Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease.

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.

Baseline glutamate levels affect group I and II mGluRs in layer V pyramidal neurons of rat sensorimotor cortex.

Possible functional roles for glutamate that is detectable at low concentrations in the extracellular space of intact brain and brain slices have not been explored. To determine whether this endogenous glutamate acts on metabotropic glutamate receptors (mGluRs), we obtained whole cell recordings from layer V pyramidal neurons of rat sensorimotor cortical slices. Blockade of mGluRs with (+)-alpha-amino-4-carboxy-alpha-methyl-benzeacetic acid (MCPG, a general mGluR antagonist) increased the mean amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), an effect attributable to a selective increase in the occurrence of large amplitude sEPSCs. 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495, a group II antagonist) increased, but R(-)-1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid (AIDA) and (RS)-hexyl-HIBO (group I antagonists) decreased sEPSC amplitude, and (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III antagonist) did not change it. The change in sEPSCs elicited by MCPG, AIDA, and LY341495 was absent in tetrodotoxin, suggesting that it was action potential-dependent. The increase in sEPSCs persisted in GABA receptor antagonists, indicating that it was not due to effects on inhibitory interneurons. AIDA and (S)-3,5-dihydroxyphenylglycine (DHPG, a group I agonist) elicited positive and negative shifts in holding current, respectively. LY341495 and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, a group II agonist) elicited negative and positive shifts in holding current, respectively. The AIDA and LY341495 elicited currents persisted in TTX. Finally, in current clamp, LY341495 depolarized cells by approximately 2 mV and increased the number of action potentials to a given depolarizing current pulse. Thus ambient levels of glutamate tonically activate mGluRs and regulate cortical excitability.

Neuropeptide Y receptors differentially modulate G-protein-activated inwardly rectifying K+ channels and high-voltage-activated Ca2+ channels in rat thalamic neurons.

1. Using whole-cell patch-clamp recordings, infrared videomicroscopy and fast focal solution exchange methods, the actions of neuropeptide Y (NPY) were examined in thalamic slices of postnatal (10-16 days) rats. 2. NPY activated a K+-selective current in neurons of the thalamic reticular nucleus (RT; 20/29 neurons) and ventral basal complex (VB; 19/25 neurons). The currents in both nuclei had activation and deactivation kinetics that were very similar to those of GABAB receptor-induced currents, were totally blocked by 0.1 mM Ba2+ and showed voltage-dependent relaxation. These properties indicate that the NPY-sensitive K+ current is mediated by G-protein-activated, inwardly rectifying K+ (GIRK) channels. 3. In RT neurons, NPY application reversibly reduced high-voltage-activated (HVA) currents to 33 +/- 5 % (n = 40) of the control level but did not affect the T-type currents. Inhibition of Ca2+ currents was voltage independent and was largely mediated by effects on N- and P/Q-type channels. 4. NPY activation of GIRK channels was mediated via NPY1 receptors, whereas inhibition of N- and P/Q-type Ca2+ channels was mediated by NPY2 receptors. 5. These results show that neuropeptide Y activates K+ channels and simultaneously inhibits HVA Ca2+ channels via different receptor subtypes.

Differential regulation of GABA release and neuronal excitability mediated by neuropeptide Y1 and Y2 receptors in rat thalamic neurons.

1. Neuropeptide Y (NPY) produced inhibitory effects on neurons of the thalamic reticular nucleus (RT; n = 18) and adjacent ventral basal complex (VB; n = 22), which included hyperpolarization (approximately 4 mV), a reduction in rebound and regular spikes and an increased membrane conductance. These effects were mediated predominantly via NPY1 receptor activation of G-protein-activated, inwardly rectifying K+ (GIRK) channels. 2. NPY reduced the frequency of spontaneous GABAA receptor-mediated inhibitory postsynaptic currents (sIPSCs) in RT (by 60 +/- 7 %, n = 14) and VB neurons (by 25 +/- 11 %, n = 16), but had no effect on the kinetic properties of sIPSCs. After removal of the RT nucleus, the inhibitory effects of NPY on sIPSCs in VB neurons remained (29 +/- 7 %, n = 5). The synaptic effects were mediated via NPY2 receptors. 3. NPY inhibited the frequency of miniature IPSCs (mIPSCs) in RT and VB neurons (by 63 +/- 7 %, n = 5, and 37 +/- 8 %, n = 10, respectively) in the presence of tetrodotoxin (TTX) (1 microM) but not TTX (1 microM) and Cd2+ (200 microM). 4. NPY inhibited evoked IPSCs in both RT (by 18 +/- 3 %, n = 6) and VB (by 5 +/- 4 %, n = 6) neurons without change in short-term synaptic plasticity. 5. We conclude that NPY1 and NPY2 receptors are functionally segregated in the thalamus: NPY1 receptors are predominantly expressed at the somata and dendrites and directly reduce the excitability of neurons in both the RT and VB nuclei by activating GIRK channels. NPY2 receptors are located at recurrent (RT) and feed-forward GABAergic terminals (VB) and downregulate GABA release via inhibition of Ca2+ influx from voltage-gated Ca2+ channels.

Adrenergic modulation of GABAA receptor-mediated inhibition in rat sensorimotor cortex.

The effect of adrenoceptor activation on pharmacologically isolated monosynaptic inhibitory postsynaptic currents (IPSCs) detected in layer V pyramidal neurons was examined by using whole cell voltage-clamp in a slice preparation of rat sensorimotor cortex. Epinephrine (EPI; 10 muM) reversibly altered the amplitude of evoked IPSCs (eIPSCs) in slices from postnatal day 9-12 (P9-12) and P15-18 rats. The effects of EPI were heterogeneous in both age groups, and in individual cases an enhancement, a depression or no effect of eIPSCs was observed, although depression was observed more commonly in the younger age group. The effects of EPI on eIPSC amplitude were likely mediated through presynaptic mechanisms because they occurred in the absence of any alteration in the current produced by direct application of gamma-aminobutyric acid (GABA), or in input resistance. EPI always elicited an increase in the frequency of spontaneous IPSCs (sIPSCs) irrespective of whether or not it induced any change in the amplitude of eIPSCs in the same neuron. The increase in sIPSC frequency was blocked by phentolamine (10 muM) but not by propranolol (10 muM), supporting the conclusion that EPI-mediated effects on sIPSC frequency result from activation of alpha-adrenoceptors located on presynaptic inhibitory interneurons. In a subpopulation of neurons (3/9) from P15-18 rats, EPI increased both the amplitude and frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin (TTX) and under conditions where postsynaptic EPI effects were blocked, suggesting activation of adrenoceptors on presynaptic terminals in these cells. Results of these experiments are consistent with an action of EPI at adrenoceptors located on presynaptic GABAergic interneurons. Adrenergic activation thus has multiple and complex influences on excitability in cortical circuits, some of which are a consequence of interactions that regulate the strength of GABAergic inhibition.

GABAA receptor-mediated Cl- currents in rat thalamic reticular and relay neurons.

GABAA receptor-mediated Cl- currents in rat thalamic reticular and relay neurons. J. Neurophysiol. 78: 2280-2286, 1997. Spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs) and responses to exogenously applied gamma-aminobutyric acid (GABA), mediated by GABA type A (GABAA) receptors, were recorded in inhibitory neurons of nucleus reticularis thalami (nRt) and their target relay cells in ventrobasal (VB) nuclei by using patch clamp techniques in rat thalamic slices. The decay of sIPSCs in both nRt and VB neurons was best fitted with two exponential components. The decay time constants of sIPSCs in nRt neurons were much slower (tau1 = 38 ms; tau2 = 186 ms) than those previously reported in a variety of preparations and two to three times slower than those in VB neurons (tau1 = 17 ms; tau2 = 39 ms). GABAA receptor-mediated Cl- currents directly evoked by local GABA application also had a much slower decay time constant in nRt (225 ms) than in VB neurons (115 ms). Slow decay of GABA responses enhances the efficacy of recurrent intranuclear inhibition in nRt. The results suggest a functional diversity of GABAA receptors that may relate to the known heterogeneity of GABAA receptor subunits in these two thalamic nuclei.

Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus.

Detailed information regarding the contribution of individual gamma-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. "Weak" inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, "strong" inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1-3 release sites, whereas stronger inhibition would require simultaneous activation of 5-70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.

Peptidergic modulation of intrathalamic circuit activity in vitro: actions of cholecystokinin.

Cholecystokinin (CCK)-mediated actions on intrathalamic rhythmic activities were examined in an in vitro rat thalamic slice preparation. Single electrical stimuli in the thalamic reticular nucleus (nRt) evoked rhythmic activity (1-15 sec duration) in nRt and the adjacent ventrobasal nucleus (VB). Low CCK concentrations (20-50 nM) suppressed rhythmic oscillations in 43% of experiments but prolonged such activities in the remaining slices. Higher CCK concentrations (100-400 nM) had a predominantly antioscillatory effect. Suppression of oscillations was associated with a relatively large membrane depolarization of nRt neurons that changed their firing mode from phasic (burst) to tonic (single-spike) output. This decreased burst discharge of nRt neurons during CCK application reduced inhibitory drive onto VB neurons from multiple peaked inhibitory postsynaptic currents (IPSCs) to single peaked inhibitory events. We hypothesize that suppression of inhibitory drive onto VB neurons decreases their probability of burst output, which, together with a reduction of nRt burst output, dampens the oscillatory activity. Low CCK concentrations, which produced little or no depolarization of nRt neurons, did not alter the firing mode of the nRt neurons. However, the probability of burst output from nRt neurons in response to subthreshold stimuli was increased in low CCK concentrations, presumably leading to an increase in the number of nRt neurons participating in the rhythmic activity. Our findings suggest that the neuropeptide CCK, by altering the firing characteristics of nRt neurons, has powerful modulatory effects on intrathalamic rhythms; the ultimate action was dependent on CCK concentration and resting state of these cells.

Cholecystokinin depolarizes rat thalamic reticular neurons by suppressing a K+ conductance.

1. The thalamic reticular nucleus (nRt) is innervated by cholecystokinin (CCK)-containing neurons and contains CCK binding sites. We used tight-seal, whole cell recording techniques with in vitro rat thalamic slices to investigate the action of CCK on neurons in nRt and ventrobasal thalamus (VB). 2. Brief applications of the CCK agonist cholecystokinin octapeptide (26-33) sulfated (CCK8S) evoked prolonged spike discharges in nRt neurons but had no direct effects on VB neuron activity. This selective excitatory action of CCK8S in nRt resulted from a long-lasting membrane depolarization (2-10 min) associated with an increased input resistance. Voltage-clamp recordings revealed that CCK8S reduced membrane conductance by 0.6-3.8 nS, which amounted to 5-54% of the resting conductance of these neurons. 3. The conductance blocked by CCK8S was linear over the range of -50 to -100 mV and reversed near the potassium equilibrium potential. Modifications of extracellular K+ concentration altered the reversal potential of the conductance as predicted by the Nernst equation. The K+ channel blocker Cs+, applied either intracellularly or combined intra- and extracellularly, blocked the response to CCK8S. 4. The CCK8S-induced depolarization persisted after suppression of synaptic transmission by either tetrodotoxin or a low-Ca2+, high-Mg2+ extracellular solution, indicating that the depolarization was primarily due to activation of postsynaptic CCK receptors and not mediated through the release of other neurotransmitters. 5. The selective CCKA antagonists L364,718 and Cam-1481 attenuated the CCK8S-induced depolarization, whereas the CCKB antagonist L365,260 had little or no effect on the depolarization. 6. Our findings indicate that CCK8S, acting via CCKA-type receptors, reduces a K+ leak current, resulting in a long-lasting membrane depolarization that can presumably modify the firing mode of nRt neurons. Through this effect, CCK actions in nRt may strongly influence thalamocortical function.

Intrathalamic rhythmicity studied in vitro: nominal T-current modulation causes robust antioscillatory effects.

Thalamocortical oscillations mediate both physiological and pathophysiological behaviors including sleep and generalized absence epilepsy (GA). Reciprocal intrathalamic circuitry and robust burst firing, dependent on underlying transient Ca current (IT) in thalamic neurons, support generation of such rhythms. In order to study the regulation of intrathalamic rhythm generation and the effects of GA anticonvulsants previously shown to reduce IT in acutely isolated thalamic neurons, we developed an in vitro rat thalamic slice preparation that retains sufficient intrathalamic circuitry to support evoked oscillations (range = 2.0-4.6 Hz, average = 2.7, n = 38), associated with burst firing in the thalamic reticular nucleus (nRt) and thalamic relay neurons. Extracellular stimulation of nRt evoked in relay neurons a biphasic inhibitory response with prominent GABAA and GABAB receptor-mediated components. The GABAA component was picrotoxin sensitive, outwardly rectifying and Cl- dependent, with a very negative reversal potential (-94 mV), indicating that an active extrusion mechanism exists in these cells to keep [Cl-]i < 5 mM. The GABAB component had a linear conductance, a reversal potential of -103 mV, and was quite long lasting (about 300 msec) so that rebound bursts often were generated on its decay phase, presumably leading to reexcitation of nRt through known excitatory connections. GABAB-mediated responses thus provide a timing mechanism for promoting slow intrathalamic oscillations. Reduction of IT (30-40%) by succinimides slightly increased the threshold for burst generation in relay and nRt cells, but there was little effect on either number of spikes/burst or intraburst frequency, and there were no other direct effects on other measures of cellular excitability. Intrathalamic oscillations were significantly reduced by these agents through a slight decrease in burst probability of thalamic neurons. We conclude that interactions between the intrinsic properties of thalamic neurons and intrathalamic circuitry lead to generation of slow oscillations. A similar mechanism may underlie the pathophysiological 3 Hz spike and wave EEG activity that characterizes GA. Furthermore, anti-GA drugs such as ethosuximide probably exert their action by reducing the burst-firing probability of neurons within populations of reciprocally interconnected relay and nRt neurons, thus producing a desynchronization of the thalamic circuit that prevents spike/wave generation.

Transient enhancement of low-threshold calcium current in thalamic relay neurons after corticectomy.

1. The alterations of voltage-sensitive calcium currents produced in thalamic cells by injury were investigated under voltage clamp using patch-clamp recordings in the whole-cell configuration. 2. One day after unilateral cortical ablation in immature rats (postnatal day 7), low-threshold transient calcium (T) currents in acutely isolated thalamic relay neurons (RNs) were increased by 68% compared with contralateral controls (P < 0.001). Three days after the operation, T currents in injured neurons were at 44% of control levels (P < 0.001). On the other hand, high-threshold (L) calcium currents in RNs did not change over the same interval. 3. To investigate the mechanism for the increase of T current, both kinetics and voltage dependency of activation and inactivation were examined. At a test voltage of -40 mV, the activation time constant decreased from 4.1 to 3.2 ms (P < 0.05); however, this small change was insufficient to explain the large increase in T current. Time constants for both fast and slow inactivation did not change significantly, nor did voltage dependence of activation or inactivation of thalamic T currents. 4. Methyl-phenyl-succinimide (MPS, 1 mM), a compound known to block T currents, was used to examine possible alterations in the pharmacological properties of T channels after injury. MPS was more effective in reducing T currents in normal versus injured RNs (24 and 20% reductions, respectively; P < 0.05), suggesting that pharmacological properties of T channels in the injured RNs may be different from those of the normal RNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow inactivation of a TEA-sensitive K current in acutely isolated rat thalamic relay neurons.

1. Voltage-gated K currents were studied in relay neurons (RNs) acutely isolated from somatosensory (VB) thalamus of 7- to 14-day-old rats. In addition to a rapidly activated, transient outward current, IA, depolarizations activated slower K+ currents, which were isolated through the use of appropriate ionic and pharmacological conditions and measured via whole-cell voltage-clamp. 2. At least two slow components of outward current were observed, both of which were sensitive to changes in [K+]o, as expected for K conductances. The first, IK1, had an amplitude that was insensitive to holding potential and a relatively small conductance of 150 pS/pF. It was blocked by submillimolar levels of tetraethylammonium [TEA, 50%-inhibitory concentration (IC50 = 30 microM)] and 4-aminopyridine (4-AP, 40 microM). In the absence of intracellular Ca2+ buffering, the amplitude of IK1 was both larger and dependent on holding potential, as expected for a Ca(2+)-dependent current. Replacement of [Ca2+]o by Co2+ reduced IK1, although the addition of Cd2+ to Ca(2+)-containing solutions had no effect. 3. The second component, IK2, had a normalized conductance of 2.0 nS/pF and was blocked by millimolar concentrations of TEA (IC50 = 4 mM) but not by 4AP. The kinetics of IK2 were analogous to (but much slower than) those of IA in that both currents displayed voltage-dependent activation and voltage-independent inactivation. IK2 was not reduced by the addition of Cd2+ to Ca(2+)-containing solutions or by replacement of Ca2+ by Co2+. 4. IK2 had a more depolarized activation threshold than IA and attained peak amplitude with a latency of approximately 100 ms at room temperature. IK2 decay was nonexponential and could be described as the sum of two components with time constants (tau) near 1 and 10 s. 5. IK2 was one-half steady-state inactivated at a membrane potential of -63 mV, near the normal resting potential for these cells. The slope factor of the Boltzman function describing steady-state inactivation was 13 mV-1, which indicates that IK2 varies in availability across a broad voltage range between -100 and -20 mV. 6. Activation kinetics of IK2 were voltage dependent, with peak latency shifting from 300 to 50 ms in the voltage range -50 to +30 mV. Deinactivation and deactivation were also voltage dependent, in contrast to inactivation, which showed little dependence on membrane potential. Increase in temperature sped the kinetics of IK2, with temperature coefficient (Q10) values near 3 for activation and inactivation. Heating increased the amplitude of IK2 with a Q10 value near 2.(ABSTRACT TRUNCATED AT 400 WORDS)

A fast transient potassium current in thalamic relay neurons: kinetics of activation and inactivation.

1. Whole-cell voltage-clamp techniques were used to record K+ currents in relay neurons (RNs) that had been acutely isolated from rat thalamic ventrobasal complex and maintained at 23 degrees C in vitro. Tetrodoxin (TTX; 0.5 microM) was used to block Na+ currents, and reduced extracellular levels of Ca2+ (1 mM) were used to minimize contributions from Ca2+ current (ICa). 2. In RNs, depolarizing commands activate K+ currents characterized by a substantial rapidly inactivating (time constant approximately 20 ms) component, the features of which correspond to those of the transient K+ current (IA) in other preparations, and by a smaller, more slowly activating K+ current, "IK". IA was reversibly blocked by 4-aminopyridine (4-AP, 5 mM), and the reversal potential varied with [K+]o as predicted by the Nernst equation. 3. IA was relatively insensitive to blockade by tetraethylammonium [TEA; 50%-inhibitory concentration (IC50) much much greater than 20 mM]; however, two components of IK were blocked with IC50S of 30 microM and 3 mM. Because 20 mM TEA blocked 90% of the sustained current while reducing IA by less than 10%, this concentration was routinely used in experiments in which IA was isolated and characterized. To further minimize contamination by other conductances, 4-AP was added to TEA-containing solutions and the 4-AP-sensitive current was obtained by subtraction. 4. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzman function with a slope factor (k) of -6.5 and half-inactivation (V1/2) occurring at -75 mV. Activation of IA was characterized by a Boltzman curve with V1/2 = -35 mV and k = 10.8. 5. IA activation and inactivation kinetics were best fitted by the Hodgkin-Huxley m4h formalism. The rate of activation was voltage dependent, with tau m decreasing from 2.3 ms at -40 mV to 0.5 ms at +50 mV. Inactivation was relatively voltage independent and nonexponential. The rate of inactivation was described by two exponential decay processes with time constants (tau h1 and tau h2) of 20 and 60 ms. Both components were steady-state inactivated with similar voltage dependence. 6. Temperature increases within the range of 23-35 degrees C caused IA activation and inactivation rates to become faster, with temperature coefficient (Q10) values averaging 2.8. IA amplitude also increased as a function of temperature, albeit with a somewhat lower Q10 of 1.6. 7. Several voltage-dependent properties of IA closely resemble those of the transient inward Ca2+ current, IT. (ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of petit mal anticonvulsants and convulsants on thalamic neurones: calcium current reduction.

1. Succinimide derivatives can be either convulsant (tetramethylsuccinimide (TMS)), or anticonvulsant (ethosuximide (ES); alpha-methyl-alpha-phenylsuccinimide (MPS)). ES, an anticonvulsant succinimide, has previously been shown to block calcium currents of thalamic neurones, while the convulsant succinimide TMS blocks gamma-aminobutyric acid (GABA) responses in a similar fashion to the convulsant pentylenetetrazol (PTZ). 2. Using voltage-clamp techniques, we analysed the effects of the anticonvulsant succinimides ES and MPS and the convulsants TMS and PTZ on calcium currents of acutely isolated thalamic relay neurones of the rat. 3. MPS and ES reduced low-threshold calcium current (LTCC) in a voltage-dependent manner, without affecting steady-state inactivation. MPS was less potent than ES (IC50 of 1100 vs 200 microM) but greater in efficacy (100% maximal reduction vs 40% for ES). 4. PTZ had no effect on calcium currents, and TMS only reduced LTCC at very high concentrations, and did not occlude MPS effects when applied concurrently. 5. These results, which demonstrate that anticonvulsant, but not convulsant, succinimides block LTCC, provide additional support for the hypothesis that LTCC reduction is a mechanism of action of the anticonvulsant succinimides related to their effects in petit mal epilepsy.

Differential effects of petit mal anticonvulsants and convulsants on thalamic neurones: GABA current blockade.

1. Currents evoked by applications of gamma-aminobutyric acid (GABA) to acutely dissociated thalamic neurones were analysed by voltage-clamp techniques, and the effects of the anticonvulsant succinimides ethosuximide (ES) and alpha-methyl-alpha-phenylsuccinimide (MPS) and the convulsants tetramethylsuccinimide (TMS), picrotoxin, pentylenetetrazol (PTZ), and bicuculline methiodide were assessed. 2. TMS (1 microM-10 microM) reduced responses to iontophoretically applied GABA, as did picrotoxin (0.1-100 microM), PTZ (1-100 mM) and bicuculline (1-100 microM). 3. ES, in high concentrations (1-10 mM), reduced GABA responses to a lesser extent, and also occluded the reductions in GABA-evoked currents produced by TMS, picrotoxin, and PTZ. ES did not occlude the effects of bicuculline on GABA responses. Therefore, we propose that ES acts as a partial agonist at the picrotoxin GABA-blocking receptor. 4. MPS had no effect on GABA responses (at a concentration of 1 mM), and, like ES, occluded the GABA-blocking actions of TMS, apparently acting as a full antagonist. 5. The anticonvulsant actions of ES and MPS against TMS and PTZ-induced seizures may thus involve two independent mechanisms: (1) the occlusion of TMS and PTZ GABA-blocking effects; and (2) the previously described specific effect of ES and MPS on low-threshold calcium current of thalamic neurones. The latter cellular mechanism may be more closely related to petit mal anticonvulsant activity.

Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current.

1. Calcium currents were recorded with whole-cell voltage-clamp procedures in relay neurones of the rat thalamus which had been acutely isolated by an enzymatic dissociation procedure. 2. Low-threshold and high-threshold Ca2+ currents were elicited by depolarizing voltage steps from holding potentials more negative than -60 mV. A transient current, analogous to the T-current in sensory neurones, was activated at low threshold near -65 mV and was completely inactivating at command steps up to -35 mV. Voltage steps to more depolarized levels activated a high-threshold current that inactivated slowly and incompletely during a 200 ms step depolarization. 3. The high-threshold current contained both non-inactivating and slowly inactivating components which were insensitive and sensitive to holding potential, respectively. 4. A 'T-type' current was prominent in relay neurones, in both absolute terms (350 pA peak current average) and in relation to high-threshold currents. The average ratio of maximum transient to maximum sustained current was greater than 2. 5. T-current could be modelled in a manner analogous to that employed for the fast Na+ current underlying action potential generation, using the m3h format. The rate of activation of T-current was voltage dependent, with a time constant (tau m) varying between 8 and 2 ms at command potentials of -60 to -10 mV at 23 degrees C. The rate of inactivation was also voltage dependent, and the time constant tau h varied between 50 and 20 ms over the same voltage range. With command potentials more positive than -35 mV, the inactivation of Ca2+ current could no longer be fitted by a single exponential. 6. Steady-state inactivation of T-current could be well fitted by a Boltzman equation with slope factor of 6.3 and half-inactivated voltage of -83.5 mV. 7. Recovery from inactivation of T-current was not exponential. The major component of recovery (70-80% of total) was not very voltage sensitive at potentials more negative than -90 mV, with tau r of 251 ms at -92 mV and 23 degrees C, compared to 225 ms at -112 mV. A smaller, voltage-sensitive component accounted for the remainder of recovery. 8. All kinetic properties, including rates of activation, inactivation, and recovery from inactivation, as well as the amplitude of T-current, were temperature sensitive with Q10 (temperature coefficient) values of greater than 2.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons.

The mechanism by which ethosuximide reduces thalamic low-threshold calcium current (LTCC) was analyzed using voltage-clamp techniques in acutely isolated ventrobasal complex neurons from rats and guinea pigs. The ethosuximide-induced reduction of LTCC was voltage dependent: it was most pronounced at more-hyperpolarized potentials and did not affect the time course of activation or inactivation of the current. Ethosuximide reduced LTCC without altering the voltage dependence of steady-state inactivation or the time course of recovery from inactivation. Dimethadione reduced LTCC by a similar mechanism, while valproic acid had no effect on LTCC. We conclude that ethosuximide reduction of LTCC in thalamic neurons is consistent with a reduction in the number of available LTCC channels or in the single LTCC channel conductance, perhaps indicating a direct channel-blocking action of this drug. Given the importance of LTCC in thalamic oscillatory behavior, a reduction in this current by ethosuximide would be a mechanism of action compatible with the known anticonvulsant effects of this drug in typical absence seizures.

Specific petit mal anticonvulsants reduce calcium currents in thalamic neurons.

Low-threshold calcium current (LTCC) in thalamic neurons is important in generation of normal thalamocortical rhythms, and may be involved in the genesis of abnormal activities such as spike-wave discharges that characterize petit mal epilepsy. Ethosuximide and dimethadione, anticonvulsants effective in petit mal, reduced the LTCC when applied to thalamic neurons at clinically relevant concentrations. Therapeutic concentrations of phenytoin and carbamazepine, drugs ineffective in the control of petit mal, had minimal effects on calcium conductances. Reduction in LTCC may be an important mechanism of action by which specific petit mal anticonvulsants depress spike-wave activity.

Noradrenergic modulation of firing pattern in guinea pig and cat thalamic neurons, in vitro.

1. The electrophysiological actions of norepinephrine (NE) in the guinea pig and cat thalamus were investigated using intracellular recordings from neurons of in vitro thalamic slices. 2. Application of NE to neurons of the lateral and medial geniculate nuclei, nucleus reticularis, anteroventral nucleus, and the parataenial (PT) nucleus resulted in a slow depolarization associated with a 2- to 15-nS decrease in input conductance and an increase in the slow membrane time constant from an average of 27.7 to 37.7 ms. The slow depolarization was not abolished by blockade of synaptic transmission, indicating that it was a direct (postsynaptic) effect. 3. The reversal potential of the NE-induced slow depolarization varied as a Nernstian function of extracellular potassium concentration ([K]o), indicating that it is due to a decrease in potassium conductance. This conclusion was supported by the finding that the amplitude of the NE-evoked depolarization was affected by changes in [K]o between 0.5 and 5.0 mM as expected for a K-mediated response. 4. Neurons of the PT nucleus displayed unusually large afterhyperpolarizations (AHPs) in comparison to cells in other thalamic nuclei. NE application to PT neurons caused not only a marked slow depolarization and decreased conductance, but also selectively reduced the slow AHP. 5. The NE-induced slow depolarization effectively suppressed burst firing and promoted the occurrence of single spike activity. NE-induced reduction of the slow AHP in PT neurons was accompanied by a decrease in spike frequency accommodation and the emergence of a slow afterdepolarization. 6. We suggest that through these electrophysiological actions, NE can effectively inhibit the generation of thalamocortical rhythms and greatly facilitate the faithful transfer of information through the thalamus to the cerebral cortex.