Electrically silent neurons in the substantia gelatinosa of the rat spinal cord.

Substantia gelatinosa (SG) neurons are usually categorized on three main types: tonic, adapting and delayed firing (DFNs), based on characteristic firing response evoked by sustained stimulation. Here, the existence of electrically silent neurons (ESNs, 9.3%) is reported by using patch-clamp recording and confocal microscopy in spinal cord slices from 3-5 weeks-old rats. Those neurons does not generate spikes at their resting membrane potential (approximately -69 mV) but only at preliminary depolarization to > -60 mV In the latter case, spikes appeared starting from the end of stimulation, which is characteristic feature of DFNs. With the exception of APs block, all other passive and active electrophysiological properties of ESNs and DFNs were similar. Their main morphological properties (vertical orientation of dendritic tree and axonal trajectory) were close too. A distinctive feature of ESNs was larger amplitude of outward A-type K+ current (K(A)). The results suggest that the latter might cause a block of APs in ESNs, while these cells likely are a functional (i.e., non-firing) subtype of DFNs. The role of DFNs in descending control of pain transmission via modulation of its K(A) is hypothesized.

[Morphophysiologic properties of delayed firing neurons in substantia gelatinosa of the rat spinal cord.

Substantia gelatinosa (SG) neurons of the spinal cord are highly heterogeneous in their morphologic and physiologic properties. Based on characteristic firing response evoked by sustained depolarization, neurons can be categorized on three main types: tonic, adapting and delayed firing (DFNs). Here, properties of DFNs in spinal cord slices from 3-5 weeks-old rats were studied with the use of patch-clamp recording and confocal microscopy. Distinctive features of DFNs were increased rheobase (95.7 +/- 11.2 pA) and depolarized threshold to evoke action potential (-37.8 +/- 0.7 mV) than in neurons of other types. In voltage-clamp mode all DFNs expressed high amplitude outward A-type potassium current (K(A)), which started activation at approximately -70 mV, before Na+ current. Structurally, the majority of DFNs had vertically oriented dendritic tree, while their axons were not restricted to SG and projected predominantly to lamina I. Possible physiological functions of DFNs are discussed.

Postsynaptic mechanism may contribute to inhibitory acetylcholine effect on GABAergic synaptic transmission in hippocampal cell cultures.

The effect of acetylcholine (ACh) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. Spontaneous IPSCs and IPSCs evoked by extracellular stimulation of a single presynaptic neuron were recorded. ACh inhibited the evoked IPSCs in most of the connections, although facilitation was also observed. Regardless of inhibitory or facilitatory effects on the evoked IPSCs, an enhanced spontaneous synaptic input to the postsynaptic neurons was usually observed. ACh-induced changes in the evoked IPSCs were usually accompanied by changes in paired pulse depression (PPD), which are thought to reflect presynaptic mechanisms of modulation. However, the time course of PPD changes did not always match that of the IPSC changes, suggesting a contribution of other, possibly postsynaptic, mechanism(s). To analyze this possibility, effects of ACh on responses to direct application of exogenous GABA were studied. In a proportion of the neurons (40%) ACh reversibly decreased GABA responses, indicating that postsynaptic mechanisms may also contribute to the inhibitory ACh effect on GABAergic transmission. We conclude that several different modulatory mechanisms of ACh action participate in the regulation of GABAergic transmission at the level of synaptic connection of a single GABAergic neuron.

Rat hippocampal neurons maintain their own GABAergic synaptic transmission in culture.

The whole-cell patch-clamp technique was used to record monosynaptic inhibitory postsynaptic currents (IPSCs) from pairs of hippocampal neurons cultured for 2-3 weeks. The application of fresh physiological solution for 2-3 min reversibly reduced the amplitude of evoked GABAergic IPSCs to 72.5% of control value. The amplitude and frequency of spontaneous IPSCs decreased too. The depression of evoked IPSCs was significantly smaller or absent if conditioned solution was applied (physiological solution which had been previously in contact with neurons for 30 min). Currents evoked by exogenously applied GABA were unaffected by fresh solution. These results suggest that hippocampal neurons release some endogenous substance(s), by which they up regulate presynaptically their own inhibitory synaptic transmission.

Development of spontaneous synaptic activity in culture of the chick spinal cord neurons.

Spontaneous postsynaptic currents in chick spinal cord neurons cultured for up to three weeks were recorded by using standard whole-cell patch-clamp techniques. Beginning with approximately the 7th to 14th day in vitro, giant postsynaptic currents, mediated presumably by glycine, were single synaptic events (inhibitory postsynaptic currents). After the 14th day in vitro excitatory postsynaptic currents appeared. Both types of currents were predominantly arranged in bursts. This pattern of synaptic activity did not change appreciably during further cultivation. Characteristics of inhibitory postsynaptic currents were studied. Decay of the majority of giant inhibitory postsynaptic currents was two-exponential. Time-constants of the decay (fast and slow) increased with depolarization and decreased with increasing temperature. Decay of miniature inhibitory postsynaptic currents was single exponential and did not depend on the membrane potential. Strychnine at concentrations of 1-2 microM was found not only to reduce the amplitude of giant inhibitory postsynaptic currents but also to prolong their decay. The time-constant of the slow component of the decay was mostly affected during the inhibitor action. Repeated binding of glycine to postsynaptic receptors due to a large presynaptic release is proposed as an explanation for the properties of giant inhibitory postsynaptic currents decay. Correlations between the development of synaptic networks under in vivo and in vitro conditions are discussed.

Glycine conductance changes in chick spinal cord neurons developing in culture.

Whole-cell glycine-activated currents were investigated in chick spinal cord neurons cultivated for up to three weeks. Based on the morphological and electrophysiological characteristics of neurons, two different types of nerve cells were distinguished during the first few days in culture. The first type consisted of "mature" nerve cells which appear to be motoneurons. They died by five to seven days in vitro. Immature neurons or neuroblasts constituted another type of nerve cell. They developed in culture and became differentiated neurons. Glycine-activated currents were elicited in both types of neurons during different periods in vitro. Sensitivity to glycine of "mature" neurons decreased from two to five days in vitro: ED50 for agonist action increased from 0.4 to 1.3 mM. The sensitivity of neuroblasts to this transmitter increased during differentiation: ED50 decreased from 1.4 to 0.12 mM on three to 14 days in vitro, respectively. Changes in glycine-activated conductance of these developing neurons were investigated later on. The conductance in differentiated neurons was markedly sensitive to membrane potential, while neuroblasts did not show such dependence. Voltage sensitivity was due to voltage-dependent kinetics of the ion channel. Desensitization kinetics of the glycine-activated currents were double-exponential. The time constant for the slow desensitizing component was dependent on glycine concentration, which was not the case for the fast component. The increase in glycine sensitivity of the neuroblasts was accompanied by deceleration of desensitization kinetics of the agonist-activated currents. A remarkable feature of the currents elicited in neuroblasts was their extremely long time course after rapid agonist removal from the cells. The properties of these long-term currents suggest that a large fraction of the receptors are desensitized, even during quite short applications of the transmitter. The presence of glycine in the culture medium did not affect the increase of neuronal sensitivity to the agonist. The block of spontaneous bioelectric activity by adding tetrodotoxin to the culture medium abolished developmental changes in glycine-activated conductance. Possible mechanisms for the changes in transmitter sensitivity of the neurons are considered.