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Despite the reduced life expectancy and staggering financial burden of medical treatment associated with tobacco smoking, the molecular, cellular, and ensemble adaptations associated with chronic nicotine consumption remain poorly understood. Complex circuitry interconnecting dopaminergic and cholinergic regions of the midbrain and mesopontine tegmentum are critical for nicotine associated reward. Yet our knowledge of the nicotine activation of these regions is incomplete, in part due to their cell type diversity. We performed double immunohistochemistry for the immediate early gene and surrogate activity sensor, c-Fos, and markers for either cholinergic, dopaminergic or GABAergic cell types in mice treated with nicotine. Both acute (0.5 mg/kg) and chronic (0.5 mg/kg/day for 7 days) nicotine strongly activated GABAergic neurons of the interpeduncular nucleus and medial terminal nucleus of the accessory optic tract (MT). Acute but not chronic nicotine also activated small percentages of dopaminergic and other neurons in the ventral tegmental area (VTA) as well as noncholinergic neurons in the pedunculotegmental and laterodorsal tegmental nuclei (PTg/LDTg). Twenty four hours of nicotine withdrawal after chronic nicotine treatment suppressed c-Fos activation in the MT. In comparison to nicotine, a single dose of cocaine caused a similar activation in the PTg/LDTg but not the VTA where GABAergic cells were strongly activated but dopaminergic neurons were not affected. These results indicate the existence of drug of abuse specific ensembles. The loss of ensemble activation in the VTA and PTg/LDTg after chronic nicotine represents a molecular and cellular tolerance which may have implications for the mechanisms underlying nicotine dependence., (© 2018 Wiley Periodicals, Inc.)

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family that has been strongly implicated in development and regeneration of autonomic nerves and modulation of nociception. Whereas other members of this family (GDNF and neurturin) primarily target parasympathetic and nonpeptidergic sensory neurons, the artemin receptor (GFRα3) is expressed by sympathetic and peptidergic sensory neurons that are also the primary sites of action of nerve growth factor, a powerful modulator of bladder nerves. Many bladder sensory neurons express GFRα3 but it is not known if they represent a specific functional subclass. Therefore, our initial aim was to map the distribution of GFRα3-immunoreactive (-IR) axons in the female rat bladder, using cryostat sections and whole wall thickness preparations. We found that GFRα3-IR axons innervated the detrusor, vasculature, and urothelium, but only part of this innervation was sensory. Many noradrenergic sympathetic axons innervating the vasculature were GFRα3-IR, but the noradrenergic innervation of the detrusor was GFRα3-negative. We also identified a prominent source of nonneuronal GFRα3-IR that is likely to be glial. Further characterization of bladder nerves revealed specific structural features of chemically distinct classes of axon terminals, and a major autonomic source of axons labeled with neurofilament-200, which is commonly used to identify myelinated sensory axons within organs. Intramural neurons were also characterized and quantified. Together, these studies reveal a diverse range of potential targets by which artemin could influence bladder function, nerve regeneration, and pain, and provide a strong microanatomical framework for understanding bladder physiology and pathophysiology., (© 2014 Wiley Periodicals, Inc.)

Mutations in human LIS1 cause abnormal neuronal migration and a smooth brain phenotype known as lissencephaly. Lis1+/− (Pafah1b1) mice show defective lamination in the cerebral cortex and hippocampal formation, whereas homozygous mutations result in embryonic lethality. Given that Lis1 is highly expressed in embryonic neurons, we hypothesized that sympathetic and parasympathetic preganglionic neurons (SPNs and PPNs) would exhibit migratory defects in Lis1+/− mice. The initial radial migration of SPNs and PPNs that occurs together with somatic motor neurons appeared unaffected in Lis1+/− mice. The subsequent dorsally directed tangential migration, however, was aberrant in a subset of these neurons. At all embryonic ages analyzed, the distribution of SPNs and PPNs in Lis1+/− mice was elongated dorsoventrally compared with Lis1+/+ mice. Individual cell bodies of ectopic preganglionic neurons were found in the ventral spinal cord with their leading processes oriented along their dorsal migratory trajectory. By birth, Lis1+/− SPNs and PPNs were separated into distinct groups, those that were correctly, and those incorrectly positioned in the intermediate horn. As mispositioned SPNs and PPNs still were detected in P30 Lis1+/− mice, we conclude that these neurons ceased migration prematurely. Additionally, we found that a dorsally located group of somatic motor neurons in the lumbar spinal cord, the retrodorsolateral nucleus, showed delayed migration in Lis1+/− mice. These results suggest that Lis1 is required for the dorsally directed tangential migration of many sympathetic and parasympathetic preganglionic neurons and a subset of somatic motor neurons.

The ventrolateral bed nucleus of the stria terminalis (BSTvl) receives direct input from two specific subpopulations of neurons in the nucleus tractus solitarius (NTS). It is heavily innervated by aldosterone-sensitive NTS neurons, which are selectively activated by sodium depletion, and by the A2 noradrenergic neurons, which are activated by visceral and immune- and stress-related stimuli. Here, we used a retrograde neuronal tracer to identify other brain sites that innervate the BSTvl. Five general brain regions contained retrogradely labeled neurons: cerebral cortex (infralimbic and insular regions), rostral forebrain structures (subfornical organ, organum vasculosum of the lamina terminalis, taenia tecta, nucleus accumbens, lateral septum, endopiriform nucleus, dorsal BST, substantia innominata, and, most prominently the amygdala--primarily its basomedial and central subnuclei), thalamus (central medial, intermediodorsal, reuniens, and, most prominently the paraventricular thalamic nucleus), hypothalamus (medial preoptic area, perifornical, arcuate, dorsomedial, parasubthalamic, and posterior hypothalamic nuclei), and brainstem (periaqueductal gray matter, dorsal and central superior raphe nuclei, parabrachial nucleus, pre-locus coeruleus region, NTS, and A1 noradrenergic neurons in the caudal ventrolateral medulla). In the arcuate hypothalamic nucleus, some retrogradely labeled neurons contained either agouti-related peptide or cocaine/amphetamine-regulated transcript. Of the numerous retrogradely labeled neurons in the perifornical hypothalamic area, few contained melanin-concentrating hormone or orexin. In the brainstem, many retrogradely labeled neurons were either serotoninergic or catecholaminergic. In summary, the BSTvl receives inputs from a variety of brain sites implicated in hunger, salt and water intake, stress, arousal, and reward., (Copyright 2008 Wiley-Liss, Inc.)

The immunohistochemical expression of a novel chromogranin-like protein, neuroendocrine secretory protein 55 (NESP55), in the rat spinal cord was investigated. NESP55-immunoreactive cells were detected in the ventral horn, intermediate laminae, and deep dorsal horn, comprising motoneurons, autonomic neurons, and interneurons throughout all spinal segments. Within laminae I-II of the dorsal horn, one or two NESP55-positive cells were often seen. Nerve fibers also contained NESP55 immunoreactivity (IR) and were particularly prominent in the ventral horn. No nerve terminals/varicosities appeared to contain NESP55 in any spinal lamina. Double-staining experiments revealed that a high proportion of the NESP55-positive neurons were cholinergic. Moreover, NESP55-IR in the motoneurons was evenly distributed in the whole cytoplasm with a finely granular appearance. In contrast, the fluorescent material in the preganglionic neurons was concentrated in the perinuclear region and largely overlapped with the trans-Golgi network marker TGN38. Our data provide detailed morphological information on the distribution of NESP55-IR in the rat spinal cord. Also, the differential intracellular expression of NESP55-IR in the spinal motoneurons and autonomic neurons suggests that NESP55 may be processed into different secretory granules and may be involved in both constitutive and regulated pathways in these neurons., ((c) 2007 Wiley-Liss, Inc.)

We used immunohistochemistry and confocal microscopy applied to fingertip punch biopsy to study glabrous skin innervation in 14 healthy subjects. In addition to epidermal nerve fibers, we quantified mechanoreceptors and their myelinated afferents. Using digital images and dedicated software, we calculated caliber, internodal and nodal length, and G-ratio of the last four internodes of the myelinated endings. In our skin samples, we found a mean density of 59.0 +/- 29.3 myelinated endings per square millimeter with a mean diameter of 3.3 +/- 0.5 microm and an internodal length of 79.1 +/- 13.8 microm. These findings indicate that Abeta fibers undergo drastic changes in their course from the nerve trunk to the target organ, with repeated branching and consequent tapering and shortening of internodal length. Our work demonstrates that skin biopsy can give information on the status of large myelinated endings as well as unmyelinated sensory and autonomic nerves. Since distal endings are primarily involved in distal axonopathy, skin biopsy can be more suitable than sural nerve biopsy to detect early abnormalities. In addition to diagnostic applications, this technique allows clarification of the mode of termination of Abeta fibers and their relationship with mechanoreceptors, leading to relevant electrophysiological speculations.

The coeliac ganglion of guinea pigs displays a unique topographical arrangement of neurochemically and functionally distinct populations of sympathetic neurons. The authors used multiple-labeling immunohistochemistry to investigate the neurochemical differentiation of these neurons during embryonic and fetal development. Sympathoadrenal precursors, located on either side of the abdominal aorta, were intensely immunoreactive for tyrosine hydroxylase (TH-IR), neurofilament, and the human natural killer 1 antibody at midembryonic stages (Carnegie stages 16-19). During late embryonic stages (stages 20-23), a single bilobed ganglion had formed. At this time, neuropeptide Y immunoreactivity (NPY-IR) was widely expressed in sympathetic neurons (with moderate TH-IR) and chromaffin cells (with intense TH-IR). The onset of somatostatin (Som-IR) expression followed that of NPY-IR and was restricted to sympathetic neurons. However, at late embryonic stages, most TH-IR neurons with Som-IR also expressed NPY-IR (a combination of peptides not found in the mature coeliac ganglion). Between late embryonic stages and the end of the early fetal period, there was a significant increase in the proportion of neurons in lateral regions that had both NPY-IR and TH-IR. At the same time, there was an increase in the proportion of neurons in medial regions that had both Som-IR and TH-IR. Neurons expressing both Som-IR and TH-IR were rarely observed in lateral regions of the coeliac ganglion. Thus, a clear topography within the coeliac ganglion is established during late embryonic and early fetal stages of development and reflects that found in the mature animal by the end of the early fetal period., (Copyright 2000 Wiley-Liss, Inc.)

Decussations of descending fibers of the hypothalamic paraventricular nucleus (PVN) were investigated by using Phaseolus vulgaris-leucoagglutinin (PHA-L) in intact and brainstem-operated rats. Fibers descend ipsilaterally along the brainstem and spinal cord and decussate at four levels: 1) Supramamillary decussations (SM). PVN fibers reach this area through the lateral hypothalamus and along the third ventricle in the dorsal hypothalamus. In the posterior hypothalamus some fibers crossover in the SM and terminate in the supramamillary region bilaterally. 2) Pontine tegmentum. PVN fibers run in the lateral part of the tegmentum arching to the basis of the pons. Some fibers crossover under the fourth ventricle. The locus ceruleus and the Barrington's nucleus receive bilateral innervation with ipsilateral dominance. 3) Commissural part of the nucleus of the solitary tract (NTS). The major crossover of PVN fibers is found here. The decussated fibers form a dense network here, and loop rostralward to innervate the entire NTS. A midsagittal knife-cut through the NTS eliminated paraventricular-fibers on the contralateral side. Synaptic contacts between PHA-L-labeled boutons and tyrozine hydroxilase-positive neurons were verified in the NTS. The caudal ventrolateral medulla also receives bilateral innervation. 4) Lamina X of the thoracic spinal cord. Paraventricular fibers enter the lateral funiculus ipsilaterally and innervate the intermediolateral cell column (IML). Some fibers cross the midline ventral and dorsal to the central canal running to the contralateral IML, at the level of the decussation. Our results demonstrated that paraventricular projections form a continuous descending pathway on their side of origin, and provide crossover fibers which may terminate segmentally without forming long tracts after crossover., (Copyright 1999 Wiley-Liss, Inc.)

We have used the neurotropic Bartha strain of pseudorabies virus (PRV) to characterise the pathway linking the endogenous circadian pacemaker of the suprachiasmatic nucleus (SCN) to the pineal gland. This low virulent strain of virus replicates within synaptically linked neurones and is ideally suited to visualise the multisynaptic pathways through which the SCN modulates the activity of the rat pineal gland. Using specific antibodies against PRV, we could follow the immunohistochemical pattern of the spatiotemporal passage of virus through the sympathetic trunk and the neuraxis. The time course of virus infection indicated that the most prominent pathway from the SCN to the pineal gland is via a final sympathetic innervation from the superior cervical ganglion (SCG). The pathway arises in the dorsomedial portion of the SCN from where neurones project to the dorsal parvicellular subdivision of the hypothalamic paraventricular nucleus (PVN) to form synaptic contact with neurones descending to the intermediolateral nucleus (IML) of the upper thoracic spinal cord. The neurones of the IML constitute the presynaptic sympathetic input synaptically connected to postsynaptic sympathetic neurones in the SCG which constitute the final input to the pineal gland. Removal of the superior cervical ganglion (SCGX) prior to viral infection completely abolished infection of neurones in this circuit. However, an additional parasympathetic projection from the superior salivatory nucleus via the sphenopalatine ganglion to the pineal gland was observed in SCGX animals., (Copyright 1999 Wiley-Liss, Inc.)

Metabotropic glutamate receptors (mGluRs) in the medulla oblongata have been suggested to be involved in the regulation of autonomic function. The aim of the present study was to examine the localization and expression of four types of mGluRs: mGluRla, mGluR2/3, mGluR5, and mGluR7 in the dorsal and ventral autonomic nuclei of the medulla of the rat. The four mGluR subtypes studied were differentially distributed in distinct subnuclei in the nucleus of the solitary tract (NTS). mGluRla immunoreactivity was identified in cell bodies, dendrites, and axonal processes in the intermediate, dorsal lateral, and interstitial subnuclei of the NTS. No mGluRla immunoreactivity was observed in the commissural or medial NTS subnuclei. Immunoreactivity for mGluR2/3 and mGluR5 as observed in fibers and putative axonal processes in the interstitial, intermediate, and dorsolateral subnuclei of the NTS. In contrast, mGluR7 was expressed primarily in fibers and terminals in the central and commissural NTS subnuclei. Expression of mGluR2/3 was clearly evident in cell bodies, dendrites, and axonal processes within the area postrema. The vagal outflow nuclei were also studied. The dorsal motor nucleus of the vagus (DMN) contained mGluRla cell bodies, dendrites, and axonal fibers and light mGluR2/3 processes. Throughout the rostral-caudal extent of the compact and semicompact formation nucleus ambiguus, mGluRla was found in cell bodies and fibers. Within the caudal and rostral regions of the ventral lateral medulla, mGluRla was observed in cell bodies and fibers. Cell bodies containing mGluRla were found adjacent to cells staining positive for tyrosine hydroxylase (TH) in these regions but were not colocalized with the TH staining. However, mGluRla-expressing neurons in the ventral lateral medulla did appear to receive innervation from TH-containing fibers. These results suggest that the mGluRla-expressing neurons within the ventral lateral medulla are predominantly not catecholaminergic but may be innervated by catecholamine-containing fibers. These data are the first to provide a mapping of the different mGluR subtypes within the medulla and may facilitate predictions regarding the function of L-glutamate neurotransmission in these regions.

The paratrigeminal nucleus (PTN) receives primary visceral afferent projections through cranial nerves IX and X and somatic afferent projections through cranial nerve V and dorsal roots as far caudally as C7. Pressure injections of the anterograde tracer tetramethylrhodamine dextran into the PTN in the rat resulted in bilateral labeling in the nucleus of the tractus solitarius, dorsal motor nucleus of the vagus nerve, and parabrachial nucleus. Anterograde labeling in the parabrachial nucleus was strongest in the external medial, external lateral, and ventral lateral subnuclei. Anterograde labeling was also found in the contralateral paratrigeminal nucleus, lamina I of the spinal trigeminal nucleus subnucleus caudalis, and ventroposteromedial nucleus of the thalamus. The collateral organization of PTN neurons was demonstrated by injecting different fluorescent retrograde tracers into the terminal fields of PTN projections as determined by the anterograde tracing experiments. Double-labeled neurons were found in the paratrigeminal nucleus following all combinations of injection sites. The most prominent PTN efferent projections and the most highly collateralized were to the nucleus of the tractus solitarius and parabrachial nucleus. The efferent and collateral connections of the paratrigeminal nucleus may provide a neuroanatomical substrate for integrating convergent visceral and somatic afferent information used to modulate autonomic function and behavior related to thermoregulation, nociception, and gustation.

The origin and termination of prefrontal cortical projections to the periaqueductal gray (PAG) were defined with retrograde axonal tracers injected into the PAG and anterograde axonal tracers injected into the prefrontal cortex (PFC). The retrograde tracer experiments demonstrate projections to the PAG that arise primarily from the medial prefrontal areas 25, 32, and 10m, anterior cingulate, and dorsomedial areas 24b and 9, select orbital areas 14c, 13a, Iai, 12o, and caudal 12l, and ventrolateral area 6v. Only scattered cells were retrogradely labeled in other areas in the PFC. Caudal to the PFC, projections to the PAG also arise from the posterior cingulate cortex, the dorsal dysgranular, and granular parts of the temporal polar cortex, the ventral insula, and the dorsal bank of the superior temporal sulcus. Cells were also labeled in subcortical structures, including the central nucleus and ventrolateral part of the basal nucleus of the amygdala. The anterograde tracer experiments indicate that projections from distinct cortical areas terminate primarily in individual longitudinal PAG columns. The projections from medial prefrontal areas 10m, 25, and 32 end predominantly in the dorsolateral columns, bilaterally. Fibers from orbital areas 13a, Iai, 12o, and caudal 12l terminate primarily in the ventrolateral column, whereas fibers from dorsomedial areas 9 and 24b terminate mainly in the lateral column. The PFC areas that project to the PAG include most of the areas previously defined as the "medial prefrontal network." The areas that comprise this network represent a visceromotor system, distinct from the sensory related "orbital network."

The organization of projections from the macaque orbital and medial prefrontal cortex (OMPFC) to the hypothalamus and related regions of the diencephalon and midbrain was studied with retrograde and anterograde tracing techniques. Almost all of the prefrontal cortical projections to the hypothalamus arise from areas within the "medial prefrontal network," as defined previously by Carmichael and Price ([1996] J. Comp. Neurol. 371:179-207). Outside of the OMPFC, only a few neurons in the temporal pole, anterior cingulate and insular cortex project to the hypothalamus. Axons from the OMPFC also innervate the basal forebrain, zona incerta, and ventral midbrain. Within the medial prefrontal network, different regions project to distinct parts of the hypothalamus. The medial wall areas 25 and 32 send the heaviest projections to the hypothalamus; axons from these areas are especially concentrated in the anterior hypothalamic area and the ventromedial hypothalamic nucleus. Orbital areas 13a, 12o, and Iai, which are related to the medial prefrontal network, selectively innervate the lateral hypothalamic area, especially its posterior part. The cellular regions of the paraventricular, supraoptic, suprachiasmatic, arcuate, and mammillary nuclei are conspicuously devoid of cortical axons, but many axons abut the borders of these nuclei and may contact dendrites that extend from them. Areas within the orbital prefrontal network on the posterior orbital surface and agranular insula send only weak projections to the posterior lateral hypothalamic area. The rostral orbital surface does not contribute to the cortico-hypothalamic projection.

The efferent projections from the periaqueductal gray matter (PAG) to the parabrachial nucleus (PB) were studied in the rat following microinjections of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into restricted regions of the PAG. The dorsomedial and dorsolateral PAG columns project almost exclusively to the superior lateral PB subnucleus, whereas the lateral and ventrolateral PAG columns project to five lateral PB sites: dorsal lateral subnucleus, medial and lateral crescent areas (which flank the dorsal lateral PB subnucleus), central lateral subnucleus (rostral portion), and superior lateral subnucleus. The PAG region lying near the cerebral aqueduct projects to five lateral PB sites: external lateral subnucleus (inner subdivision), medial and lateral crescent areas, central lateral subnucleus (rostral portion), and dorsal lateral subnucleus. The internal lateral PB subnucleus, which projects exclusively to the intralaminar thalamic nuclei, and the Kölliker-Fuse nucleus were not innervated by the PAG. The PAG selectively innervates individual PB subnuclei that may be part of the spino-parachio-forebrain pathway. All PAG columns, including the aqueductal region, project to the superior lateral PB subnucleus, a presumed nociceptive relay site that receives inputs from multiple spinal cord regions (laminae I, V, and VIII) and projects to the ventromedial and retrochiasmatic hypothalamic areas-two regions that have been implicated in complex goal-directed behavior (e.g., food intake and reproductive function). Earlier studies demonstrated that the dorsal lateral and external lateral PB subnuclei (inner division) receive overlapping inputs from the superficial dorsal horn (laminae I and II) and the nucleus tractus solitarius, and both PB subnuclei send projections to limbic forebrain areas (e.g., hypothalamus, preoptic region, amygdala). Because the PAG projects to both of these PB subnuclei, this projection system possibly functions as a behavioral state-dependent filter system that modulates ascending nociceptive and/or visceral information as it is relayed through the PB to forebrain sites.

Interactions of developing neurons with their postsynaptic targets play a significant role in neuronal differentiation. The goal of the present study was to determine if target contact affected the migration or differentiation of autonomic motor neurons (AMNs) in developing rat spinal cord. The peripheral targets of AMNs were excised microsurgically from histotypic spinal slices before the arrival of AMN axons. The migration of AMNs was assessed in DiI retrogradely labeled preparations, and the differentiation of these cells was evaluated by beta-nicotinamide adenine dinucleotide phosphate reduced diaphorase (NADPH-d) histochemistry. In target-deprived specimens, NADPH-d expression in AMNs was virtually eliminated. In addition, DiI-labeled AMNs were scattered throughout the intermediate spinal gray matter instead of being aggregated in the intermediolateral nucleus as in control slices. This observation indicated that migration of AMNs had occurred, but that it had been disorganized significantly by target removal on embryonic day 13 (E13). In sham, "incision-only" specimens from which peripheral target tissue was not removed, AMNs expressed NADPH-d and migrated normally, indicating that axotomy alone was not sufficient to disrupt AMN development. Previous studies have shown that target removal after the arrival of AMN axons at their postsynaptic targets on E14 has no affect on the organized migration of AMNs (Barber et al. [1993] J. Neurosci. 13:4898-4907). This observation together with the present results indicate that initial target contact is necessary for both the differentiation and directed migration of AMNs, and that this contact does not need to be sustained for these developmental events to progress normally.

Considerable knowledge concerning developmental cell death has come from the study of somatic motor neurons (SMNs), but a related set of spinal neurons, the autonomic motor neurons (AMNs), have been studied less extensively in this respect. In the present study, we used three different approaches to determine the amount of AMN cell death during normal development in the rat. First, target dependency was studied in organotypic slice cultures, and it was found that AMNs survived for at least 12 days after removal of their postsynaptic targets. No factors were added to the serum-free medium to substitute for the ablated targets, indicating that AMNs were able to survive without target-derived trophic factors. Such target-independent survival is not characteristic of neurons that undergo typical developmental cell death. Second, AMNs were counted in double-stained choline acetyltransferase immunocytochemical and NADPH diaphorase histochemical preparations at ages (postnatal days 4-22) encompassing the period when AMN postsynaptic target cells undergo developmental death. Neuron numbers were essentially identical at all ages examined, indicating that no AMN cell death occurred postnatally. Finally, from embryonic day 13 to postnatal day 22, animals were analyzed by using terminal transferase-mediated nick-end labeling to identify dying cells. Many fewer labeled cells were observed among AMNs than among SMNs. Thus, all three approaches indicated that there is a significant SMN/AMN difference in developmental cell death. The phenotypic trait(s) that underlies this difference may also be important in the relative resistance of AMNs to pathological conditions that induce death of SMNs, e.g., those involved in amyotrophic lateral sclerosis and excitotoxicity.

Immunolocalization of Fos protein was used to identify and characterize hypothalamic visceromotor populations responsive to acute and chronic intermittent footshock stress, and candidate afferent mediators of hypothalamic effects. Exposure to a single 30 minute footshock session induced maximal Fos expression in the paraventricular hypothalamic nucleus (PVH) 2 hours after the challenge; activated cells corresponded principally to hypophysiotropic neurons expressing corticotropin-releasing factor, with secondary involvement of magnocellular oxytocinergic and autonomic-related projection neurons. Extrahypothalamic cell groups activated in response to acute footshock included ones associated with the processing or modulation of somatosensory/nociceptive inputs, the limbic region of the telencephalon, and visceral sensory mechanisms. Rats with constant corticosterone levels displayed enhanced footshock-induced Fos expression in the parvicellular compartment of the PVH, as well as in certain limbic and somatosensory cell groups, the locus coeruleus, but not in medullary catecholaminergic cell groups. Animals subjected to chronic intermittent stress (2 sessions/day for 7 days) showed only modest evidence of habituation of cellular activation responses in the PVH and most extrahypothalamic regions. Rats bearing retrograde tracer deposits in the PVH and killed 2 hours after acute footshock displayed Fos-positive retrogradely labeled neurons principally in medullary catecholaminergic cell groups, with secondary foci in the hypothalamus, limbic region, and pontine tegmentum. This characterization of footshock-responsive systems identifies cell groups that are in a position to (1) mediate acute stress effects on hypothalamic visceromotor neurons, (2) comprise targets for corticosteroid negative feedback effects, and/or (3) underlie habituation of the neuroendocrine limb of the stress response.

The regulation of the vasculature in the skin is a complex process involving both perivascular nerves and local endothelial-mediated control. In this study, the perivascular innervation in the mystacial pad of the rat was characterized based upon immunochemical and lectin binding characteristics and distribution. All of the innervation labeled with anti-protein gene product 9.5 (PGP 9.5), which was used in double- and triple-labeling combinations with the Griffonia simplicifolia lectin (GSA) and antibodies against a variety of neuropeptides, enzymes, and structural proteins. GSA histofluorescence revealed an intricate microvasculature within the rows of tactile vibrissae, which form a natural grid to standardize analyses. Specific features of the vascular organization were confirmed by scanning electron microscopy. Each interval between adjacent vibrissae contained a predictably organized microvascular module composed of separate arterial channels and capillary networks for each of several different structures: papillary muscles, facial muscles, the interior of vibrissal follicle-sinus complexes, vibrissal papillae, and the upper dermis of the intervibrissal fur. Each module was innervated by at least two sets of sensory, at least two sets of sympathetic, and at least one possible set of parasympathetic. These sets not only differed in their biochemical characteristics, but also in their relative position within the arterial walls and their distribution among the microvasculature to the various structures. As such, the microvasculature to each type of structure had a particular combination of innervation, suggesting that separate neuronal mechanisms may be involved in regulating the blood flow to different types of targets even within the confines of a small territory of tissue.

Nitric oxide (NO) is a gaseous neurotransmitter that may mediate a decrease in sympathetic output to the periphery. This implication predicts that NO-producing neurons in the brain are activated in animals experiencing increased levels of sympathetic activity. To test this prediction, we subjected three groups of experimental rats to differing levels of environmental stimulation for 1 hour: minimal stimulation, moderate stimulation, and restraint stress. NO-producing neurons were histochemically visualized in sections of the brain, and activation of these neurons was assessed according to the neuronal expression of the immediate early gene c-fos. Constitutive activation of NO-producing neurons was found in the hypothalamus (paraventricular and supraoptic nuclei), dorsal raphe nuclei, and spinal nucleus of the trigeminal nerve of minimally stimulated rats. When animals were subjected to a novel environment (moderate stimulation), additional NO-producing neurons were activated in the medial septum, medial amygdala, hypothalamic nuclei (lateral, periventricular, and posterior), colliculi, nucleus raphe obscurus, medial vestibular nucleus, nucleus of the tractus solitarius, and several components of the ventrolateral medulla. Restraint stress caused the activation of NO-producing neurons in all of these areas, often in increasing numbers, and the activation of additional NO-producing neurons in the diagonal band of Broca, lateral and medial preoptic areas, basomedial and basolateral amygdalar nuclei, hypothalamic nuclei (dorsomedial, retrochiasmatic supraoptic, and circularis), nucleus raphe pontus, lateral parabrachial nucleus, and pontine nuclei. Expressed as a proportion of NO-producing neurons per section, the largest percentages (>20%) of double-stained neurons were found in the basolateral amygdala (46%), hypothalamic paraventricular nucleus (35%), corpora quadrigemina (estimated at 40%), dorsal raphe (45%), nuclei raphe pontus (33%) and obscurus (63%), lateral parabrachial nucleus (22%), medial vestibular nucleus (25%), lateral division of the nucleus paragigantocellularis (26%), and lateral reticular nucleus (35%). Evidence from other studies increasingly supports the concept that NO plays a generalized role in autonomic regulation by decreasing sympathetic output. Our results show that more NO-producing neurons were activated during stress than during minimal or moderate levels of stimulation. Together, the evidence suggests that NO is a neurochemical messenger that is utilized by individual autonomic neurons as the organism responds to increased levels of sympathetic activity.

An anatomical basis was sought for the postulated roles of nitric oxide (NO) as a labile transcellular messenger in the dorsal vagal complex (NTS-X). The diaphorase activity of NO synthase was used as a marker of neurons in NTS-X that are presumed to convert L-arginine to L-citrulline and NO. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) staining patterns in the nucleus tractus solitarii (NTS) were spatially related to terminal sites of primary visceral afferents from 1) orosensory receptors (e.g., rostral-central nucleus); 2) soft palate, pharynx, larynx, and tracheobronchial tree (e.g., dorsal, intermediate, and interstitial nuclei); 3) esophagus (nucleus centralis); 4) stomach (nucleus gelatinosus); 5) hepatic and coeliac nerves (nucleus subpostrema); and 6) carotid body and baroreceptors (medial commissural and dorsal-lateral nuclei). Primary visceral afferents were identified as sources of NADPHd-stained fiber plexuses in the NTS-X based on three findings: 1) the presence of NADPHd in nodose ganglion cells with morphological features of first-order sensory relay neurons; 2) retrograde transport of Fluoro-Gold (FG) or cholera toxin B (CT-B) from NTS-X to NADPHd-positive nodose ganglion neurons; and 3) striking reductions of NADPHd-stained processes within primary vagal projection fields ipsilateral to unilateral nodose ganglionectomy. A central origin of NADPHd-stained processes in NTS-X was identified in the medial parvicellular subdivision of the paraventricular hypothalamic nucleus. We conclude that NO of peripheral and central origin may modulate viscerosensory signal processing in the NTS-X and autonomic reflex function.

In an immunohistochemical study, we find that galanin is much more widely distributed in the peripheral innervation of the cat eye than in other animals so far examined. Previous studies of rat and pig eyes have revealed sparse galanin-positive nerves that presumably originate in the trigeminal ganglion. In contrast, the cat has a rich supply of galanin-containing nerve fibers throughout the uvea. Galanin-positive varicose nerves concentrate densely in iris muscles and distribute more sparsely in the ciliary muscle. The ciliary processes have a plexus of galanin-positive nerves underlying the ciliary epithelium at their base and positive nerve fibers coursing within their stroma. The ciliary artery and its branch vessels in the uvea are invested with a dense plexus of galanin-positive nerves. All autonomic ganglia supplying the eye contain cells that express galanin. It is present in 97% of superior cervical ganglion cells, coexisting with both tyrosine hydroxylase and neuropeptide Y; in 80% of pterygopalatine ganglion cells, most of which also contain vasoactive intestinal peptide; and in approximately 25% of ciliary ganglion cells. After unilateral superior cervical ganglionectomy, galanin-positive nerves almost totally disappear from the iris muscles, demonstrating that they are predominantly of sympathetic origin. Galanin-positive nerves investing the ciliary artery and choroidal blood vessels are not detectably reduced by sympathectomy, indicating that perivascular parasympathetic nerves from the pterygopalatine ganglion also express galanin. Other galanin-containing nerves in the eye can originate from the trigeminal and ciliary ganglia. The prominence of galanin in the ocular autonomic innervation of the cat provides an opportunity to explore the physiological effects of this neuropeptide in the eye.

The time at which a neuron is "born" appears to have significant consequences for the cell's subsequent differentiation. As part of a continuing investigation of cholinergic neuronal development, we have combined ChAT immunocytochemistry and [3H]thymidine autoradiography to determine the generation patterns of somatic and autonomic motor neurons at upper thoracic (T1-3), upper lumbar (L1-3), and lumbosacral (L6-S1) levels of the rat spinal cord. Additionally, the generation patterns of two subsets of cholinergic interneurons (partition cells and central canal cluster cells) were compared with those of somatic and autonomic motor neurons. Embryonic day 11 (E11) was the first day of cholinergic neuronal generation at each of the three spinal levels studied, and it also was the peak generation day for somatic and autonomic neurons in the upper thoracic spinal cord. The peak generation of homologous neurons at upper lumbar and lumbosacral spinal levels occurred at E12 and E13, respectively. Somatic and autonomic motor neurons were generated synchronously, and their production at each rostrocaudal level was virtually completed within a 2-day period. Cholinergic interneurons were generated 1 or 2 days later than motor neurons at the same rostrocaudal level. In summary, the birthdays of all spinal cholinergic neurons studied followed the general rostrocaudal spatiotemporal gradient of spinal neurogenesis. In addition, the generation of cholinergic interneurons also followed the general ventrodorsal gradient. In contrast, however, autonomic motor neurons disobeyed the rule of a ventral-to-dorsal progression of spinal neuronal generation, thus adding another example in which autonomic motor neurons display unusual developmental patterns.

Unlabelled: Acetylcholine (ACh) plays a major role in central autonomic regulation, including the control of arterial blood pressure (AP). Previously unknown neuroanatomic substrates of cholinergic-autonomic control were mapped in this study. Cholinergic perikarya and bouton-like varicosities were localized by an immunocytochemical method employing a monoclonal antiserum against choline acetyltransferase (ChAT), the enzyme synthesizing ACh. In the forebrain, bouton-like varicosities and/or perikarya were detected in the septum, bed nucleus of the stria terminalis, amygdala (in particular, autonomic projection areas AP1 and AP2 bordering the central subnucleus), hypothalamus (rostrolateral/innominata transitional area, perifornical, dorsal, incertal, caudolateral, posterior [PHN], subparafascicular, supramammillary and mammillary nuclei). Few or no punctate varicosities were labeled in the paraventricular (PVN) or supraoptic (SON) hypothalamic nuclei. In the mid- and hindbrain, immunoreactive cells and processes were present in the nucleus of Edinger-Westphal, periaqueductal gray, parabrachial complex (PBC), a periceruleal zone avoiding the locus ceruleus (LC), pontine micturition field, pontomedullary raphe, paramedian reticular formation and periventricular gray, A5 area, lateral tegmental field, nucleus tractus solitarii (NTS), nucleus commissuralis, nucleus reticularis rostroventrolateralis (RVL), and the ventral medullary surface (VMS). In the PBC, immunoreactive varicosities identified areas previously unexplored for cholinergic autonomic responsivity (superior, internal, dorsal, and central divisions of the lateral subnucleus, nucleus of Koelliker-Fuse and the medial subnucleus). In the NTS, previously undescribed ChAT-immunolabeled cells and processes were concentrated at intermediate and subpostremal levels and distributed viscerotopically in areas receiving primary cardiopulmonary afferents. In the nucleus RVL, cholinergic perikarya were in proximity to the VMS and medial to adrenergic cell bodies of the C1 area. Punctate varicosities of unknown origin and dendrites extending ventrally from the nucleus ambiguus overlapped the C1 area and immediate surround of RVL., In Conclusion: 1) Cholinergic perikarya and putative terminal fields, overlap structures that are rich in cholinoreceptors and express autonomic, neuroendocrine, or behavioral responsivity to central cholinergic stimulation (PHN, NTS, RVL). The role of ACh in most immunolabeled areas, however, has yet to be determined. Overall, these data support the concept that cholinergic agents act at multiple sites in the CNS and with topographic specificity.(ABSTRACT TRUNCATED AT 400 WORDS)