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.

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.

Chronic phencyclidine induces behavioral sensitization and apoptotic cell death in the olfactory and piriform cortex.

In this study, we tested the hypothesis that chronic administration of phencyclidine (PCP), an N-methyl-D-aspartate (NMDA) receptor antagonist, would cause a long-lasting behavioral sensitization associated with neuronal toxicity. Female Sprague-Dawley rats were administered PCP (20 mg/kg, i.p.) once a day for 5 days, withdrawn for 72 hr, placed in locomotor activity chambers, and challenged with 3.2 mg/kg PCP. Following assessment of locomotor activity, the rats were killed and their brains processed for analysis of apoptosis by either electron microscopy or terminal dUTP nick-end labeling (TUNEL). In study I, PCP challenge produced a much more robust and long-lasting increase in locomotor activity in rats chronically treated with PCP than in those chronically treated with saline. In study II, clozapine pretreatment blunted the degree of sensitization caused by PCP. In study I, a marked increase in TUNEL-positive neurons was found in layer II of the olfactory tubercle and piriform cortex of rats chronically treated with PCP. Many of these neurons had crescent-shaped nuclei consistent with apoptotic condensation and margination of nuclear chromatin under the nuclear membrane. Acute PCP had no effect. Electron microscopy revealed that PCP caused nuclear condensation and neuronal degeneration consistent with apoptosis. Cell counts in layer II of the piriform cortex revealed that chronic PCP treatment resulted in the loss of almost 25% of the cells in this region. However, an increase in glial fibrillary acidic protein (GFAP)-positive cells in the molecular layer suggests that this neurotoxicity also may involve necrosis. In study II, the PCP-induced neuronal degeneration was essentially completely abolished by clozapine pretreatment. This pattern of degeneration was found to coincide with the distribution of the mRNA of the NR1 subunit of the NMDA receptor. The relevance of these data to a PCP model of chronic NMDA receptor hypofunction is discussed.

Vestibular type I and type II hair cells. 1: Morphometric identification in the pigeon and gerbil.

Classically, type I and type II vestibular hair cells have been defined by their afferent innervation patterns. Little quantitative information exists on the intrinsic morphometric differences between hair cell types. Data presented here define a quantitative method for distinguishing hair cell types based on the morphometric properties of the hair cell's neck region. The method is based initially on fixed histological sections, where hair cell types were identified by innervation pattern, type I cells having an afferent calyx. Cells were viewed using light microscopy, images were digitized, and measurements were made of the cell body width, the cuticular plate width, and the neck width. A plot of the ratio of the neck width to cuticular plate width (NPR) versus the ratio of the neck width to the body width (NBR) established four quadrants based on the best separation of type I and type II hair cells. The combination of the two variables made the accuracy of predicting either type I or type II hair cells greater than 90%. Statistical cluster analysis confirmed the quadrant separation. Similar analysis was performed on dissociated hair cells from semicircular canal, utricle, and lagena, giving results statistically similar to those of the fixed tissue. Additional comparisons were made between fixed tissue and isolated hair cells as well as across species (pigeon and gerbil) and between end organs (semicircular canal, utricle, and lagena). In each case, the same morphometric boundaries could be used to establish four quadrants, where quadrant 1 was predominantly type I cells and quadrant 3 was almost exclusively type II hair cells. The quadrant separations were confirmed statistically by cluster analysis. These data demonstrate that there are intrinsic morphometric differences between type I and type II hair cells and that these differences can be maintained when the hair cells are dissociated from their respective epithelia.

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.

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.

Morphometric studies of type I and type II hair cells in the gerbil's posterior semicircular canal crista.

The existence of separate subtypes of type I vestibular hair cells according to morphological criteria in situ was investigated. Gerbils were anesthetized and perfused with mixed aldehydes. The crista ampullaris of the posterior canal was dissected, fixed in osmium, dehydrated, and embedded in epon. Five-micron sections were cut orthogonal to the long axis of each crista. Measurements were made on camera lucida drawings of individual cells located in the apical, middle, and basal 1/3 of the crista. Measurements for each hair cell included the circumference, greatest width of the body, length, width of the apical surface (cuticular plate region, P), width at narrowest portion of the neck (NW), neck width to plate ratio (NPR), length at a point 2 times NW from the apical surface (L2N). Type I hair cells were subgrouped into three classes (long -1, intermediate -i, and short -s) based on a subjective determination of neck length. Statistical comparisons were made between type I (n = 612) and type II (n = 74) hair cells and the type I subtypes (l, i, s). Statistically significant differences were found between type I and II hair cells for NPR, width, and length, but not perimeter. Thus, as in pigeons, NPR distinguishes type I and type II hair cells in the gerbil crista. While type I hair cells are wider and longer than type IIs, the circumference is the same, due to the restricted neck in type I hair cells. The L2N statistic separates three subtypes of type I hair cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

Projections from the sacculus to the cochlear nuclei in the Mongolian gerbil.

The projections of the saccule, an otolith end organ, to the cochlear nuclei were studied using both transganglionic transport and intracellular injection techniques. Labeled fibers and terminals were observed in the anterior and posterior portions of the ventral cochlear nucleus and the dorsal cochlear nucleus. Most terminals were present in the granule cell domain, especially in the subpeduncular corner between the anteroventral cochlear nucleus and the floccular peduncle of the cerebellum. It has been hypothesized that the cochlea in mammals may have developed phylogenetically from the saccule. The projections from the saccule to the cochlear nuclei were investigated in a mammalian species, the Mongolian gerbil, in an attempt to obtain initial information supporting or refuting this hypothesis. The presence of an otolith end organ projection to the cochlear nuclei in rodents should encourage comparative studies in additional aspects of the evolution of the auditory system.

Identification of vestibular efferent neurons in the gerbil: histochemical and retrograde labelling.

The efferent neurons of the gerbil vestibular system were investigated by retrograde tracing techniques and cytochemical staining for acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and a number of peptides. The location, bilateral distribution, cell area and number of neurons in two identified groups of retrogradely labelled cells were described and quantified. The larger of the two groups was located dorsolateral to the facial nerve genu, ventral and medial to the vestibular nuclei. Unilateral tracer injection in the vestibular end organs labelled cells bilaterally in this and the smaller group, which was located immediately ventral to the genu. No cells were found that individually projected bilaterally to both labyrinths. After injections of horseradish peroxidase (HRP) in the utricle or saccule, significantly more cells were located on the contralateral side of the brainstem. The average (+/- SD) cross sectional area of labelled cell bodies associated with the otolith organs was 259.8 (+/- 75.2) microns 2. ChAT immunoreactive and AChE positive cells were found in an area coextensive with the location of the dorsal efferent group. In double-labelling studies, cell bodies in the same group that had been retrogradely labelled with a utricular injection of HRP, were immunocytochemically stained for calcitonin gene-related peptide and met-enkephalin. In contrast, the ventral group of efferents did not have cells that were cytochemically stained for either of the acetylcholine-related enzymes or either peptide. The significance of the existence of peptidergic vestibular efferent neurons is discussed.

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.

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.