Frank L. Margolis

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.

Carnosine in the Primary Olfactory Pathway

Carnosine (β-alanyl-L-histidine) is present in mouse olfactory bulbs and nasal olfactory epithelium at concentrations exceeding that previously reported for any brain region of any species. After peripheral deafferentation, carnosine concentrations in the olfactory bulbs decrease to less than 10 percent that of normal, while other amino compounds are unaffected. Carnosine appears to be highly localized to the primary olfactory pathway.

Denervation in the primary olfactory pathway of mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section

Unilateral olfactory nerve section was performed in the mouse. Three biochemical markers of the olfactory chemoreceptor neurons: carnosine, carnosine synthetase activity and the olfactory marker protein, were measured in the olfactory bulb and epithelium. Parallel observations were made by light microscopy as well as at the ultrastructural level. The specific biochemical markers decrease rapidly in both bulb and epithelium and reach a minimum by the end of the first week after surgery. They then slowly return to 80% of control values by one month. Carnosinase activity in epithelium was essentially unaffected. These biochemical observations coincide temporally with the onset of degenerative changes seen morphologically, in both the bulb and epithelium. The degenerative changes persist for up to two weeks in the bulb and for about one week in the epithelium. At this time basal cell division and differentiation begins in the epithelium with subsequent regrowth of olfactory axons into the glomerular layer of the olfactory bulb with ther reappearance of olfactory axon terminals. The temporal coincidence of these biochemical and morphological observations suggests they are manifestations of the same process, and is consistent with the idea that the olfactory chemoreceptor neurons are perhaps unique in being able to be replaced from undifferentiated stem cells.

Immunological studies of the rat olfactory marker protein.

Abstract— Antisera against the rat olfactory marker protein were prepared by injection of the homogeneous protein into a goat and a rabbit. When the antisera were tested by immunodiffusion against olfactory tissue extracts, many but not all mammalian species cross‐reacted against these antisera. Immunoprecipitin titrations with the goat antiserum generally showed higher cross‐reactivity against olfactory extracts from species more closely related to the rat. Human olfactory bulb extracts and non‐mammalian olfactory tissue extracts did not cross‐react with the antisera by either immunodiffusion tests or immunoprecipitin titrations, however, they did cross‐react when tested by a competitive binding radioimmunoassay using tritium‐labelled purified rat protein and the goat antibody.

DNA (Cell Number) and Protein in Neonatal Brain: Alteration by Maternal Dietary Protein Restriction

Female rats were maintained on 8 or 27 percent protein diet by a pair-feeding schedule for 1 month before mating and throughout gestation. The brains of newborn rats from females on the 8 percent protein diet contained significantly less DNA and protein compared to the progeny of the females on the 27 percent diet. The data on DNA indicate that there are fewer cells; the protein content per cell was also lower. If, at birth, the brain cells are predominantly neurons, and their number becomes final at that time, then such dietary restriction may result in some permanent brain-neuron deficiency. This quantitative alteration in number as well as the qualitative one (protein per cell) may constitute a basis for the frequently reported impaired behavior of the offspring from protein-deprived mothers.

Immunocytochemistry of the olfactory marker protein.

The olfactory marker protein has been localized, by means of immunohistochemical techniques in the primary olfactory neurons of mice. The olfactory marker protein is not present in the staminal cells of the olfactory neuroepithelium, and the protein may be regarded as indicative of the functional stage of the neurons. Our data indicate that the olfactory marker protein is present in the synaptic terminals of the olfactory neurons at the level of the olfactory bulb glomeruli. The postsynaptic profiles of both mitral and periglomerular cells are negative.

Chemical deafferentation of the olfactory bulb: Plasticity of the levels of tyrosine hydroxylase, dopamine and norepinephrine

The laminar distribution of tyrosine hydroxylase activity, dopamine and norepinephrine was determined in the dog olfactory bulb. The levels of tyrosine hydroxylase activity and dopamine were highest in the glomerular layer, whereas norepinephrine appeared to be more uniformly distributed across the layers. A similar distribution was observed within the mouse olfactory bulb. Following deafferentation of the mouse olfactory bulb, the levels of tyrosine hydroxylase activity and dopamine declined, while norepinephrine levels showed a transient increase. Subsequent to regeneration of the olfactory nerve, these levels returned to control values. The levels of tyrosine hydroxylase activity and of dopamine were very low or non-detectable in the olfactory epithelium, which contains the olfactory receptor neuron perikarya. The data obtained indicate that tyrosine hydroxylase activity and dopamine content in the bulb are more tightly coupled to each other than either is to norepinephrine content. Since the two catecholamines are in two different classes of neurons, this implies that the bulk of the tyrosine hydroxylase activity in the bulb is associated with the dopamine-containing neurons. Finally, our data are consistent with a transsynaptic control mechanism of the tyrosine hydroxylase activity and dopamine level in the olfactory bulb.

Denervation of the primary olfactory pathway in mice. V. Long-term effect of intranasal ZnSO4 irrigation on behavior, biochemistry and morphology.

Intranasal irrigation of mice with 0.17 M ZnSO4 solution results in the immediate and total loss of the ability to find a buried food pellet. This anosmia persists for 6 weeks in at least 80% of the treated mice and for 4 months in half of the animals. This marked behavioral effect is matched by a long-term reduction of the levels of carnosine synthesis and transport in the primary olfactory pathway. These biochemical parameters are virtually undetectable at two weeks after treatment and even at one year after treatment do not exceed 5-10% of average control values. Light microscopic observations of tissues of the primary olfactory pathway at various times after treatment are consistent with these observations and indicate a substantial destruction of the olfactory epithelium with subsequent atrophy of the olfactory bulb. At very long intervals after treatment, some receptor regeneration is apparent with accompanying reinnervation of the olfactory bulb. Estimates from microscopy and biochemistry suggest that much less than 10% of the normal complement of functioning receptor cells is adequate to give apparently normal food-finding behavior.

Denervation in the primary olfactory pathway of mice: Biochemical and morphological effects

Abstract Peripheral deafferentation of the primary olfactory pathway by stringent intranasal irrigation with a zinc sulfate solution results in a marked decrease in olfactory bulb weight, shrinkage of the glomerular layer of the olfactory bulb and specific disappearance of the unique olfactory marker protein from the olfactory bulb. These changes are irreversible for several months. In contrast, no alterations were seen in the activities of 3 neurotransmitter-metabolizing enzymes, or in 16 high affinity ‘neurotransmitter’ uptake systems, or in the levels of cyclic nucleotides in the olfactory bulbs. Surgical removal of the olfactory bulb results in the rapid specific decrease of the level of the unique marker protein in the olfactory epithelium. This treatment does not cause any alteration in the normally low levels of 3 neurotransmitter-metabolizing enzymes in the olfactory epithelium. Perhaps the primary olfactory chemoreceptor neurons utilize a novel transmitter candidate at their synapses in the olfactory bulb.

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb: prominent differences in the intraglomerular dendritic arborization and their relationship to olfactory nerve terminals

In the glomerular layer of the rat main olfactory bulb, we previously reported three chemically defined interneuron groups: GABA-like immunoreactive, calretinin-immunoreactive and Calbindin-D28k-immunoreactive groups [Kosaka K. et al. (1995) Neurosci. Res. 23, 73-88]. In the present study, we analysed the structural features of these three neuron groups using confocal laser scanning light microscopy, focusing on their dendritic arborization pattern, especially on their close apposition to olfactory receptor terminals labeled by olfactory marker protein. Each glomerulus consisted of two zones, the olfactory nerve zone and the non-olfactory nerve zone. The former was mainly occupied by olfactory nerve preterminals and terminals as well as their targets, postsynaptic fine dendritic portions of intrinsic neurons. The latter non-olfactory nerve zone was occupied mainly by olfactory marker protein-negative profiles. Processes of GABAergic neurons and those of one of their subpopulations, tyrosine hydroxylase-immunoreactive neurons, were numerous both in the olfactory nerve and non-olfactory nerve zones, resulting in their frequent close apposition to olfactory marker protein-immunoreactive elements. Combined confocal laser scanning light microscopic electron microscopic examination revealed synaptic contacts from olfactory nerve terminals on tyrosine hydroxylase-immunoreactive processes at these sites of close apposition. In contrast, calretinin-immunoreactive and Calbindin-D28k-immunoreactive processes, particularly Calbindin-D28k-immunoreactive ones, were distributed almost exclusively in the non-olfactory nerve zone, as if they avoided the olfactory nerve zone, showing a net or honeycomb pattern. Thus, calretinin-immunoreactive and Calbindin-D28k-immunoreactive processes were not or very rarely closely apposed to olfactory nerve terminals. These findings suggested that there might be some differences among chemically defined interneuronal groups in their synaptic contacts from olfactory nerves. Further quantitative image analysis clearly exhibited the prominent differences among these neuron groups in their intraglomerular dendritic arborization in relation with the olfactory nerve zone, i.e. the percentages of the area in the olfactory nerve zone occupied by GABAergic and tyrosine hydroxylase-immunoreactive processes were about 10%, respectively, whereas those of calretinin-immunoreactive and Calbindin-D28k-immunoreactive processes were only about 1% and 0.3%, respectively. These findings suggested that so-called periglomerular cells in glomeruli might be heterogeneous not only in their chemical nature, but also in their dendritic arborization pattern and synaptic contacts from olfactory nerve terminals.