Phosphorylation of adenylyl cyclase III at serine1076 does not attenuate olfactory response in mice.

Feedback inhibition of adenylyl cyclase III (ACIII) via Ca(2+)-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine(1076) of ACIII, the only residue responsible for Ca(2+)-induced phosphorylation and inhibition of ACIII (Wei et al., 1996, 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wild-type and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine(1076) plays a far less important role in olfactory response attenuation than previously thought.

Matrix metalloproteinase-9 is associated with acute inflammation after olfactory injury.

We previously reported an increase in matrix metalloproteinase-9 (MMP-9) levels in the olfactory bulb immediately after nerve transection; however, its role remains unknown. In this study, we determined the source of MMP-9 by monitoring the infiltration of inflammatory leukocytes in the olfactory bulb after nerve transection. We used myeloperoxidase to identify neutrophils and CD68 to identify macrophages at days 1, 7, and 10. MMP-9 colocalized with neutrophils at all three time points but was not contained in macrophages. This is the first study to demonstrate that MMP-9 is associated with early inflammatory response after olfactory injury, and provides insight into mechanisms underlying olfactory injury and recovery processes.

Telomerase protects adult rodent olfactory ensheathing glia from early senescence.

Adult olfactory bulb ensheathing glia (OB-OEG) promote the repair of acute, subacute, and chronic spinal cord injuries and autologous transplantation is a feasible approach. There are interspecies differences between adult rodent and primate OB-OEG related to their longevity in culture. Whereas primate OB-OEG exhibit a relatively long life span, under the same culture conditions rodent OB-OEG divide just three to four times, are sensitive to oxidative stress and become senescent after the third week in vitro. Telomerase is a "physiological key regulator" of the life span of normal somatic cells and also has extratelomeric functions such as increased resistance to oxidative stress. To elucidate whether telomerase has a role in the senescence of rodent OB-OEG, we have introduced the catalytic subunit of telomerase mTERT into cultures of these cells by retroviral infection. Native and modified adult rat OB-OEG behaved as telomerase-competent cells as they divided while expressing mTERT but entered senescence once the gene switched off. After ectopic expression of mTERT, OB-OEG resumed division at a nonsenescent rate, expressed p75 and other OEG markers, and exhibited the morphology of nonsenescent OB-OEG. The nonsenescent period of mTERT-OEG lasted 9weeks and then ectopic mTERT switched off and cells entered senescence again. Our results suggest a role of telomerase in early senescence of adult rodent OB-OEG cultures and a protection from oxidative damage. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Neuronal nitric oxide synthase in the olfactory system, forebrain, pituitary and retina of the adult teleost Clarias batrachus.

Immunocytochemical application of antibodies against nNOS to the brain sections of Clarias batrachus revealed intense immunoreactivity in several olfactory receptor neurons (ORNs), in their axons over the olfactory nerve, and terminals in the olfactory glomeruli. Several basal cells in the olfactory epithelium showed NOS immunoreactivity. Application of post-embedding immunoelectron microscopy showed nNOS labeled gold particles in apical cilia, dendrites and soma of the ORNs and also in the axon terminals in the glomeruli of the olfactory bulb. nNOS containing fibers were also encountered in the medial olfactory tracts (MOTs). Bilateral ablation of the olfactory organ resulted in total loss of nNOS immunoreactivity in the fascicles of the olfactory nerve layer and also in the MOT. nNOS immunoreactivity was seen in several cells of the nucleus preopticus (NPO) and their axons that innervate the pituitary gland. Some cells in the floor of the tuberal area were stained positive with nNOS antibodies. nNOS immunolabeled cells were seen in all the three components of the pituitary gland with light as well as post-embedding immunoelectron microscopy. While several nNOS immunoreactive fibers were seen in rostral pars distalis, a much limited fiber population was seen in the proximal pars distalis. In addition, conspicuous immunoreactivity was noticed in some ganglion cells in the retina and in some fibers of the optic nerve traceable to the optic tectum. The NO containing system in this fish appears to be similar to that in other fishes.

Localization of 5alpha-reductase in the rat main olfactory bulb.

The enzyme steroid 5alpha-reductase catalyzes the production of dihydroprogesterone and dihydrotestosterone, which were recently recognized as neurosteroids in the brain with variably potential neuroactivity. The present study reports for the first time detailed localization of 5alpha-reductase type 1 in the rat main olfactory bulb. The occurrence of 5alpha-reductase in the olfactory bulb was detected by reverse transcription-polymerase chain reaction and Western blotting analyses. In addition, the enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed that 5alpha-reductase immunoreactive cells of variable intensity were present in all layers of the olfactory bulb. Multiple immunolabeling revealed that 5alpha-reductase was mainly localized in glial cells, namely, in S-100beta- and glial fibrillary acidic protein-immunoreactive astrocytes, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-immunoreactive oligodendrocytes, and in S-100beta- and neuropeptide-Y-immunoreactive olfactory ensheathing cells, whereas the bulbar neurons exhibited little immunoreactivity. Quantitative analysis revealed that the number of 5alpha-reductase-immunoreactive cells was greatest in the olfactory nerve layer. The most intense 5alpha-reductase-immunoreactivity was found in the olfactory ensheathing cells, and next in the CNPase-immunoreactive cells. The 5alpha-reductase in the olfactory bulb was expressed constantly throughout different ages and sexes and in neutered and hypophysectomized rats. Thus, 5alpha-reductase may contribute via 5alpha-reduced metabolites to the formation and maintenance of olfactory inputs and outputs, which were closely associated with the olfactory ensheathing cells and the oligodendrocytes, respectively.

Nitric oxide synthase containing periglomerular cells are GABAergic in the rat olfactory bulb.

In the olfactory glomeruli of the rat olfactory bulb, there is a population of periglomerular cells (PG) that contains the neuronal isoform of the nitric oxide synthase (nNOS). To date, these PG have not been characterized neurochemically and it has not been determined whether they are type 1 (GABAergic PG that receive synaptic contacts from the olfactory axons) or type 2 PG (non-GABAergic PG that do not receive synapses from the olfactory axons). Combining pre-embedding NADPH-diaphorase histochemistry and post-embedding immunoperoxidase detection of GABA, we demonstrate that nNOS-containing PG are GABAergic and therefore, belong to the type 1 PG. The possible actions of nitric oxide in the olfactory glomeruli are discussed.

The role of serine proteases and serine protease inhibitors in the migration of gonadotropin-releasing hormone neurons.

BACKGROUND: Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH) neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. RESULTS: Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. CONCLUSIONS: These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

Developmental pattern of NADPH-diaphorase activity in the peripheral nervous system of the cichlid fish Tilapia mariae.

The distribution of NADPH-diaphorase activity was studied in the cichlid fish Tilapia mariae, during the first developmental stages by means of the tetrazolium salt technique. The reaction product was first found, 48 hours after fertilization (stage 10), in the cells of the olfactory placodes and in the superficial neuromasts. A faint positivity was seen in some hair cells of the otic vesicles. The epithelial cells of the most caudal part of the intestinal tract showed a strong labeling. At stage 12 (hatch), the reaction product was in addition detected in scattered enteric neurons surrounding the digestive tract. At stage 13 (4.5 days after spawning), the reaction product was also found in the putative sympathetic trunk, which supplies the gill arches and digestive tract. The epithelial cells of the gastrointestinal canal showed a more strong positive labeling and two large clusters of cells near the pronephritic tubules (the putative adrenomedullar tissue) were also labeled. The present results indicate an early activity of NADPH-diaphorase during the development of the peripheral nervous system of Tilapia and reveal a gradual maturation of NADPH-diaphorase positive structures.

Development of NADPH-diaphorase expression in chemosensory systems of the opossum, Monodelphis domestica.

Using NADPH-diaphorase histochemistry, the present study describes development of olfactory and vomeronasal systems in postnatal opossums, Monodelphis domestica. NADPH-dependent staining is absent at and around the time of birth. By 2 weeks of age and through adulthood, intense staining is seen along the luminal surface of the olfactory epithelium (OE) and of the vomeronasal sensory epithelium (VNE), as well as in Bowman's glands of the OE. Staining of the adult VNE is not homogeneous; it is restricted to the superficial 2/3 of the epithelium. At 2 weeks of age, staining in the brain is seen only at the surface of the ventricles and in blood vessels. At 1 month of age and through adulthood, staining of varying intensity is seen in individual olfactory bulb glomeruli, although the incoming olfactory axons are relatively unstained. Interestingly, whereas at 30 days of age, staining of the accessory olfactory bulb (AOB) glomeruli is uniform, 2 weeks later and in the adult, NADPH staining is concentrated in the rostral half, with little or no staining observed in the posterior portion. Darkly stained periglomerular cells are seen throughout the extent of the differentially-stained glomerular layer. From 30 days of age and through adulthood, intense NADPH staining is also observed in the islands of Calleja, as well as in cells of the dorsal cortex, often associated with the path of the rostral migratory stream.

Localization and biochemical characterization of acid phosphatase isoforms in the olfactory system of adult rats.

Localization of acid phosphatases was studied with the use of beta-glycerophosphate and p-nitrophenyl phosphate as substrates in the brain with special emphasis on the olfactory system of adult rat at light and electron microscopic level. With the use of beta-glycerophosphate, a selective substrate for the lysosomal acid phosphatase, lead-containing reaction product was found in primary and secondary lysosomes of neurons, glial cells and perivascular macrophages as well as in the cytoplasm of olfactory sensory axons. Incubation with p-nitrophenyl phosphate as substrate additionally revealed a cytoplasmic isoform of acid phosphatase, which could not be inhibited by tartrate or fluoride and was predominantly located in dendrites. Acid phosphatase isoforms were biochemically characterized in samples prepared separately from the olfactory mucosa, olfactory nerve layer, olfactory bulb and its dendrodendritic synaptosomes isolated by subcellular fractionation. In the olfactory mucosa and olfactory nerve layer the lysosomal type (high molecular weight form) was the most prominent acid phosphatase form, whereas the isoform located in dendrites corresponded to the tartrate-resistant extralysosomal, cytosolic type (low molecular weight form). The functional significance of different isoforms of acid phosphatase in the olfactory sensory axons and dendritic elements is discussed.

Olfactory transduction: cross-talk between second-messenger systems.

Chemosensory cilia of olfactory receptor neurons contain an adenylate cyclase which is stimulated by high concentrations of odorants. Cyclic AMP produced by this enzyme has been proposed to act as second messenger in olfactory transduction. Here we report that olfactory cilia contain calmodulin and that calmodulin potently activates olfactory adenylate cyclase by a mechanism additive to and independent from direct stimulation by odorants. Activation by calmodulin is calcium dependent and enhanced by GTP. Thus, olfactory transduction may involve a second-messenger cascade in which an odorant-induced increase in intracellular calcium concentration leads to activation of adenylate cyclase by calmodulin.

Isolated frog olfactory cilia: a preparation of dendritic membranes from chemosensory neurons.

The recently introduced frog olfactory cilia preparation (Chen and Lancet, 1984; Pace et al., 1985) has been useful for studies of molecular chemosensory mechanisms. Here we describe in detail the properties of this cilia preparation. The "calcium shock" procedure leads to a complete removal of the cilia from the olfactory epithelial surface. Isolated cilia constitute segments of proximal regions with 9 X 2 + 2 microtubular arrangement and a large proportion of membrane vesicles, probably derived from the ciliary distal segments. Polypeptides unique to the olfactory cilia preparation, compared to a control preparation of palate respiratory cilia, are identified by Coomassie brilliant blue staining, silver staining, and radiolabeled lectin overlays, as well as by biosynthetic labeling with 35S-methionine in epithelial explants and protein phosphorylation in isolated cilia. The olfactory cilia preparation contains odorant-sensitive adenylate cyclase, which is absent in control membranes from deciliated epithelium. High activities of tyrosine and serine/threonine protein kinases are also present. The olfactory cilia preparation described should be instrumental in the further elucidation of the biochemistry and molecular biology of vertebrate olfaction.

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulbs.

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat olfactory bulb was obtained with sheep antiserum to glutamate decarboxylase (GAD) and rabbit antiserum to tyrosine hydroxylase (TH). GAD-positive neurons include periglomerular cells, granule cells, superficial and deep short axon cells. TH-positive neurons represent periglomerular cells. Two-color immunocytochemistry shows that GABA and dopamine periglomerular cells are separate populations. The accessory olfactory bulb has rare dopamine cells and few superficial short axon cells. Radial gradients of GAD-immunostaining are evident in the main but not in the accessory olfactory bulb.

Partial purification and characterization of (Na+ + K+)-ATPase from garfish olfactory nerve axon plasma membrane.

The (Na+ + K+)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na+ + K+)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na+ + K+)-ATPase, such as the Km for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme. The partially purified garfish (Na+ + K+)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent 22Na+ transport. The ratio of 22Na+ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na+ + K+)-ATPase, however, gave a 22Na+ pumped to ATP hydrolyzed ratio of nearly 3.

The response of ornithine decarboxylase during neuronal degeneration and regeneration in olfactory epithelium.

Mature olfactory neurons are continually replaced from a population of progenitor cells. Olfactory nerve section, bulbectomy, or treatment with certain chemicals induces degeneration of olfactory neurons followed in some cases by regeneration. Ornithine decarboxylase (ODC) activity was measured in mouse olfactory tissues as an indicator of cellular regeneration. ODC activity in olfactory tissue (0.2-0.4 nmol/mg protein/h) is 10-30 times higher than in a variety of other cerebral tissues. Within 3 h after unilateral olfactory nerve section, ODC activity in the epithelium declines to 50% of control followed by a slow return to basal activity by 6 days. In the same animals, ODC activity increases severalfold in bulb (1 day) with a gradual decline to normal (9 days). Except for an early transient increase, the effects of unilateral bulbectomy on epithelial ODC activity are similar to those seen after nerve section. The changes in ODC activity following intranasal irrigation with 10 mM-colchicine also closely mimic those seen after nerve section. The effects of intranasal irrigation on ODC activity with 0.5% Triton X-100 or 0.17 M-ZnSO4 are more complex. Thus, when the mature neuronal population is degenerating after surgery or chemical treatments, ODC activity decreases in the epithelium. The subsequent increase of ODC activity prior to reconstitution of the mature neuronal population probably reflects the regeneration mechanism of the olfactory epithelium. The increase of ODC activity in the olfactory bulb after nerve section is best interpreted as a cellular injury response. These alterations in ODC activity in olfactory tissues after chemical and surgical treatments constitute the earliest biochemical events observed in these tissues in response to cellular damage.

[The activities of the transmitter metabolizing enzymes monoamineoxidase and acetylcholinesterase and of succinic dehydrogenase were investigated in the telencephalon of Salmo irideus (teleostei) (author's transl)]. / Zur Enzymhistochemie des Vorderhirns von Salmo irideus (Teleostei).

The activities of the transmitter metabolizing enzymes monoamineoxidase and acetylcholinesterase and of succinic dehydrogenase were investigated in the telencephalon of Salmo irideus (teleostei). There is a high reaction intensity of the MAO in the whole telencephalon with graded differences between the various nuclear areas. It is highest in the area dorsalis telencephali; the larger fiber tracts (Tr. olfactorius medialis et lateralis; medial and lateral forebrain bundle) are strong MAO-positive, too. There is an extremely weak AchE-reaction throughout the telencephalon. The only nuclear area which is clearly histochemically demonstrable is the regio Dm2 in the area dorsalis telencephali. The SDH-activity is demonstrable in the whole telencephalon. The activity is higher in the area dorsalis as compared with the area ventralis telencephali. In the ependym there are only a few traces of MAO- and SDH-reaction products. The recent findings may support, from a histochemical view, the hypothesis that the telencephalon of Salmo irideus represents brain structures which show structural and functional similarities with limbic telencephalic structures of higher vertebrates.

Regional distribution of choline acetyltransferase and acetylcholinesterase within the amygdaloid complex and stria terminalis system.

The distribution of "marker" enzymes for cholinergic neurons has been studied in 10 subdivisions of the amygdaloid complex of the rat brain. Choline acetyltransferase activity was measured using a radiochemical method in samples dissected from fresh serial sections. Acetylcholinesterase was studied using a histochemical procedure. Both enzymes had similar patterns of distribution within the amygdaloid complex and were most concentrated in the posterior lateral and basolateral nuclei and in the nucleus of the lateral olfactory tract. These enzymes were much less concentrated in the cortical, medial, central, and basomedial nuclei. Large differences in acetylcholinesterase staining were found within the lateral posterior and the basolateral nuclei and within the pyriform cortex. Biochemical studies showed a parallel distribution of choline acetyltransferase within these nuclei. The results indicate that cholinergic neural elements in the amygdala are concentrated primarily in the basolateral complex and suggest that this region may be innervated by cholinergic fibers traveling in the ventral amygdalo-fugal pathway.

Post-tetanic hyperpolarization, sodium-potassium-activated adenosine triphosphatase and high energy phosphate levels in garfish olfactory nerve.

1. While much is now known about the Na-K-ATPase and the posttetanic hyperpolarization of nervous tissue, they have yet to be studied together in the same preparation. 2. The post-tetanic hyperpolarization was studied in desheathed garfish olfactory nerve. The rate constant of decay of the post-tetanic hyperpolarization was determined by monitoring difference potentials after stimulation at 1/sec for 2-3 min. 3. In membrane fractions prepared from these nerves, the ouabain-sensitive ATPase activity (Na-K-ATPase) was determined by spectrophotometric measurements. 4. Both the post-tetanic hyperpolarization and the Na-K-ATPase showed a similar sigmoidal dependence on K+ concentration. The sequence of cation specificities measured at the K-site of the enzyme was the same as that determined by post-tetanic hyperpolarization measurements in whole nerve. 5. The rate constants of the enzyme showed a dependence on Na+ concentration that paralleled the way in which the post-tetanic hyperpolarization rate constants varied as a function of the number of impulses. When Na+ was completely replaced by Li+, neither enzyme activity nor post-tetanic hyperpolarization could be measured. 6. The pH optimum for enzyme activity was between pH 7-0 and 7-8, while the optimal pH for post-tetanic hyperpolarization was above pH 8-0. 7. Metabolite levels in preparations of this nerve studied in vitro correspond to levels found in vivo. 8. High energy phosphate levels were measured fluorometrically in extracts of nerve samples that had been stimulated in air at 1/sec for various intervals. 9. During the first 2 min of stimulation, there was a significant accumulation of inorganic phosphate, and the ATP/ADP.Pi ratio dropped appreciably. 10. The accumulation of ATPase products was commensurate with the approach of post-tetanic hyperpolarization rate constants to their maximum level. This provides direct evidence for an ATPase functioning in active Na+ transport in nerve. 11. The garfish Na-K-ATPase is sensitive to the ATP/ADP ratio of the incubating medium, but is relatively insensitive to orthophosphate, Pi. The fall in post-tetanic hyperpolarization rate constants observed with continued nerve stimulation may have been partially due to the falling ATP/ADP ratio measured in nerve under similar conditions.

Distribution along the axon and into various subcellular fractions of molecules labeled with (3H)leucine and rapidly transported in the garfish olfactory nerve.

The distribution of molecules labeled with [3H]leucine by fast axoplasmic transport in vivo has been studied in the garfish olfactory nerve after incorporation of the amino acid by the olfactory mucosa. Owing to the size of the nerve, it has been possible to follow the fate of the labeled molecules in 10 different subcellular fractions of 6 consecutive nerve segments. Each segment represents a different part of the profile developed by the transported radioactive molecules. In order to determine the influence of the perikaryon (rate of protein synthesis and rate of protein release into the axon) transport was studied under 3 different conditions: (1) intact nerves (simply labeled with [3H]leucine); (2) nerves cut from the cell bodies 6 h after application of [3H]leucine; and (3) nerves pulse-chase labeled for 1 h. Several conclusions can be drawn. (1) The bulk of the rapidly transported molecules are membranous axonal proteins, as determined by enzyme markers. Most are found in subcellular fractions representing 17% of the total axonal protein. They are synthesized very rapidly in the cell bodies (less than 1 h after isotope deposition) and exhibit the highest specific activities measured. These high specific activities were found in the same axonal membrane fractions in both plateau and crest, suggesting that the membrane precursors are transported as particles rather than as subunits. (2) The majority of these proteins are released into the axon immediately after synthesis; however, at least 30% of the labeled axonal membranous proteins are not released with the fast wave itself but progressively over a long period of time. (3) The majority of the moving material, particularly in membranous fractions, is left behind the fast wave and is deposited in the axon. When the front base of the fast wve has covered 70% of the total nerve length, only 19% of the labeled material of the main axonal membranous fraction appears still to be moving. (4) Proteins with high specific activities are found near the cell bodies and may be the result of early axonal transport of amino acids, diffusing later into the surrounding cells and being incorporated into proteins. Some free amino acids are also transported along the axon.