Single ionic channels of two Caenorhabditis elegans chemosensory neurons in native membrane.

The genome of Caenorhabditis elegans contains representatives of the channel families found in both vertebrate and invertebrate nervous systems. However, it lacks the ubiquitous Hodgkin-Huxley Na+ channel that is integral to long-distance signaling in other animals. Nematode neurons are presumed to communicate by electrotonic conduction and graded depolarizations. This fundamental difference in operating principle may require different channel populations to regulate transmission and transmitter release. We have sampled ionic channels from the somata of two chemosensory neurons (AWA and AWC) of C. elegans. A Ca2+-activated, outwardly rectifying channel has a conductance of 67 pS and a reversal potential indicating selectivity for K+. An inwardly rectifying channel is active at potentials more negative than -50 mV. The inward channel is notably flickery even in the absence of divalent cations; this prevented determination of its conductance and reversal potential. Both of these channels were inactive over a range of membrane potentials near the likely cell resting potential; this would account for the region of very high membrane resistance observed in whole-cell recordings. A very-large-conductance (> 100 pS), inwardly rectifying channel may account for channel-like fluctuations seen in whole-cell recordings.

Olfactory receptor neurons express D2 dopamine receptors.

The role of the dopamine (DA) in the olfactory bulb (OB) was explored by determining which of the potential target cells express dopamine receptors (DARs). Previously, it was reported that D2-like DAR (D2, D3, and D4 subtypes) radioligand binding is restricted to the outer layers of the OB. The neuronal elements present only in these layers are the axons of the olfactory receptor neurons (ORNs) and the juxtaglomerular (JG) neurons of the glomerular layer. Based on this pattern of D2-like ligand binding, it was suggested that D2-like receptors might be located presynaptically on ORN terminals. The present study was undertaken to investigate this hypothesis. In the outer bulb layers of rats in which the ORNs were destroyed by nasal lavage with ZnSO(4), D2-like radioligand binding was reduced severely. The receptor subtype D2 mRNA, but not D3 mRNA, was detected in adult rat olfactory epithelial tissue. By using in situ hybridization, this D2 mRNA was located preferentially in epithelial layers that contain ORN perikarya. D2 mRNA was eliminated after bulbectomy, a manipulation known to cause retrograde degeneration of the mature ORNs. Taken together, the surgical manipulations indicate that mature ORNs express D2 DARs and are consistent with the hypothesis that functional receptors are translocated to their axons and terminals in the bulb. This suggests that dopamine released from JG interneurons could be capable of presynaptically influencing neurotransmission from the olfactory nerve terminals to OB target cells through the D2 receptor.

Orthodromic synaptic activation of rat olfactory bulb mitral cells in isolated slices.

Axons of olfactory receptor neurons terminate in the glomerular layer of the olfactory bulb, where they synapse with the apical dendrites of mitral cells. Although the mitral cell and its excitation by the olfactory nerve have been the subject of numerous experimental investigations, in vitro studies of these neurons have primarily used nonmammalian preparations. We have recorded the responses of rat olfactory bulb mitral cells to stimulation of the olfactory nerve layer in vitro using extracellular and whole cell patch techniques. Olfactory bulbs were cut into 400-microns thick slices in approximately horizontal section and submerged in a recording chamber. Patch clamp electrodes were guided into the mitral cell layer, which was visible under a dissecting microscope. A stimulating electrode was placed onto the olfactory nerve layer (ONL) rostral to the recording electrode. In extracellular recordings, mitral cells typically responded to ONL stimulation with a prolonged excitation lasting 1 s or longer. With whole cell patch recordings, membrane resistances (mean 272 M omega) were substantially higher than those reported in previous intracellular studies that used sharp electrodes. Small spontaneous excitatory potentials were present in some mitral cells. ONL stimulation caused a prolonged depolarization comparable to the duration of the period of excitation observed in extracellular recordings. At membrane potentials near -55 mV, ONL stimulation evoked a train of spikes. All but the first of these spikes were blocked by hyperpolarization of the membrane to -65 mV.

Evidence for GABAB-mediated inhibition of transmission from the olfactory nerve to mitral cells in the rat olfactory bulb.

The GABAB agonist baclofen blocks transmission from the olfactory nerve to second order neurons in the frog olfactory bulb, and GABAB receptors in the rat olfactory bulb are selectively located in the glomerular layer. A reasonable hypothesis, therefore, is that inhibition in the glomerular layer is mediated, at least in part, by GABAB receptors. Here, we investigated the role of GABAB receptors in regulating the responses of mitral cells to activation of the olfactory nerve in the rat. Topical application of baclofen to the surface of the rat olfactory bulb reduced the amplitude of field potentials evoked by olfactory nerve stimulation (orthodromic response). Baclofen reduced the orthodromic response in a dose-dependent manner but the drug had no effect on the field potential evoked by antidromic activation of mitral cell axons (antidromic response). Baclofen also reduced olfactory nerve-evoked responses of mitral cells in an olfactory bulb slice preparation. The pharmacological specificity of the inhibition was confirmed by showing that the GABAB antagonist, CGP 55845A, blocked the inhibitory action of baclofen. These results suggest that transmission from olfactory nerve terminals to second order neurons is negatively regulated by periglomerular GABAergic interneurons; this inhibition is mediated, at least partially, by GABAB receptors.

Evidence for presynaptic inhibition of the olfactory commissural pathway by cholinergic agonists and stimulation of the nucleus of the diagonal band.

We have investigated the role of the projection from the magnocellular basal forebrain to the olfactory bulb in regulating synaptic transmission in the commissural connection between the two olfactory bulbs. Commissural fibers arise in the contralateral anterior olfactory nucleus, travel in the anterior wing of the anterior commissure (AC), and terminate in the granule cell layer of the olfactory bulb. Electrical stimulation of the commissure causes synaptic activation of granule cells in the granule cell layer of the bulb; the resulting field potential is a reliable indicator of this synaptic current. Microinjections of cholinergic agonists, but not of identical, or larger, quantities of vehicle, reduced the amplitude of this AC field potential. Systemic injection of scopolamine reversed this depression and returned the AC response amplitude to control levels. Irreversible AChE inhibition also reduced the amplitude of the AC response, and muscarinic blockade reversed this effect. Cholinergic terminals in the olfactory bulb arise entirely from the axons of magnocellular basal forebrain neurons in the nucleus of the diagonal band (NDB). Electrical stimulation of NDB, which should release ACh, as well as other transmitters, depressed the AC response. Brief trains of NDB shocks caused a moderate decrease in the AC response that lasted 1-2 sec. Longer shock trains, which caused marked potentiation of the NDB field potential, caused a profound, prolonged (> 20 sec) inhibition of the AC response. Antidromic tests demonstrated that NDB stimulation significantly decreased the excitability of AC terminals. This and other characteristics of the inhibition strongly suggest that the decrease in amplitude of the field potential response to AC stimulation caused by cholinergic agonists and stimulation of NDB is due to presynaptic inhibition leading to reduced release of transmitter from AC terminals. These results suggest that one function of the basal forebrain projection to the olfactory bulb is inhibition of the commissural connection between the two olfactory bulbs. As NDB has been implicated in theta pacemaker input to the olfactory bulb, phasic NDB inhibition of centrifugal afferents to the bulb could function to coordinate signal processing temporally in the olfactory system. Temporal coordination may be particularly important to olfactory circuit function, as this system lacks the point-to-point topographical organization characteristic of other sensory systems.

Brain norepinephrine reductions in soman-intoxicated rats: association with convulsions and AChE inhibition, time course, and relation to other monoamines.

The organophosphate chemical nerve agent, soman, causes convulsions, neuropathology, and, ultimately, death. A major problem in treating soman intoxication is that peripherally acting pharmacological agents which prevent death do not prevent seizures. Although a primary cause of these symptoms is the excess of acetylcholine which follows acetylcholinesterase (AChE) inhibition, centrally acting muscarinic blockers, such as atropine, alleviate, but do not block, the convulsive actions of soman. Moreover, there is a relatively weak relationship between CNS reductions of AChE and the incidence of convulsions. There is evidence suggesting that soman intoxication stimulates the release of norepinephrine (NE) in the brain. Recent evidence has implicated NE in the induction and/or maintenance of seizures. Thus, in the present study the relations among soman-induced convulsions, AChE inhibition, and brain NE and other monoamine changes were examined. The time course of brain NE recovery was also determined. Rats were injected (im) with a single dose (78 micrograms/kg) of soman. At this dose 68% of the injected rats developed convulsions. Both convulsive and nonconvulsive rats were sacrificed between 1 and 96 h following soman injection and NE levels in the rostral forebrain and olfactory bulb were determined by HPLC with electrochemical detection. In all convulsive rats NE levels declined substantially. Forebrain NE levels were decreased by 50% at 1 h and 70% at 2 h following soman injection. Recovery of NE began at 8 h and was complete by 96 h following soman administration. Although nonconvulsive rats showed other signs of intoxication, NE levels in these rats were unchanged. Dopamine (DA) and serotonin (5-HT) levels were not significantly affected in either convulsive or nonconvulsive rats. However, 5-hydroxyindoleacetic acid, the major metabolite of 5-HT, and homovanillic acid and 3,4-dihydroxyphenylacetic acid, the two major metabolites of DA, were increased significantly in the forebrain of convulsive, but not nonconvulsive rats, indicating an increase in 5-HT and DA turnover. However, in contrast to the abrupt decline in NE, these increases in DA and 5-HT metabolites were slow and progressive. Taken together, the present results and other recent findings suggest that rapid, sustained NE release could play a role in the induction and/or maintenance of soman-induced convulsions, whereas increased release of 5-HT and DA may be a consequence of seizures. Further investigation of the role of NE in soman-induced convulsions may lead to improved treatment of soman intoxication and a better understanding of the role of NE in other forms of seizures, including human epilepsy.

Olfactory bulb DA receptors may be located on terminals of the olfactory nerve.

The glomerular layer of the olfactory bulb contains a substantial population of dopaminergic neurons. We determined the quantity and location of D1 and D2 dopamine receptors which are the presumed targets of these neurons. Binding of the D1 selective ligand [3H]SCH23390 was slightly above background and was distributed through all layers of the bulb except the olfactory nerve layer. In contrast there were relatively high levels of [3H]spiperone binding to D2 DA receptors in the glomerular and olfactory nerve layers. The presence of relatively high concentrations of D2 DA receptors in both the nerve layer and glomerular layer suggests the novel hypothesis that these receptors may be localized on terminals of the olfactory nerve.

Organization of projections from olfactory epithelium to olfactory bulb in the frog, Rana pipiens.

One hypothesis for the coding of olfactory quality is that regions of the olfactory epithelium are differentially sensitive to particular odor qualities and that this regional sensitivity is conveyed to the olfactory bulb in a topographic manner by the olfactory nerve. A corollary to this hypothesis is that there is a sufficiently orderly connection between the epithelium and the olfactory bulb to convey this topographical coding. Thus we examined topography in the projection from epithelium to bulb in the frog, which has been the subject of numerous electrophysiological studies but has not yet been examined using modern neuroanatomical techniques. The tracer WGA-HRP was applied to the ventral or to the dorsal olfactory epithelium, or both. Anterograde transport of label to the olfactory bulb was seen after as few as 2 days; label was still present in the bulb as long as 21 days postinjection. In cases where WGA-HRP was applied to the entire epithelium, there was dense anterograde labelling of the ipsilateral olfactory bulb. In addition, a small medial portion of the contralateral bulb was labelled. Injections limited to either the ventral or dorsal epithelium produced patterns of anterograde labelling in the glomerular layer of the olfactory bulb, which varied with the size and location of the injection. With very large injections in either the dorsal or ventral epithelium, label appeared to be evenly distributed in the glomerular layer. With smaller injections in the ventral epithelium, there was heavier labelling in the lateral than in the medial portions of the glomerular layer, although light labelling was found in all regions of the glomerular layer. In contrast, injection sites restricted to the dorsal epithelium produced more anterograde labelling in the medial than lateral portions of the glomerular layer. These patterns extended throughout the dorsal-ventral extent of the bulb. Within the limits of the anterograde tracing technique used, we were unable to detect any systematic relationship between the pattern of labelling in the glomerular layer and the medial-lateral or rostral-caudal location of the injection site in either the ventral or dorsal epithelium. We conclude that in the frog, as in other amphibia, there is only a limited degree of topographic order between the epithelium and the olfactory bulb.

Chemoanatomical organization of the noradrenergic input from locus coeruleus to the olfactory bulb of the adult rat.

The locus coeruleus contains noradrenergic neurons which project widely throughout the CNS. A major target of locus coeruleus projections in the rat is the olfactory bulb (Shipley et al.: Brain Res. 329:294-299, '85) but the organization of the projections within the bulb has not been systematically examined. In this study, the laminar distribution and densities of locus coeruleus-noradrenergic fibers in the main and accessory olfactory bulbs were determined with anterograde tracing and immunocytochemical techniques. Following iontophoretic injections of 1% wheat germ agglutinin-horseradish peroxidase into the locus coeruleus, the densest anterograde label in the accessory olfactory bulb was observed in the external plexiform layer, granule cell layer, and especially in the internal part of the mitral cell layer. Virtually no label was observed in the glomerular layer. In the main olfactory bulb, labelled axons were observed in the granule cell layer, in the internal and external plexiform layers, occasionally in the mitral cell layer, and least often in the glomerular layer. Noradrenergic fibers in the olfactory bulb were identified by using immunocytochemistry with an antibody to dopamine-beta-hydroxylase. Laminar patterns and densities of noradrenergic innervation were determined with quantitative image analysis. In the accessory olfactory bulb, the densest innervation was in the innermost portion of the mitral cell layer followed by the granule cell layer, the superficial part of the mitral cell layer, and the external plexiform layer. The density of fibers in the glomerular layer was least. The laminar pattern of noradrenergic fiber distribution in the main olfactory bulb was similar to that in accessory olfactory bulb. The present studies demonstrate that locus coeruleus-noradrenergic fibers terminate preferentially in the internal plexiform, granule cell, and external plexiform layers. This suggests that the major influence of the locus coeruleus input to both the main and accessory the olfactory bulbs is on the predominant neuronal element in those layers, the granule cells. Additional studies are needed to resolve how this input influences specific olfactory bulb circuits.

Two anatomically specific classes of candidate cholinoceptive neurons in the rat olfactory bulb.

The pharmacohistochemical technique of Butcher (1978) and choline acetyltransferase (ChAT) immunocytochemistry were used to demonstrate the presence, morphology, and differential distribution of 2 classes of acetylcholinesterase (AChE)-positive, ChAT-negative neurons in the rat olfactory bulb. One population of these neurons is located preferentially in a stratum just deep to the mitral cell layer (mcl). These AChE-positive intramitral neurons are significantly larger than the predominant inframitral neuronal type, the granule cell. Inframitral AChE-positive neurons appear to send processes deeper into the granule cell layer and superficially into the external plexiform layer (epl), above the mcl. Neurophysiological experiments reported in a companion article are consistent with the existence of a population of cholinoceptive neurons with the location and characteristics of these large inframitral interneurons. A second class of AChE-positive, ChAT-negative neurons is found exclusively in the glomerular layer. These neurons are located primarily in the periglomerular region and the superficial third of the epl; they are somewhat larger than typical periglomerular cells. Juxtaglomerular AChE neurons are smaller than inframitral AChE neurons. Since there are no neurons in the olfactory bulb that produce ChAT, the synthetic enzyme for ACh, the AChE-positive cells reported here are hypothesized to be cholinoceptive neurons for the cholinergic projection from the basal forebrain to the olfactory bulb. Anatomical, physiological, and receptor-ligand binding data are consistent with this interpretation.

Neurophysiology of magnocellular forebrain inputs to the olfactory bulb in the rat: frequency potentiation of field potentials and inhibition of output neurons.

Basal forebrain nuclei send projections, including cholinergic fibers, to forebrain cortical targets. These systems have been associated with several important functions, but their physiological actions are poorly understood. We have studied the neurophysiological characteristics of one of these systems, the projection from the nucleus of the horizontal limb of the diagonal band (HDB) to the main olfactory bulb (MOB) in the rat. Single shocks to HDB produce modest field potentials in MOB with no detectable effect on the discharge characteristics of the principal output neurons of the MOB, the mitral cells. By contrast, continuous stimulation at 10 Hz for several seconds causes dramatic changes in the HDB field potential and mitral cell firing. During this period of stimulation, there is an initial facilitation of the field potential followed by a period of moderately reduced response amplitude that lasts a few seconds. This brief period of depression is succeeded by a sudden and marked potentiation of response amplitude and duration. This potentiated response can be maintained indefinitely by stimulation at lower frequencies than those required to initiate the potentiation effect. Coincident with the onset of the potentiated response, the spontaneous activity of the mitral cells is completely inhibited. Both the potentiation and mitral cell inhibition can be maintained indefinitely by continued stimulation at frequencies as low as 6 Hz. These observations demonstrate that magnocellular basal forebrain neurons exert powerful regulatory actions on specific neuronal populations in cortical targets.

Frequency potentiation of a central cholinergic circuit.

We have used the cholinergic projection from the nucleus of the horizontal limb of the diagonal band (HDB) to the olfactory bulb as a model for studying the neurophysiology of cholinergic projections from the basal forebrain to cortical structures. Although single shocks to HDB have little effect upon function of the target structure, short periods of repetitive stimulation produce profound changes. These observations are discussed in terms of the known physiology of central cholinergic systems and the significance of cholinergic synaptic potentiation for Alzheimer's disease.

The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network.

Dense, focal injections of wheat germ agglutinin conjugated-horseradish peroxidase in the locus coeruleus of rats labeled afferent neurons in unexpectedly few brain regions. Major inputs emanate from only two nuclei--the paragigantocellularis and the prepositus hypoglossi, both in the rostral medulla. The dorsal cap of the paraventricular hypothalamic nucleus and the spinal intermediate gray are possible minor afferents to locus coeruleus. Other areas reported to project to locus coeruleus (for example, amygdala, nucleus tractus solitarius, and spinal dorsal horn) did not exhibit consistent retrograde labeling. Anterograde tracing and electrophysiologic experiments confirmed the absence of input to locus coeruleus from these areas, which instead terminate in targets adjacent to locus coeruleus. These findings redefine the anatomic organization of the locus coeruleus, and have implications for hypotheses concerning the functions of this noradrenergic brain nucleus.

Transducers for ultrasonic limb plethysmography.

Shifts in blood content of tissues cause dimension changes which can be measured with an ultrasonic dimension gauge. We have previously described the construction of a limb plethysmograph which uses this principle. Although other workers have described the construction of ultrasonic dimension gauge transducers intended for use inside animals, these transducers are not ideal for plethysmographic application. In this report we describe the design, construction, and performance characteristics of transducers suitable for limb plethysmography. Good signal quality is obtained with minimum care in transducer placement. These transducers incorporate a rugged external housing which provides for simple attachment to the calf by use of double-sided adhesive collars.

A practical ultrasonic plethysmograph.

An ultrasonic plethysmograph, which gives improved performance over the standard Whitney Strain Gauge, is described. This instrument monitors dimension changes in human limbs by measuring the transit times of acoustic pulses across two chords of the limb. In the case of a small uniform expansion, the percentage change in limb volume is shown to be proportional to twice the percentage change in either of the measured chords. Measurement of two chords allows correction for possible non-uniform expansion. In addition, measurement of two chords allows an estimate of the absolute cross-sectional area of the limb. The developed instrument incorporates a microprocessor, which performs necessary calculation and control functions. Use of the microprocessor allows the instrument to be self-calibrating. In addition, the device can be easily reprogrammed to incorporate improvements in operating features or computational schemes.