M. Steven Evans

Dimethylsulfoxide (DMSO) blocks conduction in peripheral nerve C fibers: a possible mechanism of analgesia

Dimethylsulfoxide (DMSO) is readily absorbed through skin, and relieves musculoskeletal pain when applied topically to painful areas. We studied the effects of DMSO on C-type nerve fibers, which mediate pain sensation. DMSO was applied directly to exposed cat sural nerves. C fiber conduction velocity was slowed by DMSO, even in low concentrations (5-7% v/v). Higher concentrations completely blocked C fiber conduction, with a minimum blocking concentration of 9%. Onset of nerve block was almost immediate with 15% DMSO or higher concentrations. C fiber blockade may account for analgesia with DMSO.

Electrophysiology of embryonic, adult and aged rat hippocampal neurons in serum-free culture.

Methods were recently developed for culturing neurons from adult rat hippocampus using the serum-free medium Neurobasal with B27 supplement. To determine whether adult cultured neurons have normal electrical properties, we studied cultures from rats of three age groups: (1) embryonic; (2) 10-11 months old and (3) 35-36 months old. Neurons had a polarized morphology with a large branching apical dendrite and small basal dendrites. Mean resting potentials were similar in the three age groups. All neurons had nonlinear current-voltage relationships, indicating the presence of voltage-sensitive ion channels. Most neurons had a voltage-sensitive inward current followed by a sustained voltage-sensitive outward current. Tetrodotoxin blocked the inward current, which is likely to be a sodium current. The sustained outward current, which is likely to be a potassium current, reversed at -71 mV. Most neurons exhibited anomalous rectification. Calcium currents were present in both embryonic and adult neurons. Embryonic neurons would sometimes fire multiple action potentials but adult neurons fired only single action potentials. Our results indicate that both embryonic and adult cultured neurons retain a clearly neuronal electrophysiological phenotype in Neurobasal/B27 serum-free medium.

Midazolam inhibits long-term potentiation through modulation of GABAA receptors

Benzodiazepine drugs (BZ) are used for anxiety, insomnia, and seizures. They worsen memory, especially in large doses, but the mechanism of this action is uncertain. In micromolar concentrations, benzodiazepines have been shown to reduce long-term potentiation (LTP), which could be a cellular basis for their amnesic action. We have found that the LTP-inhibiting effects of BZ occur in the nanomolar concentrations attained in humans, and that this effect occurs through modulation of GABAA receptor function. We recorded extracellular synaptic input/output (I/O) curves for population spikes (PS) and EPSPs in rat hippocampal slices before and after induction of LTP. LTP increased maximal PS and EPSPs and shifted I/O curves for PS and EPSPs to the left, reflecting increased synaptic responsiveness after LTP. Curves relating EPSPs to PS were also shifted, so that after LTP larger PS were elicited for the same size EPSP (E-S potentiation). Midazolam (0.5 microM) markedly inhibited the left-shift in PS I/O curves due to E-S potentiation but did not significantly affect other parameters. 8-Phenyltheophylline (10 microM), an adenosine receptor antagonist, did not prevent midazolam inhibition of LTP. Bicuculline, a GABAA receptor antagonist, caused a dose-dependent antagonism of midazolams LTP inhibition. Our results suggest that benzodiazepines reduce LTP primarily through reduction of E-S potentiation, and that this effect occurs through modulation of GABAA receptor function. This could in part account for the ability of benzodiazepines to disturb new memory formation.

Loss of synaptic inhibition during repetitive stimulation in genetically epilepsy-prone rats (GEPR)

Genetically epilepsy-prone rats (GEPR) are an animal model of generalized motor seizures. The underlying causes of the predisposition to seizures in GEPR have not been fully determined. The brainstem auditory system is critical for audiogenic seizures in GEPR, and neurophysiological abnormalities have been observed in these areas, but recent evidence suggests that non-auditory brain areas may also be abnormal. This may account for the lowered threshold in GEPR for various non-audiogenic seizures. Because the normal responses of the hippocampal Schaffer collateral/CA1 synapse are relatively well understood, we studied single and repetitive synaptic responses in hippocampal slices of GEPR in vitro. Our hypothesis was that altered excitatory or inhibitory synaptic transmission may contribute to GEPR non-audiogenic seizure predisposition. We recorded extracellular EPSPs, population spikes, and afferent volleys in hippocampal area CA1, and compared GEPR responses to those of Sprague-Dawley (SD) rats, the strain from which GEPR were derived. GEPR responses to single synaptic stimuli were not significantly different from SD. The second of a pair of closely spaced EPSPs or population spikes was larger in both GEPR and SD (paired pulse facilitation), but the magnitude of population spike facilitation was significantly increased in GEPR. Short trains of four stimuli caused inhibition of population spike firing in SD, an effect that was much reduced in GEPR. When SD slices were treated with bicuculline, a GABAA receptor antagonist, enhanced paired pulse facilitation and loss of inhibition during trains of stimuli were seen, similar to the patterns seen in GEPR.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased GABA and increased glutamate receptor-mediated activity on inferior colliculus neurons in vitro are associated with susceptibility to ethanol withdrawal seizures.

Cessation of ethanol administration in ethanol-dependent rats results in an ethanol withdrawal (ETX) syndrome, including audiogenic seizures (AGS). The inferior colliculus (IC) is the initiation site for AGS, and membrane properties of IC neurons exhibit hyperexcitability during ETX. Previous studies observed that ETX alters GABA and glutamate neurotransmission in certain brain sites. The present study evaluated synaptic properties and actions of GABA or glutamate antagonists during ETX in IC dorsal cortex (ICd) neurons in brain slices from rats treated with ethanol intragastrically 3 times daily for 4 days. A significant increase of spontaneous action potentials (APs) was observed during ETX. The width, area and rise time of excitatory postsynaptic potentials (EPSPs) evoked by stimulation in the commissure of IC were significantly elevated during ETX. A fast EPSP was sensitive to block by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and a slow EPSP was sensitive to the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP5). However, during ETX the concentration of CNQX or AP5 needed to block these EPSPs was elevated significantly. Inhibitory postsynaptic potentials (IPSPs) in ICd neurons evoked in both normal and ETX rats were blocked by the GABA(A) antagonist, bicuculline. However, IPSPs during ETX displayed a significantly greater sensitivity to bicuculline. These data indicate that decreased GABA(A)-mediated inhibition and increased glutamate-mediated excitability in IC may both be critical mechanisms of AGS initiation during ETX, which is similar to observations in a genetic form of AGS. The common changes in IC neurotransmission in these AGS forms may be general mechanisms subserving AGS and other forms of auditory system pathophysiology in which the IC is implicated.

Evidence for decreased calcium dependent potassium conductance in hippocampal CA3 neurons of genetically epilepsy-prone rats

The genetically epilepsy-prone rat (GEPR) has become an important model to study genetic predisposition to epilepsy involving not only the brainstem but also forebrain structures. Previous work in CA1 hippocampal cells showed a reduction in spike frequency adaptation and only subtle changes in slow afterhyperpolarization (AHP). As important differences exist in calcium dependent potentials in the CA1 and CA3 hippocampal cells, we compared the membrane properties of hippocampal CA3 cells in GEPRs and Sprague-Dawley (SD) rats. There was no significant difference in the resting membrane potential, input resistance, charging time constant or rheobase between GEPRs and SD rat neurons. The action potential amplitude and the width at half maximal amplitude did not differ. A marked reduction in spike frequency adaptation accompanied by a very significant reduction in AHP was seen in the GEPR rats. Since calcium dependent potassium conductance produces both spike frequency adaptation and AHP, our results suggest that this conductance is reduced in the GEPR CA3 neurons.

Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception

Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.

Inferior colliculus neuronal membrane and synaptic properties in genetically epilepsy-prone rats

Previous studies using single-unit recording techniques have shown that the inferior colliculus is critical for audiogenic seizure initiation in genetically epilepsy-prone rats (GEPR). In order to investigate cellular abnormalities that may be important in causing audiogenic seizure susceptibility, intracellular recordings were made from neurons of inferior colliculus dorsal cortex (ICd) in a GEPR variety that exhibits severe audiogenic seizures (GEPR-9). GEPR neuronal membrane and synaptic properties were compared to those of normal Sprague-Dawley rats (SD), the strain from which GEPR were derived. We found six electrophysiological differences between GEPR and normal SD ICd neurons, all of which could promote seizures in GEPR. (1) Input resistance was higher in GEPR than in normal ICd neurons. (2) Threshold for repetitive action potential firing was closer to resting membrane potential in GEPR ICd neurons. (3) GEPR neurons showed faster repetitive spike firing than normal SD neurons. (4) Anode break spikes occurred at the offset of a hyperpolarizing pulse more often in GEPR than in normal SD neurons. (5) Stimulation of the commissure of the inferior colliculus caused synaptic paired pulse inhibition in normal ICd neurons, but paired pulse facilitation was always observed in GEPR neurons. (6) In GEPR, a large epileptiform depolarizing event could be elicited by strong electrical stimulation of the commissure of the inferior colliculus. In normal SD rats, similar epileptiform activity was seen only after application of bicuculline or NMDA. Our results suggest that both abnormal neuronal membrane properties and altered synaptic transmission are likely to contribute to seizure predisposition and audiogenic seizure initiation in GEPR.

Age-related differences in NMDA responses in cultured rat hippocampal neurons

The N-methyl-D-aspartate receptor (NMDAR) is expressed in the cerebral cortex and hippocampus and is important in learning and memory. NMDARs are influenced by aging and implicated in neurodegenerative disorders. We investigated age-related differences in NMDAR ionic currents and intracellular calcium in embryonic (E18), middle-age (9-10 month) and old (26 month) rat hippocampal neurons cultured in serum-free medium for 7-12 days. Responses to 200 microM NMDA with 50 microM glycine were measured using whole cell voltage clamp and fura-2 fluorescence. Embryonic neurons exhibited significantly larger and faster NMDA responses than adults. Old rats had 1.5 fold greater normalized NMDA peak current compared to middle-age rats, while intracellular calcium rose 1.3 fold higher. Differences in regression slopes generated from the integral of NMDA current versus normalized NMDA current indicate age-related differences are not exclusively due to changes in receptor density but likely influenced by changes in receptor function. Corresponding age-related measures of intracellular calcium by fura-2 fluorescence in response to NMDA showed a strong correlation with peak current (r(2)=0.996). Our data support the hypothesis that NMDAR responsiveness is altered during aging with an enhanced NMDA peak current in both old and embryonic neurons compared to middle-age neurons.

N-Type Ca2+ Channels in Cultured Rat Sphenopalatine Ganglion Neurons: An Immunohistochemical and Electrophysiological Study

Results from pharmacological studies have suggested that presynaptic N-type Ca2+ channels play an important role in regulating neuronal Ca2+ influx and transmitter nitric oxide (NO) release in isolated cerebral arteries. However, the presence of N-type Ca2+ channels in cerebral perivascular nerves has not been directly demonstrated. As a major source of cerebral perivascular NOergic innervation is the sphenopalatine ganglion (SPG), adult rat SPGs were cultured and examined by whole-cell patch-clamp technique. One week after growing in the culture medium, significant neurite outgrowth from the SPG neuronal cells was observed. Both soma and neurites of these cells were immunoreactive for N-type Ca2+ channels, transmitter-synthesizing enzymes (choline acetyltransferase and NO synthase), and several neuropeptides (vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and pituitary adenylate cyclase-activating peptide-38) that had been found in cerebral perivascular nerves in whole-mount vascular preparations. In current-clamp recordings, injection of a small depolarizing current caused action potential firing. In voltage-clamp recordings, the fast inward currents were blocked by tetrodotoxin and outward currents by tetraethylammonium, which is typical for neurons. Most Ca2+ currents isolated by blockade of sodium and potassium currents were blocked by ω-conotoxin, indicating that N-type Ca2+ channels are the dominant voltage-dependent Ca2+ channels regulating Ca2+ influx during membrane depolarization of SPG neurons. The ability to culture postganglionic SPG neurons provides an opportunity to directly study the electrophysiological and pharmacological properties of these neurons.