Mechanisms of postinhibitory rebound and its modulation by serotonin in excitatory swim motor neurons of the medicinal leech.

Postinhibitory rebound (PIR) is defined as membrane depolarization occurring at the offset of a hyperpolarizing stimulus and is one of several intrinsic properties that may promote rhythmic electrical activity. PIR can be produced by several mechanisms including hyperpolarization-activated cation current (I(h)) or de-inactivation of depolarization-activated inward currents. Excitatory swim motor neurons in the leech exhibit PIR in response to injected current pulses or inhibitory synaptic input. Serotonin, a potent modulator of leech swimming behavior, increases the peak amplitude of PIR and decreases its duration, effects consistent with supporting rhythmic activity. In this study, we performed current clamp experiments on dorsal excitatory cell 3 (DE-3) and ventral excitatory cell 4 (VE-4). We found a significant difference in the shape of PIR responses expressed by these two cell types in normal saline, with DE-3 exhibiting a larger prolonged component. Exposing motor neurons to serotonin eliminated this difference. Cs+ had no effect on PIR, suggesting that I(h) plays no role. PIR was suppressed completely when low Na+ solution was combined with Ca2+-channel blockers. Our data support the hypothesis that PIR in swim motor neurons is produced by a combination of low-threshold Na+ and Ca2+ currents that begin to activate near -60 mV.

Single-cell analysis reveals cell-specific patterns of expression of a family of putative voltage-gated sodium channel genes in the leech.

To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.