Patch-clamp analysis of nicotinic synapses whose strength straddles the firing threshold of rat sympathetic neurons.

Neurons in paravertebral sympathetic ganglia are innervated by converging nicotinic synapses of varying strength. Based upon intracellular recordings of excitatory postsynaptic potentials (EPSPs) with sharp microelectrodes these synapses were classified in the past as either primary (strong) or secondary (weak) by their ability to trigger postsynaptic action potentials. Here we present an analysis of 22 synapses whose strength straddled threshold, thereby distinguishing them from the original classification scheme for primary and secondary synapses.

HCN hyperpolarization-activated cation channels strengthen virtual nicotinic EPSPs and thereby elevate synaptic amplification in rat sympathetic neurons.

The influence of hyperpolarization-activated cation current (h-current; Ih) upon synaptic integration in paravertebral sympathetic neurons was studied together with expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunit isoforms. All four HCN subunits were detected in homogenates of the rat superior cervical ganglion (SCG) using the PCR to amplify reverse-transcribed messenger RNAs (RT-PCR) and using quantitative PCR. Voltage clamp recordings from dissociated SCG neurons at 35°C detected Ih in all cells, with a maximum hyperpolarization-activated cation conductance of 1.

Topoisomerase 1 inhibition reversibly impairs synaptic function.

Topotecan is a topoisomerase 1 (TOP1) inhibitor that is used to treat various forms of cancer. We recently found that topotecan reduces the expression of multiple long genes, including many neuronal genes linked to synapses and autism. However, whether topotecan alters synaptic protein levels and synapse function is currently unknown.

Virtual leak channels modulate firing dynamics and synaptic integration in rat sympathetic neurons: implications for ganglionic transmission in vivo.

Key Points: The synaptic organization of paravertebral sympathetic ganglia enables them to relay activity from the spinal cord to the periphery and thereby control autonomic functions, including blood pressure and body temperature. The present experiments were done to reconcile conflicting observations in tissue culture, intact isolated ganglia and living animals. By recording intracellularly from dissociated neurons and intact ganglia, we found that when electrode damage makes cells leaky it could profoundly distort cellular excitability and the integration of synaptic potentials.

Vasomotor sympathetic neurons are more excitable than secretomotor sympathetic neurons in bullfrog paravertebral ganglia.

We compared the excitability of secretomotor B and vasomotor C neurons using virtual nicotinic synapses implemented with the dynamic clamp technique. In response to fast synaptic conductance (g(syn)) waveforms modeled after B cell synaptic currents, it took 17.1+/-1.

Homeostatic regulation of M-current modulates synaptic integration in secretomotor, but not vasomotor, sympathetic neurons in the bullfrog.

We compared how vasomotor C neurons and secretomotor B neurons integrated identical patterns of virtual synaptic activity using dynamic clamp, perforated-patch recordings from dissociated bullfrog sympathetic ganglion cells. The synaptic template modelled one strong nicotinic synapse and nine weak synapses, each firing randomly at 5 Hz, with strength normalized to each cell. B neurons initially fired at 12 Hz, but this declined within seconds, decreasing 27% after 40 s and recovering slowly as evidenced by the threshold synaptic conductance for firing (tau(recovery) = 136 + or - 23 s).

NMDAR-Mediated Calcium Transients Elicited by Glutamate Co-Release at Developing Inhibitory Synapses.

Before hearing onset, the topographic organization of the inhibitory sound localization pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) is refined by means of synaptic silencing and strengthening. During this refinement period MNTB-LSO synapses not only release GABA and glycine but also release glutamate. This co-released glutamate can elicit postsynaptic currents that are predominantly mediated by NMDA receptors (NMDARs).

Cav1.3 channel voltage dependence, not Ca2+ selectivity, drives pacemaker activity and amplifies bursts in nigral dopamine neurons.

Ca(v)1.3 (alpha 1D) L-type Ca(2+) 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(2+) elevation produced by Ca(v)1.

Dopamine neuron responses depend exponentially on pacemaker interval.

Midbrain dopamine neuron activity results from the integration of the responses to metabo- and ionotropic receptors with the postsynaptic excitability of these intrinsic pacemakers. Interestingly, intrinsic pacemaker rate varies greatly between individual dopamine neurons and is subject to short- and long-term regulation. Here responses of substantia nigra dopamine neurons to defined dynamic-clamp stimuli were measured to quantify the impact of cell-to-cell variation in intrinsic pacemaker rate.

Excitatory muscarinic modulation strengthens virtual nicotinic synapses on sympathetic neurons and thereby enhances synaptic gain.

Acetylcholine excites many neuronal types by binding to postsynaptic m1-muscarinic receptors that signal to ion channels through the G(q/11) protein. To investigate the functional significance of this metabotropic pathway in sympathetic ganglia, we studied how muscarinic excitation modulated the integration of virtual nicotinic excitatory postsynaptic potentials (EPSPs) created in dissociated bullfrog B-type sympathetic neurons with the dynamic-clamp technique. Muscarine (1 muM) strengthened the impact of virtual synapses by reducing the artificial nicotinic conductance required to reach the postsynaptic firing threshold from 20.

D2 autoreceptors chronically enhance dopamine neuron pacemaker activity.

Activation of D2 autoreceptors on midbrain dopamine neurons has been shown previously to acutely open K+ channels to inhibit intrinsically generated pacemaker activity. Here we report that D2 autoreceptors act chronically to produce an opposite action: to increase the speed and regularity of repetitive action potential firing. Voltage-, current-, and dynamic-clamp experiments, using conventional whole-cell and perforated patch-clamp recording, with cultured rat midbrain dopamine neurons show that a change in the number of functional A-type K+ channels alters firing rate and susceptibility to irregularity produced by other channels.

Synthesis, photophysical, photochemical and biological properties of caged GABA, 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy) carbonyl] amino] butanoic acid.

The photorelease of a caged neurotransmitter can be used to investigate the function of neuronal circuits in tissues. We have designed and synthesized a stable, caged gamma-aminobutyric acid (GABA) derivative, 4-[[(2H-1-benzopyran-2-one-7-amino-4-methoxy)carbonyl]amino] butanoic acid (BC204), that releases the neurotransmitter in physiological medium when irradiated with UV light at 300-400 nm in PBS at pH 7.4.

Estimating use-dependent synaptic gain in autonomic ganglia by computational simulation and dynamic-clamp analysis.

Biological gain mechanisms regulate the sensitivity and dynamics of signaling pathways at the systemic, cellular, and molecular levels. In the sympathetic nervous system, gain in sensory-motor feedback loops is essential for homeostatic regulation of blood pressure and body temperature. This study shows how synaptic convergence and plasticity can interact to generate synaptic gain in autonomic ganglia and thereby enhance homeostatic control.

Implementation of a fast 16-Bit dynamic clamp using LabVIEW-RT.

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.

Glutamatergic calcium responses in the developing lateral superior olive: receptor types and their specific activation by synaptic activity patterns.

The lateral superior olive (LSO) is a binaural auditory brain stem nucleus that plays a central role in sound localization. Survival and maturation of developing LSO neurons critically depend on intracellular calcium signaling. Here we investigated the mechanisms by which glutamatergic afferents from the cochlear nucleus increase intracellular calcium concentration in LSO neurons.

Excitatory action of an immature glycinergic/GABAergic sound localization pathway.

Most mammals determine the azimuthal direction of incoming sound using auditory cues arising from differences in interaural sound intensity. The first station in the ascending auditory pathway, which processes interaural intensity differences, is the lateral superior olive (LSO), a binaural nucleus in the auditory brainstem. LSO neurons encode interaural intensity differences by integrating excitatory input from the ipsilateral cochlea and inhibitory input from the contralateral cochlea.

Glycinergic and GABAergic calcium responses in the developing lateral superior olive.

The lateral superior olive (LSO), a binaural nucleus involved in sound localization, receives tonotopically organized inhibitory inputs from the medial nucleus of the trapezoid body (MNTB). During development, the tonotopic organization of this glycinergic/GABAergic MNTB-LSO pathway is established by activity-dependent axonal reorganization. However, the underlying mechanisms by which this reorganization takes place have remained largely unknown.