Characterization of the primary spinal afferent innervation of the mouse colon using retrograde labelling

Visceral pain is the most common form of pain produced by disease and is thus of interest in the study of gastrointestinal (GI) complaints such as irritable bowel syndrome, in which sensory signals perceived as GI pain travel in extrinsic afferent neurones with cell bodies in the dorsal root ganglia (DRG). The DRG from which the primary spinal afferent innervation of the mouse descending colon arises are not well defined. This study has combined retrograde labelling and immunohistochemistry to identify and characterize these neurones. Small to medium-sized retrogradely labelled cell bodies were found in the DRG at levels T8-L1 and L6-S1. Calcitonin gene-related peptide (CGRP)- and P2X3-like immunoreactivity (LI) was seen in 81 and 32%, respectively, of retrogradely labelled cells, and 20% bound the Griffonia simplicifolia-derived isolectin IB4. CGRP-LI and IB4 were co-localized in 22% of retrogradely labelled cells, whilst P2X3-LI and IB4 were co-localized in 7% (vs 34% seen in the whole DRG population). Eighty-two per cent of retrogradely labelled cells exhibited vanilloid receptor 1-like immunoreactivity (VR1-LI). These data suggest that mouse colonic spinal primary afferent neurones are mostly peptidergic CGRP-containing, VR1-LI, C fibre afferents. In contrast to the general DRG population, a subset of neurones exist that are P2X3 receptor-LI but do not bind IB4.     

Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor.

Although the gastric tension receptor has been characterized behaviorally and electrophysiologically quite well, its location and structure remains elusive. Therefore, the vagal afferents to the rat fundus (forestomach or nonglandular stomach) were anterogradely labeled in vivo with injections of the carbocyanine dye Dil into the nodose ganglia, and the nerves and ganglia of the enteric nervous system were labeled in toto with intraperitoneal Fluorogold injection. Dissected layers and cryostat cross sections of the fundic wall were mounted in glycerin and analyzed by means of conventional and laser scanning confocal microscopy. Particularly in the longitudinal, and to a lesser extent in the circular, smooth muscle layers, Dil-labeled fibers and terminals were abundant. These processes, which originated from fibers coursing through the myenteric ganglia and connectives, entered either muscle coat and then ran parallel to the respective muscle fibers, often for several millimeters. They ran in close association with the Fluorogold-labeled network of interstitial cells of Cajal, upon which they appeared to form multiple spiny appositions or varicosities. In the myenteric plexus, two different types of afferent vagal structures were observed. Up to 300 highly arborizing endings forming dense accumulations of small puncta similar to the esophageal intraganglionic laminar endings (Rodrigo et al., '75 Acta Anat. 92:79-100) were found in the fundic wall ipsilateral to the injected nodose ganglion. They often covered small clusters of myenteric neurons or even single isolated ganglion cells (mean = 5.8 neurons) and tended to extend throughout the neuropil of the ganglia. In a second pattern, fine varicose fibers with less profuse arborizations innervated mainly the central regions of myenteric ganglia. Camera lucida analyses established that single vagal afferent fibers had separate collaterals in both a smooth muscle layer and the myenteric ganglia. Finally, Dil-labeled afferent vagal fibers were also found in the submucosa and mucosa. Control experiments in rats with supranodose vagotomy as well as rats with Dil injections directly in the distal cervical vagus ruled out the possibility of colabeling of afferent fibers of passage. In triple labeling experiments, in conjunction with Dil labeling of afferents and Fluorogold labeling of enteric neurons, the carbocyanine dye DiA was injected into the dorsal motor nucleus of the vagus to anterogradely label the efferent vagal fibers and terminals. The different distributions and morphological characteristics of the vagal afferents and efferents could be simultaneously compared. In some instances the same myenteric ganglion was apparently innervated by an afferent laminar ending and an efferent terminal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light and electron microscopic immunocytochemical localization of AMPA-selective glutamate receptors in the rat spinal cord

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors are probably the most widespread excitatory neurotransmitter receptors of the central nervous system, and they play a role in most normal and pathological neural activities. However, previous detailed studies of AMPA subunit distribution have been limited mainly to the brain. Thus, a comprehensive study of AMPA receptor subunit distribution was carried out on sections of rat spinal cord and dorsal root ganglia, which were immunolabeled with antibodies made against peptides corresponding to C-terminal portions of the AMPA receptor subunits: GluR1, GluR2/3, and GluR4. In the spinal cord. Labeling was most prominent in the superficial dorsal horn, motoneurons, and nuclei containing preganglionic autonomic neurons. Immunostaining also was observed in neurons in other regions including those known to contain Renshaw cells and Ia Inhibitory cells. Although overall immunostaining was lighter with antibody to GluR1 than with GluR2/3 and 4, there were neurons were neurons that preferentially stained with antibody to GluR1. These “GluR1 intense” neurons were usually fusiform and most concentrated in lamina X. In dorsal root ganglia, immunostaining of ganglion cell bodies was moderate to dense with antibody to GluR2/3 and light to moderate with antibody to GluR4. Possible neuroglia in the spinal cord (mainly GluR2/3 and 4) and satellite cells in dorsal root ganglia (GliR4) were immunostained. Electron microscopic studies of the the superficial dorsal horn and lateral motor column showed staining that was restricted mainly to postsynaptic densities and associated dendritic and cell body cytoplasm. In dorsal horn, colocalization of dense-cored vesicles with clear, round synaptic vesicles was observed in unstained presynaptic terminals apposed to stained postsynaptic densities. Subsynaptic dense bodies (Taxi-bodies) were associated with some stained postsynaptic densities in the superficial dorsal horn and lateral motor column. Based on several morphological features including vesicle structure and presence of Taxi-bodies, it is likely that at least some of the postsynaptic staining seen in this study is apposed to glutamatergic input from primary sensory afferent terminals. © 1994 Wiley-Liss, Inc.     

Development of neurons and fibers containing calcium binding proteins in the pallial amygdala of mouse, with special emphasis on those of the basolateral amygdalar complex

We studied the development of neurons and fibers containing calbindin, calretinin, and parvalbumin in the mouse pallial amygdala, with special emphasis on those of the basolateral amygdalar complex. Numerous calbindin-immunoreactive (CB+) cells were observed in the incipient basolateral amygdalar complex and cortical amygdalar area from E13.5. At E16.5, CB+ cells became more abundant in the lateral and basolateral nuclei than in the basomedial nucleus, showing a pattern very similar to that of gamma-aminobutyric acid (GABA)ergic neurons. Many CB+ cells observed in the pallial amygdala appeared to originate in the anterior entopeduncular area/ganglionic eminences of the subpallium. The density of CB+ cells gradually increased in the pallial amygdala until the first postnatal week and appeared to decrease later, coinciding with the postnatal appearance of parvalbumin cells and raising the possibility of a partial phenotypic shift. Calretinin (CR) immunoreactivity could be observed in a few cells and fibers in the pallial amygdala at E14.5, and by E16.5 it became a good marker of the different nuclei of the basolateral amygdalar complex. Numerous CB+ and CR+ varicosities, part of which have an intrinsic origin, were observed in the basolateral amygdalar complex from E16.5, and some surrounded unstained perikarya and/or processes before birth, indicating an early formation of inhibitory networks. Each calcium binding protein showed a distinct spatiotemporal expression pattern of development in the mouse pallial amygdala. Any alteration in the development of neurons and fibers containing calcium binding proteins of the pallial amygdala may result in important disorders of emotional and social behavior.     

Simultaneous labeling of vagal innervation of the gut and afferent projections from the visceral forebrain with dil injected into the dorsal vagal complex in the rat.

The vagal innervation of the different layers of the rat gastrointestinal wall was identified with the fluorescent carbocyanine dye Dil, injected into the dorsal motor nucleus of the vagus (dmnX). Multiple, bilateral injections were used to label all dmnX preganglionic motoneurons, and as a consequence, most of the vagal primary afferents that terminate in the adjacent nucleus of the solitary tract (nts) were also retrogradely and transganglionically labeled. With Fluorogold used to label the enteric nervous system completely and specifically, the Dil-labeled vagal profiles could be visualized and quantified in their anatomical relation to the neurons of the myenteric and submucous ganglia. In the myenteric plexus, vagal fibers and terminals were found throughout the gastrointestinal tract as far caudal as the descending colon, but there was a general decreasing proximodistal gradient in the density of vagal innervation. All parts of the gastric myenteric plexus (fundus, corpus, antrum), as well as the proximal duodenum, were extremely densely innervated, with vagal fibers and terminals in virtually every ganglion and connective. Further caudally, both the percentage of innervated myenteric ganglia and the average density of label within the ganglia rapidly decreased, with the exception of the cecum and proximal colon, where up to 65% of the ganglia were innervated. In the gastric and duodenal submucosa very few and in the mucosa no vagal fibers and terminals were found. With both normal epifluorescence and laser scanning confocal microscopy, highly varicose or beaded terminal structures of various size and geometry could be identified. The Dil injections, which impregnated the dmnX as well as the adjacent nts, resulted in retrograde and anterograde labeling of all the previously reported forebrain connections with the dorsal vagal complex. We conclude that the myenteric plexus is the primary target of vagal innervation throughout the gastrointestinal tract, and that its innervation is more complete than previously assumed. In contrast, vagal afferent (and efferent) innervation of mucosa and submucosa seems conspicuously sparse or absent. Furthermore, the use of more focal injections of Dil offers the prospect to simultaneously identify specific subsets of vagal preganglionics and their central nervous inputs.     

Vagal afferent innervation of the proximal gastrointestinal tract mucosa: chemoreceptor and mechanoreceptor architecture

The vagus nerve supplies low-threshold chemo- and mechanosensitive afferents to the mucosa of the proximal gastrointestinal (GI) tract. The absence of a full characterization of the morphology and distributions of these projections has hampered comprehensive functional analyses. In the present experiment, dextran (10K) conjugated with tetramethylrhodamine and biotin was injected into the nodose ganglion and used to label the terminal arbors of individual vagal afferents of both rats and mice. Series of serial 100-μm thick sections of the initial segment of the duodenum as well as the pyloric antrum were collected and processed with diaminobenzidine for permanent tracer labeling. Examination of over 400 isolated afferent fibers, more than 200 from each species, indicated that three vagal afferent specializations, each distinct in morphology and in targets, innervate the mucosa of the proximal GI tract. One population of fibers, the villus afferents, supplies plates of varicose endings to the apical tips of intestinal villi, immediately subjacent to the epithelial wall. A second type of afferent, the crypt afferent, forms subepithelial rings of varicose processes encircling the intestinal glands or crypts, immediately below the crypt-villus junction. Statistical assessment of the isolated fibers indicated that the villus arbors and the crypt endings are independent, issued by different vagal afferents. A third vagal afferent specialization, the antral gland afferent, arborizes along the gastric antral glands and forms terminal concentrations immediately below the luminal epithelial wall. The terminal locations, morphological features, and regional distributions of these three specializations provide inferences about the sensitivities of the afferents.     

Melanocortin-4 receptor expression in different classes of spinal and vagal primary afferent neurons in the mouse

Melanocortin‐4 receptor (MC4R) ligands are known to modulate nociception, but the site of action of MC4R signaling on nociception remains to be elucidated. The current study investigated MC4R expression in dorsal root ganglia (DRG) of the MC4R‐GFP reporter mouse. Because MC4R is known to be expressed in vagal afferent neurons in the nodose ganglion (NG), we also systematically compared MC4R‐expressing vagal and spinal afferent neurons. Abundant green fluorescent protein (GFP) immunoreactivity was found in about 45% of DRG neuronal profiles (at the mid‐thoracic level), the majority being small‐sized profiles. Immunohistochemistry combined with in situ hybridization confirmed that GFP was genuinely produced in MC4R‐expressing neurons in the DRG. While a large number of GFP profiles in the DRG coexpressed Nav1.8 mRNA (84%) and bound isolectin B4 (72%), relatively few GFP profiles were positive for NF200 (16%) or CGRP (13%), suggesting preferential MC4R expression in C‐fiber nonpeptidergic neurons. By contrast, GFP in the NG frequently colocalized with Nav1.8 mRNA (64%) and NF200 (29%), but only to a moderate extent with isolectin B4 (16%). Lastly, very few GFP profiles in the NG expressed CGRP (5%) or CART (4%). Together, our findings demonstrate variegated MC4R expression in different classes of vagal and spinal primary afferent neurons, and underscore the role of the melanocortin system in modulating nociceptive and nonnociceptive peripheral sensory modalities. J. Comp. Neurol. 520:3933–3948, 2012. © 2012 Wiley Periodicals, Inc.     

Cortical and brainstem neurons containing calcium-binding proteins in a murine model of Duchenne's muscular dystrophy: selective changes in the sensorimotor cortex

In the muscular dystrophic (mdx) mouse, which is characterized by deficient dystrophin expression and provides a model of Duchenne's muscular dystrophy, we previously demonstrated marked central nervous system alterations and in particular a quantitative reduction of corticospinal and rubrospinal neurons and pathologic changes of these cells. Prompted by these findings and in view of the relations between calcium ions and dystrophin, we analyzed with immunohistochemistry the neurons containing the calcium-binding proteins parvalbumin, calbindin D28k, and calretinin in cortical areas and brainstem nuclei of mdx mice. In the sensorimotor cortex, parvalbumin-positive and calbindin-positive neurons, which represent a subset of cortical interneurons, were significantly more numerous in mdx mice than in wild-type ones. In addition, the laminar distribution of parvalbumin-positive neurons in the motor and somatosensory cortical areas of mdx mice was altered with respect to wild-type animals. No alterations in the number and distribution were found in the parvalbumin- or calbindin-expressing cell populations of the visual and anterior cingulate cortices of mdx mice. The pattern of calretinin immunoreactivity was normal in all investigated cortical areas. The cell populations containing either calcium-binding protein were similar in brainstem nuclei of mdx and wild-type mice. The present findings demonstrated selective changes of subsets of interneurons in the motor and somatosensory cortical areas of mdx mice. Therefore, the data showed that, in the cortices of these mutant animals, the previously demonstrated pathologic changes of corticospinal cell populations are accompanied by marked alterations in the local circuitry.     

Zinc-containing afferent projections to the rat corticomedial amygdaloid complex: A retrograde tracing study

The mammalian amygdaloid complex is densely innervated by zinc-containing neurons. The distribution of the terminals throughout the region has been described, but the origins of these zinc-containing fibers have not. The present work describes the origins of one major component of the zinc-containing innervation of the amygdaloid complex, namely, the component that innervates the corticomedial complex. Selective labeling of zinc-containing axons was accomplished by intracerebral microinfusion of selenium anions (SeO₃²⁻), a procedure that produces a ZnSe precipitate in zinc-containing axonal boutons with subsequent retrograde transport to the neurons of origin. After infusions of SeO₃²⁻ into combinations of cortical, medial, or amygdalohippocampal regions, retrogradely labeled zinc-containing somata were found in all amygdaloid nuclei except for the medial and central nuclei, the bed nucleus of the accessory olfactory tract, the nucleus of the lateral olfactory tract, and the anterior amygdaloid area. Extrinsic zinc-containing projections to the same amygdaloid terminal fields were found to originate from the infralimbic, cingulate, piriform, perirhinal and entorhinal cortices, and from the prosubiculum and CA1. Commissural zinc-containing projections were found to originate from the posterolateral and posteromedial cortical nuclei and from the posterior part of the basomedial nucleus. Zinc-containing neurons have been implicated in the pathophysiology of epilepsy, in cell death after seizure or stroke, and in Alzheimer's disease, all clinical conditions that involve the amygdaloid complex. Identification of the zinc-containing pathways is a prerequisite to the elucidation of zinc's role in these disorders. J. Comp. Neurol. 400:375–390, 1998. © 1998 Wiley-Liss, Inc.     

Fine structure of rat septohippocampal neurons: I. Identification of septohippocampal projection neurons by retrograde tracing combined with electron microscopic immunocytochemistry and intracellular staining.

In this report the normal dendritic organization and fine structure of identified septohippocampal projection neurons is described as a prerequisite for a time course analysis of retrograde changes in these neurons following axotomy (see Naumann et al., J. Comp. Neurol. 325:219-242, 1992). Septohippocampal projection neurons were retrogradely labeled by injection of the fluorescent tracer Fluoro-Gold into the hippocampus. Next, retrogradely labeled cells in Vibratome sections of the medial septum/diagonal band complex were intracellularly stained with the fluorescent dye Lucifer Yellow (LY). Photooxidation of LY resulted in a stable electron-dense reaction product, which allowed us to study these double-labeled neurons by electron microscopy. Another series of sections containing retrogradely labeled neurons were immunostained for choline acetyltransferase (ChAT) or parvalbumin (PARV). In this way the fine structure of two different chemically characterized subpopulations of septohippocampal neurons could be compared with that of the LY-injected neurons. Intracellular filling of retrogradely labeled neurons with LY stained the cell body and the entire dendritic arbor. Essentially, three classes of neurons could be distinguished, i.e., bipolar cells, multipolar neurons, and an intermediate group. All these neurons displayed smooth, often varicose dendrites lacking spines. Mainly located close to the midline, there was a group of cells with only very few if any LY-stained dendrites. In the electron microscope, the double-labeled neurons were easily identified by numerous electron-dense lysosomes associated with transported Fluoro-Gold and the diffuse reaction product resulting from photooxidation. They displayed fine-structural characteristics as previously described for cholinergic neurons. In fact, our fine-structural analysis of ChAT-positive Fluoro-Gold-labeled neurons, but also of back-filled PARV-positive cells, gave very similar results. All these neurons had infolded nuclei, abundant cytoplasmic organelles, and a few axosomatic synapses. Thus, a plain electron microscopic study does not allow one to distinguish between subpopulations of septohippocampal projection neurons.     

Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat

Endomorphin-2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu-opioid receptor. In the present study, we used double-fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2-expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small-to-medium size neurons, most of which co-expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 microM in diameter (mean 24.3 +/- 4.3 microM). Double-labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2-immunoreactive cell bodies also label with SP antisera, 83% co-express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non-peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin-A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double-labelling showed co-localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu-opioid autoreceptors.     

Different types of spinal afferent nerve endings in stomach and esophagus identified by anterograde tracing from dorsal root ganglia

In visceral organs of mammals, most noxious (painful) stimuli as well as innocuous stimuli are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRGs). One of the major unresolved questions is the location, morphology, and neurochemistry of the nerve endings of spinal afferents that actually detect these stimuli in the viscera. In the upper gastrointestinal (GI) tract, there have been many anterograde tracing studies of vagal afferent endings, but none on spinal afferent endings. Recently, we developed a technique that now provides selective labeling of only spinal afferents. We used this approach to identify spinal afferent nerve endings in the upper GI tract of mice. Animals were anesthetized, and injections of dextran‐amine were made into thoracic DRGs (T8–T12). Seven days post surgery, mice were euthanized, and the stomach and esophagus were removed, fixed, and stained for calcitonin gene‐related peptide (CGRP). Spinal afferent axons were identified that ramified extensively through many rows of myenteric ganglia and formed nerve endings in discrete anatomical layers. Most commonly, intraganglionic varicose endings (IGVEs) were identified in myenteric ganglia of the stomach and varicose simple‐type endings in the circular muscle and mucosa. Less commonly, nerve endings were identified in internodal strands, blood vessels, submucosal ganglia, and longitudinal muscle. In the esophagus, only IGVEs were identified in myenteric ganglia. No intraganglionic lamellar endings (IGLEs) were identified in the stomach or esophagus. We present the first identification of spinal afferent endings in the upper GI tract. Eight distinct types of spinal afferent endings were identified in the stomach, and most of them were CGRP immunoreactive. J. Comp. Neurol. 524:3064–3083, 2016. © 2016 Wiley Periodicals, Inc.     

Cochlear innervation in the developing rat: an immunocytochemical study of neurofilament and spectrin proteins

We have studied the innervation of the developing cochlea by immunocytochemical staining of the cytoskeletal proteins, neurofilament (NF), and spectrin (brain spectrin and erythrocyte spectrin). NF immunoreactivity was seen in spiral ganglion cell bodies and their processes and in fibers of the intraganglionic spiral bundle (IGSB) on gestational day 16. NF immunoreactivity with monoclonal antibodies to NF160 and NF68 was present beneath both inner hair cells (the IHC) and outer hair cells (OHCs) on gestational day 20. NF200 immunostaining was located only in the IGSB and in fibers reaching the IHC. The first NF200 immunoreactivity beneath the OHCs was seen in the basal turn at birth. NF labelling began to decrease on postnatal day 9 and its intensity became more like that of the adult. Brain spectrin immunostaining was first seen in the IGSB of the basal turn on gestational day 18. It reached the fibers between the spiral ganglion and the IHC on gestational day 20. Brain spectrin immunoreactivity was first seen beneath the OHCs in the basal turn at birth. It reached all the OHCs of the cochlea by postnatal day 4, and began to decrease 9 days after birth. Erythrocyte spectrin immunostaining was first observed during the second postnatal week, when it labelled spiral ganglion cells. The distribution of NF200 and brain spectrin immunoreactivity suggested that efferent innervation of OHCs is present at birth in the rat, and confirms previous studies showing the early efferent innervation of the OHCs of the mouse and the rat at birth, and the time lag between the appearance of the two spectrin isoforms during development.     

Organization of the dorsocaudal neostriatal complex: A retrograde and anterograde tracing study in the domestic chick with special emphasis on pathways relevant to imprinting

In the forebrain of domestic chicks, a network of distinct regions is crucially involved in auditory and visual filial imprinting. Among these areas, a distinct part of the dorsocaudal neostriatal complex (dNC complex), termed neostriatum dorsocaudale (Ndc), was recently discovered by its enhanced metabolic activity during the presentation of auditory and visual imprinting stimuli. Since there is evidence that the dNC complex consists of several distinct functional subareas, we investigated the neural connections of different parts of the dNC complex by retro- and anterograde pathway tracing. Special emphasis was put on the connections of the dNC complex with other imprinting relevant regions in the rostral telencephalon, such as the mediorostral neostriatum/hyperstriatum ventrale (MNH) and the intermediate and medial part of the hyperstriatum ventrale (IMHV). By anterograde and multiple retrograde pathway tracing, we found that the dNC complex may at least be subdivided into three major constituents. The most medial part of the dNC complex, termed neostriatum dorsale (Nd), is characterized by strong reciprocal connections with the neostriatal part of the MNH and by its auditory related inputs, including those from the output layers L1 and L3 of field L, and the shell region of the thalamic n. ovoidalis. The Ndc, which occupies the central aspects of the dNC complex, is mainly characterized by reciprocal connections with the ectostriatal belt (Ep) and the adjacent neostriatum (N). Furthermore, Nd and Ndc receive strong thalamic input from the n. dorsolateralis posterior (DLP), both project to the IMHV, and both are reciprocally connected with the archistriatum intermedium (AI). The most lateral aspect of the dNC complex, termed Ndl, is characterized by afferents from the neostriatum frontale, pars trigeminalis (NFT), and by the lack of a thalamic input. Results indicate that the dNC complex comprises distinct subregions, which are characterized by their specific afferents from parasensory areas of different sensory modalities. These different subregions may be integral components of a general pattern of sensory processing in the avian telencephalon. The strong interconnections between Nd, Ndc, and MNH as well as IMHV may constitute essential parts of auditory and visual imprinting circuits. J. Comp. Neurol. 395:380–404, 1998. © 1998 Wiley-Liss, Inc.     

Hyperpolarization-activated, cyclic nucleotide-gated cation channels: roles in the differential electrophysiological properties of rat primary afferent neurons

The large, medium-sized, and small neurons of the dorsal root ganglion (DRG) have different functions in the processing of various senses. Hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) contribute greatly to neuronal excitability. In the present study, which used whole-cell patch clamp techniques and immunohistochemical staining methods, the electrophysiological properties of DRG neurons were systematically compared, and the roles of HCN-1, -2, and -4 were examined. The main results were as follows. 1) The large neurons had significantly higher V0.5 values (membrane potential at which the HCN channels were half-activated) and shorter time constants (tau) than small or medium-sized DRG neurons. However, large DRG neurons had higher Ih density (HCN neuron current). 2) HCN-1 was found predominantly, but not exclusively, in large and medium-sized DRG neurons; HCN-2 was found in all DRG neurons; and HCN-4 was poorly visualized in all DRG neurons. HCN-1 and HCN-2 were colocalized in large and medium-sized neurons with immunostaining of adjacent sections. In the dorsal horn of the spinal cord, HCN-1, HCN-2, and HCN-4 were all expressed in laminae I-IV, although HCN-1 was not detectable in lamina II. 3) Blockade of Ih current in DRG neurons caused a significant decrease in V0.5, resting membrane potential, and repetitive firing number of action potential and a significant increase in time of rising phase of action potential. These results suggest that the different HCN channels in the three types of DRG neurons might contribute to their differential electrophysiological properties.     

Origin of the dopaminergic innervation of the central extended amygdala and accumbens shell: a combined retrograde tracing and immunohistochemical study in the rat

The origin of the dopaminergic innervation of the central extended amygdala (EAc; i.e., the lateral bed nucleus of the stria terminalis [BSTl]-central amygdaloid nucleus [Ce] continuum) and accumbens shell (AcSh) was studied in the rat by combining retrograde transport of Fluoro-Gold (FG) with tyrosine hydroxylase (TH) immunofluorescence. Perikaryal profiles (PP) immunoreactive to FG and to both FG and TH were counted in A8-A14 dopaminergic districts. Our results suggest that dopaminergic inputs to the EAc and AcSh arise from the ventral tegmental area-A10, substantia nigra, pars compacta-A9, and retrorubral nucleus-A8 groups as well as from the dorsal raphe nucleus and periaqueductal gray substance, housing the dorsocaudal part of A10 group (A10dc). Quantitative estimates reveal that the A10dc group contains approximately half of the total number of FG/TH double-labeled PP projecting to Ce and BSTl. By using an anti-dopamine serum, DR/PAG projections to Ce were confirmed to be in part dopaminergic. In contrast, modest numbers of FG/TH double-labeled PP were seen in the A10dc group after injections in the sublenticular extended amygdala, interstitial nucleus of the posterior limb of the anterior commissure or AcSh. Ventral mesencephalic projections to the EAc display a crude mediolateral topographic organization, whereas those to the AcSh are topographically organized along a mediolateral and an inverted dorsoventral dimension. The diencephalic dopaminergic groups do not innervate the EAc or AcSh, except for the periventricular gray-A11 which sends light dopaminergic projections to Ce and BSTl. Overall, the present results provide additional details on the organization of the mesolimbic dopaminergic system that critically controls behavioral responsiveness to salient environmental stimuli.     

Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions.

Three neuroanatomical tracers have been employed to map the axonal projections formed between transplants of fetal spinal cord tissue and the surrounding host spinal cord in adult rats. Solid pieces of embryonic day 14 (E14) rat spinal cord were placed into hemisection aspiration cavities in the lumbar spinal cord. Injections of either (1) a mixture of horseradish peroxidase and wheat germ agglutinin- conjugated horseradish peroxidase, (2) Fluoro-Gold, or (3) Phaseolus vulgaris leucoagglutinin (PHA-L) were made into the transplants or the neighboring segments of the host spinal cord at 6 weeks to 14 months post-transplantation. Injections of anterograde and retrograde tracers into the transplants revealed extensive intrinsic projections that often spanned the length of the grafts. Axons arising from the transplants extended into the host spinal cord as far as 5 mm from the host-graft interface, as best revealed by retrograde labeling with Fluoro-Gold. Consistent with these observations, iontophoretic injections of PHA-L into the transplants also produced labeled axonal profiles at comparable distances in the host spinal cord, and in some instances elaborate terminals fields were observed surrounding host neurons. The majority of these efferent fibers labeled with PHA-L, however, were confined to the immediate vicinity of the host-graft boundary, and no fibers were seen traversing cellular partitions between host and transplant tissues. Host afferents to the transplants were also revealed by these tracing methods. For example, the injection of Fluoro-Gold into the grafts resulted in labeling of host neurons within the spinal cord and nearby dorsal root ganglia. In most cases, retrogradely labeled neurons in spinal gray matter were located within 0.5 mm of the graft site, although some were seen as far as 4-6 mm away. The distance and relative density of ingrowth exhibited by host axons into the grafts, however, appeared modest based upon the results of HRP and Fluoro-Gold retrograde labeling. This was further confirmed with the PHA-L anterograde method. Whereas some host fibers were seen extending into the transplants, the majority of PHA-L containing axons formed terminal-like profiles at or within 0.5 mm of the host-graft interface. The comprehensive view of intrinsic connectivity and host-graft projections obtained in these studies indicates that intraspinal grafts of fetal spinal cord tissue can establish a short-range intersegmental circuitry in the injured, adult spinal cord. These observations are consistent with the view that such grafts may contribute to the formation of a functional relay between separated segments of the spinal cord after injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

PACAP-containing intrapineal nerve fibers originate predominantly in the trigeminal ganglion: a combined retrograde tracing- and immunohistochemical study of the rat

Pituitary adenylate-cyclase activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus and located in many neuronal systems in both the central and peripheral nervous system. PACAP is also found in nerve fibers innervating the pineal gland, where it stimulates the secretion of the pineal hormone, melatonin, by binding to specific PACAP-receptors located on the cell membrane of the pinealocyte. In this study we have investigated the origin of PACAP-containing nerve fibers innervating the rat pineal gland by combined retrograde tracing with Fluorogold and immunohistochemistry for PACAP. A solution of 2% Fluorogold was injected iontophoretically into the superficial pineal gland of Wistar rats, and the animals were allowed to survive for 1 week. After perfusion fixation of the rats, the location of the tracer was investigated in the brain and the sphenopalatine, otic, and trigeminal ganglia. The tracer was found in all the investigated ganglia. However, colocalization with PACAP was predominantly found in the trigeminal ganglion and only occasionally in the sphenopalatine and otic ganglia. Due to the stimulatory function of PACAP on pineal melatonin secretion, the PACAP-containing neurons of this ganglion could be considered a subset of the parasympathetic nervous system. The presence of neurons with a parasympathetic function in a ganglion that has been considered a purely sensory ganglion, is a new concept in neuroanatomy.     

A combined immunocytochemical and retrograde tracing study of noradrenergic connections between the caudal medulla and bed nuclei of the stria terminalis

Region-specific noradrenergic inputs to the bed nuclei of the stria terminalis (BST) from the caudal medulla were studied using combined Fast Blue injections and tyrosine hydroxylase immunoreactivity (TH-ir). Injections into the rostral, dorsal, ventral and lateral BST resulted in predominantly ipsilateral retrograde labelling restricted to the mediodorsal and ventrolateral caudal medulla. Mediodorsal projecting neurones comprised the A2 TH-ir and a second non-aminergic group medial to A2. All ventrolateral retrogradely labelled neurones showed TH-ir and corresponded to A1. Injections into the caudal BST did not label the A2 and very few A1 neurones, indicating a paucity of noradrenergic inputs from this area of the medulla.