Chronic membrane depolarization-induced morphological alteration of developing neurons.

During development of CNS, young neurons experience various stimuli, and thereafter differentiate to mature neurons in an activity-dependent manner. Membrane depolarization acts as an inducer of excitability and various signals in the neurons, which can be used as a model of neuronal activity. However, the mechanisms of the influence of membrane depolarization on neuronal differentiation have not been fully understood. Therefore, we investigated the effect of membrane depolarization on morphology of spines and generation of valid electrical activity. Using rat hippocampal cultures treated from the plating day with or without high KCl (35 mM, termed HK), we directly observed living neurons transfected with green fluorescence protein-expressing plasmid through a two-photon laser scanning confocal microscope and electrophysiological recording using a patch-clamp technique. Compared with controls, the neurons cultured with HK for 3 days in vitro (DIV) showed marked filopodia-like protrusions as well as an increase in the number of spines, but those cultured with HK for 6 DIV profoundly lost these spines, resulting in a small number of fine filopodia-like protrusions proximally and on the cell body, and a smooth surface of distal dendrites. Electrophysiological recordings showed no spontaneous responses in 6 DIV HK-treated neurons. Moreover, addition of an N-methyl-D-aspartate receptor (NMDAR) antagonist to HK-treated neurons blocked the shrinkage and decrease in the number of filopodia-like protrusions significantly. These findings suggest that membrane depolarization of developing neurons induces synaptogenesis in the early stages of development but chronic treatment with HK causes pathological changes through NMDAR, and that there may be alternative mechanisms for the physiological differentiation of neurons in later developmental stages.

Brain-derived neurotrophic factor increases inhibitory synapses, revealed in solitary neurons cultured from rat visual cortex.

To elucidate chronic actions of brain-derived neurotrophic factor (BDNF) on GABAergic synapses, we examined effects of a long-term application of BDNF for 10-15 days on autapses (synapses) of solitary GABAergic neurons cultured from rat visual cortex. Solitary neuron preparations were used to exclude a possible contamination of BDNF actions on excitatory neurons in dissociated neuron culture or slice preparations. Neurons were confirmed to be GABAergic pharmacologically with bicuculline, a selective antagonist for GABAA receptors and immunocytochemically with antibody against glutamic acid decarboxylase 65, a GABA synthesizing enzyme. To evaluate GABAergic synaptic function, evoked and/or miniature inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell voltage-clamp mode. The treatment with BDNF at a concentration of 100 ng/ml enhanced the amplitude of evoked IPSCs and the frequency of miniature IPSCs. In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by two, successive activations. To evaluate morphological changes, neurons were immunocytochemically stained with antibodies against microtubule-associated protein 2, to visualize somatodendritic region and synapsin I, to visualize presynaptic sites. The quantitative analysis indicated that BDNF increased the area of soma, the numbers of primary dendrites and dendritic branching points, the total length of dendrites and the number of synaptic sites. Such an action of BDNF was seen in both subgroups of GABAergic neurons, parvalbumin-positive and -negative neurons. To visualize functionally active presynaptic sites, neurons were stained with a styryl dye, FM1-43. BDNF increased the number of stained sites that was correlated with the frequency of miniature IPSCs. These results suggest that the chronic treatment with BDNF promotes dendritic and synaptic development of GABAergic neurons in visual cortex.