Ca v 1.3 (␣1D) L-type Ca2ϩ channels have been implicated in substantia nigra (SN) dopamine (DA) neuron pacemaking and vulnerability to Parkinson's disease. These effects may arise from the depolarizing current and cytoplasmic Ca 2ϩ elevation produced by Ca v 1.3 channels at subthreshold membrane potentials. However, the assumption that the Ca 2ϩ selectivity of Ca v 1.3 channels is essential has not been tested. In this study the properties of SN DA neuron L-type Ca 2ϩ channels responsible for driving pacemaker activity in juvenile rat brain slices were probed by replacing native channels blocked with the dihydropyridine nimodipine with virtual channels generated by dynamic clamp. Surprisingly, virtual L-type channels that mimic native and recombinant Ca v 1.3 channels supported pacemaker activity even though dynamic clamp currents are not carried by Ca 2ϩ . This effect is specific because pacemaker activity could not be restored by tonic current injection, virtual nonselective leak channels or virtual NMDA receptors, which share with L-type channels a negative slope conductance region in their current-voltage (I-V) curve. Altering virtual channels showed that the production of pacemaker activity depended on the characteristic voltage dependence of DA neuron L-type channels, while activation kinetics and reversal potential were not critical parameters. Virtual L-type channels also supported slow oscillatory potentials and enhanced firing rate during evoked bursts. Thus, Ca v 1.3 channel voltage dependence, rather than Ca 2ϩ selectivity, drives pacemaker activity and amplifies bursts in SN DA neurons.

SUMMARY1. The cellular organization of the ninth and tenth paravertebral sympathetic ganglia in the bullfrog was studied with intracellular and extracellular recording methods. An isolated preparation was used in which anatomical details of individual cells could be resolved while making physiological measurements. This permitted the characterization of neurones in terms of their size, the segmental origin of their cholinergic innervation, and their orthodromic and antidromic conduction velocities. With these criteria, three classes of sympathetic neurones were identified.2. As in previous studies, C cells were distinguished from B cells by the origin of their innervation. C cells are innervated by slowly conducting axons (0 4 m/sec) from spinal nerves 7 and 8 and B cells are innervated by rapidly conducting axons (2-4 m/sec) from the sympathetic chain above ganglion 7.3. In earlier work it has been suggested that the conduction velocity of a preganglionic axon generally matches that ofits target neurone. In this study we have characterized a large group of B cells for which this is not true. The axons of B cells fall into a rapidly conducting group (2-0 m/sec) and a slowly conducting group (0-6 m/sec). In contrast, C neurones, like their preganglionic inputs, have only slowly conducting axons (0-3 m/sec). Consequently

The strength and number of nicotinic synapses that converge on secretomotor B neurons were assessed in the bullfrog by recording intracellularly from isolated preparations of paravertebral sympathetic ganglia 9 and 10. One input to every B neuron invariably produced a suprathreshold EPSP and was defined as the primary nicotinic synapse. In addition, 93% of the cells received one to four subthreshold inputs that were defined as secondary nicotinic synapses. This contradicts the prevailing view, which has long held that amphibian B neurons are singly innervated. More important, the results revealed that B cells provide the simplest possible experimental system for examining the role of secondary nicotinic synapses on sympathetic neurons. Combining the convergence data with previous estimates of divergence indicates that the average preganglionic B neuron forms connections with 50 ganglionic B neurons and that the majority of these nicotinic synapses are secondary in strength. Secondary EPSPs evoked by low-frequency stimulation ranged from 0.5 to 10 mV in amplitude and had an average quantal content of 1. Nonetheless, secondary synapses could trigger action potentials via four mechanisms: spontaneous fluctuations of EPSP amplitude, two-pulse facilitation, coactivation with other secondary synapses, and coactivation with a slow peptidergic EPSP. The data were used to formulate a stochastic theory of integration, which predicts that ganglia function as amplifiers of the sympathetic outflow. In this two-component scheme, primary nicotinic synapses mediate invariant synaptic gain, and secondary nicotinic synapses mediate activity-dependent synaptic gain. The model also provides a common framework for considering how facilitation, metabotropic mechanisms, and preganglionic oscillators regulate synaptic amplification in sympathetic ganglia.

The dynamic-clamp method provides a powerful electrophysiological tool for creating virtual ionic conductances in living cells and studying their influence on membrane potential. Here we describe G-clamp, a new way to implement a dynamic clamp using the real-time version of the Lab-VIEW programming environment together with a Windows host, an embedded microprocessor that runs a real-time operating system and a multifunction data-acquisition board. The software includes descriptions of a fast voltage-dependent sodium conductance, delayed rectifier, M-type and A-type potassium conductances, and a leak conductance. The system can also read synaptic conductance waveforms from preassembled data files. These virtual conductances can be reliably implemented at speeds < or =43 kHz while simultaneously saving two channels of data with 16-bit precision. G-clamp also includes utilities for measuring current-voltage relations, synaptic strength, and synaptic gain. Taking an approach built on a commercially available software/hardware platform has resulted in a system that is easy to assemble and upgrade. In addition, the graphical programming structure of LabVIEW should make it relatively easy for others to adapt G-clamp for new experimental applications.