Tokio Sugai

Subdivisions of the guinea-pig accessory olfactory bulb revealed by the combined method with immunohistochemistry, electrophysiological, and optical recordings.

The presence of subgroups in vomeronasal sensory neurons has been known in various animals. To elucidate possible functional subdivisions in the guinea-pig accessory olfactory bulb, the combined studies with GTP-binding protein immunohistochemistry, electrophysiological and optical recordings were carried out. Gi2 alpha and Go alpha proteins were immunohistochemically localized, respectively, in the anterior and posterior regions of the vomeronasal nerve and glomerular layers, indicating that the guinea-pig accessory olfactory bulb receives at least two different inputs. This suggests that an anatomical boundary exists in these two layers. A mapping study of field potentials in sagittal slice preparations demonstrated that stimulation of the anterior vomeronasal nerve layer elicited field potentials with weak oscillatory responses exclusively in the anterior region of the external plexiform layer, whereas shocks to the posterior vomeronasal nerve layer provoked distinct oscillatory responses within the posterior one. The damping factors of oscillations in the anterior and posterior regions were 0.064+/-0.028 and 0.025+/-0.014, respectively. These electrophysiological results suggest that the accessory olfactory bulb consists of two functionally different subdivisions. Real-time optical imaging showed that anterior vomeronasal nerve layer shocks produced neural activity which spread horizontally from anterior to posterior only within the anterior region of the external plexiform and mitral cell layers, whereas shocks to the posterior vomeronasal nerve layer evoked periodic neural activity which spread horizontally from posterior to anterior only within the posterior region. Furthermore, the most posterior extent of the optical response evoked in the anterior region immediately adjoined the most anterior extent of that evoked in the posterior region. The maximal distance of signal propagation in the granule cell layer corresponded to that in the overlying external plexiform and mitral cell layers, indicating that the granule cell layer also has a similar boundary. Thus, these optical imaging studies not only demonstrated a precise boundary in each layer of the accessory olfactory bulb, which was positioned right beneath the boundary defined by GTP-binding protein immunohistochemistry, but also confirmed the observations from electrophysiological mapping that evoked field potentials are independently distributed in each of two subdivisions. The presence of the functional subdivision in each layer leads us to conclude that the accessory olfactory bulb in the guinea-pig is distinctly segregated into the anterior and posterior subdivisions, and to suggest that there are at least two different input output pathways in the vomeronasal system.

Selective suppression of horizontal propagation in rat visual cortex by norepinephrine

The release of norepinephrine in the cerebral cortex from axon terminals of locus coeruleus neurons was suggested to be involved in the control of attention. Accumulating data indicate that the responses of cortical neurons are varied when norepinephrine is applied iontophoretically in the vicinity of the cells being recorded. However, it is not known how the pattern of excitatory propagation is modified when norepinephrine is applied over a wide area in the visual cortex. By applying optical imaging to rat visuocortical slices, we found a new mode of norepinephrine action; a prominent suppression of the horizontal propagation in layers II/III. This action of norepinephrine was confirmed by the simultaneous recording of field potentials from multiple sites by use of a multi‐electrode dish. Furthermore, our electrophysiological recordings showed that this norepinephrine action is exerted through suppression of excitatory neural transmission and enhancement of inhibitory transmission to the pyramidal neurons in these layers. Because the release of norepinephrine in the visual cortex is regulated by the level of attention, the neural basis of visual attention may relate partially to the suppression of the integration of visual information by norepinephrine resulting in a state‐dependent restructuring of the receptive field.

Actions of cholinergic agonists and antagonists on the efferent synapse in the frog sacculus

Intracellular recordings were made from hair cells in the frog saccular epithelium isolated with its innervating nerves. Inhibitory post-synaptic potentials (IPSPs) were recorded from hair cells when the efferent fibers were activated by electrical stimulation. The effects of acetylcholine (ACh), cholinomimetics, and cholinergic antagonists on the efferent synapse were studied in a preparation where the IPSPs can be observed directly. ACh or carbachol (CCh) produced a transient membrane hyperpolarization with a decrease in input resistance followed by an abolition or reduction of the IPSP. In a low Ca2+ medium where efferent synaptic activity was abolished, ACh or CCh still induced hyperpolarization, though the response appeared to be smaller than that in normal medium. Neither nicotinic (dimethyl-4-phenyl-piperazinium (DMPP), phenyltrimethylammonium (PTMA) and nicotine) nor muscarinic (muscarine, methacholine, bethanechol and oxotremorine) agonists induced the membrane hyperpolarization, but the former drugs inhibited the IPSPs while the latter drugs did not. Both d-tubocurarine and atropine inhibited the IPSP, but the d-tubocurarine was more potent, causing inhibition even at a dose of 0.5 microM while 2 microM or more atropine was needed. The ACh- or CCh-induced hyperpolarization was inhibited completely by d-tubocurarine (5 microM), but only slightly by atropine (5 microM). These results may indicate that the IPSP and the effects of ACh or CCh are based on a direct interaction between ACh or CCh and ACh receptors on the hair cells.

Somatotopic organization and columnar structure of vibrissae representation in the rat ventrobasal complex

SummaryThe region of vibrissae representation in the ventrobasal complex (VB) of the rat was systematically mapped, based on receptive fields of many single neurons. Results showed that the ventralmost row of vibrissae projected to the rostral part of VB, that the dorsal-most row projected to the caudal part, and that the caudalmost vibrissae of each row projected to the most dorsolateral part of VB and more rostral vibrissae to the more ventromedial part. Further, it was revealed that the clusters of neurons receiving projections from any individual vibrissae formed corresponding columns extending from the anterodorsomedial to the posteroventrolateral direction, and that these columns piled up dorsoventrally and anteroposteriorly, with ventral ones shifted progressively medially. When cross sections of these columns were viewed on an oblique horizontal section of VB, a group of columns corresponding to each row lined up from the dorsolateral to the ventromedial direction with a rostral convexity, which means that the third or fourth vibrissa in each row projected most rostrally in that row. These results confirmed previous physiological mapping studies of vibrissal representation and are in good agreement with anatomical studies on barreloid structure in VB.

Optical imaging of the in vitro guinea pig piriform cortex activity using a voltage-sensitive dye.

The spatio-temporal patterns of signal processing in guinea pig piriform cortex (PC) slices were analyzed by optical imaging using a voltage-sensitive dye. Slices (400 microns thick) were cut in a plane parallel to the lateral olfactory tract and perpendicular to the cortical surface. In all the anterior PC and the majority of the posterior PC preparations, neural activity elicited by electrical stimulation of layer Ia propagated along the same layer, then it invaded into layers II and III and propagated along them. In addition to the above pattern, invasion of activity into the deeper area than layer III was observed in some posterior PC preparations. Real-time imaging of an active zone evoked by Ia shocks and its spatio-temporal behavior will contribute to resolving olfactory information processing.

Increase in Cortical Pyramidal Cell Excitability Accompanies Depression-Like Behavior in Mice: A Transcranial Magnetic Stimulation Study

Clinical evidence suggests that cortical excitability is increased in depressives. We investigated its cellular basis in a mouse model of depression. In a modified version of forced swimming (FS), mice were initially forced to swim for 5 consecutive days and then were treated daily with repetitive transcranial magnetic stimulation (rTMS) or sham treatment for the following 4 weeks without swimming. On day 2 through day 5, the mice manifested depression-like behaviors. The next and last FS was performed 4 weeks later, which revealed a 4 week maintenance of depression-like behavior in the sham mice. In slices from the sham controls, excitability in cingulate cortex pyramidal cells was elevated in terms of membrane potential and frequencies of spikes evoked by current injection. Depolarized resting potential was shown to depend on suppression of large conductance calcium-activated potassium (BK) channels. This BK channel suppression was confirmed by measuring spike width, which depends on BK channels. Chronic rTMS treatment during the 4 week period significantly reduced the depression-like behavior. In slices obtained from the rTMS mice, normal excitability and BK channel activity were recovered. Expression of a scaffold protein Homer1a was reduced by the FS and reversed by rTMS in the cingulate cortex. Similar recovery in the same behavioral, electrophysiological, and biochemical features was observed after chronic imipramine treatment. The present study demonstrated that manifestation and disappearance of depression-like behavior are in parallel with increase and decrease in cortical neuronal excitability in mice and suggested that regulation of BK channels by Homer1a is involved in this parallelism.

Convergence of olfactory and gustatory connections onto the endopiriform nucleus in the rat

Electrical and optical recordings were made from slice preparations including the piriform and gustatory cortices. Electrical stimulation of the gustatory cortex evoked a characteristic field potential in the endopiriform nucleus. A field potential was induced in the endopiriform nucleus by stimulation of the piriform cortex. Voltage-sensitive dye studies showed that stimulation of the piriform cortex induced signal propagation from the piriform cortex to endopiriform nucleus, whereas stimulation of the gustatory cortex did the same from the gustatory cortex to endopiriform nucleus via the agranular division of the insular cortex. After stimulation of the endopiriform nucleus, optical signals propagated not only to the piriform cortex but also to the gustatory cortex via the agranular division of the insular cortex. The olfactory and gustatory pathways appeared to be reciprocally connected. Unit recordings indicated that olfactory and gustatory activity converged onto a single neuron of the endopiriform nucleus. It is suggested that the cortical integration of olfactory and gustatory information could modulate mechanisms involved in food selection and emotional reactions relating to the chemical senses.

Odor-concentration coding in the guinea-pig piriform cortex

By optical imaging of intrinsic signals, we demonstrated a possible code for odor concentration in the anterior piriform cortex of the guinea-pig. Odor-induced cortical activation, which primarily originated in layer II, appeared in a narrow band beneath the rhinal sulcus over the lateral olfactory tract, corresponding to the dorsal part of the anterior piriform cortex. Lower concentrations activated the rostral region of the band, whereas higher ones generated caudally spreading activation, and the site at which neural activation reached its maximum extent depended upon odor concentration. Different odors with low concentrations generated distinct but somewhat overlapping patterns in the rostral region of the band; the limited extent of cortical activity may be one focal domain for each odor. It was hard to judge, however, that odor-specific domains appeared in the anterior piriform cortex, because the strong stimuli induced largely overlapping patterns. Furthermore, the total area activated increased in proportion to concentrations raised to a power of 0.5-0.9. Importantly, these imaging results were confirmed with unit recordings which indicated a rostro-caudal gradient in odor-sensitivity among cortical neurons. Our results suggest that the dorsal part of the anterior piriform cortex may be associated with odor concentration. Therefore, in addition to recruitment of more olfactory sensory cells and glomeruli in response to stronger stimuli, a rostro-caudal gradient in axonal projections from mitral/tufted cells and/or in association fibers may play an important role in odor-concentration coding in the anterior piriform cortex.

Novel subdivisions of the rat accessory olfactory bulb revealed by the combined method with lectin histochemistry, electrophysiological and optical recordings.

Wistaria floribunda agglutinin and peanut agglutinin were found to bind histochemically to the anterior and posterior regions, respectively, of the vomeronasal nerve and glomerular layers in the rat accessory olfactory bulb. Furthermore, Ricinus communis agglutinin showed strong binding to the anterior region of the vomeronasal nerve and glomerular layers, whereas it bound weakly and/or moderately to the rostral two-thirds of the posterior glomerular layer but not at all to the caudal one-third. This suggests that the posterior region is further divided into two subregions. An electrophysiological mapping study in sagittal slice preparations demonstrated that stimulation given within the anterior vomeronasal nerve layer elicited field potentials within the anterior region of the external plexiform layer, whereas shocks to the rostral two-thirds and the caudal one-third of the posterior vomeronasal nerve layer provoked field responses within the rostral two-thirds and within the caudal one-third of the posterior external plexiform layer, respectively, indicating that the posterior external plexiform layer is also divided into two subregions. Real-time optical imaging showed similar results as above, except that neural activity also spread into mitral cell layers. Furthermore, the most anterior and posterior ends of the neural activity evoked in the rostral two-thirds of the posterior region immediately adjoined the posterior border of that evoked in the anterior region and the anterior border of that evoked in the caudal one-third of the posterior region, respectively. Moreover, the granule cell layer was also found to have similar boundaries. Thus, optical imaging studies demonstrated individual precise boundaries of these subdivisions, which were positioned right beneath those defined by Ricinus communis agglutinin histochemistry. The presence of functional segregation in each layer leads us to conclude that there are at least three different input-output pathways in the rat vomeronasal system.

Synchronized population oscillation of excitatory synaptic potentials dependent of calcium-induced calcium release in rat neocortex layer II/III neurons.

We examined the roles played by calcium-induced calcium release from ryanodine-sensitive calcium stores in induction of neocortical membrane potential oscillation by using caffeine, an agonist of ryanodine receptors. Intracellular recordings were made from neurons in layer II/III of rat visual cortex slices in a caffeine-containing medium. White matter stimulation initially evoked monophasic synaptic potentials. As low-frequency stimulation continued for over 10 min, an oscillating synaptic potential gradually became evoked, in which a paroxysmal depolarization shift was followed by a 8-10-Hz train of several depolarizing wavelets. This oscillating potential was not induced in a medium containing no caffeine with 2 or 0.5 mM [Mg2+](o). Under blockade of N-methyl-D-aspartate receptors, induction of this oscillating potential failed even with caffeine application. Experiments with the calcium store depletor, thapsigargin, revealed that this oscillating potential is induced in a manner dependent on intracellular calcium release. Dual intracellular recordings revealed that the oscillation was synchronized in pairs of layer II/III neurons. The oscillating potential was detectable by field potential recordings also, suggesting that the present oscillation seems to reflect a network property.