Thomas V. Getchell

Perireceptor and receptor events in vertebrate olfaction

In this article we have summarized the basic information which identifies several key issues in the study of perireceptor and receptor events in vertebrate olfaction. We have emphasized the biophysical and biochemical data which have established a pivotal role for the olfactory mucus in the access of odorants to receptor sites as well as their clearance from the micro-environment. In addition, based on initial reports in the literature, we have postulated that the uptake of odorants by cells in the olfactory epithelium and their subsequent enzymatic degradation is an important mechanism in odorant removal. Hence, the pre- and post-interactive events in vertebrate olfaction play a key role in molecular recognition, sensory transduction and receptor desensitization. Study of the primary events in vertebrate olfaction is an increasingly active area of research in neurobiology. Application of contemporary techniques in cell and molecular biology as well as biochemistry and cellular biophysics is yielding new insights into the process and into establishing new hypotheses to be tested.

Mechanosensitive afferent units in the lumbar facet joint.

The purpose of this study was to characterize somatosensory units of the lumbar facet joint, which may play a central role in idiopathic low-back pain. A laminectomy was performed on the lumbar spine of adult male New Zealand White rabbits. Receptive fields of mechanosensitive afferent units were investigated in the lumbar facet joint and adjacent surrounding tissues, and electrophysiological recordings were obtained from filaments of the dorsal root. Twenty-four units were identified in the region of the facet joint: ten, in the capsule of the joint; twelve, in the border regions between capsule and muscle or tendon; and two, in the ligamentum flavum. Of these units, two had a conduction velocity that was slower than 2.5 meters per second (group IV), fifteen had a velocity ranging from 2.5 to twenty meters per second (group III), and seven had a velocity faster than twenty meters per second. Seven units had a von Frey threshold of more than 6.0 grams, thirteen had a threshold of less than 6.0 grams, and four were not examined. Seven units in the facet joint responded to movement of the joint. Fourteen other mechanosensitive units were found in the muscle, tendon, and interspinous ligament; seven had a conduction velocity of 2.5 to twenty meters per second, and seven had a velocity that was faster than twenty meters per second.

Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice

The brain is susceptible to oxidative stress, which is associated with age-related brain dysfunction, because of its high content of peroxidizable unsaturated fatty acids, high oxygen consumption per unit weight, high content of key components for oxidative damage, and the relative scarcity of antioxidant defense systems. Protein oxidation, which results in functional disruption, is not random but appears to be associated with increased oxidation in specific proteins. By using a proteomics approach, we have compared the protein levels and specific protein carbonyl levels, an index of oxidative damage in the brains of old mice, to these parameters in the brains of young mice and have identified specific proteins that are altered as a function of aging. We show here that the expression levels of dihydropyrimidinase-like 2 (DRP2), alpha-enolase (ENO1), dynamin-1 (DNM1), and lactate dehydrogenase 2 (LDH2) were significantly increased in the brains of old versus young mice; the expression levels of three unidentified proteins were significantly decreased. The specific carbonyl levels of beta-actin (ACTB), glutamine synthase (GS), and neurofilament 66 (NF-66) as well as a novel protein were significantly increased, indicating protein oxidation, in the brains of old versus young mice. These results were validated by immunochemistry. In addition, enzyme activity assays demonstrated that oxidation was associated with decreased GS activity, while the activity of lactate dehydrogenase was unchanged in spite of an up-regulation of LDH2 levels. Several of the up-regulated and oxidized proteins in the brains of old mice identified in this report are known to be oxidized in neurodegenerative diseases as well, suggesting that these proteins may be particularly susceptible to processes associated with neurodegeneration. Our results establish an initial basis for understanding protein alterations that may lead to age-related cellular dysfunction in the brain.

Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages

Aged humans and rodents are susceptible to infection with Streptococcus pneumoniae bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines and increased production of interleukin (IL)‐10 by macrophages (MΦ) from aged mice. To understand the molecular basis of cytokine dysregulation in aged mouse MΦ, a microarray analysis was performed on RNA from resting and lipopolysaccharide (LPS)‐stimulated MΦ from aged and control mice using the Affymetrix Mouse Genome 430 2.0 gene chip. Two‐way ANOVA analysis demonstrated that at an overall P < 0.01 level, 853 genes were regulated by LPS (169 in only the young, 184 in only the aged, and 500 in both). Expression analysis of systematic explorer revealed that immune response (proinflammatory chemokines, cytokines, and their receptors) and signal transduction genes were specifically reduced in aged mouse MΦ. Accordingly, expression of Il1 and Il6 was reduced, and Il10 was increased, confirming our previous results. There was also decreased expression of interferon‐γ. Genes in the Toll‐like receptor‐signaling pathway leading to nuclear factor‐κB activation were also down‐regulated but IL‐1 receptor‐associated kinase 3, a negative regulator of this pathway, was increased in aged mice. An increase in expression of the gene for p38 mitogen‐activated protein kinase (MAPK) was observed with a corresponding increase in protein expression and enzyme activity confirmed by Western blotting. Low doses of a p38 MAPK inhibitor (SB203580) enhanced proinflammatory cytokine production by MΦ and reduced IL‐10 levels, indicating that increased p38 MAPK activity has a role in cytokine dysregulation in the aged mouse MΦ.

Amphibian terminal nerve: distribution revealed by LHRH and AChE markers

Immunocytochemical and histochemical studies in the tiger salamander and bullfrog demonstrated the presence of luteinizing hormone-releasing hormone-like immunoreactive (LHRH-ir) material and acetylcholinesterase (AChE) in the terminal nerve (TN). Immunoreactive perikarya and processes were found within the olfactory, vomeronasal and trigeminal nerves and in the nasal epithelium. Central TN projections consisted of fibers terminating in the olfactory bulb and bundles that projected to another group of LHRH-ir perikarya in the preoptic region. Up to 4 weeks following hypophysectomy, the labeling intensity and number of TN-immunoreactive neurons were not altered. Acetylcholinesterase histochemistry in the salamander revealed two distinct groups of neurons associated with the TN: a lightly labeled group of fusiform perikarya was located in the olfactory nerve proper and a more heavily labeled group of larger oval perikarya was found within AChE-positive trigeminal fascicles in the ventral mucosa. This study has demonstrated that the amphibian TN follows olfactory, vomeronasal and trigeminal nerves to reach peripheral targets in the nasal mucosa. The projection of TN fibers to discrete olfactory bulb glomeruli, especially evident in the bullfrog, suggests that the TN functions in odor processing. The TN projection to the preoptic region in both of these amphibians implicates the TN in reproductive processes.

Immunolocalization of two cytochrome P450 isozymes in rat nasal chemosensory tissue

The NMa and NMb isoforms of cytochrome P450 enzymes are expressed in three nasal chemosensory organs: the olfactory, septal and vomeronasal mucosae. The NMa isoform is widely distributed throughout the nasal mucosa whereas the NMb isoform is present primarily in the chemosensory mucosae. The localization of cytochromes P450 demonstrates that sustentacular cells in the olfactory and septal epithelia, the mucus of the vomeronasal organ and the acinar cells of glands in the lamina propria of all three chemosensory systems engage in xenobiotic metabolism and participate in odorant/pheromone clearance, a perireceptor process associated with chemosensory transduction.

Leptin regulates olfactory-mediated behavior in ob/ob mice.

We have investigated olfactory-mediated pre-ingestive behavior in leptin (ob/ob) and leptin receptor (db/db) mutant mice compared to age- and gender-matched wild-type (wt) mice. Olfactory-mediated behavior was tested using a buried food paradigm 5 times/day at 2-h intervals for 6 days. Mean food-finding times of ob/ob and db/db mice were approximately 10 times shorter than those of wt mice. To test the effect of leptin replacement in ob/ob mice, leptin (1 or 5 microg/g body weight in sterile saline) or carrier was injected i.p. once daily prior to testing. Mean food finding times in ob/ob mice injected with carrier or with 1 microg/g leptin were similar and were 2-3 times faster than in wt mice. Mean food finding times in ob/ob mice injected with 5 microg/g leptin tripled compared to carrier-injected ob/ob mice and were of the same order of magnitude as those of wt mice, suggesting functional leptin replacement. A 3-factor repeated measures ANOVA demonstrated significant differences between the 6 cohorts (P = 0.0001), food finding times (P< or = 0.0001), and cohort by day interaction (P< or = 0.0001). Post hoc tests suggested that the ob/ob+5 mug/g leptin cohort performed more like the wt cohort in the food-finding test than like the ob/ob or ob/ob+carrier cohort. Potential local sites of leptin production and action were identified with immunohistochemistry and in situ hybridization in epithelial and gland cells of the olfactory and nasal mucosae. Our results strongly suggest that leptin acting through leptin receptors modulates olfactory-mediated pre-ingestive behavior.

Leukemia inhibitory factor, interleukin-6, and their receptors are expressed transiently in the olfactory mucosa after target ablation.

Removal of the synaptic targets of olfactory receptor neurons by olfactory bulb ablation results in apoptosis of olfactory receptor neurons and up‐regulation of proliferation of their progenitors. This study focuses on the expression of the neuropoietic cytokines leukemia inhibitory factor (LIF) and its receptor (LIFR) and interleukin 6 (IL‐6) and its receptor (IL‐6R) in intercellular signaling pathways in the olfactory mucosa after target ablation. Olfactory bulbectomy (OBX) resulted in several transient, early‐onset, temporally integrated events that were detected immunohistochemically. Macrophages infiltrated the olfactory epithelium (OE) by 16 hours post‐OBX. LIF expression was up‐regulated transiently at 2 days post‐OBX, when up‐regulated expression of LIFR also was detected on globose basal cells (GBCs), a subpopulation of which are immediate progenitors of olfactory receptor neurons. GBC proliferation peaked at 3–4 days post‐OBX. In the olfactory nerve (ON), LIF‐positive and IL‐6‐positive macrophage infiltration was followed by the transient up‐regulation of expression of LIFR, IL‐6, and IL‐6R in ensheathing cells by 3 days post‐OBX. The mRNAs for LIF/LIFR, IL‐6/IL‐6R, and their common signal‐transduction molecule, gp130, in olfactory‐nasal mucosa from control mice and from 3‐day post‐OBX mice were detected with reverse transcriptase‐polymerase chain reaction (RT‐PCR). Analysis of Northern blot and relative quantitative RT‐PCR demonstrated similar temporal patterns of changes in relative mRNA levels for both LIF and IL‐6, which were up‐regulated by 16 hours post‐OBX and peaked at 2–3 days post‐OBX. These data indicate that LIF from infiltrating macrophages acts as a mitogen for GBCs and that LIF from infiltrating macrophages and IL‐6 from infiltrating macrophages and ensheathing cells act as repair factors in the ON. J. Comp. Neurol. 435:60–77, 2001.

The effects of controlled mechanical loading on group-II, III, and IV afferent units from the lumbar facet joint and surrounding tissue. An in vitro study.

An in vitro model was developed to investigate the responses of afferent units in the lumbar spine to controlled loading as measured by a load-cell. The neuronal discharge was recorded simultaneously with loading. Three types of neuronal responses were observed. The first type of response involved phasic-type mechanoreceptors, which responded to movement, regardless of direction or initial position. The response did not outlast the movement phase of loading. These units may serve as velocity detectors. The second type of response was seen in slowly adapting low-threshold mechanoreceptors, which tended to respond to loading in the 0.3 to 0.5-kilogram range with an immediate and sustained increase in the rate of firing. This type of response appears to be associated with the activation of low-threshold group-II and group-III fibers, which were located in muscles and tendons inserting into the facet joint. The third type of response involved slowly adapting high-threshold mechanoreceptors, which could not be activated until a threshold of three to five kilograms had been exceeded. It appears that this type of response is at least partially due to the activation of high-threshold group-III and group-IV capsular afferent units, which may signal noxious mechanical stimulation.

Immunohistochemical Localization of a Cytochrome P-450 Isozyme in Human Nasal Mucosa: Age-Related Trends

Immunoperoxidase staining with an antibody to cytochrome P-450 (NMa) was used to investigate the localization of this isozyme in the human nasal mucosa. Olfactory mucosa was identified by staining of olfactory receptor cells with an antibody to olfactory marker protein. Immunoreactivity to NMa was localized in sustentacular cells in the olfactory epithelium, and in Bowmans gland acinar cells and vascular endothelial cells in the lamina propria. In the respiratory mucosa, ciliated epithelial cells, as well as serous gland acinar cells and vascular endothelial cells in the lamina propria, were immunoreactive for this isozyme. An age-related decrement in the intensity and extent of immunoreactivity within these cells was noted in nasal tissue from patients 60 years of age and over when compared with that of patients under 60 years of age. These results identify sites of xenobiotic metabolism or activation in human nasal mucosa.