Search

Mature functional hypothalamic neurons of male and female rats (21-day postnatal) were successfully cultured without attachment to non-neuronal cells in serum-free astrocyte-conditioned medium (ACM). A novel cell-collecting method was designed for these vulnerable cells by allowing the dissociated cell suspension to stand in a vertically held, wide-tipped syringe so that the cells were concentrated near the lower liquid surface, from which position they could be easily dropped into the medium, leaving most of the small debris in the syringe. This method made it possible to study statistically the survival of cultured neurons. It was impossible to collect many viable cells by the commonly used dissociating technique for fetal rat brain. However, neuron-like cells with a few processes could be isolated from sliced hypothalamic tissues by means of enzymatic and mechanical treatments. The original processes disappeared within 1-2 days and some new processes were generated after 2-3 days in vitro; the cells survived for 28 days in vitro. The cells were identified as neurons by the immunostaining method for microtubule-associated protein 2 (MAP2) and neurofilament (NF). Such neurons were obtained from every site of hypothalamic tissue sampled. These phenomena were not observed in chemically-defined medium (CDM), CDM supplemented with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) or nerve growth factor (NGF).

Dorsal root ganglion cells of adult guinea-pigs in vitro were inoculated with herpes simplex virus (HSV). Intracellular recording with a glass microelectrode revealed that HSV-infection caused a marked decrease in membrane excitability of the nerve cells within 24 h, which could be explained as a decrease in Na+ channel activity in the plasma membrane; while changes in other physiological properties (resting membrane potential, membrane resistance and capacitance) remain small. Viral antigens were detected in only 10-15% of the HSV-infected nerve cells. These indicate that the loss of the membrane excitability occurs much earlier than any other change in the HSV-infected 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.

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.

A new technique for primary culture of nerve cells isolated from peripheral ganglia of adult mammals has been developed. Nerve cells were isolated from trigeminal ganglia, nodose ganglia, sympathetic ganglia or dorsal root ganglia of adult guinea pigs by collagenase or dispase, and then were grown on collagen-coated plastic dishes for more than 4 weeks. Intracellular recording with a glass microelectrode reveals that the nerve cells in vitro generate not only Na spikes but also tetrodotoxin-resistant Na and Ca spikes. An exception was that the sympathetic ganglion cells in vitro failed to generate tetrodotoxin-resistant Na spikes. Difference in values of the Vmax of these spike components is observed among nerve cells (3-8 days in vitro) of different origin, suggesting that distribution of ionic channels on the plasma membrane may be different among species of nerve cells.

Dissociated culture of adult mouse dorsal root ganglion cells on glass plates, on which grating-associated microstructures (a repetition of microgrooves [mGRV] and microsteps [mSTP] of 0.1-10 micron) are fabricated by the conventional lithographic techniques, represents a remarkable bi-directional growth of their nerve fibers in the axial direction of the grating. Microscopical observation shows that the nerve fibers prefer to grow in the mGRV (70%), while their growth cones exhibit an even distribution onto the mGRV and mSTP. The efficiency of the nerve fibers to grow along the grating-axis are highly sensitive to a fine alteration of the width and depth of the mGRV. The preferential growth of the nerve fibers is thus due to a mechanical recognition of the microstructures by the growth cones and neurites.