Response properties of neurones in the rat olfactory bulb to various parameters of odour stimulation

Carbon fibre filled microelectrodes were used for extracellular recording of spikes discharges from 345 neurones of the lateral aspect of the left olfactory bulb of the rat under pentobarbitone anaesthesia. The identity of the neurones was determined by antidromic activation of the cell axons by electrical stimuli applied to the lateral olfactory tract (LOT) using collision blocking and twin stimuli. The bulbar units were classified as mitral (269) or tufted (16) cells; units in a third class were not identified (60). Application of stimuli (21) was made over a period of 15 s while slowly increasing the odorant concentration monitored by a flame ionization device (FID). The stimulating efficiency reached a maximum at a nasal flow of about 8 cm3 s-1. The response type excitation was seen in 12.0% and inhibition in 11.8% of the 7245 odour presentations. Each response type was subdivided into four classes according to the reactions to ramp stimulation. In the majority of cases the spike discharge remained constant or followed the odour concentration for the entire stimulation period. In 18% of the cases, the response adapted quickly towards the spontaneous activity level.     

Some properties of receptive fields of olfactory mitral/tufted cells in the frog.

1. Different regions of the frog's olfactory epithelium were stimulated with nine glass micropipettes either individually or simultaneously in different combinations. The stimulus was a positive electrical pulse (4 s) consisting of a progressive increase, a plateau, and a progressive decrease in current intensity. Extra- and intracellular recordings were made from olfactory bulb mitral/tufted cells. Some of these cells were identified by intracellular injection of Lucifer yellow. 2. The action potential response patterns of mitral/tufted cells during the different phases of the stimulation were coded according to whether the activity was increased or decreased compared with its spontaneous level just before stimulation. Neural responses were classified into 11 types: individual neurons responded with different response types to stimuli delivered at different epithelial sites. On the basis of these response types, it was found that neurons could be classified into two groups. All response types in one group included an initial phase of increased discharge (excitation), whereas all types in the other group included an initial phase of decreased activity (suppression). Neurons that displayed response types belonging to one group never displayed those of the other group. It was thus concluded that a given neuron responded either always with an increased activity or always with a decreased activity, whatever the location of the stimulus. 3. The receptive field of a mitral/tufted cell appeared to be homogenous and not divided into areas of different properties, at least under the present experimental conditions. The extent of a receptive field was estimated by determining the number of effective epithelial sites (where an electrical stimulus evoked a response from a bulbar neuron).(ABSTRACT TRUNCATED AT 400 WORDS)     

The functional organization of the fish olfactory system

Recent developments in the functional anatomy and physiology of the fish olfactory system reveal three parallel pathways from the sensory epithelium, via the olfactory bulb to the telencephalon. There are three morphological types of sensory neurones spread in a seemingly overlapping arrangement in the olfactory epithelium. The axons of each type of sensory neurones converge to a specific region of the olfactory bulb and connect to separate sets of relay neurones. The axons of these relay neurones leave in three bundles to the telencephalon. Each bundle conveys specific information that elicits sets of characteristic behaviour in response to odours involved in essential life processes in the fish. One pathway is tuned to social cues, another to sex pheromones, and the third to food odours.     

Response patterns of amphibian olfactory bulb neurones to odour stimulation

1. Responses of 199 single olfactory bulb neurones in the frog were observed with odour stimulation.2. To effect control over the concentration, flow rate and time course of the stimulus application, a simple olfactometer was developed and calibrated using a gas chromatograph. The stimulus pulses were monitored by recording the electro-olfactogram from the olfactory mucosa.3. Several categories of unit activity became evident after delivering many odour applications (637). Some odour applications did not cause any change in the spontaneous activity of the unit. This was classified as type N (no response) activity. Some applications caused a suppression of ongoing activity (type S activity). Some applications caused an excitation during the rising concentration phase of the stimulus pulse (type E activity). Three temporal patterns of excitatory responses were seen and these were categorized as subgroups of activity type E.4. Since each response type was seen with about the same frequency regardless of the odour used, this suggested that the temporal patterns of response per se were not coding for odour type.5. When odour concentration was manipulated for a unit showing suppressive (type S) activity at one concentration, then the unit tended to show qualitatively similar suppression at other concentrations as long as they were above threshold.6. When peak concentrations were manipulated for units showing excitatory (type E) activity, the temporal patterns of response could change dramatically. In many cases the units fired after one level of concentration had been reached and were subsequently suppressed with increase in concentration. Such ;concentration tuning' suggested that excitatory temporal patterns, in contrast to suppressive ones, were coding odour concentration information.7. The significance of the changes in temporal response patterns seen here, for experiments examining the relationships between odours using single unit response data, is discussed.     

Expression of connexin 57 in the olfactory epithelium and olfactory bulb

In the visual system, deletion of connexin 57 (Cx57) reduces gap junction coupling among horizontal cells and results in smaller receptive fields. To explore potential functions of Cx57 in olfaction, in situ hybridization and immunohistochemistry methods were used to investigate expression of Cx57 in the olfactory epithelium and olfactory bulb. Hybridization signal was stronger in the olfactory epithelial layer compared to the connective tissue underneath. Within the sensory epithelial layer, hybridization signal was visible in sublayers containing cell bodies of basal cells and olfactory neurons but not evident at the apical sublayer comprising cell bodies of sustentacular cells. These Cx57 positive cells were clustered into small groups to form different patterns in the olfactory epithelium. However, individual patterns did not associate with specific regions of olfactory turbinates or specific olfactory receptor zones. Patched distribution of hybridization positive cells was also observed in the olfactory bulb and accessory olfactory bulb in layers where granule cells, mitral cells, and juxtaglomerular cells reside. Immunostaining was observed in the cell types described above but the intensity was weaker than that in the retina. This study has provided anatomical basis for future studies on the function of Cx57 in the olfactory system.     

An electrophysiological study of odour similarities of homologous substances

1. The activity of single bulbar units in the olfactory system of the frog was recorded extracellularly by means of micro-electrodes. The electro-olfactogram was recorded simultaneously from the receptor epithelium.2. The olfactory epithelium was stimulated with substances of homologous series of normal aliphatic alcohols, acetates and ketones.3. The effect on a bulbar unit was classified as excitatory or inhibitory, and the chi-square values calculated with one degree of freedom for all pairs in a given series. The statistical values obtained indicated the degree of similarity in olfactory stimulative properties between the odours.4. The results show that the degree of similarity in olfactory properties is greatest among neighbouring substances and gradually decreases with increasing separation in chain length.5. Significant rank correlation coefficients were found between the chi-square values and the molecular weight ratios for alcohols and acetates. The results are discussed in relation to psychophysical findings obtained with the same substances.     

Complex neural representation of odour information in the olfactory bulb

The most important task of the olfactory system is to generate a precise representation of odour information under different brain and behavioural states. As the first processing stage in the olfactory system and a crucial hub, the olfactory bulb plays a key role in the neural representation of odours, encoding odour identity, intensity and timing. Although the neural circuits and coding strategies used by the olfactory bulb for odour representation were initially identified in anaesthetized animals, a large number of recent studies focused on neural representation of odorants in the olfactory bulb in awake behaving animals. In this review, we discuss these recent findings, covering (a) the neural circuits for odour representation both within the olfactory bulb and the functional connections between the olfactory bulb and the higher order processing centres; (b) how related factors such as sniffing affect and shape the representation; (c) how the representation changes under different states; and (d) recent progress on the processing of temporal aspects of odour presentation in awake, behaving rodents. We highlight discussion of the current views and emerging proposals on the neural representation of odorants in the olfactory bulb.     

Amplifying role of convergence in olfactory system a comparative study of receptor cell and second-order neuron sensitivities

1. Extracellular unitary responses of receptor cells of second-order neurons identified as output cells were recorded in the frog. Four odorants of defined concentrations distributed over a wide range were delivered in the form of 2-s square pulses to the olfactory mucosa with a multistage dynamic flow dilution olfactometer. Bulbar responses were studied under two conditions, the stimuli being delivered either to the ventral or to the entire mucosa. 2. Overall responsiveness of the cells was compared. For the second-order neurons, the response ratio (excitation or inhibition) clearly depended on the condition of stimulation when the entire mucosa was stimulated, the bulbar response ratio was increased, as compared with that obtained when only the ventral mucosa received stimuli. Furthermore, when the stimuli were delivered to the whole mucosa, the bulbar excitation ratio was found to be similar to those of receptor cells and second-order neurons. 3. Response thresholds were determined from a comparison of the interspike interval values in the 30-s pre- and in the 12-s poststimulus time periods, using the Mann-Whitney U test (Table 2). The distribution of response thresholds of receptor cells as a function of stimulus concentration did not significantly differ from that of second-order neurons as excited by stimulating the ventral mucosa. These two distributions differed significantly from the distribution of second-order neurons as stimulated through the entire mucosa. In this last experimental condition, the bulbar neurons displayed an improved sensitivity. 4. The overall recruitment process, represented by the cumulative percentage of cells responding with excitation as a function of concentration, was found to be continuous over the entire concentration range. At the bulbar level, when the entire mucosa was stimulated, the recruitment occurred at lower concentrations than when only the ventral mucosa was stimulated. In this last case, the dynamics of the bulbar recruitment did not differ from that of receptor cells. 5. The recruitment process was further studied for each stimulus, for receptor cells as well as for second-order neurons. Differences in recruitment were found between stimuli and, as for the bulbar neurons, they depended on the condition of stimulation. 6. The main outcome of these results is the demonstration that the convergence of receptor cells onto second-order neurons is functionally implicated in an amplification process of the primary signal in olfaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Occurrence of neuropeptides and tyrosine hydroxylase in the olfactory epithelium of the lesser-spotted catshark (Scyliorhinus canicula Linnaeus, 1758)

Immunohistochemical studies using antisera against various neuropeptides (Substance P, vasoactive intestinal polypeptide, and cholecystokinin octapeptide) and tyrosine hydroxylase revealed both olfactory sensory neuron (OSN) polymorphisms and transepithelial-subepithelial nerves in the olfactory epithelium of the cartilaginous fish, Scyliorhinus canicula. This study provides the first evidence of three morphological types of OSNs within the olfactory epithelium of cartilaginous fish that are similar to those found in the teleosts. In fishes there is evidence that OSNs differ functionally, including their differential olfactory bulb projections and molecular properties. The Substance P positive olfactory neurons in S. canicula may have a separate bulbar projection site that is not known, but may indicate a characteristic found in olfactory neuron subtypes in both lampreys and teleost fish. Numerous Substance P immunopositive nerves are found at the base of and in the olfactory epithelium. Some of them were observed to extend outwards almost reaching the epithelial surface. Their presumptive origin from the trigeminal nerve and their interrelationship with chemosensory cells in the nasal passages of vertebrates are discussed.     

The spatial distribution of odour induced potentials in the olfactory bulb of char and trout (Salmonidae)

Monopolar DC-recordings were made of the gross responses from the olfactory bulb of char (Salvelinus alpinus syn. Salmo alpinus L.) and trout (Salmo trutta L.) during stimulation with different odours. The response features studied were: the magnitude and polarity of the slow potential shift, the amplitude of the induced waves and their asymmetrical waveform. Amino acids elicited the largest responses in the lateral part of the bulb. Water containing “crude fish odour” caused the largest responses in the rostral and medial parts. The results demonstrate odour specific differences in the localization of the bulb responses and the separate origin of the slow potential and the induced waves.     

Morphology of the nasal cavities and associated structures in Artibeus jamaicensis and Myotis lucifugus

Anatomical differences in olfactory structures point to a more highly developed olfactory sense in the frugivorous bat Artibeus jamaicensis than in the insectivorous bat Myotis lucifugus. Artibeus' nostrils show greater potential for differential control of nasal airstreams. Its nasal cavities, in contrast to Myotis, are clearly demarcated into olfactory and respiratory portions. Both species have seven turbinals, but of the five ethmoturbinals, Artibeus has four endoturbinals and one ectoturbinal whereas Myotis has three and two, respectively. The configuration and histology of the more complex turbinals of Artibeus indicates a greater surface area of olfactory epithelium. The nasal epithelia are thicker in Artibeus. Olfactory receptors appear similar in both species but the thickness and composition of the olfactory epithelium in Artibeus suggests the presence of about twice as many receptors per unit area. This is also reflected in the relative prominence of olfactory nerve bundles. From these and other bulbar relationships we conclude that the proportion of fila olfactoria to large mitral cells, as reflected by the olfactory bulb glomeruli, is some function of a fundamental 2:1 relationship. Nasal glands, with flaps covering two of their more prominent ducts, and blood vessels are more pronounced in Artibeus. This presumably correlates with the nasal type of phonation exhibited by this bat as well as a greater ability to condition air per se. Nasopalatine ducts exist in both species but only Artibeus has a vomeronasal organ. A diffuse ganglion accompanies the organ; some of its neurons are associated with the unmyelinated vomeronasal nerve, whereas others are intraepithelial. The vomeronasal nerve terminates in a prominent accessory olfactory bulb.     

A multimodal MR-compatible olfactometer with real-time controlling capability

Abstract

Design of an MR-compatible and computer-controlled odour stimuli system is essential in the studies of human olfactory function. Olfactometers are used to deliver odours to the subjects in an objective manner. We present a portable, computer-controlled eight channels olfactometer able to stimulate olfaction by employing liquid odorant stimuli. We used a high-pressure pump to generate medical grade airflow. After passing through solenoid valve-controlled odour reservoirs, odorant stimulus is conveyed to the nasal mask. The odour delivery delay of the device was measured using photo-ionisation detectors. To assess the application of the designed olfactometer, an fMRI experiment was done with 9 healthy subjects. Two odour stimuli (Vanillin and Rose) were presented to each subject in an alternating block design task of odour and non-odour conditions. The response time of each subject was gathered using the response box. Group analysis revealed a significant BOLD signal change in some regions of olfactory and trigeminal networks including the orbitofrontal cortex, insula, inferior frontal gyrus, hippocampus, cingulate gyrus and piriform cortex. The odour delivery delay measured by photo-ionisation detector was 190?ms, and the subjects’ response showed 205?ms for the Vanillin and 243?ms for the Rose odour stimuli. Our portable MR-compatible olfactometer as a stimulation device is capable of creating adequate stimulation suitable for olfactory fMRI experiments.     

Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactory epithelium to the brain of the adult rat

Summary The sensory neurons of the olfactory epithelium, as a consequence of their odor detection function, contact both the external environment and the central nervous system. The possibility that substances applied to the epithelium might reach the central nervous system was investigated by the intranasal application of peroxidase-conjugated wheat germ agglutinin (WGA-HRP). WGA-HRP was transported through olfactory receptor axons to the glomerulus of the olfactory bulb. Reaction product was localized electron microscopically to tubulovesicular profiles and dense bodies in sensory axons. Evidence of transneuronal transport was indicated by reaction product localized in dense bodies in dendrites postsynaptic to receptor cell axons. Periglomerular, tufted and mitral cells in the olfactory bulb also were transneuronally labeled. Anterograde transneuronal labeling occured in the olfactory tubercle, piriform cortex and surrounding the lateral olfactory tract. Retrograde transneuronal label was found in neurons of the basal forebrain with the largest number of perikarya in the lateral nucleus of the horizontal limb of the diagonal band, a major source of cholinergic afferents to the olfactory bulb. These data suggest that substances, specifically those which bind to receptors, are transported from the olfactory receptor neurons in the nasal epithelium to the brain. Thus, the olfactory system may provide a route of entry for exogenous substances to the basal forebrain.Abbreviations AC anterior commissure - CC corpus callosum - CI internal capsule - CP caudate putamen - DBB diagonal band of Broca - FX fornix - GP globus pallidus - IC island of Callelae - LV lateral ventricle - MS medial septum - OC optic chiasm - PIR piriform cortex - RF rhinal fissure - SON supraoptic nucleus - SCN suprachiasmatic nucleus - SM stria medullaris - ST stria terminalis - TOL lateral olfactory tract - TUO olfactory tubercle - III third ventricle     

Immunolocalization of G protein α subunits in the olfactory system of Polypterus senegalus (Cladistia, Actinopterygii)

In vertebrates, the receptor neurons of the olfactory/vomeronasal systems express different receptor gene families and related G-protein types (in particular the G protein alpha subunit). There are no data in the literature about the molecular features of the olfactory/vomeronasal systems of Cladistia thus, in this work, the presence and distribution of different types of G protein alpha subunits were investigated in the olfactory organs of the bichir Polypterus senegalus, using immunohistochemistry. Gαo-like immunoreactivity was detected in the microvillous receptor neurons, with the cell body in the basal zone of the sensory epithelium, and in the crypt neurons. Gαo-like ir glomeruli were mainly localized in the anterior part of the olfactory bulb. Gαolf-like immunoreactivity in the sensory epithelium was detected in the ciliated receptor neurons, while the immunoreactive glomeruli in the olfactory bulb were mainly localized in the ventral-posterior part. No Gαq nor Gαi3 immunoreactivity was detected. These data are partially in agreement with studies that show the distribution of G protein alpha subunits in teleosts, allowing to hypothesize a common organization of the olfactory/vomeronasal systems in the group of Actinopterigians.     

Spontaneous activity of single neurons in the olfactory bulb of the rainbow trout (Salmo gairdneri) and its modulation by olfactory stimulation with amino acids

Summary Spontaneous action potentials recorded from 126 neurons in the olfactory bulb of Salmo gairdneri show a higher tendency towards bursting patterns of activity than those shown in previous reports about other fish. Granule cells and periglomerular cells are more likely to fire in bursts than mitral cells. Natural chemical stimulation of the olfactory mucosa with amino acid solutions produced a unique pattern of excitatory and inhibitory responses across all units. Chi-square values were calculated for stimulatory effectiveness between forty-five pairs of odours. L-serine and L-alanine consistently showed a high degree of similarity with several other odours. The converse was true for GABA and L-histidine, although this pair had a high chi-square value when mutually compared. Enantiomeric pairs of amino acids were often found to have opposite stimulatory effects on bulbar units. These results are discussed in relation to the possible properties and configurations of odorant receptor sites for amino acids in the fish olfactory mucosa.     

Olfactory function in zinc-deficient adult mice

Summary Adult zinc deficiency reportedly leads to degeneration of the olfactory epithelium in the rat. Human zinc deficiency can cause reduced olfactory sensitivity. Given the importance of zinc in embryonic neural development its primary action on the adult olfactory system may be to disrupt olfactory receptor neurogenesis. We report here on the effects of zinc deficiency on the olfactory system of the adult mouse. After 42 days of dietary restriction of zinc, mice were tested behaviourally for olfactory function and general activity. Their olfactory epithelia were examined histologically using [³H]-thymidine autoradiography to identify recently-divided cells, and immunohistochemistry for olfactory marker protein to identify mature receptor neurones. Zinc deficient mice failed to show a food odour preference but they Were as active as controls and their olfactory epithelia appeared normal. Basal cell proliferation and postmitotic survival were similar to controls and the epithelia were of normal thickness and were positive for olfactory marker protein. It was concluded that zinc deficiency did not affect the turnover of cells in the olfactory epithelium. It may disrupt olfactory function through interference with zinc-containing neurones in higher olfactory centres.     

Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade

Summary The influences of centrifugal projections to the olfactory bulb were examined on the bulbar EEG and mitral-tufted cell activity in waking rabbits. Each of 6 rabbits was implanted, under surgical anesthesia, with fine wire electrodes for recording of the EEG and mitral-tufted cell unit activity and for stimulating the lateral olfactory tract. Two cooling probes, for reversible cryogenic blockade, were implanted on either side of the left olfactory peduncle. Records of EEG and unit activity were taken for 200 s before, during and after cooling of the probes to 3 degrees centigrade. Antidromic evoked potentials were used to assess the efficacy of the blockade. During the cryogenic blockade bursts of EEG activity, evoked in the bulb by inspiration through the nose, were augmented in amplitude and reduced in frequency. Mitral-tufted cell unit activity was reduced in rate but was more highly correlated with the phase and amplitude of the EEG bursts. Analysis of individual EEG bursts revealed that the variance in frequency of bulbar activity was significantly reduced in the isolated state. The data demonstrate that oscillatory bursting activity in the olfactory bulb is intrinsically maintained within a relatively fixed frequency range during receptor input and does not depend on centrifugal projections for its electrogenesis. Changes in EEG frequency, amplitude and correlation with unit activity support the hypothesis that centrifugal projections act in part to inhibit mitral-tufted cell output by direct excitation of granule cells. These findings are supported by a theoretical model in which distributed feedback to the granule cells from more central olfactory structures acts to regulate the coherency of bulbar activity.     

Differential expression of N-CAM, vimentin and MAP1B during initial pathfinding of olfactory receptor neurons in the mouse embryo

Olfactory receptor neurons extend their primary axons from the nasal epithelium to the olfactory bulb primordium via the frontonasal mesenchyme. In the present study, expression of neuronal markers (vimentin and MAP1B) and N-CAM was immunohistochemically investigated in the development of the olfactory system in mouse embryos. Expression of vimentin and MAP1B was first observed at early day 10 of gestation (D10) in the posterosuperior region of the medial nasal epithelium, while N-CAM was initially detected in the mesenchyme adjacent to the vimentin- and MAP1B-positive nasal epithelium. As development proceeded, the N-CAM positive region in the nasal mesenchyme became broader and reached the olfactory bulb primordium late on day 10. In comparision with adjacent sections, the localization of neuronal marker-positive cells was mostly included in the N-CAM positive region. In addition, we adopted in situ labelling with vital dye (DiI) to directly determine the localization of the olfactory nerve and N-CAM on the same sections. We demonstrated that most extending axons were located in the N-CAM positive region. These results suggest that the expression of N-CAM plays a crucial role in the initial pathfinding of the olfactory nerve.     

Analysis of the onset phase of olfactory bulb unit responses to odour pulses in the salamander

1. A method for delivering odour pulses of controlled onset, steady plateau and abrupt termination, has been developed and applied to a single unit study of mitral cell responses in the olfactory bulb of the salamander. The pulses have been monitored during the experiments near the site of stimulation on the olfactory mucosa.

2. Responses have been categorized as excitatory or suppressive based on the initial response to the odour pulse.

3. Initially excitatory responses had sustained discharges near threshold. With increasing concentration, the discharge changed to a brief burst followed by suppression. The briefest latency of a unit response was 120 msec, using stimulation of medium concentration, after the start of the pulse; the majority of units appeared to be excited within 200-300 msec. Ramp stimuli gave increasing periods of excitation as the rise time of the odour front became less abrupt.

4. Initially suppressive responses showed suppression at all levels of concentration. The majority of units appeared to have an onset of suppression about 300-400 msec after the start of the pulse.

5. These basic responses, involving suppression or excitatory—suppressive sequences, can be correlated with some basic properties of the synaptic circuits in the olfactory bulb. The time courses of the initial responses appear to be within the time periods of the inhalation cycle of the salamander, and therefore may reflect mechanisms of processing of natural olfactory stimuli.

     

Olfactory experience decreases responsiveness of the olfactory bulb in the adult rat

We recently reported the existence of dramatic modifications of the olfactory bulb reactivity following a very simple manipulation of the olfactory input as an exposure to an odorant. The present study aimed at testing the possibility that such effects could depend on the nature of the exposure odour. For this purpose, rats were exposed 20 min per day during six consecutive days to cineole, methyl-amyl ketone, isoamyl acetate or with no odour in the control group. On day 7, rats were anaesthetized and the spontaneous activity of mitral/tufted cells was recorded along with their responses to the familiar odour and to four novel odours. Results revealed that: (i) the firing frequencies were not significantly different in the four groups; (ii) the proportion of excitatory responses was considerably decreased in the exposed groups while the number of non-responses was significantly enhanced; (iii) excitatory responses were decreased not only to the familiar odour but also to four other novel odours; (iv) this lower responsiveness was long lasting at least for isoamyl acetate exposure; and (v) increasing concentration of test odours was not enough to allow mitral/tufted cells to recover control responsiveness. All of these effects have a differential importance according to the exposure odour. In particular, the more powerful an odour is in activating control cells, the more non-specific the decrease in mitral/tufted cell reactivity is. Hypotheses on the underlying mechanisms are advanced.