Dopaminergic modulation of olfactory-evoked motor output in sea lampreys (Petromyzon marinus L.).

Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor-evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory-driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole-brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor-evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.

[Olfactory ensheathing cell tumour: case report and literature review]. / Tumor de las células envolventes del olfatorio: caso clínico y revisión de la literatura.

Olfactory ensheathing cells are glial cells located in the olfactory bulb and nerve. Microscopically, both olfactory ensheathing cells and Schwann cells have similar morphological and immunohistochemical features. However, olfactory ensheathing cells are negative for Leu-7(CD-57), whereas Schwann cells are positive. We present the case of a 49 year-old male with a history of visual impairment and hyposmia. Radiological CT and MRI studies showed a subfrontal cystic extra-axial mass, which eroded the right cribriform plate, with heterogeneous contrast enhancement. Total excision of the tumour was performed by bifrontal craniotomy. Histological examination initially suggested a schwannoma, with immunohistochemical staining being positive for S-100 protein and negative for epithelial membrane antigen (EMA). However, the tumour was negative for Leu-7. Accordingly, the final diagnosis was olfactory ensheathing cell tumour. Herein, we describe the sixth case of intracranial olfactory ensheathing cell tumour and stress the important role of immunohistochemical techniques in obtaining a definitive diagnosis.

Development of the olfactory system in turbot (Psetta maxima L.).

We have examined the histogenesis of the olfactory system during turbot development using histological and immunohistochemical methods. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was used to detect dividing cells, whereas calretinin (CR) immunohistochemistry was used to distinguish some neuronal components of the olfactory system. Around hatching, the olfactory placode of embryos transforms into an olfactory pit, which enlarges progressively during development. In metamorphic turbots, the right olfactory organ moves to the tip of the head. Each olfactory chamber opens to the external medium by two nostrils and accessory nasal sacs develop during metamorphosis. The order of birth of olfactory receptor cells in the sensory epithelium follows the pattern of most teleosts: ciliated cells differentiate prior to microvillous cells in turbot larvae, and crypt cells are generated during metamorphosis. Axons of olfactory sensory neurons reach the rostral forebrain by hatching, and calretinin-immunoreactive (CR-ir) glomerular fields were apparent during the subsequent larval development. During metamorphosis olfactory bulbs become strongly distorted by head torsion and glomeruli acquire asymmetric organization. The spatio-temporal course of proliferation in the olfactory system reveals changes in the distribution of dividing cells in the sensory epithelium throughout the developmental period investigated. In the olfactory bulb, proliferative activity becomes restricted to the ventral periventricular zone in turbot larvae, as well as in metamorphic specimens.

Olfactory system involvement in natural scrapie disease.

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

Chemical properties of type 1 and type 2 periglomerular cells in the mouse olfactory bulb are different from those in the rat olfactory bulb.

We analyzed the cellular composition of the juxtaglomerular region in the main olfactory bulb of C57B/6J strain mice, focusing on 1) the compartmental organization of the glomerulus and the presence of type 1 and 2 periglomerular cells, 2) the colocalization relationships among the 4 major chemically identified groups of periglomerular cells, glutamic acid decarboxylase (GAD)/gamma-aminobutyric acid (GABA), tyrosine hydroxylase, calretinin and calbindin D28k positive periglomerular cells, and 3) the chemical properties of the nitric oxide synthase (NOS)-positive juxtaglomerular cells. We confirmed the compartmental organization of the glomerulus and the presence of both type 1 and 2 periglomerular cells in the mice. Similar to rat periglomerular cells, the tyrosine hydroxylase-positive cells were type 1 and GAD/GABA-positive. On the other hand, both the calbindin D28k-positive and calretinin-positive cells were type 2 periglomerular cells, but in contrast to those in rats, which are GAD/GABA-negative, all of the calbindin D28k-positive periglomerular cells and 65% of the calretinin-positive periglomerular cells were GAD/GABA-positive. The GAD/GABA-positive cells thus included both type 1 and type 2 periglomerular cells. Juxtaglomerular NOS-positive cells have been proposed as a subgroup of type 1 periglomerular cells that are separate from the calretinin-positive and calbindin D28k-positive cells in rats. However, in the mice, about 70% of the NOS-positive cells were calretinin-positive, and 50% of the calretinin-positive cells were NOS-positive. We herein reveal the significant species differences in the chemical properties of periglomerular cells and suggest that the cellular organization of the mouse main olfactory bulb cannot be extrapolated from that of rats.

Olfactory nerve stimulation-induced calcium signaling in the mitral cell distal dendritic tuft.

Olfactory receptor neuron axons form the olfactory nerve (ON) and project to the glomerular layer of the olfactory bulb, where they form excitatory synapses with terminal arborizations of the mitral cell (MC) tufted primary dendrite. Clusters of MC dendritic tufts define olfactory glomeruli, where they involve in complex synaptic interactions. The computational function of these cellular interactions is not clear. We used patch-clamp electrophysiology combined with whole field or two-photon Ca2+ imaging to study ON stimulation-induced Ca2+ signaling at the level of individual terminal branches of the MC primary dendrite in mice. ON-evoked subthreshold excitatory postsnaptic potentials induced Ca2+ transients in the MC tuft dendrites that were spatially inhomogeneous, exhibiting discrete "hot spots." In contrast, Ca2+ transients induced by backpropagating action potentials occurred throughout the dendritic tuft, being larger in the thin terminal dendrites than in the base of the tuft. Single ON stimulation-induced Ca2+ transients were depressed by the NMDA receptor antagonist D-aminophosphonovaleric acid (D-APV), increased with increasing stimulation intensity, and typically showed a prolonged rising phase. The synaptically induced Ca2+ signals reflect, at least in part, dendrodendritic interactions that support intraglomerular coupling of MCs and generation of an output that is common to all MCs associated with one glomerulus.

The alpha6 integrin subunit in the developing mouse olfactory bulb.

Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the alpha 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the alpha 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an alpha 6 knockout (alpha 6-/-). Immunohistochemical analyses and confocal microscopy were used to examine alpha 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the alpha 6-/- mouse. In CD-1 mice from E13 to E17, alpha 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; approximately E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the alpha 6-/- mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for alpha 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.

X-ray scattering study of pike olfactory nerve: intensity of the axonal membrane, solution of the phase problem and electron density profile.

Synchrotron radiation X-ray scattering experiments were performed on unmyelinated pike olfactory nerves. The difference between the meridional and the equatorial traces of the 2-D spectra yielded the 1-D equatorial intensity of the macromolecular components oriented with respect to the nerve: axonal membranes, microtubules and other cytoskeletal filaments. These 1-D spectra display a diffuse band typical of bilayer membranes and, at small s, a few sharper bands reminiscent of microtubules. All the spectra merge at large s. The intensity of the axonal membrane was determined via a noise analysis of the nerve-dependent spectra, involving also the notion that the thickness of the membrane is finite. The shape of the intensity function indicated that the electron density profile is not centrosymmetric. The knowledge of intensity and thickness paved the way to the electron density profile via an ab initio solution of the phase problem. An iterative procedure was adopted: (i) choose the lattice D of a 1-D pseudo crystal, interpolate the intensity at the points sh = h/D, adopt an arbitrary set of initial phases and compute the profile; (ii) determine the phases corresponding to this profile truncated by the thickness D/2; (iii) repeat the operation with the updated phases until a stable result is obtained. This iterative procedure was carried out for different D-values, starting in each case from randomly generated phases: stable results were obtained in less than 10,000 iterations. Most importantly, for D in the vicinity of 200 A, the overwhelming majority of the profiles were congruent with each other. These profiles were strongly asymmetric and otherwise typical of biological membranes.

X-ray scattering study of pike olfactory nerve: elastic, thermodynamic and physiological properties of the axonal membrane.

The effects of several agents, sugars, isotonic KCl, and a variety of drugs, on the structure of the axonal membranes of unmyelinated pike olfactory nerve have been studied by synchrotron radiation X-ray scattering experiments. The main effects of the sugars are: (i) to increase the electron density of the extra-axonal space and thereby yield the absolute scale of the electron density profile; (ii) to osmotically stress the membrane and thus yield its elastic modulus of area compressibility, since the related strain, thickness dilation, is directly determined by the X-ray scattering experiments. Exposure to isotonic KCl, a depolarizing agent, induces membrane thickness to increase. The energy liberated in this process is a function of the amplitude of the dilation and of the elastic modulus of the membrane. This energy turns out to be close to the thermal energy liberated by the pike olfactory nerve during the initial phase of action potential that has previously been measured by others. Electrical depolarization thus seems to be accompanied by a thickness dilation of the axonal membrane. Another effect of isotonic KCl is to induce a large fraction of the membranes to pair by tight apposition of their extra-axonal faces. Local anaesthetics and some drugs have the effect of altering membrane thickness. All these observations are interpreted in terms of a modulation of the conformational disorder of the hydrocarbon chains of the lipid molecules.

Microglia in the olfactory bulb of rats during postnatal development and olfactory nerve injury with zinc sulfate: a lectin labeling and ultrastrucutural study.

Using isolectin (GSA I-B4) as a marker, this study examined the possible alterations of lectin-labeled membranous glycoproteins in microglial cells in the olfactory bulb of normal development and under experimentally induced degeneration. In light microscopy, several morphological types of microglial cells representing different degrees of cell differentiation were distributed in the bulb laminae. A gradient of microglial differentiation extending from the intermediate to superficial and intermediate to deep occurs in the bulb layers. The differentiation gradient and lectin labeling pattern of microglial cells in the developing bulb resembled those in other areas of the brain tissues. Differentiating microglia showed a gradual diminution of lectin staining when the nascent round cells transformed into the mature ramified cells. Microglia in the external plexiform layer of the olfactory bulb were the first to mature and the cells expressed very weak lectin reactivity. In mature or adult rats, some microglial cells showing intense lectin labeling were observed in the olfactory nerve layer, granule cell layer and subependymal layer. Ultrastructurally, lectin labeling was localized at the trans saccules of the Golgi apparatus. Microglial cells in other bulb laminae, however, exhibited a negative reaction for the isolectin at the Golgi apparatus. Following intranasal irrigation of zinc sulfate, some microglial cells in the olfactory nerve layer and glomerular layer were activated to become phagocytic cells with increased lectin labeling at their ramified processes. GSA I-B4 staining was also localized at their trans saccules of the Golgi apparatus. The lectin labeling pattern of these phagocytic cells resembled that of differentiating microglia in postnatal bulbs, suggesting that bulb microglia in the lesioned sites were activated through cell dedifferentiation into macrophages.

Changes in immunoreactivity to calcium-binding proteins in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effects of olfactory deprivation on the density of neuronal populations expressing the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin in the anterior olfactory nucleus of the rat were studied immunohistochemically in 60-day-old rats subjected to unilateral naris closure on the day of birth. The neuronal populations were characterized morphologically and topologically, and the density of each cell type was calculated in each subdivision of the anterior olfactory nucleus at seven rostrocaudal levels. Data were gathered into three groups: data from either the ipsilateral or contralateral anterior olfactory nucleus of experimental animals and data from control animals. Statistical analysis indicated that disruption of the normal afferent activity to one olfactory bulb affects the expression of the calcium-binding proteins investigated in the anterior olfactory nucleus, as revealed by variations in the density of certain neuronal populations. The observed effects were very heterogeneous and could not be related to any specific neuronal type, location, or to the expression of a given calcium-binding protein. Nevertheless, as a general rule the most affected neuronal populations were those expressing calbindin D-28k located in the rostral subdivisions. These subdivisions are the latest to develop in mammals and are those that receive the largest amount of inputs from the olfactory bulb.

The terminal nerve in turbot, Psetta maxima: a developmental immunocytochemical study.

The ontogeny and organization of the terminal nerve (TN) during turbot development was studied using an antiserum to neuropeptide Y. First immunoreactive cells were detected in the olfactory placode at hatching time. At 1 day after hatching, a loose group of labeled neurons form an extracranial primordial ganglion of the TN. During the subsequent larval development, more perikarya displaying increased immunoreactivity were found along the course of the olfactory nerve. Moreover, labeled cells cross the meninx of the forebrain gathering in the olfactory bulb of larval turbot. Projections from these cells, directed both to the caudal brain and to the retina, develop when the cells become established in the olfactory bulb. The generation of immunoreactive cells in the olfactory organ extends into the metamorphic period, when a pronounced asymmetry affects the turbot morphology. At this time, the topological location of the immunoreactive cells in the TN becomes distorted. This developmental pattern was compared with those found in other teleosts and in other vertebrates. Preabsorption experiments of anti-neuropeptide Y serum with neuropeptide Y and FMRF-amide suggests that immunoreactive material observed in TN cells was not neuropeptide Y, and raises the possibility that other peptides, e.g. FMRF-amide-like peptides, exist in this neural system.

Migration of LHRH neurons into the spinal cord: evidence for axon-dependent migration from the transplanted chick olfactory placode.

In the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons originate in the olfactory placode and migrate along the olfactory nerve to the forebrain. In previous studies, we demonstrated that LHRH neurons followed the trigeminal nerve when the olfactory nerve was physically interrupted. To examine whether LHRH neurons possess the capacity to migrate along the different type of axons, the olfactory placode was transplanted into the base of the forelimb. Three to five days after the transplantation, LHRH neurons were detectable in the spinal nerve, the dorsal root ganglion, the sympathetic ganglion and the spinal cord. Double or triple labelling studies for LHRH, somatostatin and/or axonin-1 showed that LHRH neurons entered the spinal nerve in contact with the olfactory axons, which are specifically immunoreactive to somatostatin. Migrating LHRH neurons continued to associate closely with the olfactory axons in the spinal nerve. However, some LHRH neurons often migrated along with the axonin-1 positive spinal sensory axons, maintaining a distance from the olfactory axons. Furthermore, a few LHRH neurons were observed in the ventral root and the ventral funiculus independent of olfactory axons. As LHRH neurons were observed in the motor component of the spinal nerve, it is probable that LHRH neurons also invaded the spinal cord using the motor axons as a guiding substrate for their migration. These results suggest that the migration mode of LHRH neurons is axon dependent in the peripheral region, however, chemical identity with regard to axonal substrate choice for migration was not specified in the present study.

Ontogenetic organization of the FMRFamide immunoreactivity in the nervus terminalis of the lungfish, Neoceratodus forsteri.

The development of the nervus terminalis system in the lungfish, Neoceratodus forsteri, was investigated by using FMRFamide as a marker. FMRFamide immunoreactivity appears first within the brain, in the dorsal hypothalamus at a stage around hatching. At a slightly later stage, immunoreactivity appears in the olfactory mucosa. These immunoreactive cells move outside the olfactory organ to form the ganglion of the nervus terminalis. Immunoreactive processes emerge from the ganglion of the nervus terminalis in two directions, one which joins the olfactory nerve to travel to the brain and the other which courses below the brain to enter at the level of the preoptic nucleus. Neither the ganglion of the nervus terminalis nor the two branches of the nervus terminalis form after surgical removal of the olfactory placode at a stage before the development of FMRFamide immunoreactivity external to the brain. Because this study has confirmed that the nervus terminalis in lungfish comprises both an anterior and a posterior branch, it forms the basis for discussion of homology between these branches and the nervus terminalis of other anamniote vertebrates.

The glucose transporter GLUT1 and the tight junction protein occludin in nasal olfactory mucosa.

The nervous cells in the brain and the peripheral nerves are isolated from the external environment by the blood-brain, blood-cerebrospinal fluid and blood-nerve barriers. The glucose transporter GLUT1 mediates the specific transfer of glucose across these barriers. The olfactory system is unique in that its sensory cells, olfactory receptor neurons, are embedded in the nasal olfactory epithelium and send their axons directly to the olfactory bulb of the brain. Only the apical parts of the olfactory receptor neurons are exposed to the lumen, and these serve as sensors for smell. Immunohistochemical examination showed that the tight junction protein occludin was present in the junctions of the olfactory epithelium. Endothelial cells in the blood vessels in the lamina propria of the olfactory mucosa were also positive for occludin. These observations suggest that the olfactory system is guarded from both the external environment and the blood. GLUT1 was abundant in these occludin-positive endothelial cells, suggesting that GLUT1 may serve in nourishing the cells of the olfactory system. Taken together, GLUT1 and occludin may serve as part of the machinery for the specific transfer of glucose in the olfactory system while preventing the non-specific entry of substances.

Seasonal changes in beta-endorphin-like immunoreactivity in the olfactory system of the female catfish, Clarias batrachus (Linn).

In the olfactory system of the catfish Clarias batrachus, beta-endorphin-like immunoreactivity was seen in several olfactory receptor neurons (ORN) and their fiber projections extending caudally over the olfactory nerve to the olfactory bulb (OB). With beta-endorphin-like immunoreactivity as a cellular marker, the olfactory system in the female fish was investigated at different stages of its annual reproductive cycle. The reproductive cycle of the fish is divisible into four distinct phases: preparatory (February-April), prespawning (May-June), spawning (July-August), and postspawning (September-January). The gonosomatic index and the immunocytochemical profile of beta-endorphin-like immunoreactivity showed distinct changes as the fish progressed from one phase to another. In the preparatory phase, limited immunoreactivity was seen in the periphery of the bulb. However, the immunoreactivity showed a robust increase as the immunolabeled fibers extended progressively deeper into the bulb toward the mitral cell layer during the prespawning and spawning phases. Significant reduction in the immunoreactivity was noticed in the olfactory nerve layer of the fish in the postspawning phase. Several granule cells showed poor to moderate immunoreactivity during the spawning phase, although no immunoreactivity was seen in the inner cell layer during the rest of the year. The beta-endorphin-like immunoreactivity in the ORN also showed season-related changes, although these were less distinct. Whereas weak immunoreactivity confined to a few ORN was noticed in the fish collected in the preparatory phase, those in the prespawning phase showed conspicuous augmentation in immunoreactivity. During the spawning phase, the sensory layer of the olfactory epithelium showed reduced, homogenous immunoreactivity. In the postspawning phase, several ORN revealed distinct granular immunoreactivity, suggesting possibilities of de novo synthesis. These annual cyclic changes in the beta-endorphin-like immunoreactivity were consistently observed over a 30-month study period that spanned three consecutive spawning phases. The results suggest that the beta-endorphin-containing ORN, their fiber projections to the OB, and several granule cells in the inner cell layer may be involved in the processing of reproduction/reproductive behavior-related signals.

Glucagon-like immunoreactivity in the forebrain and pituitary of the teleost, Clarias batrachus (Linn.).

The organization of glucagon-like immunoreactivity (GLI) in the olfactory system, forebrain, and pituitary was investigated in the teleost Clarias batrachus. Weak to moderate GLI was seen in some olfactory receptor neurons and basal cells of the olfactory epithelium. Intense GLI was seen in the olfactory nerve fascicles that ran caudally to the bulb, spread over in the olfactory nerve layer, and profusely branched in the glomerular layer to form tufts organized as spherical neuropils; some of the immunoreactive fibers seem to closely enfold the mitral cells. In the inner cell layer of the bulb, some granule cells were intensely immunoreactive. Although there were thick fascicles of immunoreactive fibers in the medial olfactory tracts (MOT), the lateral olfactory tracts were generally devoid of immunoreactivity. Immunoreactive fibers in the medial olfactory tract penetrated into the telencephalon from its rostral pole and entered into the area ventralis telencephali/pars ventralis where the compact fiber bundles loosen somewhat and course dorsocaudally into the area ventralis telencephali/pars supracommissuralis just above the anterior commissure. While some immunoreactive fibers decussated in the anterior commissure, fine fibers were seen in the commissure of Goldstein. Isolated immunoreactive fibers of the medial olfactory tract were traced laterally into the area dorsalis telencephali/pars lateralis ventralis and mediodorsally into the area dorsalis telencephali/pars medialis. However, a major component of the MOT continued dorsocaudally into the thalamus and terminated in the habenula. Two immunoreactive neuronal groups and some isolated cells were seen in the periventricular region of the thalamus. Although nucleus preopticus showed no immunoreactivity, some neurons of the nucleus lateralis tuberis displayed moderate GLI. Several immunoreactive cells were seen in the pars intermedia of the pituitary gland; few were encountered in the rostral pars distalis and proximal pars distalis. Immunoreactive fibers were seen throughout the pituitary gland.

Physical structure of the excitable membrane of unmyelinated axons: X-ray scattering study and electrophysiological properties of pike olfactory nerve.

The aim of this work was to elicit correlations between physical structure and physiological functions in excitable membranes. Freshly dissected pike olfactory nerves were studied by synchrotron radiation X-ray scattering experiments and their physiological properties were tested by electrophysiological techniques. The scattering spectra contained a sharply oriented equatorial component (i.e. normal to the nerve axis), and an isotropic background. After background subtraction, the equatorial component displayed a weak and fairly sharp spectrum of oriented microtubules, and a strong and diffuse band of almost the same shape and position as the band computed for an isolated myelin membrane. We ascribed this spectrum to the axonal membranes. Under the action of temperature and of two local anesthetics, the spectrum underwent a contraction (or expansion) in the s-direction, equivalent to the structure undergoing an expansion (or contraction) in the direction perpendicular to the plane of the membrane. The main observations were: (i) with increasing temperature, membrane thickness decreased with a thermal expansion coefficient equal to -0.97(+/-0.19) 10(-3) degrees C(-1). The polarity and amplitude of this coefficient are typical of lipid-containing systems with the hydrocarbon chains in a disordered conformation. The amplitude and propagation velocity of the compound action potentials were drastically and reversibly reduced by lowering the temperature from 20 degrees C to 5 degrees C. (ii) Exposing the nerve to two local anesthetics (tetracaine and dibucaine) had the effect of decreasing membrane thickness. Action potentials were fully inhibited by these anesthetics. (iii) Upon depolarization, induced by replacing NaCl with KCl in the outer medium, approximately 25 % of the membranes were found to associate by apposing their outer faces. Electrophysiological activity was reversibly impaired by the KCl treatment. (iv) No detectable structural effect was observed upon exposing the nerves to tetrodotoxin or veratridine. Electrophysiological activity was fully impaired by tetrodotoxin and partially impaired by veratridine. The main conclusions of this work are that axonal membranes yield highly informative X-ray scattering spectra, and that these spectra are sensitive to the functional state of the nerve. These results pave the way to further studies of more direct physiological significance.

Immunohistochemical evidence for the Na+/Ca2+ exchanger in squid olfactory neurons.

The olfactory organs from the squid Lolliguncula brevis are composed of a pseudostratified epithelium containing five morphological subtypes of chemosensory neurons and ciliated support cells. Physiological recordings have been made from two of the subtypes and only the type 4 neuron has been studied in detail. Odour-stimulated increases in intracellular calcium and rapid activation of an electrogenic Na+/Ca2+ exchanger current in type 4 neurons suggest that the exchanger proteins are localized very close to the transduction machinery. Electrophysiological studies have shown that olfactory signal transduction takes place in the apical ciliary regions of olfactory neurons. Using polyclonal antiserum against squid Na+/Ca2+ proteins, we observed specific staining in the ciliary region of cells that resemble type 2, 3, 4 and 5 neurons. Staining was also observed in axon bundles, and in muscle tissue. Collectively, these data support the model that Na+/Ca2+ exchanger proteins are localized to transduction machinery in cilia of type 4 neurons and suggest that the other olfactory subtypes also use Ca2+ during chemosensory responses.