoked action potentials with a latency of < 10 ms. Antagonists to ionotropic (AP5, memantine and 6,7-dinitroquinoxaline-2,3-dione (DNQX)) and metabotropic glutamate receptors (N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) and (RS)-a-methyl-4-phosphono-phenylglycine (MPPG)) did not affect mechano-transduction. Glutamate, NMDA and the selective mGluR group II and III agonists, (2R, 4R)-APDC and L-AP4, had no effect on spontaneous or stretch-induced firing. The P2X purinoreceptor agonist, alpha,beta-methylene ATP, caused concentration-dependent excitation of vagal mechanoreceptors (EC50 = 22.2 microM) which was blocked by the non-selective P2 antagonist PPADS (30 microM). On its own, PPADS affected neither stretch-induced firing nor spontaneous firing. Neither Ca(2+)-free solution (1 mM EDTA, 3.6 mM Mg(2+)) solution nor Cd(2+) (100 microM) blocked stretch-induced firing. Thus chemical transmission is not involved in activation of vagal mechanoreceptors. The blocker of stretch-activated channels, Gd(3+) (300 microM), did not inhibit stretch-induced firing. However, benzamil (100 microM) significantly inhibited spontaneous and distension-evoked firing in a stretch-dependent manner; proportionally greater inhibition was seen with larger stretches. The results suggest that IGLEs of vagal tension receptors directly transduce mechanical stimuli probably via benzamil-sensitive, Gd3+-insensitive, stretch-activated ion channels, and that chemical transmission is not involved in transduction.

Auditory afferents terminating as "large myelinated club endings" on goldfish Mauthner cells are identifiable "mixed" (electrical and chemical) synaptic terminals that offer the unique opportunity to correlate physiological properties with biochemical composition and specific ultrastructural features of individual synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we demonstrate that gap junctions at these synapses contain connexin35 (Cx35). This connexin is the fish ortholog of the neuron-specific human and mouse connexin36 that is reported to be widely distributed in mammalian brain and to be responsible for electrical coupling between many types of neurons. Similarly, connexin35 was found at gap junctions between neurons in other brain regions, suggesting that connexin35-mediated electrical transmission is common in goldfish brain. Conductance of gap junction channels at large myelinated club endings is known to be dynamically modulated by the activity of their colocalized glutamatergic synapses. We show evidence by confocal microscopy for the presence of the NR1 subunit of the NMDA glutamate receptor subtype, proposed to be a key regulatory element, at these large endings. Furthermore, we also show evidence by FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor is present at postsynaptic densities closely associated with gap junction plaques containing Cx35 at mixed synapses across the goldfish hindbrain. Given the widespread distribution of electrical synapses and glutamate receptors, our results suggest that the plastic properties observed at these identifiable junctions may apply to other electrical synapses, including those in mammalian brain.

Effects of a single local dose of gentamicin upon sensory and nonsensory cells throughout the cochlea were assessed by changes in immunostaining patterns for a broad array of functionally important proteins. Cytochemical changes in hair cells, spiral ganglion cells, and cells of the stria vascularis, spiral ligament, and spiral limbus were found beginning 4 days post administration. The extent of changes in immunostaining varied with survival time and with cell type and was not always commensurate with the degree to which individual cell types accumulated gentamicin. Outer hair cells, types I and II fibrocytes of the spiral ligament, and fibrocytes in the spiral limbus showed marked decreases in immunostaining for a number of constituents. In contrast, inner hair cells, type III fibrocytes and root cells of the spiral ligament, cells of the stria vascularis, and interdental cells in the spiral limbus showed less dramatic decreases, and in some cases they showed increases in immunostaining. Results indicate that, in addition to damaging sensory cells, local application of gentamicin results in widespread and disparate disruptions of a variety of cochlear cell types. Only in the case of ganglion cells was it apparent that the changes in nonsensory cells were secondary to loss or damage of hair cells. These results indicate that malfunction of the ear following gentamicin treatment is widespread and far more complex than simple loss of sensory elements. The results have implications for efforts directed toward detecting, preventing, and treating toxic effects of aminoglycosides upon the inner ear.

The characteristics of the different populations of sensory nerve terminals that selectively contact pulmonary neuroepithelial bodies (NEBs) in rat lungs were investigated after chemical denervation with capsaicin and compared with control lungs. Vagal calbindin D28k and P2X(3) purinoceptor immunoreactive (IR) afferent nerve terminals contacting NEBs appeared to have their origin in the nodose ganglion. Thick CB/P2X(3)-IR nerve fibers were seen to be myelinated and to lose their myelin sheaths just before branching and protruding intraepithelially between the NEB cells. This vagal sensory component of the innervation of NEBs was not affected by capsaicin nor expressed capsaicin receptors (vanilloid receptor subtype 1). A second sensory nerve fiber population that selectively innervates pulmonary NEBs in the rat lung consists of thin unmyelinated nonvagal substance P/calcitonin gene-related peptide IR nerve fibers, contacting mainly the basal pole of pulmonary NEBs, and having their origin in dorsal root ganglia. In concordance with vanilloid receptor 1 expression on these nerve terminals, the spinal sensory substance P/calcitionin gene-related peptide-IR component of the innervation of NEBs was depleted by systemic capsaicin treatment. The complex sensory innervation pattern of pulmonary NEBs characterized in the present study strongly suggests that, physiologically, pulmonary NEBs represent a group of intraepithelial receptors that may be able to accommodate various local and central reflex actions, in relation to both chemo- and mechanosensory stimuli.

The vanilloid receptor VR1 is a nonselective cation channel activated by capsaicin as well as increases in temperature and acidity, and can be viewed as molecular integrator of chemical and physical stimuli that elicit pain. The distribution of VR1 receptors in peripheral and central processes of rat primary vagal afferent neurons innervating the gastrointestinal tract was investigated by immunohistochemistry. Forty-two percent of neurons in the nodose ganglia retrogradely labeled from the stomach wall expressed low to moderate VR1 immunoreactivity (VR1-IR). VR1-IR was considerably lower in the nodose ganglia as compared to the jugular and dorsal root ganglia. In the vagus nerve, strongly VR1-IR fibers ran in separate fascicles that supplied mainly cervical and thoracic targets, leaving only weakly VR1-IR fibers in the subdiaphragmatic portion. Vagal afferent intraganglionic laminar endings (IGLEs) in the gastric and duodenal myenteric plexus did not express VR1-IR. Similarly, VR1-IR was contained in fibers running in perfect register with vagal afferents, but was not colocalized with horseradish peroxidase in the same varicosities of intramuscular arrays (IMAs) and vagal afferent fibers in the duodenal submucosa anterogradely labeled from the nodose ganglia. Only in the gastric mucosa did we find evidence for colocalization of VR1-IR in vagal afferent terminals. In contrast, many nerve fibers coursing through the myenteric and submucosal plexuses contained detectable VR1-IR, the majority of which colocalized calcitonin gene-related peptide immunoreactivity. In the dorsal medulla there was a dense plexus of VR1-IR varicose fibers in the commissural, dorsomedial and gelatinosus subnuclei of the medial NTS and the lateral aspects of the area postrema, which was substantially reduced, but not eliminated on the ipsilateral side after supranodose vagotomy. It is concluded that about half of the vagal afferents innervating the gastrointestinal tract express low levels of VR1-IR, but that presence in most of the peripheral terminal structures is below the immunohistochemical detection threshold.

To assess whether vagal afferents in the gastrointestinal (GI) tract mature postnatally and differentiate at different rates, potentially reflecting the changing functional requirements of weaning and independence, the vagal afferent innervation of the stomach was inventoried in pre-weanling and adult rats. Wheatgerm agglutinin-horseradish peroxidase was injected into the nodose ganglia of 9-day-old and adult rats, and after tracer transport, the animals were sacrificed. Their stomachs were prepared as wholemounts and processed with tetramethylbenzidine. Inventories were obtained with a counting grid that was systematically positioned throughout the wholemounts by the use of a sampling template that was adjusted for stomach size and shape. Densities in the gastric antrum, corpus, and forestomach were determined for (1) afferent bundles, (2) individual fibers separated from the bundles and presumably located near their targets, (3) differentiated intraganglionic laminar endings (IGLEs) associated with myenteric ganglia, and (4) differentiated intramuscular arrays (IMAs) situated within the smooth muscle layers. In pre-weanling rats, which were 10 days old at perfusion, the distributions of vagal bundles and fibers were similar to those of adults, suggesting that the basic vagal architecture develops early. Differentiated IGLEs were also distributed in a mature pattern in 10 day olds, whereas few IMAs had yet been distributed and differentiated in the forestomach of the pre-weanlings. The authors hypothesize that these different developmental patterns for the two types of vagal afferents are consistent with their putative functional roles as, respectively, mechanoreceptors (IGLEs) that coordinate rhythmic motor function needed early for the digestion of milk and stretch receptors (IMAs) needed later as the GI tract natures for the transition to solid food at weaning.

Interstitial cells of Cajal in the circular (ICC-CM) and longitudinal (ICC-LM) muscle layer of the rat gastric antrum and their innervation were studied ultrastructurally. Both ICC-CM and ICC-LM are characterized by many mitochondria, rough and smooth endoplasmic reticulum, caveolae, and formation of gap junctions with each other and with muscle cells, though ICC-LM tend to show more variable cytoplasmic features depending on section profiles. Close contacts between nerve terminals and both ICC-CM and ICC-LM are observed. These possible synaptic structures are characterized by: (1) accumulation of synaptic vesicles in nerve varicosities, (2) a narrow gap (about 20 nm) between pre- and postjunctional membranes, (3) lack of a basal lamina between pre- and postjunctional membranes, and (4) the presence of an electron-dense lining on the inner aspect of prejunctional membranes. Almost the same characteristics are observed between the nerve terminals and the muscle cells of both circular and longitudinal muscle layers of the same specimens. Therefore, we conclude that the smooth muscle cells of both circular and longitudinal layers of the rat antrum are directly and indirectly innervated via ICC. Their functional significance is discussed.

Our confocal three-dimensional analyses revealed substantial differences in the innervation to vibrissal follicle-sinus complexes (FSCs) in the rat and cat. This is the first study using anti-protein gene product 9.5 (PGP9.5) immunolabeling and confocal microscopy on thick sections to examine systematically the terminal arborizations of the various FSC endings and to compare them between two species, the rat and the cat, that have similar-appearing FSCs but different exploratory behaviors, such as existence or absence of whisking. At least eight distinct endings were clearly discriminated three dimensionally in this study: 1) Merkel endings at the rete ridge collar, 2) circumferentially oriented lanceolate endings, 3) Merkel endings at the level of the ring sinus, 4) longitudinally oriented lanceolate endings, 5) club-like ringwulst endings, 6) reticular endings, 7) spiny endings, and 8) encapsulated endings. Of particular contrast, each nerve fiber that innervates Merkel cells at the level of the ring sinus in the rat usually terminates as a single, relatively small cluster of endings, whereas in the cat they terminate en passant as several large clusters of endings. Also, individual arbors of reticular endings in the rat ramify parallel to the vibrissae and distribute over wide, overlapping territories, whereas those in the cat ramify perpendicular and terminate in tightly circumscribed territories. Otherwise, the inner conical body of rat FSCs contains en passant, circumferentially oriented lanceolate endings that are lacking in the cat, whereas the cavernous sinus of the cat has en passant corpuscular endings that are lacking in the rat. Surprisingly, the one type of innervation that is the most similar in both species is a major set of simple, club-like endings, located at the attachment of the ringwulst, that had not previously been recognized as a morphologically unique type of innervation. Although the basic structure of the FSCs is similar in the rat and cat, the numerous differences in innervation suggest that these species would have different tactile capabilities and perceptions possibly related to their different vibrissa-related exploratory behaviors., (Copyright 2002 Wiley-Liss, Inc.)

Study Design: The posterior ligament of the human sacroiliac joint was examined for nerves and nerve endings using histologic and immunohistochemical techniques., Objective: To identify nerve fibers and mechanoreceptors in the posterior ligament., Summary of Background Data: According to the findings of previous studies, the human sacroiliac joint receives myelinated and unmyelinated axons that presumably conduct pain and proprioceptive impulses derived from mechanoreceptors and free nerve endings in the human sacroiliac joint., Methods: Tissue obtained from six patients was stained with gold chloride and that obtained from six additional patients was stained using antibodies specific for substance P and protein gene product 9.5., Results: The staining of joint tissue using the gold chloride technique showed myelinated and unmyelinated nerve fibers, two morphotypes of paciniform encapsulated mechanoreceptors, and a single nonpaciniform mechanoreceptor. Analysis using immunohistochemical staining for protein gene product 9.5 did not unequivocally show axons, nerve fascicles, or mechanoreceptors. Similarly, analysis based on immunohistochemical staining for substance P, one of several neurotransmitters known to signal pain from the periphery, showed reactive elements that may have been nerves, but because of background staining, could not be positively identified as such., Conclusions: The presence of nerve fibers and mechanoreceptors in the sacroiliac ligament demonstrates that the central nervous system receives information, certainly proprioceptive, and possibly pain from the sacroiliac joint. Although it is not known how the central nervous system uses such information, it seems reasonable to speculate that the proprioceptive information is used to optimize upper body balance at this joint. In addition, because the staining techniques used generally to show nerves and nerve elements in periarticular connective tissue are nonspecific, the distinction between neural and nonneural should be made on the basis of both morphologic and staining characteristics.

Introduction: To identify central neuroanatomic structure, biocytin labeling has recently been used. To date, there are no bibliographic references about the use of this molecule in investigations of the peripheral nervous system. In the present study, fresh, harvested human pancreas was used to evidence pancreatic innervations by biocytin., Aim: To investigate for the first time pancreatic innervation in harvested pancreas from human multiorgan cadaveric donors., Methodology: Biocytin labeling was used as a neuroanatomic tracing method, and confocal laser scanning microscopy was used for analysis for description by means of high-resolution images., Results: The application of biocytin-avidin staining in harvested human pancreas revealed numerous bundles of nervous fibers, intrapancreatic ganglia, few small solitary neurons, and a large number of positive supporting cells (glial-like cells). Biocytin appeared to pass through gap junctions between glial elements and neurons and among the neurons. In human pancreas, biocytin is rapidly transported in both anterograde and retrograde directions, with consequent visualization of fine details of pancreatic innervation morphology. Indeed, evidence of anterograde and retrograde transportation of biocytin has been demonstrated in the extensive labeling of pancreatic preganglionic and postganglionic fibers as well as a great number of chemical buds that wind through exocrine tissue or undetermined target cells., Conclusion: To our knowledge, this is the first report of the successful use of biocytin in neuronal retrograde and anterograde labeling in the human peripheral nervous system.

Previous studies showed immunoreactivities for neuropeptide Y (NPY), molluscan cardioexcitatory tetrapeptide (FMRFamide), and gonadotropin-releasing hormone (GnRH) in the terminal nerve of elasmobranchs. The present immunohistochemical study demonstrated two types of cells, i.e. GnRH- and NPY/FMRFamide-positive cells, in the terminal nerve ganglion of the elasmobranch Scyliorhinus torazame. The second cell type (non-GnRH element) contained a substance with a common structure or epitope recognized by anti-NPY and anti-FMRFamide antibodies. The NPY/FMRFamide-like immunoreactivity was associated with granules 70-130 nm in diameter, found in the cell bodies, axons, and axon endings.

Cholinergic activation of dopaminergic neurons in the ventral tegmental area (VTA) is thought to play a major role in cognitive functions and reward. These dopaminergic neurons differentially project to cortical and limbic forebrain regions, where their terminals differ in levels of expression of the plasmalemmal dopamine transporter (DAT). This transporter selectively identifies dopaminergic neurons, whereas the vesicular acetylcholine transporter (VAchT) is present only in the neurons that store and release acetylcholine. We examined immunogold labeling for DAT and immunoperoxidase localization of VAchT antipeptide antisera in single sections of the rat VTA to determine whether dopaminergic somata and dendrites in this region differ in their levels of expression of DAT and/or input from cholinergic terminals. VAchT immunoreactivity was prominently localized to membranes of small synaptic vesicles in unmyelinated axons and axon terminals. VAchT-immunoreactive terminals formed almost exclusively asymmetric synapses with dendrites. Of 159 dendrites that were identified as cholinergic targets, 35% contained plasmalemmal DAT, and 65% were without detectable DAT immunoreactivity. The DAT-immunoreactive dendrites postsynaptic to VAchT-labeled terminals contained less than half the density of gold particles as seen in other dendrites receiving input only from unlabeled terminals. These results suggest selective targeting of cholinergic afferents in the VTA to non-dopaminergic neurons and a subpopulation of dopaminergic neurons that have a limited capacity for plasmalemmal reuptake of dopamine, a characteristic of those that project to the frontal cortex.

Background: The aim of the present study was to identify the varieties of sensory and motor nerve endings in cat extraocular muscles., Methods: Sensory terminals were identify by injecting neuronal tracers (fast blue, biocytin, or peroxidase) into the trigeminal ganglion, which contains the sensory cells innervating the eye muscles. Motor terminals were identified by injections of horseradish peroxidase or DiI, a fluorescent carbocyanin dye, into either the oculomotor nerve or the IIIrd nuclei., Results: Injections into the trigeminal ganglion anterogradely labelled three types of sensory nerve endings for each neuronal tracer used: (1) the well-known "palisade" endings at the myotendinous junction of each extraocular muscle; (2) "compact" endings consisting of a dense terminal arborization extending up to 60 microm in length on striated muscle fibres 10-15 microm in diameter; and (3) "complex" endings on muscle fibres 15-20 microm in diameter. The complex ending issued from multiple collateral branches of the parent nerve fibre, which stretched and turned around the muscle fibre and gave off numerous terminal varicosities over a distance of about 140 microm. The sensory complex and compact endings presented strong similarities with some "atypical muscle spindles" previously described. In addition to the classic motor "plate" and "grape," we found evidence for the existence of motor "spiral" endings with each tracer., Conclusions: The sensory nature of the palisade endings was demonstrated, and two other types of sensory terminals were identified and described. The spiral nerve terminals were demonstrated to be motor in nature, and a possible function in the microsaccadic movements associated with fixation is suggested.

Three palmar wrist ligaments from fresh human cadavers were dissected from the proximal to the distal insertions and stained to identify the mechanoreceptors. Golgi organs, Pacinian corpuscles, Ruffini endings and free nerve endings were present in all three ligaments. In the radial collateral and radiolunate ligaments they were found in increased density towards the proximal and distal insertions. A more uniform distribution was found in the radioscaphocapitate ligament which has attachments to three bones. The palmar wrist ligaments may have a significant sensory role in maintaining the stability of the wrist and in controlling its movement. Although technically difficult, the surgical repair of traumatic wrist defects should attempt to preserve the innervation of the ligaments, shown to be mainly near bony attachments. This may allow improvement in postoperative outcomes by preserving some proprioception. In some painful post-traumatic or degenerative conditions, however, denervation may be advantageous.

Intraganglionic laminar endings (IGLEs) are special terminal structures of vagal afferent fibers and have been demonstrated in the myenteric plexus of esophagus and stomach. In order to quantitatively map their presence and distribution over the entire gastrointestinal tract, including the small and large intestines, vagal afferents were anterogradely labeled in vivo by microinjections of the fluorescent carbocyanine dye DiI into the left or right nodose ganglion of adult male rats. In the most successfully labeled cases the highest density of IGLEs was found in the stomach, with about half to one-third of the myenteric ganglia receiving at least one IGLE. The proportion of myenteric ganglia innervated by IGLEs decreased in the small intestine; however, because of its large surface area this gut segment was estimated to contain the highest total number of IGLEs. Both the cecum and colon also contained significant numbers of IGLEs. In the stomach, this vagal afferent innervation by IGLEs was more or less lateralized, with less than 20% of labeled IGLEs found on the contralateral side with respect to the injection. The left/ventral vagus contributed a larger proportion of IGLEs to the proximal duodenum, while the right/dorsal vagus contributed a larger proportion of IGLEs to the distal duodenum and jejunum. Laser scanning confocal microscopy on select specimens revealed further structural details. The parent axon typically formed two or more branches that flanked the ganglia laterally, and in turn produced numerous highly arborizing laminar terminal branches that covered one or both flat sides of the ganglion in a dome-like fashion. The similar distribution patterns and structural details suggest a uniform function for the IGLEs throughout the gastrointestinal tract, but there is as yet no clear proof for any of the hypothesized roles as specialized mechanosensors or local effector terminals.

The postnatal development of noradrenergic (NA) neurons and adrenergic (AD) terminals in the rat locus coeruleus (LC) was studied immunohistochemically. Cell body size was measured after staining of NA neurons with anti-tyrosine hydroxylase (TH) serum, and AD terminals were visualized with anti-phenylethanolamine N-methyltransferase serum. NA neurons in the LC were strongly TH-immunoreactive throughout the postnatal period. At birth, their mean cell body volume was 660 +/- 30 microns 3. It reached a maximum of 2580 +/- 230 microns 3 at postnatal day (PD) 14, and decreased thereafter to 930 +/- 50 microns 3 at PD 60. This transient enlargement of NA neurons may be closely related to the development of the cerebral cortex. AD afferents to the LC had terminals forming predominantly asymmetric junctions at birth (about 96% of all junctions). They occasionally made axo-somatic contact, suggesting that AD input already modulated the activity of LC neurons at this stage. AD terminals making axo-spinous synapses increased in number until PD 31, but still represented a minor proportion of these LC terminals, since there were more than 80% in contact with dendritic shafts at all ages examined.

This study describes the connectivity between horizontal cells and short-wavelength-sensitive (SWS) cones in macaque monkey retina. H1 and H2 horizontal cells were either labelled with the carbocyanine dye, DiI, or injected intracellular with Neurobiotin. The retinas were then processed with an antiserum against human SWS cone pigment, which usually stained the entire SWS cone. In these double-labelled retinas, the pattern of connectivity of H1 (n = 91) and H2 (n = 7) cells with SWS cones has been determined. About 85% of the H1 cells examined do not contact SWS cones. The dendritic terminal knobs of five H1 cells that do contact SWS cones were counted. They have, at most, 3% of their dendritic terminal knobs at SWS cones. All H2 cells examined make contact with SWS cones. The dendritic terminal knobs of one H2 cell were counted; about 11% of the dendritic terminal knobs are at the SWS cone. We conclude that horizontal cells in macaque monkey retina show specific patterns of connectivity to SWS cones.

Tree-like nerve endings in the smooth muscle layer of bronchi of the dog were examined by immunohistochemical staining with antibodies against neurofilament protein (NFP). The endings were revealed as ramified axon terminals, with arborisation at their termini. The endings were 100-300 microns in maximal length and 50-100 microns in minimal length. Most of the endings were arranged parallel to the smooth muscle strands. The endings were densely distributed in the proximal region but their density decreased towards the alveoli. In the histological sections, the endings were seen between smooth muscle cells. Terminal Schwann cells, which reacted with antibodies against glial fibrillary acidic protein and S-100 protein, and putative 'septal cells' with vimentin-like immunoreactivity were distributed near the endings. In addition, the nerve endings with NFP-like immunoreactivity were surrounded dense connective tissue that contained large amounts of fine elastic fibres. These findings indicate the nerve endings with NFP-like immunoreactivity are similar to other slowly adapting receptors (i.e. Golgi tendon organs, Ruffini endings). Some degenerated endings, which found in the unilaterally vagotomised dog, suggest the endings in the bronchi are originated from vagal nerves.

Laryngeal muscles, namely, the cricothyroid (CT), lateral crico-arytenoid (LCA), thyrio-arytenoid (TA), posterior crio-arytenoid (PCA), interarytenoid (IA) muscles, of Japanese monkeys (body weight, 4.4-8.3 kg; 3-10 years old, male) were examined histologically and by light and scanning electron microscopy. The diameter of muscle fibres with nerve terminals in the CT of the Japanese monkey was larger than that of other laryngeal muscles. However, the areas of nerve terminals varied among laryngeal muscles. The mean diameters of nerve terminals of the CT and PCA were large and basically resembled those of other laryngeal muscles. They contribute mainly to maintenance of phonation which the adductor muscles contribute mainly to postural adjustments of the cartilage. The differences in features of nerve terminals of each muscle suggest the CT and PCA may contribute mainly to the frequency or pitch of voice and intensity of the voice during vocalization and to respiration.

The neural histology of the human shoulder ligaments, glenoid labrum, and subacromial bursae were studied using a modified gold chloride stain. Two morphological types of mechanoreceptors and free nerve endings were found in the ligaments. Slow adapting Ruffini end organs and rapidly adapting Pacinian corpuscles were identified in the superior, middle, inferior, and the posterior glenohumeral ligaments. These specialized proprioceptive nerve endings were also found in the coracoclavicular, and coracoacromial ligaments. Only free nerve endings were found in the glenoid labrum and these were located in the peripheral half. Scattered free nerve endings were found throughout the subacromial bursae. This is the first histological evidence of neural receptors in the human shoulder ligaments, glenoid labrum, and the subacromial bursae. Any disruption of the labrum or these ligaments by trauma or surgery can deprive the shoulder of mechanical stability, and may cause a decrease in proprioception because of the loss of these afferent neural receptors. Removal of symptomatic, inflamed bursae may decrease pain signals from this area of the shoulder.

Toxic and nontoxic effects of ouabain were investigated on frog neuromuscular preparation by measuring the mean quantal content of endplate potentials elicited during repetitive nerve stimulation. In the untreated normal muscles, application of 10 microM ouabain gave rise to a slow exponential increase in the transmitter release (toxic ouabain effect) with a certain delay. This delay was increased with either 100 microM amiloride, a Na(+)-Ca2+ exchange blocker, or the intracellular loading of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a specific intracellular Ca2+ chelator. Measurements of frequency augmentation-potentiation (FAP) revealed a specific nontoxic effect of ouabain: 1 microM ouabain pivoted the long-linear FAP relation counter-clockwise without altering the intercept on the ordinate. Contrary to their action in the toxic effect, both 100 microM amiloride and the intracellular loading of BAPTA failed to counteract the nontoxic effect of 1 microM ouabain. The present results suggest that the toxic and nontoxic effects of ouabain are of different entities. The ouabain-sensitive subtype of Na+,K(+)-ATPase, which is abundant in neural tissues, seems to play a specific role in the process of nontoxic potentiation of transmitter release.

The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.

Modulation of ionic channel properties and synaptic functions by neurotransmitters and hormones is called neuro-modulation and may be the basis for many long-lasting changes in animal behavior, for example, changes in the arousal or motivational states. We have previously shown in a teleost, the dwarf gourami, that the terminal nerve (TN) cells are a major component of the gonadotropin-releasing hormone (GnRH) system and are structurally independent from the preoptic/hypophysial-GnRH system, which projects to the pituitary and facilitates gonadotropin release from the gonadotropes. While GnRH fibers originating from TN cells are distributed widely throughout the brain, they do not project to the pituitary. Thus, TN-GnRH system does not function as a hypophysiotropic hormone but probably as a neuro-modulator, capable of affecting widespread regions of the brain. In the present study, we used a whole-brain in vitro preparation to examine the spontaneous electrical activities of TN-GnRH cells and to determine the morphology of individual cells by intracellular injections of either neurobiotin or biocytin. The recorded cells were clearly identified as GnRH-immunoreactive TN cells by using combined intracellular injection and GnRH immunocytochemistry. Most of the TN cells showed endogenous slow (1-7 Hz), regular beating discharges. The intrinsic nature of this activity was demonstrated by the voltage dependency of the beating frequency, rhythm resetting, and persistence of rhythmicity after synaptic isolation. Only a small number of TN cells showed either irregular or bursting discharge patterns. Anatomical observation of intracellularly labeled cells showed that, regardless of discharge patterns, all the TN cells had multiple axonal branches that project to those areas where we had previously demonstrated dense GnRH-immunoreactive fibers. From the present results, we propose a hypothesis that may be relevant to the peptidergic and monoaminergic neuromodulatory systems in general. The modulator neurons have endogenous rhythmic activities that vary according to the animal's hormonal or environmental conditions, and they regulate the excitability of target neurons in a wide variety of brain regions simultaneously via multiple axonal branches.

The innervation of the inner conical body of the vibrissal follicle-sinus complex of the rat was examined by high-voltage and conventional transmission electron microscopy of serial and semi-serial sections. The inner conical body is innervated by axons supplied almost exclusively by several superficial vibrissal nerves that arise from the infraorbital branch of the trigeminal nerve and converge upon the neck of the follicle-sinus complex. Each superficial vibrissal nerve contains a few A delta myelinated axons and several bundles of 20-30 unmyelinated axons. These axons enter the inner conical body and distribute circumferentially within 7-10 ring-like arrays that encircle the vibrissal follicle and are stacked through the superficial-to-deep extent of the inner conical body. Each ring consists of 1 or 2 myelinated axons and several small bundles of 2-15 unmyelinated axons enclosed in sheaves of parallel collagen fibrils. Myelinated axons provide exclusively lanceolate endings that may arise at the termination of the axon or at nodes of Ranvier. Within the small bundles, unmyelinated axons individually terminate in succession as abrupt cytoplasmic swellings referred to as cytoplasmic blebs, which contain mitochondria or clusters of clear or dense-core vesicles. Because of their affiliation with collagen fibrils and the proximity of myelinated axons, the blebbed endings may have been misinterpreted as Ruffini endings in previous studies. Their structure, distribution, and origin from unmyelinated axons suggest that the blebbed endings may constitute a unique array of low-threshold C-mechanoreceptors.

This study is concerned with the distribution and ultrastructure of sensory nerve endings in the beak skin of adult Japanese quail (Coturnix coturnix japonica). The following nerve endings were found: free nerve endings, clusters of dermal Merkel nerve endings, Herbst corpuscles and Ruffini corpuscles. The latter were found only in the dermis of the tip of the upper beak. The remaining endings were present in the skin of all areas of upper and lower beak. Free nerve endings were supplied by either thin myelinated axons or unmyelinated C-fibers and were localized in the dermis close to the basal layer of the epidermis. Merkel cells formed clusters (up to 50) localized below and between the epidermal cones of the beak skin. Disc-shaped thickenings of nerve endings were squeezed between individual Merkel cells. Small Herbst corpuscles were found in the dermis close to the epidermal cones of the beak skin. Large Herbst corpuscles occurred in deep layers of the dermis. The Ruffini corpuscles were cylindrical in shape (80 microns x 400 microns) and arranged in groups of up to ten corpuscles. Each corpuscle was surrounded by an incomplete fibrous capsule.

The retinal projection to the basal telencephalon was studied in eight species of primates from the suborders Strepsirhini and Haplorhini, including one anthropoid primate, the gibbon. Animals received an intraocular injection of tritiated amino acids and the distribution of retinal fibers and terminals was demonstrated by autoradiographic techniques in horizontal and coronal sections. In all species a discrete group of labeled retinal fibers is observed to branch off from the dorsolateral aspect of the optic tract at the level of the suprachiasmatic nucleus. These fibers, destined to the basal telencephalon, are topographically distinct from the retinal fibers which innervate the suprachiasmatic nucleus and medial hypothalamic regions. The fibers of the retinotelencephalic tract course dorsally above the supraoptic nucleus through the lateral hypothalamic area and then proceed further rostrally and laterally below the diagonal band of Broca towards the olfactory tubercle. Within the olfactory tubercle, terminal distribution of label is observed in the mediocaudal region along the granular cell layer II. In the macaque this cellular layer shows a characteristic thickening in the region of retinal terminals which is evident in both coronal and horizontal section. In some species this labeled region is seen within the superficial bulge of the tubercle on the ventral aspect of basal telencephalon. In all primates the retinal projection to olfactory tubercle is bilateral. In prosimians label is predominantly contralateral to the injected eye, in New World monkeys label is equally distributed on both sides of the brain and in Old World monkeys label is mainly found ipsilaterally. Retinal fibers were also seen in the periamygdaloid region but never extended as far as piriform cortex. These results, in addition to previous studies in other mammalian orders, confirm that the basal telencephalon, and in particular the olfactory tubercle, constitutes a region of visual and olfactory convergence. This sensory integration may be related to photic and chemosensory modulation of reproductive physiology and behavior.

Many calcitonin gene-related peptide-like immunoreactive (CGRP-IR) fibers were found in the vestibular end-organs and the cochlea of rats. CGRP-IR fibers in the vestibular end-organs originated in the bilateral cell groups dorsolateral to the genu of the facial nerve. These fibers formed a fiber plexus at the base of the sensory epithelia in the maculae of the otolith organs and the ampullae of the semicircular canals, and formed synaptic contacts with the nerve chalice on type I vestibular sensory cells. CGRP-IR fibers in the cochlea originated in the ipsilateral lateral superior olivary nucleus. Most of them existed in the inner spiral bundle under the inner hair cells, and formed synaptic contacts with afferent terminals on the inner hair cells. These findings suggest a postsynaptic modulation of CGRP on the vestibular and cochlear information at the level of type I vestibular sensory cells and the inner hair cells.

The occurrence of nerve growth factor receptor (NGFR)-like immunoreactivity in the perineurial cells of sympathetic and sensory nerves was analyzed in normal controls and after surgical severance. In the normal nerve trunk, no cellular elements exhibited NGFR-like immunoreactivity. Immunoreactivity was induced in the innermost perineurial cells and in all Schwann cells distal to a severed nerve. The acquisition of immunoreactivity in perineurial cells was also seen in deafferented sympathetic ganglia. On the other hand, the perineurial cells as well as axonal endings showed NGFR-like immunoreactivity consistently in the terminal region of normal nerves, and this immunoreactivity was enhanced by nerve section. The present study clearly showed that different types of nerve terminals are ensheathed to different extents by perineurial cells which were first clearly identified by their NGFR-like immunoreactivity: 1) nerve terminals were free of the perineurium far away from targets in the iris, 2) they were free of the perineurium at sites close to targets in the lingual epithelium, and 3) the perineurium ensheathed nerve terminals together with targets in Meissner's sensory corpuscle. The functional significance of the three different patterns of perineurial enclosure requires further examination.

Dopamine (DA) release was simultaneously monitored in the nucleus accumbens (NAC) and ventral tegmental area (VTA) of conscious rats using in vivo microdialysis. During dialysis perfusion, rats were allowed access to food or water for 20 min following a 36 h food and water deprivation period. DA release increased significantly in the NAC and VTA in response to eating and drinking. The increases in both regions continued until 20-60 min after the end of the feeding or drinking session. These results show that the mesolimbic DA pathway is activated in response to ingestive behavior, and that DA release occurs in the cell body (A10) region as well as in the mesolimbic DA nerve terminals.

Monoclonal antibodies OM-1 to OM-4 and IM-1 [Woodhams et al. (1991) Neuroscience 46, 57-69] have complementary immunostaining patterns in the molecular (dendritic) layer of the adult rat dentate gyrus, with OM-1 to OM-4 selectively recognizing the outer (distal) two-thirds (i.e. the entorhinal afferent zone), and IM-1 the inner (proximal) one-third (i.e. the hippocampal commissural/associational zone). Immunoblotting suggests that OM-1 recognizes a single glycoprotein antigen of mol. wt around 93,000, and OM-2, OM-3, and OM-4 all recognize a second glycoprotein antigen of mol. wt around 36,000. At four weeks after removal of the ipsilateral entorhinal cortex the background OM immunostaining of the entorhinal afferent zone is abolished and replaced by a network of densely stained granules, which we interpret as degenerating entorhinal afferent axons. At the same time, the proximal, IM immunoreactive zone expands by about 10 microns in width (while the distal deafferented zone shrinks by about 80 microns). Attempts were made to restore the OM immunoreactivity of the distal zone by grafting either small pieces or cell suspensions of embryonic day 18 entorhinal cortex directly into the dentate molecular layer of entorhinally deafferented adult hosts. About half (14/26) of the animals with successfully positioned grafts showed restoration of OM-2 to OM-4 immunostaining throughout the entire width of the outer two-thirds (entorhinal afferent zone) of the dentate molecular layer. Strikingly, however, in adjacent serial sections the restoration of OM-1 immunoreactivity was restricted to the "middle" molecular layer, i.e. the most proximal part of the distal (entorhinal) two-thirds of the dentate molecular layer. In no case did the OM-1 immunoreactivity extend to the outer margin of the molecular layer. This did not appear to be associated with incompleteness of the removal of the host entorhinal projection, since it occurred in grafted cases where the hippocampus had been completely isolated from the entorhinal area. The simplest explanation of the observed pattern of OM loss and restitution is that the epitopes are located on the entorhinodentate axons, but it is not clear whether the antigens recognized by OM-1 and OM-2 to OM-4 are expressed in different parts of the same group of axons, or in different subsets of entorhinodentate axons. Nor is it clear why the pattern of OM-1 is only restored to the "middle" molecular layer, while that of OM-2 to OM-4 is restored to the entire outer two-thirds.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to study the morphological characteristics and terminating patterns of the neurons of the trigeminal mesencephalic nucleus (Vme), 55 masseteric neurons in Vme in the rat were stained by intracellular injection of horseradish peroxidase (HRP). Labeled cells were distributed throughout the nucleus. These neurons were divided into three types: uni- or pseudounipolar (type A, n = 43), bipolar (type B, n = 5), and multipolar cells (type C, n = 7). Each type was further divided into two subtypes according to the largest diameter of the perikarya (type a greater than or equal to 30 microns, type b less than 30 microns). The central processes of type Aa neurons projected to the following three groups of target nuclei: 1) nuclei functioning as interneurons, including supratrigeminal nucleus (Vsup), intertrigeminal nucleus (Vint), juxta-trigeminal region (Vjux), and parvicellular nucleus of the pontomedullary reticular formation (PcRF); 2) motor nuclei, including the trigeminal motor nucleus (Vmo), accessory facial nucleus (NVIIacs), accessory abducens nucleus (NVIacs), and a small number of labeled axons in the oculomotor nucleus and trochlear nucleus; 3) sensory nuclei, including the dorsomedial part of the principal trigeminal sensory nucleus (Vpdm) and the dorsomedial part of subnucleus oralis of the trigeminal spinal nucleus (Vodm). Labeled processes were dense in the Vsup, Vmo, and Vpdm. The proprioceptive pathway of the fifth nerve is discussed. Direct projections from type Aa neurons of Vme to the Vpdm and dorsolateral part of the Vsup contribute to conduction of the proprioceptive information from spindles of masticatory muscle to the contralateral thalamus in the rat. Different axon morphology, distribution, terminal branch density, and terminating patterns of type Aa neurons were noted in different functional groups of the projecting nuclei, especially in the Vsup, Vmo, and Vpdm. The highest terminal branching density, the most extensive distribution, and two different types of branching patterns (claw-like and comb-like) were observed in Vsup. Selective distribution and single-beaded or "Y"-shaped terminal branches were observed in Vmo. In the Vppdm the axonal branches were sparser than in the Vsup or Vmo, and had an arrangement like the branches of a weeping willow tree. These characteristics of anatomical organization might be related to the function of each projecting nucleus.

Studies on PC12 and isolated adrenal chromaffin cells have revealed that PC12 cells have a closer identity to the adrenergic nerve ending than do the chromaffin cells. This is revealed by the presence of monoamine oxidase (MAO) A and tyramine-released pool of catecholamines in PC12, resembling that in adrenergic neurones, and their absence in adrenal chromaffin cells. Indeed, chromaffin cells possess primarily MAO-B activity. Like the observations on adrenergic neurones, non-selective and selective MAO-A inhibitors potentiate the catecholamine-releasing property of tyramine in PC12 cells. This property has clearly been demonstrated to be associated with selective inhibition of MAO-A and not MAO-B. The fact that MAO-A and MAO-B are different proteins and under separate gene product control suggests that their regulation may be highly differentiated. Indeed, it has been shown that while steroids such as progesterone and hydrocortisone induce and estrogen diminishes MAO-A activity in PC12 cells, no such regulatory mechanism has been identified for MAO-B activity in chromaffin cells. In the final analysis the inter-relationship between MAO-A activity and the presence of tyramine-releasable pool of catecholamines in adrenergic neurons and PC12 cells may have a genetic basis and could be important in illuminating the differentiation of neural crest into adrenergic neurones and adrenal medulla on the one hand and chromaffin cells to PC12 cells on the other.

Morphological and morphometric features of the retinal synaptic terminals (RLP) and cortical synaptic terminals (RSD) were analyzed in the alpha E sector of the rabbit dorsal-lateral geniculate nucleus (dLGN). A methodological approach was selected which allowed us to determine volume of the neuropil and elsewhere record variations in the size and distribution of the two types of terminals found in the three zones (superior, middle, and inferior) from up to down into which the alpha E sector of the dLGN was divided. After obtaining an isotropic, uniform, and pseudorandom (IUR) sample, the terminals were examined on the basis of a set of morphometric parameters. An analysis of these data showed the retinal terminals (RLP) to be more numerous and to occupy a greater total area of the neuropil in the dorsal (superior) zone of the nucleus, whereas the number and total area occupied by cortical terminals (RSD) did not vary in the superior, middle, and inferior zones. Upon comparing the two types of terminals, the RLP were larger and more widely distributed, the greatest differences between the two appearing in the dorsal (superior) zone of the dLGN.

Close relationships between Merkel cells (MC) and nerve endings (NE) exist in the adult mouse. Because MC may serve as targets for the ingrowth of NE during embryogenesis, the purpose of the present study was to analyze the relationship between MC and NE during embryogenesis. Frozen tissue from whisker pads and backs of NMRI mouse embryos (12-17 d gestational age) were studied by double-labeling indirect immunofluorescence (IIF) with a cytokeratin monoclonal antibody that recognizes MC and with a neurofilament anti-serum. Such an approach allowed the analysis of a large number of MC (up to 5000), thus yielding quantitative data. At day 12 of gestational age, no MC were observed by IIF. From day 13 to 17, the number of MC, as well as their association with NE, progressively increased. On day 13, only 57% of whisker pad MC were NE associated, whereas by day 17, 95% were NE associated. These results were confirmed by electron microscopic (EM) observations. On the back, the same chronologic relationship between MC and NE was observed, but was later in the course of embryogenesis. There was also a time- and zone-dependent increase in MC association with NE in the epidermal zones studied (isthmic, parafollicular, interfollicular). These observations 1) establish the time course of MC and NE contacts during embryogenesis in the mouse epidermis, 2) show that MC are present in the epidermis and appendages before NE reach the epithelium, and 3) support the hypothesis that MC could act as targets for the growing NE.

The cholinergic circuitry in the nucleus basalis magnocellularis of the rat was investigated in a correlated light and electron microscopic study by using monoclonal antibodies against the acetylcholine-synthesizing enzyme, choline acetyltransferase, following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. After the immunocytochemical approach, large cholinergic cells and a few immunoreactive fibres exhibiting a varicose appearance, were detected by light microscopy in portions of the nucleus basalis magnocellularis located within the anatomical limits of the globus pallidus, mostly in its ventromedial part. Cholinergic neurons and fibre-like structures were also found within the substantia innominata on the edge of globus pallidus. The same material studied by light microscopy was analysed with the electron microscope. At the ultrastructural level, the immunopositive neurons showed the same cytological characteristics and pattern of synaptic input as cholinergic basal forebrain cells. Additionally, scarce immunoreactive preterminal axons and terminal boutons were detected in the region. The immunoreactive terminals were scattered or formed occasional clusters and appeared as heavily immunostained vesicle-filled boutons making exclusively axodendritic synaptic contacts principally with immunonegative distal dendrites. Both symmetric and asymmetric synaptic contacts established between these structures were detected, although the symmetric contacts were the more numerous. The surface of postsynaptic immunonegative dendrites in asymmetric synaptic contact with immunoreactive terminals was generally covered by terminals that lacked detectable immunoreactivity. In contrast, those in symmetric synaptic contact with labelled terminals showed much sparser input from immunonegative terminals, suggesting that they may belong to interneurons. Very rarely, cholinergic terminals were detected in asymmetric synaptic contact with dendrites which also contained positive immunoreaction product. Asymmetric contacts were frequently characterized by the presence of subjunctional dense bodies. The detection of cholinergic terminals in the region of the nucleus basalis magnocellularis of the rat indicates that this region not only contains cholinergic projecting neurons, but receives a cholinergic input itself. Results of this study provide evidence of the existence of a cholinergic transmission in the basal forebrain of the rat, and also that acetylcholine might play a role in the regulation of the extrinsic cortical cholinergic innervation. The possible sources of this innervation are discussed.

Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin within the ventromedial thalamic nucleus resulted in many filled fibres in the frontal areas of rat cerebral cortex. The fibres were restricted to the upper part of layer I except in a small area of motor cortex where terminals were also found in deeper layers. Terminals were also seen in the striatum, in parts of the mesencephalic reticular formation and occasionally in the contralateral ventromedial nucleus. There is some topographical order in the projection with medial and dorsal areas well represented in medial cortex while lateral parts of ventromedial nucleus are more directly related to the cortical area that receives the ventrolateral thalamic nucleus projection. Electron microscopic examination showed the terminals in layer I of cortex making synaptic contact with dendritic spines and small dendritic profiles that showed a very dense postsynaptic specialization. Neurons in the ventromedial nucleus could be antidromically driven from electrode positions along strips of cortex which could not be easily related to any known organizational pattern in the cortex. Thalamic neurons responding antidromically to only one stimulation site were more common when the stimulation was within motor cortical areas, suggesting that in this region a more restricted pattern of termination is the rule.

Several lines of evidence indicate that L-glutamate may be a neurotransmitter of fine myelinated and unmyelinated primary afferent fibres in the spinal cord. The aim of the present study was to determine if L-glutamate was enriched in the terminals of these fibres. We performed the post-embedding immunogold technique on sections taken from the superficial regions of the lumbar cord in two cats. An antiserum, raised against protein-conjugated L-glutamate, was employed. Several tests on tissue and on a model system indicated that the antiserum recognized a glutaraldehyde-fixed L-glutamate-like substance. Terminals of fine afferent fibres were identified in the substantia gelatinosa as central boutons of synaptic glomeruli. Central boutons were examined through serial sections following immunogold reactions and were found to be heavily labelled with gold particles in consecutive sections. Quantitative analysis indicated that central boutons were more than two and a half times as densely labelled with gold particles than the tissue average. It was concluded that this represents a genuine enrichment of L-glutamate in these structures. Comparisons were made between L-glutamate-immunoreactive properties of central terminals and immunoreactivity for GABA, aspartate and glutamine. Statistical analysis revealed that central boutons in sections incubated in GABA antiserum and glutamine antiserum were associated with significantly lower densities of gold particle labelling than the average for the same tissue. Particle densities of central boutons in sections incubated in aspartate antiserum were not significantly different from average tissue densities. It was concluded that central boutons were not enriched with these three amino acids. Central boutons of synaptic glomeruli were classified into three groups on morphological criteria: (1) dense sinusoidal boutons; (2) large dense-core vesicle-containing boutons; and (3) regular synaptic vesicle-containing boutons. Quantitative analysis revealed that all of these groups were enriched in glutamate immunoreactivity, however, there were differences between the groups; large dense-core vesicle-containing boutons were associated with significantly lower densities of particles than regular synaptic vesicle-containing and dense sinusoidal terminals. The evidence indicates that central boutons, which most probably originate from fine myelinated and unmyelinated primary afferent fibres, are enriched with L-glutamate which may serve as a neurotransmitter in such fibres.

The development of the adrenergic cardiac innervation was studied in premature dog fetuses, puppies and adult dogs by means of the formalin-induced fluorescence technique. A point-counting technique was used to evaluate the density of innervation. Two types of fluorescent profiles can be observed in the heart during development: (1) sprouting axons, and (2) beaded terminals. The axonal fluorescence disappears in adult neurons. A different morphology and a different time course of development enable to study separately the innervation of the myocardium (cardiomotor innervation) and that of the vessels (vasomotor innervation). The late prenatal innervation is very poor (0.1 hit). The first but very scant cardiomotor terminals appear in this period. A mature cardiomotor innervation is found in 4-month-old puppies [1.5 +/- 0.3 (SD) hits]. The vasomotor innervation is shifted to the right. The development of beaded vascular terminals begins and matures 1-2 weeks later. The growing fluorescent axons reveal that the myocardium is supplied by axons of the cardiac plexus and of the perivascular nerves; the vascular wall, on the other hand, is supplied by the perivascular nerves only. The developmental, spatial and morphological differences in innervation suggest that two different types of neurons exist in the sympathetic ganglia: (1) neurons innervating the vessels (coronaromotor neurons), and (2) neurons innervating the myocardium (cardiomotor neurons).

In all, 30 adult (45-day-old) Swiss Webster mice were used for light and electron microscopic examination of the presence, number, and location of adrenergic endings in the first molar teeth. Prior to sacrifice, 10 animals received i.p. injections at 8, 6, 4, and 2 hours of 0.5 cc of 20 mg/kg solution of 5-hydroxydopamine (5-OH-DA) as a label for adrenergic endings. The animals were then anesthetized, perfused with Karnovsky's fixative, and the teeth were postfixed in Osmic acid, decalcified, embedded in methacrylate, and serial-sectioned. The sections were surveyed by light microscopy, and the number and location of nerve endings containing the reduced 5-OH-DA were recorded. Ten control mice were injected with the vehicle solution and prepared in the same manner. A third series of mice were given a single injection of 5-OH-DA, sacrificed, and prepared for ultrastructural study. The molar pulps were divided into four areas to facilitate examination: pulp horns, coronal pulp, bifurcation area, and root pulp. These four areas were further divided into three zones: odontogenic, vascular-related, and nonvascular-associated. The location and number of endings were evaluated, and an average of approximately 70 endings containing the 5-OH-DA were found in each tooth using light microscopy. These represented 35.5 +/- 5.2 in the pulp horns; 26.1 +/- 2.4 in the central coronal; 5.4 +/- 0.7 in the bifurcation, and 5.6 +/- 0.9 in the root pulp per tooth. Vascular related endings were found in greatest number, the odontogenic zone next, and free endings lease. Verification of location of 5-OH-DA by ultrastructural analysis revealed the false transmitter in vesiculated endings in the four areas and zones of the pulp.

Intraepithelial fibers do occur in the mucosa of the esophagus, as demonstrated by the osmium tetroxide-zinc iodide method in cats and rhesus monkeys. The esophagus is divided into three parts, in order to study the penetration incidence, and the uppermost and the lowest show the greatest density of penetration, while in the middle portion only occasional fibers in small numbers are found. The specific characteristics observed in this type of fiber, such as their distribution along the wall of the esophagus, the levels reached by their endings within the mucous epithelium itself and this same epithelium considered as the specified destination of the endings, lead to the belief that they may be functionally regarded as structures of a sensory character.