Structural and functional changes in the cristae ampullares of aged gerbils.

We have reported that calretinin and calbindin staining of calyxes in the apical region of the cristae is reduced or absent in old gerbils (>or=35 months) that had normal numbers of hair cells [Kevetter GA, Leonard RB (2002) Decreased expression of calretinin and calbindin in the labyrinth of old gerbils. Brain Res 957:362-365]. Here we examine the ability of primary afferents in aged gerbils to carry a tracer injected into the vestibular nuclear complex to their terminals in the cristae. Calyxes throughout the cristae were well labeled in a young animal with such an injection. In the aged animals, many calyxes were only partially filled or not filled at all. In some cases labeled axons were also missing from the stroma underlying the missing calyxes. There is a strong correspondence between the region where the calyxes were not filled and the absence of calretinin immunostaining. To determine if afferents from the cristae are functionally abnormal, we recorded from their axons and attempted to activate them with natural stimulation. Among afferents that could be activated, we encountered many afferents that had spontaneous activity but could not be modulated with natural stimulation. When tested, the firing rate of these afferents could be modulated with galvanic stimulation, and/or they could be activated by pulsed electrical stimulation. We also encountered afferents that had no spontaneous activity. The presence of these axons was revealed by an injury discharge that could not be modulated with natural stimulation. When tested, these axons could be activated with pulsed electrical stimulation. In some instances we encountered two or more such afferents in a row, an event we have not seen in young animals. We suggest that the simplest explanation for these observations is that calyxes are being lost in old animals.

Muscarinic acetylcholine receptor subtype expression in avian vestibular hair cells, nerve terminals and ganglion cells.

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and peripheral nervous system and play an important role in modulating the cell activity and function. We have shown that the cholinergic agonist carbachol reduces the pigeon's inwardly rectifying potassium channel (pKir2.1) ionic currents in native vestibular hair cells. We have cloned and sequenced pigeon mAChR subtypes M2-M5 and we have studied the expression of all five mAChR subtypes (M1-M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa's) ganglion using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). We found that vestibular hair cells, nerve fibers and ganglion cells each expressed all five (M1-M5) mAChR subtypes. Two of the three odd-numbered mAChRs (M1, M5) were present on the hair cell cilia, supporting cells and nerve terminals. And all three odd numbered mAChRs (M1, M3 and M5) were expressed on cuticular plates, myelin sheaths and Schwann cells. Even-numbered mAChRs were seen on the nerve terminals. M2 was also shown on the cuticular plates and supporting cells. Vestibular efferent fibers and terminals were not identified in our studies. Results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia were consistent with the results from IH and IC. Our findings suggest that there is considerable co-expression of the subtypes on the neural elements of the labyrinth. Further electrophysiological and pharmacological studies should delineate the mechanisms of action of muscarinic acetylcholine receptors on structures in the labyrinth.

Pattern of selected calcium-binding proteins in the vestibular nuclear complex of two rodent species.

The distribution of immunoreactivity to calbindin, calretinin, and parvalbumin in the vestibular nuclear complex and the adjacent nucleus prepositus hypoglossi was studied in rats and gerbils. The distribution of stained fibers was the same for both rodent species. All three calcium-binding proteins were present in vestibular afferents. However, none of the three proteins were present in all afferent fibers. Many fibers were labeled in the vestibular nerve and in fascicles of the descending vestibular nucleus, as well as ascending fibers in the superior vestibular nucleus and fibers directed to the medial vestibular nucleus. Labeled terminals were present in the medial vestibular nucleus, especially along the ventricular border, in the neuropil of the superior vestibular nucleus, and scattered in the descending and ventral portions of the lateral vestibular nucleus. Calbindin- and parvalbumin-positive terminals, but not calretinin-positive terminals, were present in the neuropil of the dorsal lateral vestibular nucleus, especially surrounding the large neuronal somas. Some of these terminals are presumably from cerebellar Purkinje cells, which were also labeled by both antibodies. The pattern of parvalbumin immunoreactivity was slightly different from that of calbindin, indicating that parvalbumin might be contained in additional fibers. Some neurons in the vestibular nuclear complex were labeled with antibodies to calretinin, but few cells were stained with either calbindin or parvalbumin antibodies. The largest group of calretinin-positive cells was a cluster of small- to medium-sized neurons located in a densely stained mesh of dendrites and terminals in the medial vestibular nucleus, adjacent to the ventricular border.

Distribution of vestibular afferents that innervate the sacculus and posterior canal in the gerbil.

The central distribution of afferents that innervate the macula of the saccule and the crista of the posterior canal was assessed in the gerbil following the direct injection of horseradish peroxidase (HRP) separately into the sensory neuroepithelia of each peripheral receptor organ. Ganglion cells innervating the posterior canal were located in the caudal part of the inferior ganglion, while those cells innervating the saccule were located in the rostral part of the inferior ganglion, scattered in the superior ganglion, and concentrated at the junction (isthmus) between the two. The paths of the central axons of these two groups of ganglion cells through the vestibular root and their division into ascending or descending pathways were similar. However, the distributions of their terminals were different. The posterior canal projected to medial parts of the vestibular nuclear complex. Terminals were found in the medial and superior vestibular nuclei. The posterior canal also projected to the uvula of the cerebellum. The saccule projected to more lateral-lying brainstem areas. Terminal fields were located in the lateral and descending vestibular nuclei and cell group y. Saccule projections outside the vestibular complex were observed to the lateral cuneate nucleus, the N. gigantocellularis, and the cerebellar cortex. Of the eight areas receiving primary afferent projections from these two organs, only within the medial and descending vestibular nuclei and the cerebellar cortex were overlapping projections observed.

Use of calcium-binding proteins to map inputs in vestibular nuclei of the gerbil.

We wished to determine whether calbindin and/or calretinin are appropriate markers for vestibular afferents, a population of neurons in the vestibular nuclear complex, or cerebellar Purkinje inputs. To accomplish this goal, immunocytochemical staining was observed in gerbils after lesions of the vestibular nerve central to the ganglion, the cerebellum, or both. Eleven to fourteen days after recovery, the brain was processed for immunocytochemical identification of calretinin and calbindin. After lesion of the vestibular nerve, no calretinin staining was seen in any of the vestibular nuclei except for a population of intrinsic neurons, which showed no obvious change in number or staining pattern. Calbindin staining was reduced in all nuclei except the dorsal part of the lateral vestibular nuclei. The density of staining of each marker, measured in the magnocellular medial vestibular nucleus, was significantly reduced. After the cerebellar lesion, no differences in calretinin staining were noted. However, calbindin staining was greatly reduced in all nuclei. The density of staining, measured in the caudal medial vestibular nucleus, was significantly lower. After a combined lesion of the cerebellum and vestibular nerve, the distribution and density of calretinin staining resembled that after vestibular nerve section alone, whereas calbindin staining was no longer seen. This study demonstrates that calretinin and calbindin are effective markers for the identification of vestibular afferents.

Connections of the corticomedial amygdala in the golden hamster. I. Efferents of the "vomeronasal amygdala".

The medial (M) an posteromedial cortical (C3) amygdaloid nuclei and the nucleus of the accessory olfactory tract (NAOT) are designated the "vomeronasal amygdala" because they are the only components of the amygdala to receive a direct projection from the accessory olfactory bulb (AOB). The efferents of M and C3 were traced after injections of 3H-proline into the amygdala in male golden hamsters. Frozen sections of the brains were processed for autoradiography. The efferents of the "vomeronasal amygdala" are largely to areas which are primary and secondary terminal areas along the vomeronasal pathway, although the efferents from C3 and M terminate in different layers in these areas than do the projections from the vomeronasal nerve or the AOB. Specifically, C3 projects ipsilaterally to the internal granule cell layer of the AOB, the cellular layer of NAOT, and layer Ib of M. Additional fibers from C3 terminate in a retrocommissural component of the bed nucleus of the strain terminalis (BNST) bilaterally, and in the cellular layers of the contralateral C3. The medial nucleus projects to the cellular layer of the ipsilateral NAOT, layer Ib of C3, and bilaterally to the medial component of BNST. Projections from M to non-vomeronasal areas terminate in the medial preoptic area-anterior hypothalamic junction, ventromedial nucleus of the hypothalamus, ventral premammillary nucleus and possibly in the ventral subiculum. These results demonstrate reciprocal connections between primary and secondary vomeronasal areas between the secondary areas themselves. They suggest that M, but not C3, projects to areas outside this vomeronasal network. The medial amygdaloid nucleus is therefore an important link between the vomeronasal organ and areas of the brain not receiving direct vomeronasal input.

Connections of the corticomedial amygdala in the golden hamster. II. Efferents of the "olfactory amygdala".

The anterior cortical (C1) and posterolateral cortical (C2) nuclei of the amygdala are designated the "olfactory amygdala" because they each receive direct projections from the main olfactory bulb. The efferents of these nuclei were traced after stereotaxic placement of 1-5 muCi tritiated proline in the corticomedial amygdala of the male golden hamsters. Following survival times of 12, 24, or 48 hours, 20 micron frozen sections of the brains were processed for light microscopic autoradiography. Efferents from C2 terminate in layers II and III of the olfactory tubercle and in layer Ib of pars ventralis and pars medialis of the anterior olfactory nucleus. Fibers from this nucleus also project to layers I and II of the infralimbic cortex and to the molecular layer of the agranular insular cortex. More posteriorly, fibers from C2 terminate in layer I of the dorsolateral entorhinal cortex, and in the endopiriform nucleus. From C1, efferent fibers travel in the stria terminalis and terminate in the precommissural bed nucleus of the stria terminalis and in the mediobasal hypothalamus. Efferents from C1 also innervate the molecular layer of C2, the amygdalo-hippocampal area, and the adjacent piriform cortex. Neurons in both C1 and C2 project to the molecular layer of the medial amygdaloid nucleus and the posteromedial cortical nucleus of the amygdala, the plexiform layer of the ventral subiculum, and the molecular layer of the lateral entorhinal cortex.

Collaterals of spinothalamic cells in the rat.

Spinothalamic (STT) cells were investigated in the rat to determine the distribution of subpopulations with terminals in both the lateral and medial thalamus, the thalamus bilaterally, or the thalamus and the medullary reticular formation. Two or more retrogradely transported substances (fluorescent dyes, and/or horseradish peroxidase) were injected in each animal. Three combinations of injections were most commonly used: (1) injections of the medullary reticular formation and thalamus, (2) separate injections into each side of the thalamus, and (3) separate injections into the medial and lateral thalamus. The distribution of single labeled cells after each injection was compared with previously published results for rats. The distribution of cells which contained both tracers, double-labeled (DL) cells, was the focus of this study. An average of 15% of STT cells and 8% of spinoreticular cells projected to both the reticular formation and thalamus. However, only a small component of STT cells (less than 2%) projected bilaterally into the thalamus. Most DL cells were found in upper cervical segments. The laminar distribution of all three groups of DL neurons were similar. These cells were most often located in the reticulated part of lamina V and the intermediate zone, lamina VII. STT cells that had terminals in both the medial and lateral thalamus and STT cells with collaterals in the reticular formation were concentrated on the side contralateral to their terminals. These DL neurons provide an anatomical substrate for noxious stimuli to stimuli to activate the reticular formation and thalamus and/or specific sensory and intralaminar thalamus simultaneously.

Midbrain nuclei projecting to the medial medulla oblongata in the monkey.

To identify the midbrain nuclei that project to the medial part of the lower brainstem in the monkey, labeled cells were mapped in the midbrain following the injection of horseradish peroxidase into the medial medulla oblongata. After the general distribution of labeled cells was observed in three animals with large injections, more discrete injections of HRP were made in different locations in six additional animals. The small injections were centered in the nucleus raphe magnus, nucleus reticularis gigantocellularis, or nucleus medullae oblongatae centralis. The five labeled midbrain nuclei were the periaqueductal gray, nucleus cuneiformis, deep layers of the superior colliculus, nucleus of Darkschewitsch, and the interstitial nucleus of Cajal. In addition, the parvocellular division of the red nucleus and the posterior pretectal nucleus contained large numbers of cells when the injection spread into the inferior olive. No major differences in the distribution of labeled cells between different injection sites were found with the exception that the superior colliculus did not contain any labeled cells when the injection was restricted to midline structures. The functional implications of these anatomical findings are discussed in relation to the descending control of pain.

Cells of origin of the spinoreticular tract in the monkey.

The distribution of the cells of origin of the primate spinoreticular tract was determined following injections of horseradish peroxidase (HPR) into the pontomedullary reticular formation in Macaca fascicularis. Five animals received large bilateral injections which included the raphe nuclei and seven monkeys received smaller, unilateral injections. Sections sampled were from upper cervical levels, the cervical enlargement, upper and lower thoracic levels, and lumbosacral levels. The laminar distribution of spinoreticular cells in all spinal cord levels was comparable. More than half of the labeled cells were located ventromedially, in laminae VII and VIII. HRP-labeled cells were also found in the dorsal horn, primarily in the lateral reticulated part of lamina V. Some cells were also found in laminae I and X. Spinoreticular cells in the lumbosacral spinal cord mainly projected to the contralateral brainstem. In the cervical enlargement, however, a bilateral distribution of cells was observed following unilateral injections of HRP. Most spinoreticular cells were multipolar neurons with extensive dendritic ramifications. The distribution of spinoreticular cells is similar to the distribution of spinal cord neurons that project to the medial thalamus, but different from that of spinal neurons projecting to the ventrobasal complex. The anatomical organization of the spinoreticular tract is consistent with a role for this pathway in nociception.

Excitatory amino acid immunoreactivity in vestibulo-ocular neurons in gerbils.

Retrogradely labeled vestibulo-ocular neurons (VOR) that also stain with antibodies for the excitatory amino acid neurotransmitters glutamate (GLU-LI) or aspartate (ASP-LI) were studied. VOR neurons that contained GLU-LI or ASP-LI label were identified in the medial (MVN) and superior (SVN) vestibular nuclei, and cell group Y. More than half of the VOR cells in MVN were also GLU-LI positive, but less than half of the VOR cells in SVN were double labeled.

Development of the marginal zone in the rhombenecephalon of Xenopus laevis.

The marginal zone is the most superficial component of the developing central nervous system. In this study we describe the pattern of formation of the marginal zone in Xenopus laevis as seen with the light microscope and correlate its appearance with developing axon bundles seen with the electron microscope. The marginal zone was first identified at stage 26 on the ventrolateral aspects of the neural tube in restrictive foci throughout the medulla. By stage 28 this presumptive fiber area had expanded to occupy a longitudinal zone along the ventrolateral aspect of the rhombencephalon. The marginal zone continued expanding rostrocaudally, mediolaterally, and dorsoventrally coming to occupy a substantial area along the periphery of the central nervous system by the time of hatching (stage 36). Electron microscopic (EM) observations allowed us to identify axons and growth cones on the lateral borders of the neural tube at stage 22. Foci of marginal zones identified in the caudal medulla at stage 24 consisted of several fascicles grouped together on the ventrolateral surface. These fascicles, separated and surrounded by ependymal processes contained axons and growth cones. The pattern of marginal zone development reflected the addition of axons within fascicles and also the addition of new fascicles of axons.

Collateralization in the spinothalamic tract: new methodology to support or deny phylogenetic theories.

Components of the spinothalamic system that ascend in the anterolateral funiculus are reviewed. The presence of collateralization in this system in mammals is discussed with regard to theories of the phylogenetic development of pathways. The major theory investigated suggested that collateralization is an intermediate stage between a multisynaptic pathway and a direct non-collateralized lemniscus. The evidence and theories are reviewed. Methods for confirming or rejecting this theory are discussed. The literature reporting ascending spinal projections for non-mammalian vertebrates is reviewed. Certain reptiles have projections analogous to both the mammalian neospinothalamic and paleospinothalamic tracts. The presence of spinothalamic projections in elasmobranchs and amphibians is still controversial. Confirmation of earlier reports of projections in salamander and dogfish shark based on degeneration techniques have not been done. In addition, results from too few species of these classes have been reported. However, it is possible that paleospinothalamic connections are present in some species (e.g. salamander, nurse shark) and not in others (e.g. frog, dogfish shark) of the same class. Spinothalamic projections have not been reported for teleosts. A plea for new research in this area is made.

A quantitative study of the vestibular commissures in the gerbil.

Direct commissural connections between the bilateral vestibular nuclear complexes (VNC) were investigated in the gerbil using ionophoretic injections of horseradish peroxidase into individual vestibular nuclei. Labelled commissural neurons were counted, the cell counts adjusted by the relative nuclear volume, and the results treated quantitatively. The medial nucleus (MVN) contained the greatest number of commissural neurons. The MVN projected to each of the contralateral vestibular nuclei, but most strongly to the contralateral MVN and superior (SVN) nucleus. The SVN projected modestly to the contralateral VNC. Commissural connections of the descending nucleus were weak. Commissural afferents to the MVN were topographically organized. The crossed fastigiovestibular projection was also investigated.

Serotonergic projections to the caudal brain stem: a double label study using horseradish peroxidase and serotonin immunocytochemistry.

Cells of origin of serotonergic and non-serotonergic projections to the caudal brain stem in the primate were examined using a double label technique. Following HRP injections into medullary raphe nuclei and the adjacent reticular formation double labeled cells were found in the dorsal raphe nucleus, the central superior nucleus and the ventrolateral tegmentum. Retrogradely labeled cells that did not stain for serotonin-like immunoreactivity were found primarily in the periaqueductal gray (PAG) and the mesencephalic and pontine reticular formation. The results are discussed in relation to the descending pathway(s) mediating the effects of PAG stimulation.

Hair cell and supporting cell density and distribution in the normal and regenerating posterior crista ampullaris of the pigeon.

The numbers of supporting cells and the numbers and types of hair cells in three distinct longitudinal regions through the posterior canal cristae of control and streptomycin-treated pigeons were determined using stereological techniques. For control cristae, type I (3758) and type II (3517) hair cells occurred in approximately equal numbers. However, the proportions varied in different longitudinal zones: Zone I (peripheral region) had four times more type II hair cells (2083) than type I (483), while Zone II (intermediate region) had almost seven times more type I (2517) than type II (367) hair cells and Zone III (central region) had relatively equal numbers of type I (758) and type II (1067) hair cells. Novel findings included the following: (1) immediately after the post-injection sequence (PIS) of streptomycin, there was a significant reduction in both hair cells (-93%) and supporting cells (-45%); (2) by 70 days after the PIS, the population of type I hair cells returned to control values (however, the normal complement of complex calyces took 1 year to recover); (3) during the first 143 days after the PIS, the number of type I and type II hair cells across all zones returned linearly with about the same slope (46 and 43 cells per day, respectively), although the rate of return differed significantly in different zones; (4) there was a massive overproduction of hair cells (+150%) and supporting cells (+120%) during the first 5 months of recovery; and (5) during the first year after the PIS, both hair cells and supporting cells increased and their increases in numbers were correlated (r = 0.88, P < 0.01). Knowledge of the sequence and numbers of regenerating hair cells may help elucidate common modes of cell survival, recovery, and compensation from neural insult.

Aspartate-like immunoreactivity in vestibulospinal neurons in gerbils.

In an effort to further characterize vestibulospinal pathways in the gerbil, immunocytochemistry was combined with retrograde identification of neurons. Vestibulospinal neurons were retrogradely labeled following injections of horseradish peroxidase into the cervical cord of anesthetized gerbils. Sections were reacted with nickel acetate-diaminobenzidine for horseradish peroxidase, giving a black reaction product. Sections were incubated in polyclonal antisera to aspartate, incubated in an avidin-biotin-peroxidase procedure, and reacted to give a brown reaction product. Alternatively, fluoro-gold was used as a retrograde tracer and aspartate-like immunoreactivity was demonstrated with avidin conjugated to Texas red. Cells stained with aspartate-like immunoreactivity, were located in all vestibular nuclei. Double-labeled cells were located in the medial nucleus and in the lateral vestibular nucleus where many of the large cells were double labeled.

Ipsilateral and contralateral projections to the trochlear nucleus arise from different subdivisions in the vestibular nuclei.

Vestibular neurons that project to the trochlear nucleus were studied following unilateral injections of horseradish peroxidase. After 48 h, the animals were perfused, transverse sections were cut, and reacted with diaminobenzidine. After injections centered on the trochlear nucleus, one-third of the labeled neurons were located in the ipsilateral superior (S) vestibular nucleus and almost half were in the contralateral medial (M) vestibular nucleus. Labeled fibers were restricted to the medial longitudinal fasciculus ipsilateral to the injection. This study supports hypotheses, based on physiological data of two vertical vestibulo-ocular pathways; one originating in the ipsilateral S that may be inhibitory and the second originating predominantly from the contralateral M that may be excitatory.

Cytochrome oxidase histochemistry in Scarpa's ganglion after hemilabyrinthectomy.

Cytochrome oxidase histochemistry was studied in neurons in the vestibular ganglion in gerbils two weeks after hemilabyrinthectomy. This study measured the staining density in ganglion cells on both the lesioned and non-lesioned side of the brainstem. Cytochrome oxidase staining was significantly reduced in ganglion cells ipsilateral to the lesion. This decrease may have been related to the concomitant loss of spontaneous discharge and reduced energy demand for oxidative metabolism.

Central projections of vestibular afferents innervating the macula of the saccule in gerbil.

The central distribution of vestibular afferents that innervate the saccule has been investigated in the gerbil using transganglionic transport techniques. Following horseradish peroxidase injection into the saccular neuroepithelium, labeled ganglion cells were clustered at the junction of the superior and inferior ganglion. Labeled fibers entered the vestibular nuclear complex and divided into rostral and caudal branches. Terminal fields were observed in the interstitial nucleus of the vestibular nerve among the entering fibers and in the lateral vestibular nucleus. Rostrally, fibers terminated in cell group y and the nodulus; caudally, fibers ended in the descending and medial vestibular nuclei.