Behavioral and electrophysiological abnormalities in two rat models of antiretroviral drug-induced neuropathy.

Painful peripheral neuropathy due to the antiretroviral therapy used to treat HIV is one of the most prevalent side effects occurring in at least 30% of patients living with this infection. We have evaluated the electrophysiological and behavioral effects of d4T and ddC on peripheral large and small nerve fibers in male rats treated with d4T (Sprague-Dawley, 50 mg/kg, twice within 1 week), ddC (Wistar, 50 mg/kg, 3 times per week for 3 weeks), or vehicle. The effect of the interventions was assessed using behavioral measures of mechanical sensitivity, conventional nerve conduction studies, and microneurographic single nerve C-fiber recordings. To mimic as much as possible the human clinical condition, all treated animals were included in the study. No statistically significant differences were observed in behavioral parameters of mechanical sensitivity. Nerve conduction studies did not reveal any significant change in the ddC-treated group. In contrast, we observed electrophysiological evidence of significant demyelinating neuropathy 1 week after the start of d4T treatment. Additionally, spontaneous activity in mechanoinsensitive C-nociceptors was observed in both drug-treated groups. No relationship could be established between measures of spontaneous activity in C-nociceptors and the results of the behavioral tests. Our results show that both models of antiretroviral-induced neuropathy differ in their effects on peripheral nerves. However, both groups present abnormal spontaneous activity in mechanoinsensitive C-nociceptors that can be used as a model for pharmacological intervention.

Levetiracetam accelerates the onset of supply rate depression in synaptic vesicle trafficking.

OBJECTIVE: To determine if levetiracetam (LEV) enhances the impact in excitatory presynaptic terminals of a rate-limiting mechanism in vesicle trafficking termed supply rate depression that emerges to limit synaptic transmission during heavy, epileptiform use. METHODS: The effect of LEV was measured with electrophysiologic assays of monosynaptic connections in ex vivo hippocampal slices from wild-type and synapsin knockout mice, and in primary cell culture neurons from wild-type and synaptic vesicle glycoprotein 2a (SV2a) knockout mice. RESULTS: LEV enhanced the impact of supply rate depression at Schaffer collateral synapses by shortening the time course for induction. The LEV effect was selective for supply rate depression because other presynaptic vesicle trafficking mechanisms were not affected. The half maximal effective concentration (EC50 ) was ~50 µm. The maximal effect was ~15% and occurred at 100 µm, which is a clinically relevant concentration. An experimental protocol is established for distinguishing atypical antiepileptic drugs (AEDs) that affect supply rate depression, such as LEV, from typical AEDs, such as carbamazepine, that affect upstream mechanisms. The LEV effect was abolished at synapses from knockout mice lacking SV2a and from synapses lacking synapsin 1 and 2. SIGNIFICANCE: The findings are consistent with the new hypothesis that LEV acts to treat epilepsy by accelerating the induction of supply rate depression at excitatory synapses during incipient epileptic activity. The absence of the effect in the knockouts confirms that presynaptic function is the target. More specifically, the absence in SV2a knockouts is consistent with previous binding studies suggesting that SV2a is the target for LEV. The absence in synapsin knockouts indicates that the phenotypic target intersects with the biochemical pathway that is altered in synapsin knockouts. The results from synapsin knockouts additionally suggest that development of functional analogs with increased potency might be possible because induction of supply rate depression is faster in synapsin knockouts compared to wild-type synapses treated with LEV.

A new kinetic framework for synaptic vesicle trafficking tested in synapsin knock-outs.

At least two rate-limiting mechanisms in vesicle trafficking operate at mouse Schaffer collateral synapses, but their molecular/physical identities are unknown. The first mechanism determines the baseline rate at which reserve vesicles are supplied to a readily releasable pool. The second causes the supply rate to depress threefold when synaptic transmission is driven hard for extended periods. Previous models invoked depletion of a reserve vesicle pool to explain the reductions in the supply rate, but the mass-action assumption at their core is not compatible with kinetic measurements of neurotransmission under maximal-use conditions. Here we develop a new theoretical model of rate-limiting steps in vesicle trafficking that is compatible with previous and new measurements. A physical interpretation is proposed where local reserve pools consisting of four vesicles are tethered to individual release sites and are replenished stochastically in an all-or-none fashion. We then show that the supply rate depresses more rapidly in synapsin knock-outs and that the phenotype can be fully explained by changing the value of the single parameter in the model that would specify the size of the local reserve pools. Vesicle-trafficking rates between pools were not affected. Finally, optical imaging experiments argue against alternative interpretations of the theoretical model where vesicle trafficking is inhibited without reserve pool depletion. This new conceptual framework will be useful for distinguishing which of the multiple molecular and cell biological mechanisms involved in vesicle trafficking are rate limiting at different levels of synaptic throughput and are thus candidates for physiological and pharmacological modulation.

Kinetic isolation of a slowly recovering component of short-term depression during exhaustive use at excitatory hippocampal synapses.

This study examines the kinetics of the longest lasting form of short-term depression at excitatory hippocampal synapses. After initial depletion of the readily releasable pool (RRP), continued 20-Hz stimulation was found to be fast enough to maximally drive presynaptic neurotransmitter exocytosis; maximal is defined here as the rate needed to maintain the RRP in a nearly empty steady state. Induction of depression proceeded in two distinct phases. The first was caused by RRP depletion, whereas the second is shown to reflect the progressive reduction of the overall rate at which new vesicles are supplied to the RRP and is termed "supply-rate depression." Supply-rate depression is identified further with the emergence, during heavy use, of a rate-limiting vesicle trafficking step that slows the timing of RRP replenishment by switching from a fast (tau congruent with 7 s) to a slow (tau congruent with 1 min) vesicle supply mechanism. Both mechanisms apparently follow first-order kinetics. After the induction of the maximum amount of depression, individual synapses were able to output only <1 quantum of neurotransmitter per synapse per second, matching previous predictions based on cell biological measurements of synaptic vesicle cycling. Surprisingly, the onset of supply-rate depression occurred with a marked delay, not having a detectable impact on synaptic function until after several seconds of continuous use. The delayed onset is not consistent with traditional vesicle trafficking models, but may be important for limiting the impact of supply-rate depression to pathological episodes and might function as a native antiepilepsy device.

Augmentation controls the fast rebound from depression at excitatory hippocampal synapses.

Short-term plasticity occurs at most central chemical synapses and includes both positive and negative components, but the principles governing interaction between components are largely unknown. The residual Ca(2+) that persists in presynaptic terminals for several seconds after repetitive use is known to enhance neurotransmitter release under artificial, low probability of release conditions where depression is absent; this is termed augmentation. However, the full impact of augmentation under standard conditions at synapses where depression dominates is not known because of possibly complicated convolution with a variety of potential depression mechanisms. This report shows that residual Ca(2+) continues to have a large enhancing impact on release at excitatory hippocampal synapses recovering from depression, including when only recently recruited vesicles are available for release. No evidence was found for gradual vesicle priming or for fast refilling of a highly releasable subdivision of the readily releasable pool (RRP). And decay of enhancement matched the clearance of residual Ca(2+), thus matching the behavior of augmentation when studied in isolation. Because of incomplete RRP replenishment, synaptic strength was not typically increased above baseline when residual Ca(2+) levels were highest. Instead residual Ca(2+) caused single pulse release probability to rebound quickly from depression and then depress quickly during subsequent bursts of activity. Together, these observations can help resolve discrepancies in recent timing estimates of recovery from depression. Additionally, in contrast to results obtained under reduced release conditions, augmentation could be driven to a maximal level, occluding paired-pulse facilitation and other mechanisms that increase release efficiency.

On the dynamics of the spontaneous activity in neuronal networks.

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABA(A) receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics.

Spontaneous electrical activity and behavior in the leech hirudo medicinalis.

In the absence of external stimuli, animals explore the environment by performing irregular movements, but the neuronal mechanisms underlying this arrhythmic motion are largely unknown. In this paper, we studied the relationship between the spontaneous neuronal activity in the leech (Hirudo medicinalis) and its behavior. We analyzed the electrical activity of isolated ganglia, chains of two connected ganglia, and semi-intact preparations. The spontaneous electrical activity in ganglia was characterized by the occurrence of irregular bursts of spikes with variable duration and size. Properties of these bursts were modified by synaptic inputs arriving from the neighboring ganglia and from the two primitive brains located in the head and tail. In fact, in semi-intact preparations, unusually large bursts of spikes occurring spontaneously were recorded and caused the leech to move even in the absence of any external sensory stimulation. These large bursts appear to act as internal triggers controlling the spontaneous leech behavior and determining the duration of stereotypical motor patterns.

Statistics of decision making in the leech.

Animals continuously decide among different behaviors, but, even in invertebrates, the mechanisms underlying choice and decision are unknown. In this article, leech spontaneous behavior was tracked and quantified for up to 12 h. We obtained a statistical characterization, in space and time domains, of the decision processes underlying selection of behavior in the leech. We found that the spatial distribution of leech position in a uniform environment is isotropic (the same in all directions), but this isotropy is broken in the presence of localized external stimuli. In the time domain, transitions among behaviors can be described by a Markov process, the structure of which (allowed states and transitions) is highly conserved across individuals. Finally, a wide range of recurrent, deterministic motifs was identified in the apparently irregular and unstructured exploratory behavior. These results provide a rigorous description of the inner dynamics that control the spontaneous and continuous flow of behavioral decisions in the leech.

Quantitative characterization and classification of leech behavior.

This paper describes an automatic system for the analysis and classification of leech behavior. Three colored beads were attached to the dorsal side of a free moving or pinned leech, and color CCD camera images were taken of the animal. The leech was restrained to moving in a small tank or petri dish, where the water level can be varied. An automatic system based on color processing tracked the colored beads over time, allowing real-time monitoring of the leech motion for several hours. At the end of each experimental session, six time series (2 for each bead) describing the leech body motion were obtained. A statistical analysis based on the speed and frequency content of bead motion indicated the existence of several stereotypical patterns of motion, corresponding to different leech behaviors. The identified patterns corresponded to swimming, pseudo-swimming, crawling, exploratory behavior, stationary states, abrupt movements, and combinations of these behaviors. The automatic characterization of leech behavior demonstrated here represents an important step toward understanding leech behavior and its properties. This method can be used to characterize the behavior of other invertebrates and also for some small vertebrates.

Dynamics and reproducibility of a moderately complex sensory-motor response in the medicinal leech.

Local bending, a motor response caused by mechanical stimulation of the leech skin, has been shown to be remarkably reproducible, in its initial phase, despite the highly variable firing of motoneurons sustaining it. In this work, the reproducibility of local bending was further analyzed by monitoring it over a longer period of time and by using more intact preparations, in which muscle activation in an entire body segment was studied. Our experiments showed that local bending is a moderately complex motor response, composed of a sequence of four different phases, which were consistently identified in all leeches. During each phase, longitudinal and circular muscles in specific areas of the body segment acted synergistically, being co-activated or co-inhibited depending on their position relative to the stimulation site. Onset and duration of the first phase were reproducible across different trials and different animals as a result of the massive co-activation of excitatory motoneurons sustaining it. The other phases were produced by the inhibition of excitatory and activation of inhibitory motoneurons, and also by the intrinsic relaxation dynamics of leech muscles. As a consequence, their duration and relative timing was variable across different preparations, whereas their order of appearance was conserved. These results suggest that, during local bending, the leech neuromuscular system 1) operates a reduction of its available degrees of freedom, by simultaneously recruiting groups of otherwise antagonistic muscles and large populations of motoneurons; and 2) ensures reliability and effectiveness of this escape reflex, by guaranteeing the reproducibility of its crucial initial phase.

Synaptic integration in electrically coupled neurons.

Interactions among chemical and electrical synapses regulate the patterns of electrical activity of vertebrate and invertebrate neurons. In this investigation we studied how electrical coupling influences the integration of excitatory postsynaptic potentials (EPSPs). Pairs of Retzius neurons of the leech are coupled by a nonrectifying electrical synapse by which chemically induced synaptic currents flow from one neuron to the other. Results from electrophysiology and modeling suggest that chemical synaptic inputs are located on the coupled neurites, at 7.5 microm from the electrical synapses. We also showed that the space constant of the coupled neurites was 100 microm, approximately twice their length, allowing the efficient spread of synaptic currents all along both coupled neurites. Based on this cytoarchitecture, our main finding was that the degree of electrical coupling modulates the amplitude of EPSPs in the driving neurite by regulating the leak of synaptic current to the coupled neurite, so that the amplitude of EPSPs in the driving neurite was proportional to the value of the coupling resistance. In contrast, synaptic currents arriving at the coupled neurite through the electrical synapse produced EPSPs of constant amplitude. This was because the coupling resistance value had inverse effects on the amount of current arriving and on the impedance of the neurite. We propose that by modulating the amplitude of EPSPs, electrical synapses could regulate the firing frequency of neurons.