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Spikes were evoked in rat olfactory sensory neuron (OSN) populations by electrical stimulation of the olfactory bulb nerve layer in pentobarbital anesthetized rats. The latencies and recording positions for these compound spikes showed that they originated in olfactory epithelium. Dual simultaneous recordings indicated conduction velocities in the C-fiber range, around 0.5 m/s. These spikes are concluded to arise from antidromically activated olfactory sensory neurons. Electrical stimulation at 5 Hz was used to track changes in the size and latency of the antidromic compound population spike during the odor response. Strong odorant stimuli suppressed the spike size and prolonged its latency. The latency was prolonged throughout long odor stimuli, indicating continued activation of olfactory receptor neuron axons. The amounts of spike suppression and latency change were strongly correlated with the electroolfactogram (EOG) peak size evoked at the same site across odorants and across stimulus intensities. We conclude that the curve of antidromic spike suppression gives a reasonable representation of spiking activity in olfactory sensory neurons driven by odorants and that the correlation of peak spike suppression with the peak EOG shows the accuracy of the EOG as an estimate of intracellular potential in the population of olfactory sensory neurons. In addition, these results have important implications about traffic in olfactory nerve bundles. We did not observe multiple peaks corresponding to stimulated and unstimulated receptor neurons. This suggests synchronization of spikes in olfactory nerve, perhaps by ephaptic interactions. The long-lasting effect on spike latency shows that action potentials continue in the nerve throughout the duration of an odor stimulus in spite of many reports of depolarization block in olfactory receptor neuron cell bodies. Finally, strong odor stimulation caused almost complete block of antidromic spikes. This indicates that a very large proportion of olfactory axons was activated by single strong odor stimuli.

The act of sniffing increases the air velocity and changes the duration of airflow in the nose. It is not yet clear how these changes interact with the intrinsic timing within the olfactory bulb, but this is a matter of current research activity. An action of sniffing in generating a high velocity that alters the sorption of odorants onto the lining of the nasal cavity is expected from the established work on odorant properties and sorption in the frog nose. Recent work indicates that the receptor properties in the olfactory epithelium and olfactory bulb are correlated with the receptor gene expression zones. The responses in both the epithelium and the olfactory bulb are predictable to a considerable extent by the hydrophobicity of odorants. Furthermore, receptor expression in both rodent and salamander nose interacts with the shapes of the nasal cavity to place the receptor sensitivity to odorants in optimal places according to the aerodynamic properties of the nose.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

1. Electroolfactorgram (EOG) recordings were made from different regions of the rat olfactory epithelium to test for spatial distribution of odor responses. 2. The EOG recordings showed spatial distribution of the odor responses in the olfactory epithelium. While some odorants (amyl acetate, anisole, and ethyl butyrate) were more effective in evoking responses in the dorsal recess near the septum, other odorants (including limonene, cineole, cyclooctane, and hexane) were more effective in the lateral recesses among the turbinate bones. These differences were seen as statistically significant odorant-by-position interactions in analysis of variance. 3. Comparisons of recordings along the anteroposterior dimension of the epithelium produced smaller differences between the odor responses. These were not significant for 3-mm distances, but were statistically significant for 5- to 6-mm distances along the dorsomedial epithelium. 4. The latencies were significantly longer in the lateral recesses than in the medial region. This probably reflects a more tortuous air path along the turbinate bones to the lateral recesses. 5. The olfactory receptor cells were activated by antidromic stimulation via the nerve layer of the olfactory bulb. The population spikes evoked from the olfactory receptor cells could be suppressed by prior stimulation with odorants that evoked strong EOG responses. This collision of the antidromic action potentials with the odor-evoked action potentials indicates that the same population of receptor cells was activated in both cases. 6. The flow rate and duration of the artificial sniff were varied systematically in some experiments. The differential distribution of response sizes was present at all flow rates and sniff durations. Some odors (e.g., amyl acetate and anisole) produced increased responses in the epithelium of the lateral recesses when flow rates or sniff durations were high. We suggest that these changes may reflect the sorptive properties of the nasal membranes on these odors. The responses to other odors (e.g., hexane or limonene) were not greatly affected by flow rate or sniff duration. 7. Taken with existing anatomic data, the results indicate that the primary olfactory neurons that project axons to glomeruli in different parts of the olfactory bulb are responsive to different odors. The latency differences between responses at medial and lateral sites are large enough to be physiologically significant in the generation of the patterned responses of olfactory bulb neurons.

1. Intracellular recordings were made from the output neurons (mitral and tufted cells) of the rat olfactory bulb during electrical orthodromic stimulation of the olfactory nerve layer (ONL) and antidromic stimulation of the lateral olfactory tract and posterior piriform cortex (pPC) to test for physiological differences among the neuron types. Many of these neurons were identified by intracellular injections of biocytin, and others were identified by their pattern of antidromic activation. 2. Both marked and unmarked mitral cells showed large inhibitory postsynaptic potentials (IPSPs) in response to antidromic stimulation of the pPC, whereas tufted cells exhibited small IPSPs in response to pPC stimulation. Tufted cells, however, showed large IPSPs in response to ONL stimulation. In many cases, these tufted cell responses to ONL stimulation were larger than the mitral cell responses. The marked superficial tufted cells, those with basal dendrites in the superficial sublayer of the external plexiform layer (EPL), had the smallest IPSPs in response to pPC stimulation. These data support anatomic observations suggesting that the granule cell populations responsible for the IPSPs may be different for mitral and for superficial tufted cells. 3. The different types of output cells also showed differences in their responses to orthodromic stimulation. Type I mitral cells, which have basal dendrites confined to the deep sublayer of the EPL, were significantly less excitable by ONL stimulation than were the type II mitral cells, which have basal dendrites distributed within the intermediate sublayer of the EPL. Half of the type I mitral cells could not be excited at all by ONL stimulation. Superficial tufted cells showed even greater orthodromic excitability than type II mitral cells, usually responding to ONL stimulation with two or more spikes. 4. The ionic basis of the IPSPs in the superficial tufted cells appeared similar to those described for mitral cells. These IPSPs could be reversed by chloride injection and were associated with increased membrane conductance. 5. For both mitral and tufted cells, the number of ONL electrodes evoking IPSPs was greater than the number evoking spikes. These data suggest a kind of center-surround organization of inputs to these cells from the ONL, although this does not yet imply that the sensory receptive field of these output cells has a center-surround organization. 6. In conclusion, the properties of rat olfactory bulb output cells correlate with the sublayers of the EPL in which their basal dendrites lie.(ABSTRACT TRUNCATED AT 400 WORDS)

Complete understanding of the role of the mammalian main olfactory bulb in sensory processing has remained elusive despite many detailed studies on its anatomy and physiology. Several lines of recent evidence viewed in the context of earlier knowledge have provided new insights into the bulbar mechanisms of olfactory coding. The output cells of the olfactory bulb receive a localized olfactory nerve input and interneuronal input via dendrodendritic synapses on distinct sets of dendrites. The spatial arrangement of granule cell contacts on output cell basal dendrites suggests that lateral inhibitory interactions may occur between neighboring output cells. The input from olfactory receptor cell axons to the bulb also has spatial order, but does not represent a precise map of the receptor surface. Recent studies with antibodies and lectins suggest that different groups of axons from chemically similar receptor cells collect into certain glomeruli, even if the axons originate from cells that are not contiguous in the mucosa. Electrophysiological studies have begun to explore the participation of spatially organized circuits in olfactory processing. The degree to which neighboring output cells respond similarly to odor stimulation, for example, depends on the distance between the cells, with those further apart showing complementary responses. Also, a single output cell can show 2 or more different temporal response patterns when different odors are presented. Intracellular recordings indicate that these responses are shaped by IPSPs. Electrical stimulation during such recordings shows that some mitral cells are excited by nerve inputs close to their glomerular tufts, while they are inhibited by nerve inputs to other parts of the bulb. Finally, recordings from granule and periglomerular cells indicate their potential in mediating components of output cell odor responses. These considerations suggest that the olfactory bulb performs a spatially based analysis on the information coming from the receptor cells. While the spatial organization of the olfactory bulb is probably not faithfully represented in the projections to the olfactory cortex, bulbocortical projections are not random. The fact that spatial factors exist at each of these levels in the olfactory system must be considered in developing models of central olfactory processing.

Studies on the properties of olfactory receptors and of the olfactory glomeruli indicate that there is spatial segregation of response to particular characteristics of odorant molecules at the input level of the olfactory bulb. Existing anatomical information and studies of synaptic mechanisms in the olfactory bulb suggest that the bulb circuitry might act as a contrast detection mechanism analyzing a spatially organized input. Recent electrophysiological studies have supported this idea. Extracellular recordings have shown that the similarity between responses of cell pairs to the same stimulus odor depend upon the distance between those cells. Intracellular recordings from mitral and tufted cells have shown spatially organized excitatory and inhibitory responses to localized electrical stimulation of the input layer of the bulb. Some of the major interneurons of the olfactory bulb have also been identified during odor and localized electrical stimulation. These recordings are also consistent with a spatially based organization.

It has been suggested that the principal output cells of the main olfactory bulb, the mitral cells, along with the glomerulus they enter, form an anatomical and functional column. To test the extent to which mitral cell somata lying close together in the mitral cell layer are connected to the same glomerulus, we reconstructed 267 mitral cells labeled by extracellular injections of horseradish peroxidase (HRP) in the external plexiform layer. Results show that apical dendrites tilt rostrally from the somata to the glomeruli and this tilt is gradually greater as the somata are located more and more rostrally in the olfactory bulb. The apical dendrites of most mitral cells in the same region of the bulb are parallel. We analyzed the degree to which dendrites were parallel by measuring the difference in angle of all pairs of apical dendrites in each section, with the restriction that no cells were separated by more than 500 microns. About half of these pairs of dendrites differed in angle by 20 degrees or less and were therefore said to be parallel. The degree of parallelism did not vary with the cell pair location or with intercell distance. Study of glomerular connections of pairs of mitral cells as a function of intercell distance revealed that 96% of mitral cells connected to the same glomerulus were separated by less than 120 microns, while 72% of cells connected to adjacent glomeruli and only 29% of cells connected to distant, i.e., nonadjacent, glomeruli were separated by less than 120 microns.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensory systems rely upon spatially organized projections for the faithful transfer of receptor activity into the central nervous system. While the mammalian olfactory nerve shows a regional topographical organization, data on the sources of variation within this projection are scarce. We evaluated the degree of precision of olfactory bulb glomerular innervation from the olfactory nerve layer (ONL) by making small cuts across the trajectory of the olfactory nerve fibers and assessing the resulting denervation by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) labeling of primary nerve fibers. In control animals (no ONL cut), the ONL and glomerular layer showed intense labeling around the entire circumference of the bulb. Transneuronal labeling of the mitral cell layer and the external plexiform layer was also observed in animals surviving at least 2 days, which allowed us to study the nerve fiber innervation of second order cells as well. ONL cuts on the lateral face of the bulb produced a stripe of denervation, as evidenced by the absence of primary and transneuronal label in a well-defined region. Fascicles of fibers within the ONL were never observed to enter the denervated region from more than one or two glomerular widths away, indicating a relatively tight limit of variation in the spatial termination of olfactory nerve fibers on the lateral bulb surface. This limit of variation does not hold around the circumference of the bulb since similar ONL cuts on the dorsal surface failed to produce a distinct stripe of denervation. Field potential measurements from the ONL after similar cuts supported the anatomic findings. These results show that primary olfactory axons and their terminals lie in parallel along the lateral face of the olfactory bulb. These spatial relationships of olfactory input to the bulb are maintained in the second order connections. This organization allows one to study the spatial aspects of interneuronally mediated synaptic mechanisms involved in olfactory coding.

1. Intracellular recordings were made from 28 granule cells and 6 periglomerular cells of the rat olfactory bulb during odor stimulation and electrical stimulation of the olfactory nerve layer (ONL) and lateral olfactory tract (LOT). Neurons were identified by injection of horseradish peroxidase (HRP) or biocytin and/or intracellular response characteristics. Odorants were presented in a cyclic sniff paradigm, as reported previously. 2. All interneurons could be activated from a wide number of stimulation sites on the ONL, with distances exceeding their known dendritic spreads and the dispersion of nerve fibers within the ONL, indicating that multisynaptic pathways must also exist at the glomerular region. All types of interneurons also responded to odorant stimulation, showing a variety of responses. 3. Granule cells responded to electrical stimulation of the LOT and ONL as reported previously. However, intracellular potential, excitability, and conductance analysis suggested that the mitral cell-mediated excitatory postsynaptic potential (EPSP) is followed by a long inhibitory postsynaptic potential (IPSP). An early negative potential, before the EPSP, was also observed in every granule cell and correlated with component I of the extracellular LOT-induced field potential. We have interpreted this negativity as a "field effect," that may be diagnostic of granule cells. 4. Most granule cells exhibited excitatory responses to odorant stimulation. Odors could produce spiking responses that were either nonhabituating (response to every sniff) or rapidly habituating (response to first sniff only). Other granule cells, while spiking to electrical stimulation, showed depolarizations that did not evoke spikes to odor stimulation. These depolarizations were transient with each sniff or sustained across a series of sniffs. These physiological differences to odor stimulation correlated with granule cell position beneath the mitral cell layer for 12 cells, suggesting that morphological subtypes of granule cells may show physiological differences. Some features of the granule cell odor responses seem to correlate with some of the features we have observed in mitral/tufted cell intracellular recordings. Only one cell showed inhibition to odors. 5. Periglomerular (PG) cells showed a response to ONL stimulation that was unlike that found in other olfactory bulb neurons. There was a long-duration hyperpolarization after a spike and large depolarization or burst of spikes (20-30 ms in duration). Odor stimulation produced simple bursts of action potentials, Odor stimulation produced simple bursts of action potentials, suggesting that PG cells may simply follow input from the olfactory nerve.(ABSTRACT TRUNCATED AT 400 WORDS)

Several types of short axon cells of the mammalian olfactory bulb have been described after Golgi impregnation. Two of these types have been observed in our material after treatment with the NADPH-diaphorase procedure or after immunohistochemistry for neuropeptide-Y (NPY). The cells stained by the two procedures have similar morphologies and distributions. A less extensive series of observations confirms that similar cells also display somatostatin (SS)-like immunoreactivity. One of these cell types corresponds to the superficial short axon cell of Golgi and electron microscopic studies. The dendrites of this cell lie within the periglomerular region and in the superficial external plexiform layer (EPL), generally lying parallel to the glomerular layer. In some cases the axon has been traced across the EPL into the granule cell layer (GCL). This cell may provide another route of interaction between the periglomerular region and the granule cells in addition to the influences conducted by basal dendrites and axon collaterals of some mitral and tufted cells. A type of deep short axon cell is also visible with these two procedures. It lies deep in the granule cell layer, frequently near the ventricular layer and its dendrites lie parallel to that layer. This deep short axon cell is stained with much greater frequency by the NADPH-diaphorase and NPY procedures than is the superficial short axon cell. It corresponds most closely to the Blanes or Golgi cells of the Golgi impregnation literature, but it appears to differ from these cells in the position and orientation of its dendrites. No spines have been observed on either the superficial or deep cells in this series. Many glomeruli are also stained by the NADPH-diaphorase procedure, but are not NPY or SS immunoreactive. This may provide additional evidence for functional differences between glomeruli in local regions of the olfactory bulb.

The pattern of output of mitral and tufted cells of the rat olfactory bulb (OB) to layer Ia overlying the pars externa (pE) of the anterior olfactory nucleus (AON) has been studied in the rat by iontophoresis of horseradish peroxidase and Phaseolus vulgaris-leucoagglutinin. These agents labeled mitral and tufted cells and at least the proximal portion of their axons. In most cases we observed small branches from axons of the lateral olfactory tract that appear to terminate in the region of the pE AON, while the main axon could often be traced for considerable distances past these branches. These branches are assumed to terminate in the pE AON because they could not be traced to other terminal regions, because they ramify in layer Ia, and because they usually show small swellings characteristic of axons in terminal regions. Although each ramification could be extensive, we found that the positions of these small branches were related to the positions of the injections within the OB. Dorsal medial injections labeled dorsal branches. Ventral medial injections labeled ventral branches. Injections on the lateral face of the OB labeled intermediate branches. The centers of the regions within which branches were labeled were strongly correlated with the positions of the injection around the circumference. Comparison of the anterior-posterior axis of the OB produced no such strong correlation. Reconstructions of axons showed that terminal branches arise from both mitral and tufted cells, although at least some mitral cells are shown not to have such branches in the pE AON. Studies of the patterns of dendrites and terminals in the pE AON indicate that this region has the same pattern of layer Ia and Ib terminals seen in other olfactory cortical regions. The pE AON cell layer is intercalated just below the boundary between layers Ia and Ib. Since dendrites of the underlying pars lateralis of the AON (pL AON) penetrate into layer Ia over much of the pE AON, it is necessary to remember that at least part of the pL AON may also receive topographically organized inputs.

The effect of anesthetic upon olfactory bulb field potentials evoked by paired stimuli to the lateral olfactory tract (LOT) was examined in male rats. Experiments were performed on rats anesthetized with urethane or barbiturates and on unanesthetized rats with midcollicular brain stem sections. Anesthetic doses of urethane (1.5 g/kg) produced an increse in the test response for condition-test intervals of 15-60 msec or more. With barbiturate anesthesia there was a decrease in the test response for all intershock intervals up to 150 msec. In unanesthetized rats with midbrain sections there was an increase in the test response with low intensity shock pairs and a decrease in the test response with high intensity shock pairs. Simultaneous use of both anesthetics produced either an increase or a decrease in the test response, depending upon the relative doses and upon the stimulus intensity. Paired stimuli to the olfactory nerve produced results similar to those with LOT stimulation. Augmentation of the olfactory bulb field potential produced by paired olfactory nerve shocks was not abolished by lesions which sectioned the LOT and produced extensive damage of the anterior olfactory nucleus.

Olfactory bulb field potential and single unit responses to paired pulse stimulation of the lateral olfactory tract were observed in the rat. In agreement with previous reports, surgical levels of barbiturate anesthesia prolonged the period of diminution of the field potential responses to the test pulse relative to the effect seen under light anesthesia. Very deep barbiturate anesthesia (sufficient to depress the EEG) led to decreased responses to the primary pulse but augmented responses to the test pulse. Urethane and ether anesthesia produced augmented test responses without severely depressing the EEG and without producing a period of diminished test responses. Single unit recordings failed to show external plexiform layer units excited at the time of maximal augmentation of the field potential. Simultaneous single unit and field potential recordings showed that prolonged diminution of the field potential response was associated with prolonged suppression of unit responses to the test pulse, while augmented field potential responses were associated with decreased suppression of unit responses to the test pulse. These observations are most easily explained by the assumption of temporal facilitation in the synaptic output of mitral and tufted cells. This facilitation is masked by feedback inhibition under some conditions, particularly under barbiturate anesthesia.

Extracellular recordings were made from mitral cells, tufted cells, and presumed glomerular layer and external plexiform layer interneurons of the olfactory bulb of anesthetized rats during odor stimulation. Intensity responses of these cells were studied by presenting a series of six or seven concentrations, spanning a range greater than two log units, in a cyclic artificial sniff paradigm, which produced repeated response measures at each concentration. Experiments focused on obtaining a complete intensity series, including interspersed unstimulated spontaneous activity records, for a single odorant (usually amyl acetate), but concentration responses to other odorants were tested when possible. Odor responses of 46 cells were studied with two approaches. Response form was examined in an attempt to define response classes based on qualitative characteristics of the temporal pattern of response. Assessment of response magnitude was attempted, in order to construct stimulus-response functions for each cell, independent of response form. As previously reported for olfactory bulb cells, the cells in our sample responded to odor stimulation with spike trains of a variety of temporal patterns, consisting of excitatory and inhibitory components that were frequently recognizable in the responses of a cell across a range of concentrations. However, response patterns usually changed significantly with concentration, such that response form across the concentration range could not be predicted from the response at any one concentration. Responses of different cells were sometimes similar to each other in form at one concentration and quite different from each other in the rest of their concentration-response profiles. Classification of response profiles into discrete types, based on consistency of response form throughout the profile, was therefore not feasible. In agreement with other reports, response of a single cell to different odorants sometimes showed similar forms and sometimes showed very different forms across the concentration-response profiles. Since the response form depends on the stimulus intensity as well as the stimulus quality, characterization of response magnitude and of the pattern of response to different odors require testing with a series of stimulus concentrations. Because odor responses consisted of temporally patterned spike trains, whose components changed in complex ways with stimulus intensity, it was not possible to quantify response magnitude by measuring characteristics of particular response components or counting mean frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

The output cells of the main olfactory bulb, the mitral and tufted cells, can be categorized into subclasses on the basis of their intrabulbar dendritic and axonal characteristics. Their form was studied in rats following labeling by iontophoretic injection of horseradish peroxidase into the external plexiform layer (EPL). The fact that these extracellular injections labeled small numbers of neurons permitted reconstruction of individual cells. The injection depth within the EPL determined the type of cells labeled. Secondary dendrites of each cell type lay in one of three partially overlapping zones in the EPL. The deepest zone contained the secondary dendrites of one group of mitral cells (Type I), which had the deepest and longest dendrites of the output cells. An intermediate zone of the EPL contained the secondary dendrites of middle tufted and a second class of mitral cells (Type II). The superficial zone, adjacent to the glomerular layer, contained the relatively short, asymmetric dendritic fields of external tufted cells. The few labeled internal tufted cells had secondary dendrites in either the intermediate or deep zones. Every cell type, except the Type I mitral cells, had axon collaterals in the internal plexiform and upper granule cell layers. No cell types had axons re-entering the EPL. These results for output cells combined with our previous observations on granule cells point to a functional sublaminar organization of the EPL that has not previously been proposed.

The organization of the projection of olfactory bulb output cells was studied in the rat by injection of horseradish peroxidase (HRP) into the piriform cortex or olfactory tubercle. We made single HRP injections into small cuts in the fiber layer of the projection areas in order to enhance uptake by axons and to confine the region of HRP uptake. Following most of these injections, HRP-labeled axons could be traced in discrete fascicles through the fiber layer of the cortex or tubercle. These observations indicate that axons innervating the piriform cortex do not emit many long collaterals after they leave the lateral olfactory tract. HRP-labeled cells were generally observed throughout the ipsilateral olfactory bulb, but there were regions of greater density of labeled cells that differed in the various brains. The differences among the distributions of labeled mitral and tufted cells were analyzed statistically in 39 brains to test whether they varied systematically with injection site. In these analyses, the olfactory bulb was divided into 30 standard regions, and the labeled cells in each regions were counted. The distributions of labeled cells were similar for brains where injections were made into similar regions of the piriform cortex. The variations in density of labeled cells of the dorsal and anterior regions of the olfactory bulb were most strongly correlated with the positions of cortical injections. In contrast, the posterior medial regions of the bulb were heavily labeled after almost all injections. The ventral portions of the olfactory bulb were most heavily labeled after injections into the olfactory tubercle.

Cytochrome oxidase staining of the rat olfactory bulb external plexiform layer (EPL) produces a darkly stained intermediate zone bordered by lightly stained superficial and deep zones. Similar zonal staining was seen in cats, rabbits, and hamsters. These zones vary in relative thickness around the circumference of the olfactory bulb; the deep zone is proportionally thicker in the most dorsal and ventral parts of the bulb. Tufted cell somata are unevenly distributed within the EPL; the outer part of the EPL has more somata. The distribution of the cytochrome oxidase reaction product shows that the darkly stained intermediate zone is not produced by staining of tufted cell somata. Zones of cytochrome oxidase staining correspond to the sublaminar distribution of mitral and tufted cell basal dendrites. This was demonstrated by labeling mitral and tufted cells with small extracellular horseradish peroxidase injections and processing alternate sections for horseradish peroxidase and for cytochrome oxidase. Because there was cross-reaction of the cytochrome oxidase procedure with horseradish peroxidase, it was possible to trace many neurons through both series of sections. Middle tufted cells of the superficial EPL have basal dendrites confined to the superficial zone of light cytochrome oxidase staining. Internal tufted cells and middle tufted cells of the intermediate zone send their basal dendrites into the intermediate zone. One group of mitral cells (type I) has basal dendrites confined to the deep zone of lighter cytochrome oxidase staining. A second group of mitral cells (type II) and tufted cells of the intermediate EPL has basal dendrites primarily confined to the intermediate zone of dark cytochrome oxidase staining. The correlation of the enzyme staining with the dendritic laminar patterns supports the existence of three relatively distinct sublaminae in the EPL and supports the designation of two types of mitral cells. The staining pattern also provides an independent method for evaluating the sublaminae of the EPL without the necessity of labeling individual groups of cells. Finally, the staining pattern suggests that the intermediate zone of the EPL may be subjected to more tonic synaptic input, causing it to have an increased level of metabolic activity.

A technique for measurement of small samples of unit behavior during regularly repeated stimulation is described. A correlation-based measure was computed over two successive stimulus presentation by summing the products of spike counts for corrsponding time bins and normalizing to the number of bins and the number of spike counts during the two stimulus presentations. This measure was combined with the mean frequency of spike occurrence during the stimulus presentation to give a characterization of neuronal activity sensitive to changes in both strength of temporal patterns and mean frequency. Examples are given of olfactory responsive neurons with comparison of measurement techniques. The time course of the response to odor of neurons recorded under urethane anesthesia from the olfactory bulb and the lateral hypothalamus was studied. By both simple mean frequency measures and the measures proposed in this study, the neurons recorded from the lateral hypothalamus underwent more rapid temporal decrements in the odor response.

1. Intracellular and extracellular recordings were made from rat olfactory bulb mitral and tufted cells during odor stimulation and during electrical stimulation of the olfactory nerve. Neurons were identified by horseradish peroxidase injections and/or antidromic activation. The presentation of multiple concentrations of at least one odorant in a cyclic artificial sniff paradigm, as reported previously (10), allowed the study of odor responses. This approach was extended to multiple odorants to compare their concentration-response profiles. This procedure avoids the problems of interpretation resulting from nonequivalence of the effective concentrations of different odorants used as stimuli that have characterized previous studies of odor quality effects. Comparisons of intracellular events and responses to electrical stimulation with the odor-induced spike train activity allow us to begin to delineate the local circuitry involved in generating odor-induced responses. 2. The concentration-response profiles of the 72 cells in the present study are comparable to those previously reported for output neurons of the olfactory bulb, showing ordered changes in the temporal patterning of spike activity with step changes in odor concentration. However, eight of the neurons exhibited inhibitory responses to lower concentrations, but excitation, at similar latency, to higher concentrations of the same odorant. These data emphasize that to study pattern changes induced by changing odor quality the influence of stimulus intensity must also be carefully examined. The data also provide evidence that the temporal pattern evoked by an odorant is probably not in itself the code for odor quality recognition. 3. Complete concentration-response profiles, including subthreshold concentrations, to more than one odorant show that, although responses to the different odorant can evolve systematically with concentration, the responses to different odorants can evolve through very different patterns. For example, in some cells, the response patterns to different odors were complementary in form. These results demonstrate that the patterned responses of olfactory bulb neurons can reflect changes in odor quality as well as intensity. 4. Intracellular recording was employed to compare the temporal patterning of spikes during odor stimulation with membrane potential changes. In some cases, the spike pattern was closely correlated with apparent postsynaptic potentials. However, there were several clear exceptions. In five cells, a prominent hyperpolarization, seen in the first sniff of a series of 10 consecutive sniffs, was associated with pauses in spike activity. In the following

Studies on the morphological organization of the main olfactory bulb have indicated that there are subpopulations of granule cells with different dendritic patterns in the external plexiform layer (EPL). Small, extracellular injections of horseradish peroxidase (HRP) were made iontophoretically into superficial and deep parts of the EPL and the granule cell layer (GCL) in adult rats. Superficial EPL injections principally labeled superficial granule cell somata, whereas deep EPL injections labeled both superficial and deep granule cell somata. Injections in the superficial GCL labeled granule cell dendritic processes extending across the entire EPL. However, deep GCL injections labeled few granule cell dendrites in the superficial EPL, but labeled many such processes in the deep EPL. These results were the same in material processed with the Hanker-Yates procedure, where the morphology of individual neurons could be studied, and in the more sensitive tetramethyl benzidine procedure. Serial reconstructions of individual granule cells were made from both HRP and Golgi-Kopsch material. The distal dendrites of deep granule cells reached only as far as the deep EPL, where they branched extensively and had many dendritic spines. The dendrites of superficial granule cells, however, reached the most superficial part of the EPL where they ramified most extensively. The superficial granule cells typically had a higher spine density in the superficial part of the EPL than in the deep part. On the basis of these results, we conclude that the superficial granule cells predominantly innervate the superficial EPL and that the deep granule cells exclusively influences on the bulbar output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses. Since the secondary dendrites of the tufted cells ramify in the superficial EPL and the dentrites of most mitral cells ramify in deep EPL, the superficial and deep granule cells may preferentially modulate the responses of tufted and mitral cells, respectively.