Kazuhiko Yamaguchi

Field potential and intracellular potential studies of the olfactory bulb in the carp: evidence for a functional separation of the olfactory bulb into lateral and medial subdivisions

Summary1.Field potentials in the olfactory bulb and intracellular potentials from mitral cells were analyzed in the carp. Electrical shocks were applied to a part of the input (the lateral or medial bundle of the olfactory nerve: l-ON or m-ON respectively) or output pathways (lateral or medial olfactory tract: LOT or MOT respectively) of the olfactory bulb in order to activate the olfactory bulb partially.2.When shocks were applied to the regions described above, the distributions of the C2-wave component (which reflects the synaptic depolarization of the peripheral dendrites of granule cells) of field potentials were different between the lateral and medial parts of the olfactory bulb. It was suggested that the patterns of excitatory synaptic inputs to peripheral dendrites of the granule-cell population are different between the two parts of the olfactory bulb.3.The distributions of the C3- and C4-wave components (which reflect the synaptic depolarization of somata and deep dendrites of granule cells by volleys in centrifugal nerve fibers to the olfactory bulb) of field potentials were similar between the two parts of the olfactory bulb. It was suggested that the patterns of excitatory synaptic inputs from the centrifugal fibers to somata and deep dendrites of the granule-cell population are similar between the two parts of the bulb.4.Mitral cells activated antidromically by LOT shocks or synaptically by l-ON shocks were located mainly in the lateral part of the olfactory bulb. On the other hand, mitral cells activated antidromically by MOT shocks or synaptically by m-ON shocks were located mainly in the medial part of the bulb.5.Mitral cells, showing IPSPs in response to LOT, MOT and l-ON shocks, but not to m-ON shocks, were located mainly in the lateral part of the olfactory bulb. On the other hand, mitral cells, showing IPSPs to MOT and m-ON shocks, but not to LOT and l-ON shocks, were located mainly in the medial part of the bulb. Such a spatial distribution of mitral cells showing IPSPs was in accordance with that expected from the spatial distribution of the field potentials.6.These results suggest that the olfactory bulb of the carp can be functionally separated into two subdivisions (the lateral and medial parts); the activities of neurons in the one part exert little influence on neurons in the other part.7.From these results we suppose that the olfactory system of the carp is composed of two separate systems; in the lateral olfactory system, the lateral part of the olfactory bulb receives inputs mainly from the lateral bundle of the olfactory nerve and sends outputs to the LOT, while in the medial olfactory system, the medial part of the olfactory bulb receives inputs mainly from the medial bundle of the olfactory nerve and sends outputs to the MOT.

NGF-dependent and -independent growth of neurites from sympathetic ganglion cells of the aged human in a serum-free culture

Small pieces of tissue isolated from abdominal sympathetic ganglia in aged male patients were cultured in a chemically defined, serum-free medium. The growth of neurites from pieces of ganglia in cultures with and without 50 ng/ml mouse 2.5S nerve growth factor (NGF) was compared. The NGF stimulated significantly greater regeneration of neurites, causing the growth of long fibers from the ganglion pieces. Many short neurites grew, even in the absence of the NGF, but these were generally short, except for long neurites generated in several nerve cells. A method was devised for the evaluation of NGF-dependent growth of neurites in the culture. The rate of the NGF-dependent growth of neurites, which was calculated by the difference in the total lengths of the NGF-dependent neurites between 2- and 4-day-old cultures, was approximately 160 micron/day. The results indicate that although the growth of neurites from some sympathetic nerve cells of the aged human become independent of the NGF, most of the nerve cells remain dependent on the NGF, even in the stage of senescence.

Specific reduction in Na currents after infection with herpes simplex virus in cultured mammalian nerve cells

Tissue-cultured nerve cells originating from dorsal root ganglia of adult guinea-pigs were infected with type 2 herpes simplex virus (HSV) in vitro, and changes in membrane properties were examined (48 h later) by conventional electrophysiological technique using a glass microelectrode. The nerve cells infected with HSV (HSV-NC) failed to generate full-sized Na spikes, which was associated with a marked reduction in the maximum rate of rise of the Na spike, i.e. Na currents. Some SV-NC failed to generate any Na spike. By contrast, the HSV-NC generated unchanged, full-sized Ca spikes when bathed in a Na-free solution containing tetraethylammonium+. Changes in resting membrane properties, such as resting potential, input resistance and capacitance, remained small after the infection. We thus consider that HSV reduces Na currents in a specific fashion in this early period of the infection.

Morphological and physiological studies on cultured nerve cells from guinea pigs infected with herpes simplex virus in vivo

Adult guinea pigs were inoculated with type 2 herpes simplex virus (HSV) on the whole back skin, and nerve cells from the dorsal root ganglia (DRG) 6 days after infection were grown in tissue culture. Morphological and physiological properties of the cultured nerve cells from HSV-infected animals (HSV-NC) were compared to those of nerve cells of DRG from the control, non-virus infected animals (CON-NC). During the early period of the culture (0-4 days) growth of nerve cells and non-neuronal cells from the HSV infected animals was essentially the same as that from the control animals. HSV-antigen was present in only a small percentage of the HSV-NC by immunofluorescence (IF). Electrophysiological examination revealed that most of the HSV-NC exhibited a reduced capability of generating spikes, which was quantitatively described as a reduction in the Vmax of the Na spikes. This was interpreted as a reduction in the number of Na channel molecules in the plasma membrane of the HSV-NC, while the resting membrane properties of the same cells, such as the resting membrane potential, input resistance and capacitance, were essentially the same as those of the CON-NC. On 4-5 days in culture, some HSV-NC regained a full capability to generate Na spikes. We considered these nerve cells to have overcome the HSV infection and were now entering a latent period of HSV infection. About 1/3 of the HSV-NC still remained incapable of generating Na spikes. Viral antigen was detected in only 10% of the nerve cells. In the late stage of the culture, HSV infection in vitro was first observed as lysis of non-neuronal cells growing close to some HSV-NC. Nerve cells then started to lose their neurites and became spherical. Finally on 8-9 days most of the cells, including the nerve cells, were lost from the dishes as a result of a generalized infection of supporting cells, i.e. fibroblasts and Schwann cells. This study confirms our previous finding that the electrophysiological technique is much more sensitive than the IF method for the detection of HSV-infection in nerve cells. The results indicate that some nerve cells infected with HSV overcome the infection in vitro. This is interpreted as the entering of these nerve cells into a latent period of HSV infection in vitro.

Nerve cells of senescent mouse grown in tissue culture

Abstract Nerve cells isolated from dorsal root ganglia of senescent mouse (98–99 weeks old) and young adult mouse (4 and 8 weeks old) were grown in tissue culture. Number of senescent nerve cells which regenerated axons in vitro was 1 4 to 1 20 of young nerve cells, even though approximately the same number of alive nerve cells were incubated at the start of the tissue culture. Intracellular recording with a glass microelectrode revealed that resting membrane properties (membrane potential, input resistance and capacitance) and action potential components (Na and Ca spikes) were essentially the same for the senescent and young nerve cells.

Enhancement of the Ca2+-current by a serum factor in cultured dorsal root ganglia neurons of the adult guinea pig

To investigate effects of serum factor on the Ca2(+)-spike and neurite outgrowth in cultured nerve cells, dorsal root ganglia (DRG) neurons of the adult guinea pig were cultured in a serum-free N1 medium (N1 group) and a serum-containing medium (FCS group). The maximum rate of rise (MRR) of the Ca2(+)-spike, an indicator of the maximum Ca2(+)-current, was enhanced in the FCS group on day 5 in culture. The MRR of Ca2(+)-spike remained at a low level in the N1 group (2-10 days), but neurites outgrew rapidly during 2-5 days in both the FCS and N1 groups. Replacement of a serum-free medium by a serum-containing one on day 5 caused faster increase in the MRR of the Ca2(+)-spike. The active serum component for the Ca2(+)-spike enhancement was a heat-stable, small molecule. Chronic application of dibutyryl cyclic AMP (10 microM) mimicked the serum action on the Ca2(+)-spike. Whole-cell voltage-clamp experiment by a patch electrode showed that currents through the L- and T-type Ca-channels were enhanced in FCS group. Since kinetic and voltage-dependent gating properties of Ca-channels were similar between the FCS and N1 groups, available channel density might be increased by a serum factor.

Electrophysiological properties of tissue-cultured nerve cells from senescent mouse

Nerve cells were isolated from dorsal root ganglia of senescent mouse (C57 black, male) by collagenase and were grown on collagen-coated plastic dishes. The nerve cells extended their neurites and survived for more than 3 weeks in vitro. Intracellular recording with a glass microelectrode revealed that, although their resting membrane potential was small, the tissue-cultured nerve cells recovered membrane excitability after continuous hyperpolarization by DC current passage. Both Na and Ca spikes were elicited in thus hyperpolarized nerve cells. Some nerve cells generated tetrodotoxin-resistant Na spikes.

Rhythmic discharge of mitral cells in the carp olfactory bulb

We examined conditions in which rhythmic discharges of the mitral cell are obtained in the carp olfactory bulb. When the mean firing rates of the responses to various concentrations of NaCl solutions were high (more than 5 Hz), rhythmic discharges appeared. Results provide experimental support for the rhythm generation hypothesis on the dendrodendritic synapse in the vertebrate olfactory bulb.

Induced wave and its generation mechanism in the carp olfactory bulb

Abstract 1. 1. The induced wave and one component of the field potential evoked by electrical stimulation of the olfactory tract showed similar depth profiles in the carp olfactory bulb. 2. 2. The stimulation of the olfactory tract reset the rhythm of the induced wave. 3. 3. Rhythmic discharge of the mitral cell synchronized with the induced wave. 4. 4. We concluded that the induced wave mainly reflects the extracellular current caused by the synaptic depolarization of the granule cell dendrite and that the rhythm of the induced wave is due to the dendrodendritic synaptic interaction between the mitral cell and the granule cell.

The requirement of nerve growth factor for axon regeneration from sympathetic nerve cells of newborn, adult and aged mammals in a serum-free culture

Sympathetic ganglia dissected from newborn, young adult, adult and senescent mammals, including human beings, were grown in a chemically defined tissue culture environment, and axon regeneration from the nerve cells were compared with the cultures done with and without the nerve growth factor (NGF, extracted from submaxillary salivary glands of male mice). The cultures from newborn rabbits exhibited well growth of axons in the presence of 7 S NGF (200 ng/ml); while, in the absence of the NGF, no or only a few nerve cells exhibited regrowth of their fibers. In young adult and adult rabbits, however, several nerve fibers of short length regenerated, even in the absence of the NGF. The length of the regenerated fibers was approximately 1/2 that of the fibers in 200 ng/ml 7S NGF. Many nerve fibers regenerated from the ganglia of aged animals without NGF; 115 week-old rats and 55-60 years old human patients. The length of the regenerated fibers was between 1/4 and 1/2 of those regenerated in the presence of the NGF (200 ng/ml 7S NGF: the aged rats, and 50 ng/ml 2.5 S NGF: the aged human). In conclusion, I) sympathetic nerve cells of mammals of various ages can be grown in a chemically defined tissue culture condition, 2) most of the nerve cells need the NGF for axon regeneratioru 3) While some nerve cells regenerate their axons in a NGF-free medium, in which the numberof the regenerated fibers is increased in ageing. 4) The length of the regenerated axons without the NGF is increased in ageing.