High-frequency alternating current (HFAC) is known to disrupt axonal conduction in peripheral nerves, and HFAC has much potential as a therapeutic approach for a number of pathological conditions. Many previous studies have utilized motor output as a bioassay of effects of HFAC on conduction through medium- to large-diameter motor axons. However, little is known about the effectiveness of HFAC on smaller, more slowly conducting nerve fibres. The present study tested whether HFAC influences axonal conduction through sub-diaphragmatic levels of the rat vagus nerve, which consists almost entirely of small calibre axons. Using an isolated nerve preparation, we tested the effects of HFAC on electrically evoked compound action potentials (CAPs). We found that delivery of charge-balanced HFAC at 5000 Hz for 1 min was effective in producing reversible blockade of axonal conduction. Both Aδ and C components of the vagus CAP were attenuated, and the degree of blockade as well as time to recovery was proportional to the amount of HFAC current delivered. The Aδ waves were more sensitive than C waves to HFAC blockade, but they required more time to recover.

Dendritic degeneration and loss of synaptic proteins are early events correlated with functional decline in neurodegenerative disease. The temporal and mechanistic relationship between synapse loss and cell death, however, remains unclear. We used confocal microscopy and image processing to count post‐synaptic sites on rat hippocampal neurons by expressing post‐synaptic density protein 95 fused to green fluorescent protein. Fluorescent puncta co‐localized with neurotransmitter release sites, NMDA‐induced Ca2+ increases and NMDA receptor immunoreactivity. During excitotoxic neurodegeneration, synaptic sites were lost and synaptic transmission impaired. These changes were mediated by NMDA receptors and required Ca2+‐dependent activation of the proteasome pathway. Tracking synapses from the same cell following brief neurotoxic insult revealed transient loss followed by recovery. The time‐course, concentration‐dependence and mechanism for loss of post‐synaptic sites were distinct from those leading to cell death. Cells expressing p14ARF, which inhibits ubiquitination of post‐synaptic density protein 95 and prevents loss of synaptic sites, displayed an increased sensitivity to glutamate‐induced cell death. Thus, excitotoxic synapse loss may be a disease‐modifying process rather than an obligatory step leading to cell death. These results demonstrate the importance of assessing synaptic function independent of neuronal survival during neurodegeneration and indicate that this approach will be useful for identifying toxins that degrade synaptic connections and for screening for agents that protect synaptic function.

RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner G␤5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2⅐G␤5⅐R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.G protein signaling pathways mediate a wide range of critical neuronal processes (1). The normal functioning of these pathways is dependent on the tight control of signal duration mediated by regulators of G protein signaling (RGS) 2 proteins (2, 3). RGS proteins act to control the inactivation of heterotrimeric G proteins by dramatically accelerating the rate of their GTP hydrolysis (4, 5). Much recent attention has been focused on the R7 subfamily of RGS proteins due to their emerging roles in the regulation of a variety of critical neuronal functions (6, 7). The R7 subfamily contains four highly homologous proteins, RGS6, RGS7, RGS9, and RGS11, which are expressed exclusively in the nervous system (8) as constitutive complexes with a type 5 G protein ␤ subunit (G␤5) (9 -11).RGS9, with its two splice isoforms, is one of the best-studied members of the R7 subfamily. The short isoform RGS9-1 is expressed in the photoreceptor neurons where it regulates the visual signal transduction cascade (12, 13). The long splice isoform RGS9-2 is predominantly expressed in the striatum and the central nervous system structures, mediating nociceptive responses where it was shown to regulate dopamine and -opioid pathways (14 -17). Disruption of RGS9-2 in mice resulted in the stimulation of locomotor and reward behavior, increased physical dependence and analgesic effects of morphine, and movement disorders (15)(16)(17). In photoreceptors, the function ...

Dendritic pruning and loss of synaptic contacts are early events in many neurodegenerative diseases. These effects are dynamic and seem to differ mechanistically from the cell death process. Cannabinoids modulate synaptic activity and afford protection in some neurotoxicity models. We investigated the effects of cannabinoids on activity-induced changes in the number of synapses between rat hippocampal neurons in culture. Morphology and synapses were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 (PSD95) fused to enhanced green fluorescent protein (GFP). Reducing the extracellular Mg 2ϩ concentration to 0.1 mM for 4 h induced intense synaptic activity, which decreased the number of PSD95-GFP puncta by 45 Ϯ 13%. The protection was mimicked by inhibition of presynaptic Ca 2ϩ channels, and WIN55,212-2 did not prevent PSD loss elicited by direct application of glutamate, suggesting a presynaptic mechanism. Prolonged exposure to WIN55,212-2, but not THC, desensitized the protective effect. Treating cells that had undergone PSD loss with WIN55,212-2 reversed the loss and enabled recovery of a full compliment of synapses. The modulation of synaptic number by acute and prolonged exposure to cannabinoids may account for some of the effects of these drugs on the plasticity, survival, and function of neural networks.