Richard M. Costanzo

Neural regeneration and functional reconnection following olfactory nerve transection in hamster

The olfactory sensory neurons in the vertebrate nervous system are unique in that they undergo continuous neurogenesis and replacement. Anatomical studies have shown that transection of the olfactory nerves leads to a degeneration of sensory neurons followed by a neurogenesis and replacement with newly formed cells. Replacement neurons grow axonal processes that are capable of reestablishing morphological connections with cells in the olfactory bulb. To determine the functional capacity of these anatomical reconnections, single unit responses to odor stimuli were recorded from cells in the olfactory bulb following recovery from unilateral olfactory nerve transection. A total of 56 cells were studied, taken from hamsters with recovery times of 4,35,60,90,120,180 and 270 days. At day 4, although there was spontaneous activity recorded from cells on the experimental side (n = 10), they did not respond to stimulation of the olfactory epithelium with odors. Control cells (n = 9) from the unoperated side of the same animals showed normal odor responses. By day 35, some of the cells tested on the experimental side responded to odor stimulation, indicating that connections had been reestablished with sensory neurons. With longer recovery times, an increasing percentage of cells responded to odor stimuli. In addition, concentration response functions showed that cells were capable of signaling differences in stimulus intensity. The response of cells to four odors (amyl acetate, 1-butanol, ethyl acetate and ethyl butyrate) showed differences in odor selectivity, suggesting their ability to discriminate among odors.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of olfactory mediated behavior after olfactory bulb deafferentation

Transection of olfactory nerve fibers leads to deafferentation of olfactory bulbs and a loss of olfactory mediated behavior. Nerve transection studies have shown that during recovery, olfactory nerve fibers can reestablish connections with the olfactory bulbs. Two groups of experimental animals were studied to determine if olfactory mediated behavior returns after recovery. One group (n = 18) received bilateral olfactory nerve transection (BTX), while the second group (n = 4) received a sham surgical procedure. Performance on odor detection and discrimination tasks was measured during recovery periods ranging from 1-120 days. Return of olfactory mediated behavior was first observed 19 days after nerve transection. Performance levels improved with recovery time and by day 40 animals returned to criterion level (> or = 90% correct response). Sham animals maintained a criterion level of performance throughout the recovery period. Horseradish peroxidase (HRP) was used to trace reconnection of olfactory nerve fibers. The absence of HRP label in the bulbs of animals examined one day after BTX, verified the completeness of the nerve transection procedure. After 10 days of recovery, a few HRP labeled axons were observed and the amount of HRP in the bulb increased with recovery time. The results of this study demonstrate that olfactory receptor axons can reestablish functional connections with the deafferented olfactory bulb and these connections are sufficient to restore olfactory mediated behavior.

Comparison of neurogenesis and cell replacement in the hamster olfactory system with and without a target (olfactory bulb)

The olfactory sensory neurons are unique in the vertebrate nervous system in that they are replaced following experimentally induced degeneration. Unilateral removal of the olfactory bulb in hamster results in degeneration of all mature receptor neurons followed by a neurogenesis and partial replacement of the receptor cell population. To determine if full recovery requires the presence of normal target tissue, a study of sensory neuron replacement was made following a nerve transection procedure, which leaves the olfactory bulb (target) intact. A comparison of quantitative measurements of cell number and thickness in the sensory epithelium showed that the presence of the target tissue alone did not result in improved recovery. One possible explanation is that complete recovery requires that axons of newly formed receptors must first re-establish synaptic contact with the olfactory bulb. To test this possibility, it will be necessary to include longer postoperative recovery times than those used in the present study.

Three-dimensional scanning electron microscopic study of the normal hamster olfactory epithelium

SummaryThe olfactory epithelium of the adult hamster (Mesocricetus auratus) was studied using the scanning electron microscope. A method that produced fractures in the epithelium exposed structures below the surface and made it possible to examine the morphological and structural relationships among cells.Three cell types were studied: supporting cells, olfactory neurons (receptor cells) and basal cells. Supporting cells were observed spanning the full extent of the epithelium, and had basal foot processes that terminated at or near the basal lamina. Along the lateral margin of supporting cells, cellular processes were observed extending outwards, reaching olfactory neurons and adjacent supporting cells. These cellular contacts among supporting cells and olfactory neurons were present at different levels of the epithelium. Olfactory neurons were located primarily in the middle and lower epithelial regions. Their dendritic processes reached the epithelial surface in a straight or tortuous manner, passing between the supporting cells. Olfactory axons were observed as thin unbranched processes that emerged from a conical hillock region, passed basally, and fasciculated into larger sensory bundles within the lamina propria. Basal cells were observed adjacent to the basal lamina as a row of single cells or clustered in groups. Within the lamina propria connective tissue, blood vessels, axon bundles and Bowmans glands were examined. Bowmans glands were composed of pyramidal secretory cells arranged about a single duct that extended to the epithelial surface.Scanning electron microscopy provided a unique three-dimensional analysis of cell structure within the olfactory epithelium. The results provide new and different observations on the detailed morphology and intimate relationships that exist among epithelial cells, and complement previous light and transmission EM observations.

A comparative immunocytochemical study of development and regeneration of chemosensory neurons in the rat vomeronasal system

Vomeronasal neurons undergo continuous neurogenesis during development and after neuronal injury. We used immunocytochemical methods to compare different stages of the vomeronasal organ development to those of regeneration following vomeronasal nerve transection. At E15 and at 6 to 10 days after injury, nestin-positive cells were observed throughout the sensory epithelium. We did not find nestin immunoreactivity to be localized to the boundary region of the epithelium. The early appearance and wide distribution of nestin-positive cells suggests that they represent chemosensory precursor cells that develop and migrate vertically in the epithelium. Vomeronasal receptor cells degenerated 6 to 8 days after nerve transection, but axon terminals in the accessory olfactory bulb (AOB) continued to show the presence of the chemosensory specific marker (OMP) for up to ten days, a significant finding observed in this study. It is likely that the distance from the site of nerve transection may contribute to differences in the time course of anterograde and retrograde axon degradation. OMP-positive neurons were observed in the normal adult epithelium and to a much lesser extent 10-60 days after recovery from nerve transection. Axons from regenerated receptor cells did not reach the AOB during this time period. This failure to reestablish connections with target cells in the AOB could explain why OMP-positive cells were rarely observed among the regenerated cells in the vomeronasal epithelium.

Scanning electron microscopic study of degeneration and regeneration in the olfactory epithelium after axotomy

SummaryThe olfactory epithelium of the adult hamster (Mesocricetus auratus) was examined with the scanning electron microscope following olfactory nerve axotomy. Axotomy results in retrograde degeneration of mature olfactory neurons. Maximum degeneration was observed around day 4. During the degeneration period the epithelium consists primarily of supporting and basal cells. Microvillar columnar supporting cells were observed to have fine cellular processes extending from their lateral border to neighbouring cells. Supporting cells extended to the basal lamina where they terminated in foot-like processes of variable shapes (club, splay and hook). Basal cells which gave rise to new replacement olfactory neurons were observed near the basal lamina. They had a rough cellular surface covered with small granules and fine cellular extensions. Bowmans gland duct cells extended unbranched through the epithelium where they formed funnel duct openings covered with microvilli. During early recovery periods (5–30 days) the number of olfactory neurons in the lower epithelium region increased. We observed olfactory neurons with developing axon and dendritic processes. Specialized growth cone structures were seen at the tips. Olfactory neuron growth cones were elongated or club-shaped and had a ruffled membrane surface. Several thin filopodia extended from the growth cone and made contact with adjacent cells. At late recovery periods (35–120 days) there was a marked increase in the number of olfactory neurons within the middle and lower epithelium regions. Numerous dendritic processes extended to the epithelial surface and terminated in knob-like ciliated structures. Olfactory axons passed basally, forming small intra-epithelial bundles that penetrated the basal lamina then fasciculated into larger bundles within the lamina propria.This study provides detailed three-dimensional observations of the olfactory epithelium following neuron injury, and describes neural degenerative changes, replacement of olfactory neurons, development and maturation. In addition, we describe the structure and basal attachment of supporting cells and their glial-like relation with olfactory neurons.

Electrophysiological characterization of the olfactory bulb during recovery from sensory deafferentation

Axonal transection results in degeneration of the olfactory sensory neurons (OSNs). Replacement OSNs reinnervate the olfactory bulb. To document reinnervation, lateral OSN fibers were stimulated while bulb negative evoked potentials (NEPs) were recorded. For control adult hamsters and at 20, 30, and 120 days after transection, lateral fibers were connected to the lateral more than the medial bulb. The percentage of lateral bulb positions with NEPs was similar to control at 30 and 120 days, but NEP amplitude did not reach control level.

Is nestin a marker for chemosensory precursor cells

The vomeronasal and olfactory systems are unique in that their chemosensory neurons undergo continuous neurogenesis after development. Immunoreactivity to nestin, a neuronal precursor marker protein, was investigated in the developing rat vomeronasal organ to determine its potential as a cell marker. From postnatal day 1 (P1) to P22, the distribution of nestin positive cells became progressively restricted to the area adjacent to the basement membrane. By P29, the vomeronasal organ reached structural maturity and only a few nestin positive cells were observed. Results suggest that nestin may be a useful marker for neuronal precursor cells in studies of neurogenesis and development of chemosensory systems.

Identifying normosmics: a comparison of two populations.

An olfactory function test developed by the Connecticut Chemosensory Clinical Research Center (CCCRC) was administered to 65 normal subjects to determine its ability to identify normosmic subjects. The CCCRC test categorizes individuals into five distinct levels of olfactory functioning according to composite score ranges derived from odor identification and butanol threshold subtests. These categories are: normosmic (normal); mildly, moderately, and severely hyposmic (impaired functioning); and anosmic (no sensation). Comparing score distributions from CCCRC with ours revealed that a greater percentage of our subjects were categorized as hyposmic because of higher butanol thresholds. The butanol threshold subtest was studied further to determine test-retest reliability and normal day-to-day variability in scores. Norms are the basis for interpreting test scores and are important in diagnosing and treating smell disorders.

Neurosurgical Applications of Clinical Olfactory Assessmenta

The neurosurgeon, through routine neurological examination, encounters olfactory deficits in those patients with skull fractures, contusions, lesions in the anterior fossa, and intracranial tumors. We randomly sampled 25 patients attending the head injury follow-up clinic and administered the CCCRC test of olfactory function’ including a butanol detection threshold and odor identification subtest. Patients were of ages 9 to 61 and most had sustained a severe head injury as evidenced by a score of eight or less on the Glasgow coma scale. All patients were tested at least two months postinjury and were judged competent and coherent on the basis of an independent neuropsychological interview and assessment. FIGURE 1 compares olfactory function in severe head injury patients to a group of 65 normal subjects.* Almost 60% of the head injury patients were found to be anosmic. About 15% showed some decrease in olfactory function and the remaining 25% appeared normal (fell within the normosmic or mildly hyposmic range). Further analysis revealed that of the 40% of patients who could detect olfactory levels of butanol(O.1570 to 0.005%), half were unable to identify the standard number of odor stimuli. This suggests the possibility of a more central type of damage in these patients. A separate group of “surgical anosmics, ” patients lacking olfactory connections as a result of surgical procedure, were also tested and their scores validate the olfactory function test’s ability to define anosmia. All surgical anosmics fell to the left of the control distribution detecting only a 1.3% or stronger solution of butanol. Surgical anosmics were unable to identify any of the odor stimuli. An example of a head injury patient illustrates the application of olfactory function testing in diagnosing and locating neural damage. The patient was a 26-year-old white male involved in a motorcycle accident who sustained a blow to the head. The olfactory function test was administered to the patient and results indicated a detection level within the normal range (0.016% butanol). However, the patient was unable to discriminate any of the odor identification stimuli. In FIGURE 2, a CAT scan of the patient’s head revealed the presence of a large mass (hematoma) in a ventral region of the right frontal lobe (near olfactory pathways). In this case, the patient’s inability