Virginia McMillan Carr

Rapid appearance of labeled degenerating cells in the dorsal root ganglia after exposure of chick embryos to tritiated thymidine

The interval between [3H]thymidine delivery and onset of cellular degeneration in 5.5 day embryonic chick brachial dorsal root ganglia was examined autoradiographically. Of the degenerating cells, 14% were labeled by 2 h after [3H]thymidine delivery. This percentage increased for 24 h. Wing bud amputation had no effect on this percentage through 9 h. Thus, some cause of cell death other than faulty peripheral connections may exist in at least some degenerating ganglionic cells.

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis.

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19-37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 pM.

Effect of ketamine on stress protein immunoreactivities in rat olfactory mucosa

Administration of 9 mg ketamine per 100 g b.wt. to rats leads to transient enhancement of immunoreactivity to monoclonal antibodies against two stress proteins, ubiquitin and human 70 kDa heat shock protein (HSP70), in the supranuclear region of supporting cells of the olfactory epithelium and in the Bowmans gland acinar cells in the subepithelial lamina propria. In the supporting cells the enhanced immunoreactivities are not caused by other drugs used in our surgical anesthetic/antibiotic regimen (xylazine, buprenorphine, and gentamicin), but in Bowmans glands they are. Results are discussed in terms of possible ketamine binding to phencyclidine receptors (either NMDA-associated or not) and possible direct stress-inducing interactions of ketamine or ketamine breakdown products with the inhalant detoxification or secretory systems in the reactive cells.

Altered epithelial density and expansion of bulbar projections of a discrete HSP70 immunoreactive subpopulation of rat olfactory receptor neurons in reconstituting olfactory epithelium following exposure to methyl bromide

A previously described subpopulation of rat olfactory receptor neurons, the 2A4(+)ORNs, is 1) distinguished by intense constitutive cytoplasmic immunoreactivity to antibodies to the 70‐kD heat shock protein (HSP70); 2) occurs sparsely but consistently through ventral and lateral olfactory epithelium (OE); and 3) projects to just two to three consistently located glomeruli in each olfactory bulb (OB) (Carr et al. [1994] J Comp Neurol 348:150–160). Immunoreactivity appears not to be stress‐related. To examine the persistence of these features following destruction and reconstitution of the OE, rats were subjected to methyl bromide‐induced OE lesion (Schwob et al. [1995] J Comp Neurol 59:15–37; Schwob et al. [1999] J Comp Neurol 412:439–457] and their OE and OBs examined with antibodies to HSP70 6–10.5 weeks postlesion. Lesioned OE showed significantly increased 2A4(+)ORN densities but no alteration of 2A4(+)ORN zonal distribution. The OBs of lesioned animals showed marked expansions of 2A4(+)ORN bulbar projections, with 2–15‐fold increases in numbers of glomeruli showing 2A4(+)axons, and projection expansions were greater in animals maintained on chronic food restriction prior to lesioning. Examination of archival 5‐month post‐MeBr lesion material indicates that altered projection patterns are maintained. J. Comp. Neurol. 469:475–493, 2004.

Rat olfactory neurons express a 200 kDa neurofilament

Neurofilament expression in peripheral olfactory neurons of adult rats was investigated by immunoblotting and immunohistochemistry using monoclonal antibodies specific for each of the 3 neurofilament proteins. Immunoblotting analysis of olfactory epithelium extracts demonstrated the presence of only the 200 kDa (NFH) polypeptide; the 68 kDa (NFL) and 160 kDa (NFM) neurofilaments were not detected. Similarly, no immunoreactivity was observed in tissue sections using the NFL and NFM antibodies. In contrast, when sections were probed with the antibody to NFH, immunoreactivity was localized primarily in the dendritic knobs and near the cell bodies of the receptor cells.

Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70‐kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150–160). The 2A4(+)ORNs are dispersed through zones II–IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress‐inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters. J. Comp. Neurol. 404:375–386, 1999.

Tissue-Specific Effects of Allergic Rhinitis in Mouse Nasal Epithelia

Allergic rhinitis (AR) can cause significant olfactory loss, but few studies have specifically investigated AR effects on olfactory and nasal respiratory tissues per se. To address this, we used a murine AR protocol employing nasal allergen infusion for both sensitization and challenges. Seven- to 11-week BALB/c mice were bilaterally infused with 1% ovalbumin (OVA) in phosphate-buffered saline (PBS) or PBS alone for 6 or 11 weeks, given single bilateral PBS or OVA infusions 24 h before sacrifice, or left untreated. High OVA-specific IgE serum levels and eosinophil infiltration confirmed AR induction. Olfactory (OE) and respiratory (RE) epithelia showed distinctly different responses, most conspicuously, massive eosinophil infiltration of immediately RE-subjacent lamina propria. In OE, such infiltration was minimal. Significant RE hypertrophy and hyperplasia also occurred, although OE organization was generally maintained and extensive disruption localized despite a 20% reduction in sensory neurons and globose basal cells after 11 weeks OVA. Pronounced Bowmans gland hypertrophy crowded both OE and olfactory nerve bundles. Cellular proliferation was widely distributed in RE but in OE was localized to normally thinner OE and RE-proximal OE, suggesting possible indirect RE influences. Terminal deoxynucleotide transferase (TdT) nick end labeling was greater in OE than RE and, in contrast to other effects, occurred with acute infusions and chronic PBS alone, often unilaterally. Following chronic OVA, AR-related bilateral increases appeared superimposed on those. These findings indicate AR effects on olfactory function may be complex, reflecting various levels of RE/OE responses and interactions.

Induction of allergic rhinitis in mice.

We describe a method for allergic rhinitis (AR) induction in mice. Methodology involves nasal infusions of small volumes of ovalbumin for both initial sensitization and challenges. The latter are frequent and carried out over several weeks. This methodology more closely resembles natural AR induction than does the common use of systemic sensitization, often with adjuvants, followed by nasal challenges with relatively large allergen volumes. Also described are methodologies for collection of cardiac blood and perfusion for preparation of histological samples, both essential in verifying AR induction in individual animals.