Peripheral innervation patterns of vestibular nerve afferents in the bullfrog utriculus

Vestibular nerve afferents innervating the bullfrog utriculus differ in their response dynamics and sensitivity to natural stimulation. They also supply hair cells that differ markedly in hair bundle morphology. To examine the peripheral innervation patterns of individual utricular afferents more closely, afferent fibers were labeled by the extracellular injection of horseradish peroxidase (HRP) into the vestibular nerve after sectioning the vestibular nerve medial to Scarpa's ganglion to allow the degeneration of sympathetic and efferent fibers. The peripheral arborizations of individual afferents were then correlated with the diameters of their parent axons, the regions of the macula they innervate, and the number and type of hair cells they supply. The utriculus is divided by the striola, a narrow zone of distinctive morphology, into medial and lateral parts. Utricular afferents were classified as striolar or extrastriolar according to the epithelial entrance of their parent axons and the location of their terminal fields. In general, striolar afferents had thicker parent axons, fewer subepithelial bifurcations, larger terminal fields, and more synaptic endings than afferents in extrastriolar regions. Afferents in a juxtastriolar zone, immediately adjacent to the medial striola, had innervation patterns transitional between those in the striola and more peripheral parts of the medial extrastriola. Most afferents innervated only a single macular zone. The terminal fields of striolar afferents, with the notable exception of a few afferents with thin parent axons, were generally confined to one side of the striola. Hair cells in the bullfrog utriculus have previously been classified into four types based on hair bundle morphology (Lewis and Li: Brain Res. 83:35–50, 1975). Afferents in the extrastriolar and juxtastriolar zones largely or exclusively innervated Type B hair cells, the predominant hair cell type in the utricular macula. Striolar afferents supplied a mixture of four hair cell types, but largely contacted Type B and Type C hair cells, particularly on the outer rows of the medial striola. Afferents supplying more central striolar regions innervated fewer Type B and larger numbers of Type E and Type F hair cells. Striolar afferents with thin parent axons largely supplied Type E hair cells with bulbed kinocilia in the innermost striolar rows. © 1994 Wiley-Liss, Inc.     

Actions of ATP on the soma of bullfrog primary afferent neurons and its modulating action on the GABA-induced response

Adenosine 5′-triphosphate (ATP) produced a long-lasting depolarization inbullfrog spinal ganglion cells. Since the ATP-induced slow depolarization was associated with an increase in membrane resistance and a reverse in polarity (about—90mV) which was most likely brought about by an inactivation of membrane potassium conductance. In some cells, a rapid and transient depolarization followed by the long-lasting depolarization was produced by ATP and it was markedly reduced in sodium-free solution. ATP reversibly augmented the GABA-induced depolarization which was caused by ionophoresis of GABA. These observations were confirmed using a voltage clamp method. Dose-response analysis of the action of ATP on the GABA-induced response suggests that the facilitatory action of ATP on the GABA response is effected on the GABA receptor channel complexes without changing the GABA affinity.     

The development of the relationship between dorsal root afferents and motoneurons in the larval bullfrog spinal cord

The relationship of dorsal root afferents to motoneuron somata and dendrites was studied by labelling dorsal and ventral roots of the tadpole lumbar enlargement with HRP at different stages of hindlimb development. Procedures were used which allowed for sequential light and electron microscopic analysis to determine whether close appositions between labelled elements represented synaptic contacts. Lateral motor column (LMC) motoneuron dendrites grow first into the lateral funiculus, and later begin arborizing within the spinal gray, concurrent with the arrival of developing dorsal root afferent fibers. Mature-appearing synaptic contacts between dorsal root afferents and motoneuron dendrites are established first on distal dendrites, and are observed on progressively more proximal dendrites as hindlimb development proceeds. Migrating motoneurons were also labelled in some animals. Distinct dorsal and ventral migratory pathways were noted; cells migrating dorsally were contacted by developing dorsal root afferents. Migrating motoneurons were associated with radially oriented processes, and were often closely apposed to other cells. The coincident development of dorsal root projections and the motoneuron dendrites which these fibers innervate in the adult, as well as the interaction between these two systems during cell migration, suggest that these two systems may be interdependent in establishing their normal relationship during development.     

Substance P- and opioid-immunoreactive structures in olfactory centers of the cat: adult pattern and postnatal development.

Substance P (SP)-ir and opioid-ir structures were studied in the cat main olfactory bulb (MOB), accessory olfactory bulb (AOB), and olfactory peduncle. In the MOB, the opioid-ir and the majority of the SP-ir neurons belong to the granule cell type. SP-ir granule cells reside in the deeper granule cell layer, whereas opioid-ir granule cells reside in the superficial granule cell layer, internal plexiform, and mitral cell layer. Many granule cells are observed in the external plexiform and glomerular layer. Other granule cells were found in the bulbar/peduncular white matter, the taenia tecta, and the genu of the corpus callosum. A new substance P-ir cell type was identified in the glomerular layer. This cell type was also identified by using the technique of intracellular injection of Lucifer Yellow. The cell type corresponds neither to the external tufted type nor to the short axon cell types described so far. The AOB resembles the MOB with respect to large numbers of SP-ir and opioid-ir granule cells. In addition, a few opioid-ir neurons, probably superficial mitral cells, were found in the glomerular layer. The AOB is surrounded by islands of immunoreactive granule cells, which connect to the granule cell layer by extremely long processes. Opioid-ir and SP-ir beaded axons pass through the olfactory peduncle terminating on granule cells, and ascend as far as the glomerular layer. All subdivisions of the anterior olfactory nucleus (AON) contain immunoreactive terminal fields. Afferent fibers and terminal plexuses derive from a population of immunoreactive neurons located predominantly in the region of the septo-olfactory junction. They have large somata. Their axons form recurrent collaterals, some of which run rostrally in the peduncular white matter. Others ascend caudally towards the septal region. The fibers seem to remain ipsilaterally, since the olfactory limb of the anterior commissure and the commissure proper are devoid of SP-ir and opioid-ir fibers. During development SP and opioid immunoreactivity were found only in differentiated granule cells. The peptides were not detectable in migrating or immature granule cells, as identified in Golgi-impregnated material. The granule cell population largely develops during postnatal life. The number of opioid-ir granule cells increases slowly and continuously, reaching the adult level not before the sixth postnatal month. Strikingly, SP-ir granule cell number increases fast and reaches a transient peak during the second month. Thereafter it declines (40% decrease) to the adult density, which is similar to that of opioid-ir granule cells.     

Vasoactive intestinal polypeptide nullifies cytochrome reduction response to afferent activation of the perfused bullfrog brain

Redox states of cytochromes a(a3), b and c+c1 were continuously measured on the surface of the cerebral hemisphere of the perfused bullfrog brain with the aid of a scanning organ spectrophotometer. To correlate with changes of the redox state, electrical activity of the brain was simultaneously recorded in the vicinity of the tip of the light guide of the spectrophotometer. Electrical stimulation of the third dorsal root of the spinal cord induced parallel reduction of the cytochromes and burst activity in the brain. Intracerebroventricular (i.c.v.) infusion of vasoactive intestinal polypeptide (VIP; 30 pmol/100 g body weight) nullified the cytochrome reduction induced by the electrical stimulation of the dorsal root, but did not attenuate the burst activity. The i.c.v. infusion of VIP (30 pmol/100 g b.wt.) per se had little if any influence upon both redox state and burst activity. The infusion of 1 nM VIP into the internal carotid arteries also nullified the cytochrome reduction response to the afferent electrical activation. The present results provide evidence that one of the physiological roles of the VIPergic neurons in the brain may be to balance the redox states of electron transfer systems in neurons.     

Heterogeneity and dynamics of lateral line afferent innervation during development in zebrafish (Danio rerio)

The lateral line system of larval zebrafish is emerging as a model to study a range of topics in neurobiology, from hair cell regeneration to sensory processing. However, despite numerous studies detailing the patterning and development of lateral line neuromasts, little is known about the organization of their connections to afferent neurons and targets in the hindbrain. We found that as fish grow and neuromasts proliferate over the body surface, the number of afferent neurons increases linearly. The number of afferents innervating certain neuromasts increases over time, while it decreases for other neuromasts. The ratio of afferent neurons to neuromasts differs between the anterior and posterior lateral line system, suggesting potential differences in sensitivity threshold or spatial resolution. A single afferent neuron routinely contacts a group of neuromasts, suggesting that different afferent neurons can convey information about receptive fields along the body. When afferent projections are traced into the hindbrain, where a distinct somatotopy has been previously described, we find that this general organization is absent at the Mauthner cell. We speculate that directional input from the lateral line is less important at an early age, whereas the speed of the escape response is paramount, and that directional responses arise later in development. By quantifying morphological connections in the lateral line system, this study provides a detailed foundation to understand how hydrodynamic information is processed and ultimately translated into appropriate motor behaviors.     

Studies of innervation patterns in grafted chick wings

The pattern of innervation of 12 day embryonic chick wings was studied by morphological and electrophysiological techniques. Several types of chick wing grafts were performed to investigate the rules governing nerve pattern formation: whether the motor neurons actively seek out and connect with specific muscles or are passively guided and whether cell death plays any role in pattern formation. In each type of graft the outgrowing neurons were confronted with extra targets distal to the elbow. Two grafts replicated lower arm parts in series (proximodistal graft) or in parallel (forearm and hand replicated in the same pattern of asymmetry in a split limb graft); a third produced a mirror image limb. The flexor carpi ulnaris muscle (F.C.U.) was studied in these limbs. The innervation of the most distal f.c.u. muscle in proximodistal grafted limbs varied with the extent of host f.c.u. muscle present. The f.c.u. muscles in the split limb graft were always innervated. In both limb grafts the f.c.u. muscles were innervated by the appropriate neurons, as determined by retrograde HRP technique and electrophysiological recordings. This strongly suggests that the motor neurons are able to seek out and connect with their appropriate muscle. Results of the mirror image limb graft suggest that other factors may be involved. In addition, motor neuron cell counts were performed. From this one can infer that the normally occurring pattern of cell death was not affected by the grafts. Cell death is therefore excluded as a major factor in nerve pattern formation.     

Spatial and morphological differentiation of trigger zones in afferent fibers to the teleost utricle.

A morphological correlate of the trigger site (the locus of action potential initiation) was identified in afferent axons of the utricle in the ear of two species of teleost fish. These sites were identified by the ferric-ferrocyanide (Prussian blue) cytochemical procedure and they were correlated with the geometries of afferent intraepithelial arbors as visualized by means of a silver stain. The intraepithelial arbors of afferent fibers show regional distributions that correlate with axon diameter and Prussian blue staining. Afferent axons with diameters greater than 4-5 microns only innervate the striola regions of the epithelium and terminate as one of two distinct types of intraepithelial arbors. Afferent axons with diameters smaller than 4 microns are ubiquitously distributed throughout the epithelium. Arbors that stained by Prussian blue within the utricular epithelium are restricted to the striolar regions. These arbors possess nodal-like membrane in different branches as postsynaptic membrane. Afferents that innervate hair cells in the extrastriolar epithelial regions stained with Prussian blue only at the extraepithelial terminal heminode. The postsynaptic membrane of these afferents is passive or dendritic-like.     

Afferent and efferent innervation patterns of the superior olivary nucleus of the leopard frog

An HRP study of the frog's superior olivary nucleus (SON) revealed that it (1) receives reciprocal tonotopic projections from dorsal medullary nuclei and principal nuclei of the torus semicircularis (TS) bilaterally; (2) receives bilateral projections from caudalis nuclei and brainstem reticular nuclei, and unilateral projections from the ipsilateral ventral tegmental nuclei and the contralateral SON; (3) is reciprocally connected with the ipsilateral laminar and magnocellular nuclei of the TS, dorsal tegmental nuclei and the posterior thalamic nucleus; (4) projects directly to the ipsilateral central thalamic nucleus.     

Vestibular afferent inputs to lobules I and II of the cerebellar anterior lobe vermis in the cat

Vestibular nerve stimulation evoked mossy fiber responses in lobule I and a part of lobule II of the cerebellar cortex in the anesthetized cat. The latencies of the N2 and N3 potentials were in the range of 1.7–2.0 and 3.2–5.0 ms, respectively. The contribution of both primary and secondary vestibular neurons in producing these responses were indicated by electrophysiological methods.     

Neural and anatomic characteristics of peripheral afferent fibers in the milk ejection reflex

Conduction velocities were measured and certain morphologic characteristics were examined of the abdominal mammary nerve in two- to ten-day postpartum rats. This nerve enters the spinal cord at the spinal segmental level T-12. Overall conduction velocity was (Mean +/- S.D.) 18.9 +/- 2.25 m/sec with a major peak at 9.7 +/- 0.72 m/sec. The distribution of conduction velocities in the nerve was similar to that of a typical spinal nerve. Nerve fiber diameters measured between about 1 and 25 microns with peaks at 4.9, 10.5, and 18.9 microns. Injection into the peripheral nerve of fluorescent dye, Lucifer yellow CH (LY), or wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) after ventral root rhizotomy permitted study of the distribution of primary afferents in the spinal cord. The terminal field of these fibers centered around the dorsal cap of Clarke's column and the lateral spinal nucleus, bilaterally. The distribution of WGA-HRP was more restricted than that of LY. A large number of LY-staining fibers were also found ipsilaterally in the medial portion of the intermediomedial column. A smaller amount of LY-staining was present contralaterally in the area of the spinothalamic tract. It is concluded that afferent impulses resulting from mammary stimulation in the milk ejection reflex are probably carried in a mixed spinal nerve whose primary afferent field lies mainly in ipsilateral spinal structures, although there is some evidence for crossing fibers. The data suggest that considerable opportunity exists for interaction with major sensory afferent fiber systems as well as with autonomic fibers. Hence, the spinal path of afferent information relevant for the milk ejection reflex may well be diffuse and it may involve several sensory modalities.     

Cytoarchitecture and saccular innervation of nucleus y in the mouse

The cytoarchitecture and saccular innervation of the mouse nucleus y were investigated by using Golgi, Nissl, and myelin stains and anterograde axonal transport of horseradish peroxidase. Nucleus y was found to be a compact group of cells in a small fiber-free region dorsal to the restiform body. Qualitative and morphometric analyses showed that most (75%) of the nucleus y neurons could not be reliably subdivided into morphologic subgroups, but varied continuously in soma size (15-25 microns), shape (fusiform to stellate), and number of dendrites (two to four), and had sparsely branched dendrites with an average of 3 to 4 spines per 10 microns of length. Three groups of cells that were identified morphometrically accounted for 10% (type I: large stellate cells), 9% (type II: long-dendrite cells), and 6% (type III: elongated soma cells) of the y neurons. Vestibular nerve axons transporting horseradish peroxidase after injury at their origin in the saccular neuroepithelium were found to form a dense terminal meshwork that was virtually co-extensive with the cytoarchitectonic boundaries of nucleus y. Nucleus y was distinguished from the overlying infracerebellar nucleus on the basis of anatomical, cytoarchitectural, and hodological features.     

The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord

To assess the extent to which depolarization by accumulated K+ contributes to the generation of primary afferent depolarization (PAD), the isolated bullfrog spinal cord was superfused with K+-rich Ringer solutions and the resultant dorsal root depolarizations were recorded extracellularly. Action potential blockade (with tetrodotoxin) did not reduce the K+-induced depolarization of primary afferents, indicating that the depolarization was generated locally in the region around the afferents. In this respect superfusion with K+-rich solutions adequately models the localized K+ accumulation which occurs physiologically during afferent activity. K+-induced depolarizations were decreased in the presence of 20 mM Mg2+; this effect was due to a direct decrease in the membrane response to K+ and not to blockade of K+-induced transmitter release onto primary afferents. The depolarization caused by a K+ concentration comparable to a maximum estimate of the K+ accumulating around afferent terminals following a single afferent volley was found to account for no more than about one-third of the DRP height. However, higher K+ levels, comparable to those resulting from high frequency afferent stimulation, caused large depolarizations of primary afferents, sometimes greater than the DRP amplitude. Therefore, K+-induced depolarization may contribute more significantly to PAD evoked by high frequency afferent activity.     

Afferent and efferent innervation patterns of the cochlear nucleus (dorsal medullary nucleus) of the leopard frog

The afferent and efferent innervation patterns of the frog dorsal medullary nucleus (DMN; anuran homolog of the cochlear nucleus) were examined by studying the anterograde and retrograde transport patterns of horseradish peroxidase injected focally into the nucleus. It was found that this structure projected bilaterally to the superior olivary nuclei (SON) and dorsal midbrain tegmental nuclei, and contralaterally to the opposite DMN, the lateral lemniscus nucleus (LLN) and the torus semicircularis (TS). The termination sites in the TS were restricted to the laminar and principal nuclei. The DMN in turn received projections from these structures with the exception of the TS and dorsal tegmental nuclei. The projection to the ipsilateral LLN and TS was not pronounced. In addition to the above findings, the ascending projection to the DMN, SON and TS, as well as the centrifugal projection from the SON, were found to be organized tonotopically.     

Developmental expression of NMDA and AMPA receptor subunits in vestibular nuclear neurons that encode gravity-related horizontal orientations

We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements.     

Cervical primary afferent input to vestibulospinal neurons projecting to the cervical dorsal horn: An anterograde and retrograde tracing study in the cat

Vestibulospinal neurons in the caudal half of the medial and descending vestibular nuclei terminate in the cervical spinal cord, not only in the ventral horn and intermediate zone but also in the dorsal horn. The purpose of the present study was to examine whether the areas containing these vestibulospinal neurons are reached by cervical primary afferents. In one group of experiments, wheat germ agglutinin-horseradish peroxidase conjugate and horseradish peroxidase were pressure injected into spinal ganglia C₂-C₈ and revealed anterogradely labeled fibers and boutons in the caudal part (caudal to the dorsal cochlear nucleus) of the ipsilateral medial and descending vestibular nuclei. This projection was verified in experiments in which wheat germ agglutinin-horseradish peroxidase conjugate was microiontophoretically injected into the caudal half of either the medial or the descending vestibular nuclei and revealed retrogradely labeled cells only in ipsilateral spina ganglia C₂-C₇, with a maximum of cells in C₃. In another group of experiments, after microiontophoretic injections of Phaseolus vulgaris leucoagglutinin or Biocytin into either the medial or the descending vestibular nuclei, anterogradely labeled fibers and boutons were present in the cervical spinal cord, mainly bilaterally in the dorsal horn (laminae I–VI) but also, to a lesser extent, in the ventral horn and intermediate zone. The existence of a loop that relays cervical primary afferent information to vestibulospinal neurons projecting to the cervical spinal cord, in particular the dorsal horn, may have implications for vestibular control over local information processing in the cervical dorsal horn. © 1995 Wiley-Liss, Inc.     

The sensory epithelium and its innervation in the mole rat cochlea.

The mole rat (Spalax ehrenbergi) burrows throughout its life in subterranean tunnels. Several structural and functional features which adapt the mole rat to its habitat have been discerned. The goal of this study was to elucidate the structural basis for adaptation of the auditory end-organ to an environment where low-frequency acoustical signals prevail. For this purpose, cochleae of adult mole rats were studied with light and electron microscopy. Inner hair cells throughout the cochlea, and outer hair cells in the basal (high-frequency) portion of the cochlea, were similar in structure to those seen in other mammals. In contrast, outer hair cells in the apical (low-frequency) portion displayed unique structural features. These features resembled the structure of inner hair cells or immature outer hair cells. The innervation of outer hair cells was most uncommon, in that classical medial efferent innervation was not found throughout the cochlear spiral.     

Alpha-9 nicotinic acetylcholine receptor immunoreactivity in the rodent vestibular labyrinth

Vestibular tissues (cristae ampullares, macular otolithic organs, and Scarpa's ganglia) in chinchilla, rat, and guinea pig were examined for immunoreactivity to the alpha9 nicotinic acetylcholine receptor (nAChR) subunit. The alpha9 antibody was generated against a conserved peptide present in the intracellular loop of the predicted protein sequence of the guinea pig alpha9 nAChR subunit. In the vestibular periphery, staining was observed in calyces around type I hair cells, at the synaptic pole of type II hair cells, and in varying levels in Scarpa's ganglion cells. Ganglion cells were also triply labeled to detect alpha9, calretinin, and peripherin. Calretinin labels calyx-only afferents. Peripherin labels bouton-only afferents. Dimorphic afferents, which have both calyx and bouton endings, are not labeled by calretinin or peripherin. In these experiments, alpha9 was expressed in both calyx and dimorphic afferents. A subpopulation of small ganglion cells did not contain the alpha9 nAChR but did stain for peripherin. We surmise that these are bouton-only afferents. Bouton (regularly discharging) afferents also show efferent responses, although they are qualitatively different from those in irregularly discharging (calyx and dimorphic) afferents, much slower and longer lasting. Thus, regular afferents are probably more affected via a muscarinic cholinergic or a peptidergic mechanism, with a much smaller superimposed fast nicotinic-type response. This latter response could be due to one of the other nicotinic receptors that have been described in studies from other laboratories.     

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.