The development of central nervous system slice preparations for electrophysiological studies has led to an explosion of knowledge of neuronal properties in health and disease. Studies of spinal motoneurons in these preparations, however, have been largely limited to the early postnatal period, as adult motoneurons are vulnerable to the insults sustained by the preparation. We therefore sought to develop an adult spinal cord slice preparation that permits recording from lumbar motoneurons. To accomplish this, we empirically optimized the composition of solutions used during preparation in order to limit energy failure, reduce harmful ionic fluxes, mitigate oxidative stress, and prevent excitotoxic cell death. In addition to other additives, this involved the use of ethyl pyruvate, which serves as an effective nutrient and antioxidant. We also optimized and incorporated a host of previously published modifications used for other in vitro preparations, such as the use of polyethylene glycol. We provide an in-depth description of the preparation protocol and discuss the rationale underlying each modification. By using this protocol, we obtained stable whole cell patch-clamp recordings from identified fluorescent protein-labeled motoneurons in adult slices; here, we describe the firing properties of these adult motoneurons. We propose that this preparation will allow further studies of how motoneurons integrate activity to produce adult motor behaviors and how pathological processes such as amyotrophic lateral sclerosis affect these neurons.

A subtype of retinal amacrine cells displayed a distinctive array of K+ currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of −60 to −40 mV. Depolarizations above approximately −40 mV were associated with the disappearance of SMOCs and the appearance of transient (Ito) and sustained (Iso) outward K+ currents. Ito appeared at about −40 mV and its apparent magnitude was biphasic with voltage, whereas Iso appeared near −30 mV and increased linearly. SMOCs, Ito, and a component of Iso were Ca2+ dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of −20 mV and above. Their mean amplitude was Nernstian with respect to [K+]ext and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca2+-free solutions, indicative of BK-type Ca2+-activated K+ currents. Dihydropyridine Ca2+ channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca2+ permeation through the kainic acid receptor had no effect. Blockade of organelle Ca2+ channels by ryanodine, or intracellular Ca2+ store depletion with caffeine, eradicated SMOCs. Internal Ca2+ chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca2+ influx through L-type Ca2+ channels and its subsequent amplification by Ca2+-induced Ca2+ release via the ryanodine receptor leads to a localized elevation of internal Ca2+. This amplified Ca2+ signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.

Retinal ganglion cells (RGCs) display the phenomenon of rebound excitation, which is observed as rebound sodium action potential firing initiated at the termination of a sustained hyperpolarization below the resting membrane potential (RMP). Rebound impulse firing, in contrast to corresponding firing elicited from rest, displayed a lower net voltage threshold, shorter latency and was invariably observed as a phasic burst-like doublet of spikes. The preceding hyperpolarization leads to the recruitment of a Tetrodotoxin-insensitive depolarizing voltage overshoot, termed as the net depolarizing overshoot (NDO). Based on pharmacological sensitivities, we provide evidence that the NDO is composed of two independent but interacting components, including (1) a regenerative low threshold calcium spike (LTCS) and (2) a non-regenerative overshoot (NRO). Using voltage and current clamp recordings, we demonstrate that amphibian RGCs possess the hyperpolarization activated mixed cation channels/current, Ih, and low voltage activated (LVA) calcium channels, which underlie the generation of the NRO and LTCS respectively. At the RMP, the Ih channels are closed and the LVA calcium channels are inactivated. A hyperpolarization of sufficient magnitude and duration activates Ih and removes the inactivation of the LVA calcium channels. On termination of the hyperpolarizing influence, Ih adds an immediate depolarizing influence that boosts the generation of the LTCS. The concerted action of both conductances results in a larger amplitude and shorter latency NDO than either mechanism could achieve on its own. The NDO boosts the generation of conventional sodium spikes which are triggered on its upstroke and crest, thus eliciting rebound excitation.

Given that the action potential output of retinal ganglion cells (RGCs) determines the nature of the visual information that is transmitted from the retina, an understanding of their intrinsic impulse firing characteristics is critical for an appreciation of the overall processing of visual information. Recordings from RGCs within an isolated whole-mount retina preparation showed that their normal impulse firing from the resting membrane potential (RMP) was linearly correlated in its frequency with the stimulus intensity. In addition to describing the relationship between the magnitude of the current injection and the resulting impulse frequency (F/I relationship), we have characterized the properties of individual action potentials when they are elicited from the RMP. In contrast, hyperpolarizing below the RMP revealed that RGCs displayed a time dependent anomalous rectification, manifested by the appearance of a depolarizing sag in their voltage response. When an adequate period of hyperpolarization was terminated, a fast phasic period of "rebound excitation" was observed, characterized by a brief phasic burst of impulse activity. When compared to equivalent action potential firing evoked by depolarizing from the RMP, rebound spiking was associated with a lower threshold and shorter latency for impulse activation as well as a prominent, phasic, burst-like doublet, or triplet of impulses. The rebound action potential had a more positive voltage overshoot and displayed a higher peak rate of rise in its upstroke than those correspondingly generated by depolarizing current pulses from the RMP. Blocking sodium spikes with TTX confirmed that the preceding hyperpolarization led to the recruitment and subsequent generation of a transient depolarizing voltage overshoot, which we have termed the net depolarizing overshoot (NDO). We propose that the NDO boosts the generation of sodium spikes by triggering rebound spikes on its upstroke and crest, thus accounting for the observed voltage dependent change in the firing pattern of RGCs.

Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between −60 and −40 mV. At more depolarized potentials, a transient outward current (Ito) appears and SMOCs disappear. Both SMOCs and the Ito are K+ currents carried by BK channels. They both arise from Ca2+ influx through high voltage–activated (HVA) Ca2+ channels, which stimulates release of internal Ca2+ from caffeine- and ryanodine-sensitive stores. An increase in Ca2+ influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca2+ influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca2+ influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the Ito at high levels of Ca2+ influx. Therefore, low levels of Ca2+ influx, arising from low levels of activation of the HVA Ca2+ channels, produce randomly occurring SMOCs within the range of −60 to −40 mV. Further depolarization leads to greater activation of the HVA Ca2+ channels, larger Ca2+ influx, and the disappearance of discontinuous SMOCs, along with the appearance of the Ito. Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.