Group II metabotropic glutamate receptor antagonism prevents the antiallodynic effects of R-isovaline.

We previously showed that isovaline is a peripheral analgesic which acts in vivo and in brain slices as an atypical metabotropic GABA(B) agonist. Peripheral inhibitory group II and III metabotropic glutamate receptors (mGluRs) belong to the same family C as GABA(B) receptors; therefore, we hypothesized that isovaline's analgesic effects could include their activation. We examined the effects of R-isovaline on mechanical allodynia produced by prostaglandin E2 in the mouse paw. Subcutaneous R-isovaline produced dose-dependent antiallodynia restricted to the injected hindlimb. This antiallodynia was blocked by co-injection with a selective group II mGluR antagonist, LY341495, but not a group III mGluR antagonist (MAP-4). The antiallodynic effect of R-isovaline was potentiated by co-administration of a group II mGluR-positive allosteric modulator, LY487379. Injection of a group II mGluR agonist (LY354740) produced an antiallodynic effect which was completely reversed by group II antagonism, but was not affected by group III or GABA(B) (CGP35348) antagonism. Similarly, group II mGluR antagonism did not alter the antiallodynia produced by the prototypical GABA(B) agonist, baclofen. Hence, there was no apparent crosstalk between group II mGluRs and GABA(B) receptors. Previous studies have demonstrated that peripheral GABA(B) receptor activation by isovaline produces antiallodynia. In addition, the present results indicate that activation of peripheral group II mGluRs by R-isovaline produces antiallodynia.

GABA(B) receptor-mediated selective peripheral analgesia by the non-proteinogenic amino acid, isovaline.

Peripherally restricted analgesics are desirable to avoid central nervous system (CNS) side effects of opioids. Nonsteroidal anti-inflammatory drugs produce peripheral analgesia but have significant toxicity. GABA(B) receptors represent peripheral targets for analgesia but selective GABA(B) agonists like baclofen cross the blood-brain barrier. Recently, we found that the CNS-impermeant amino acid, isovaline, produces analgesia without apparent CNS effects. On observing that isovaline has GABA(B) activity in brain slices, we examined the hypothesis that isovaline produces peripheral analgesia mediated by GABA(B) receptors. We compared the peripheral analgesic and CNS effect profiles of isovaline, baclofen, and GABA (a CNS-impermeant, unselective GABA(B) agonist). All three amino acids attenuated allodynia induced by prostaglandin E2 injection into the mouse hindpaw and tested with von Frey filaments. The antiallodynic actions of isovaline, baclofen, and GABA were blocked by the GABA(B) antagonist, CGP52432, and potentiated by the GABA(B) modulator, CGP7930. We measured Behavioural Hyperactivity Scores and temperature change as indicators of GABAergic action in the CNS. ED(95) doses of isovaline and GABA produced no CNS effects while baclofen produced substantial sedation and hypothermia. In a mouse model of osteoarthritis, isovaline restored performance during forced exercise to baseline values. Immunohistochemical staining of cutaneous layers of the analgesic test site demonstrated co-localization of GABA(B1) and GABA(B2) receptor subunits on fine nerve endings and keratinocytes. Isovaline represents a new class of peripherally restricted analgesics without CNS effects, mediated by cutaneous GABA(B) receptors.

R-Isovaline: a subtype-specific agonist at GABA(B)-receptors?

The R-enantiomer of isovaline, an analgesic amino acid, has a chemical structure similar to glycine and GABA. Although its actions on thalamic neurons are strychnine-resistant and independent of the Cl(-) gradient, R-isovaline increases membrane conductance for K(+). The purpose of this study was to determine if R-isovaline activated metabotropic GABA(B) receptors. We used whole-cell voltage-clamp recordings to characterize the effects of R-isovaline applied by bath perfusion and local ejection from a micropipette to thalamic neurons in 250 µm thick slices of rat brain. The immunocytochemical methods that we employed to visualize GABA(B1) and GABA(B2) receptor subunits showed extensive staining for both subunits in ventrobasal nuclei, which were the recording sites. Bath or local application of R-isovaline caused a slowly developing increase in conductance and outward rectification in 70% (54/77) of neurons, both effects reversing near the K(+) Nernst potential. As with the GABA(B) agonist baclofen, G proteins likely mediated the R-isovaline effects because they were susceptible to blockade by non-hydrolyzable substrates of guanosine triphosphate. The GABA(B) antagonists CGP35348 and CGP52432 prevented the conductance increase induced by R-isovaline, applied by bath or local ejection. The GABA(B) allosteric modulator CGP7930 enhanced the R-isovaline induced increase in conductance. At high doses, antagonists of GABA(A), GABA(C), glycine(A), µ-opioid, and nicotinic receptors did not block R-isovaline responses. The observations establish that R-isovaline increases the conductance of K(+) channels coupled to metabotropic GABA(B) receptors. Remarkably, not all neurons that were responsive to baclofen responded to R-isovaline. The R-isovaline-induced currents outlasted the fast baclofen responses and persisted for a 1-2-h period. Despite some similar actions, R-isovaline and baclofen do not act at identical GABA(B) receptor sites. The binding of R-isovaline and baclofen to the GABA(B) receptor may not induce the same conformational changes in receptor proteins or components of the intracellular signaling pathways.

Isovaline causes inhibition by increasing potassium conductance in thalamic neurons.

The rare amino acid isovaline has analgesic properties in pain models and is a structural analogue of the inhibitory neurotransmitter glycine. Glycinergic inhibition is prevalent in pain pathways. In this paper, we examined the possibility that isovaline inhibits neurons by activating strychnine (Str)-sensitive glycine(A) receptors in ventrobasal thalamus. Sagittal brain sections containing ventrobasal nuclei were prepared from P10-P15 rats. Whole-cell recordings were made in current-clamp and voltage-clamp modes. R-isovaline (R-Iva) increased input conductance and hyperpolarized the membrane. The conductance increase shunted action potentials and low-threshold Ca(2+) spikes evoked by current pulse injection. Unlike the Cl(-)-mediated responses to glycine, isovaline responses were insensitive to Str antagonism and usually not reversible. The concentration-response curve was non-sigmoidal, rising to a maximum at approximately 100 microM, and thereafter declining in amplitude. Current-voltage relationships showed that isovaline increased inward and outward rectification. The isovaline current reversed polarity close to the K(+) equilibrium potential. The relationships were negligibly affected by tetrodotoxin (TTX), chelation of intracellular Ca(2+) or blockade of the hyperpolarization-activated current, I(h). Internal Cs(+) and external Ba(2+) or Cs(+) prevented isovaline responses. In conclusion, isovaline inhibited firing mainly by activating rectifying and possibly leak K(+) currents. Isovaline-induced changes shunted action potentials and suppressed rebound excitation in ventrobasal neurons, as expected for analgesic actions.

Pentobarbital induces thalamic oscillations in brain slices, modulated by GABA and glycine receptors.

We studied the effects of pentobarbital and antagonists of glutamate, gamma-aminobutyrate (GABA), and glycine receptors on extracellular activity in ventrobasal thalamic slices. Pentobarbital at sedative-hypnotic concentration (20 microM) reversibly induced 1-15 Hz oscillations. Sustained oscillations required electrical stimulation of internal capsule, but not elevated temperature or low [Mg2+]. Anesthetic concentration (200 microM) of pentobarbital evoked only transient oscillations. Kynurenate-sensitive glutamate receptors were essential for oscillations. GABA(A) antagonism (bicuculline, 50 microM or gabazine, 20 microM) suppressed oscillations at 5-15 Hz. GABA(B) antagonism (CGP 35348, 100 nM), or antagonism of glycine receptors (strychnine, 1 microM) suppressed oscillations at 1-4 and 11-15 Hz. GABA and glycine receptors modulated oscillation frequency. For elimination, oscillations required GABA antagonists and strychnine. Receptors for glutamate and glycine mediated oscillations during GABA receptor blockade in ventrobasal nuclei, or on disconnection from nRT. Glycine receptors were critical for oscillations in dorsal thalamic network, divested of GABAergic inhibition. Glutamate and GABA receptors mediated pentobarbital-induced oscillations in nRT, disconnected from ventrobasal nuclei. Hence, pentobarbital oscillogenesis occurred in isolated networks of the ventrobasal and reticularis nuclei mediated by glutamate receptors, with frequency modulation by GABA(A), GABA(B), and glycine receptors. These stationary oscillations represent a model of sedation-hypnosis, amenable to pharmacological analysis.

Selective GABA-receptor actions of amobarbital on thalamic neurons.

1. We studied amobarbital's effects on membrane properties and currents, and electrically evoked inhibitory postsynaptic currents (IPSCs) mediated by gamma-aminobutyric acid (GABA) in rat thalamic slices. Using concentration-response relationships, we compared amobarbital's effects in nociceptive nuclei and non-nociceptive nucleus reticularis thalami (nRT). 2. Amobarbital decreased input resistance by activating GABA(A) receptors. Amobarbital produced a larger decrease in ventrobasal than nRT neurons. 3. Amobarbital depressed burst and tonic firing. Depression of burst firing was more effective, particularly in ventrobasal and intralaminar neurons. Depression was reversed by GABA(A) antagonists, and surmountable by increasing current injection, implicating a receptor-mediated shunt mechanism. 4. Amobarbital did not affect the tetrodotoxin-isolated low threshold Ca(2+) spike during GABA(A) blockade. Amobarbital reduced excitability without altering outward leak, or hyperpolarisation-activated inward currents. 5. Amobarbital increased mean conductance and burst duration of single GABA(A) channels. Consistent with this, amobarbital increased amplitude and decay time of IPSCs with distinct EC(50)s, implicating actions at two GABA(A) receptor sites. 6. Activation of GABA(A) receptors by low concentrations, fast IPSC amplitude modulation, and failure to affect intrinsic currents distinguished amobarbital's mechanism of action from previously characterised barbiturates. The selective actions of amobarbital on GABA(A) receptor may have relevance in explaining anaesthetic and analgesic uses.

Pentobarbital modulates intrinsic and GABA-receptor conductances in thalamocortical inhibition.

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 microM, pentobarbital increased input conductance and induced net outward current, I(PB), at potentials near action potential threshold. The reversal potential of I(PB) was -75 mV, implicating K+ and other ions. Cs+ (3 mM) which inhibits both K+ currents and inward rectifier (Ih), completely blocked IPB, whereas the selective Ih blocker, ZD-7288 (25 microM), or Ba2+ (2 mM) which suppresses only K+ currents, reduced IPB. Pentobarbital inhibited the Ih, consistent with a ZD-7288-induced shift in reversal potential for IPB toward K+ equilibrium potential. Pentobarbital increased the inward K+ rectifier, IKir, and leak current, Ileak. We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC50 = 53 microM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABAA channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 microM, pentobarbital did not directly activate GABAA receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, Ih, IKir, and Ileak, as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones.

1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.

Metabotropic transmitter actions in auditory thalamus.

Neurons in the ventral partition of the medial geniculate body (MGBv), the primary auditory thalamus, receive afferent input from the inferior colliculus via excitatory glutamate-ergic and inhibitory GABA-ergic input fibres. The feedback from the auditory cortex to the thalamic relay also is mediated via neuron systems using glutamate and GABA as transmitters. We studied effects on excitability mediated by these transmitters via G-protein coupled metabotropic receptors. In a slice preparation of rat thalamus we investigated the membrane responses of MGBv neurons using the whole cell recording technique. Application of a metabotropic glutamate receptor (mGluR) agonist, ACPD (5-100 microM), depolarized MGBv neurons. As a result, the burst mode of firing, which characterizes states of sleep at hyperpolarized potentials was replaced by the tonic mode, which is compatible with sound signal transmission during alertness. The depolarization was caused by an inward current (I(ACPD)) that persisted during blockade of Na+ channels with tetrodotoxin (TTX) and of Ca2+ channels with Cd2+. The I(ACPD) depended, however, on extracellular Na+, which could be replaced with Li+, excluding a major contribution of the Na+/Ca2+ exchange current. ACPD application also inhibited an inwardly rectifying K+ current at hyperpolarized potentials and activated an outward current in the depolarized range. Application of the GABA(B) agonist, baclofen (10 microM), hyperpolarized MGBv neurons by activation of an inwardly rectifying K+ current. The corresponding membrane conductance acted as a powerful shunt that reduced voltage responses and inhibited firing in both the tonic and burst modes. Thus, the effects of GABA(B) receptor activation would suppress auditory signal transfer, whereas mGluR activation enhances excitability, possibly accounting for the alerting effects of certain auditory stimuli.

Effects of metabotropic glutamate receptor activation in auditory thalamus.

Metabotropic glutamate receptors (mGluRs) are expressed predominantly in dendritic regions of neurons of auditory thalamus. We studied the effects of mGluR activation in neurons of the ventral partition of medial geniculate body (MGBv) using whole cell current- and voltage-clamp recordings in brain slices. Bath application of the mGluR-agonist, 1S,3R-1-aminocyclopentan-1,3-dicarboxylic acid or 1S,3R-ACPD (5-100 microM), depolarized MGBv neurons (n = 67), changing evoked response patterns from bursts to tonic firing as well as frequency responses from resonance ( approximately 1 Hz) to low-pass filter characteristics. The depolarization was resistant to Na(+)-channel blockade with tetrodotoxin (TTX; 300 nM) and Ca(2+)-channel blockade with Cd(2+) (0.1 mM). The application of 1S, 3R-ACPD did not change input conductance and produced an inward current (I(ACPD)) with an average amplitude of 84.2 +/- 5.3 pA (at -70 mV, n = 22). The application of the mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (0.5 mM), reversibly blocked the depolarization or I(ACPD). During intracellular application of guanosine 5'-O-(3-thiotriphosphate) from the recording electrode, bath application of 1S,3R-ACPD irreversibly activated a large amplitude I(ACPD). During intracellular application of guanosine 5'-O-(2-thiodiphosphate), application of 1S, 3R-ACPD evoked only a small I(ACPD). These results implicate G proteins in mediation of the 1S,3R-ACPD response. A reduction of external [Na(+)] from 150 to 26 mM decreased I(ACPD) to 32.8 +/- 10. 3% of control. Internal applications of a Ca(2+) chelator, 1, 2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 10 mM), suppressed I(ACPD), implying a contribution of a Ca(2+) signal or Na(+)/Ca(2+) exchange. However, partial replacement of Na(+) with Li(+) (50 mM) did not significantly change I(ACPD). Therefore it seemed less likely that a Na(+)/Ca(2+) exchange current was a major participant in the response. A reduction of extracellular [K(+)] from 5.25 to 2.5 mM or external Ba(2+) (0.5 mM) or Cs(+) (2 mM) did not significantly change I(ACPD) between -40 and -85 mV. Below -85 mV, 1S,3R-ACPD application reversibly attenuated an inward rectification, displayed by 11 of 20 neurons. Blockade of an inwardly rectifying K(+) current with Ba(2+) (1 mM) or Cs(+) (2-3 mM) occluded the attenuation. In the range positive to -40 mV, 1S, 3R-ACPD application activated an outward current which Cs(+) blocked; this unmasked a voltage dependence of the inward I(ACPD) with a maximum amplitude at approximately -30 mV. The I(ACPD) properties are consistent with mGluR expression as a TTX-resistant, persistent Na(+) current in the dendritic periphery. We suggest that mGluR activation changes the behavior of MGBv neurons by three mechanisms: activation of a Na(+)-dependent inward current; activation of an outward current in a depolarized range; and inhibition of the inward rectifier, I(KIR). These mechanisms differ from previously reported mGluR effects in the thalamus.

Mechanism of anesthesia revealed by shunting actions of isoflurane on thalamocortical neurons.

By using thalamic brain slices from juvenile rats and the whole cell recording technique, we determined the effects of aqueous applications of the anesthetic isoflurane (IFL) on tonic and burst firing activities of ventrobasal relay neurons. At concentrations equivalent to those used for in vivo anesthesia, IFL induced a hyperpolarization and increased membrane conductance in a reversible and concentration-dependent manner (ionic mechanism detailed in companion paper). The increased conductance short-circuited the effectiveness of depolarizing pulses and was the main cause for inhibition of tonic firing of action potentials. Despite the IFL-induced hyperpolarization, which theoretically should have promoted bursting, the shunt blocked the low-threshold Ca2+ spike (LTS) and associated burst firing of action potentials as well as the high-threshold Ca2+ spike (HTS). Increasing the amplitude of either the depolarizing test pulse or hyperpolarizing prepulse or increasing the duration of the hyperpolarizing prepulse partially reversed the blockade of the LTS burst. In voltage-clamp experiments on the T-type Ca2+ current, which produces the LTS, IFL decreased the spatial distribution of imposed voltages and hence impaired the activation of spatially distant T channels. Although IFL may have increased a dendritic leak conductance or decreased dendritic Ca2+ currents, the somatic shunt appeared to block initiation of the LTS and HTS as well as their electrotonic propogation to the axon hillock. In summary, IFL hyperpolarized thalamocortical neurons and shunted voltage-dependent Na+ and Ca2+ currents. Considering the importance of the thalamus in relaying different sensory modalities (i.e., somatosensation, audition, and vision) and motor information as well as the corticothalamocortical loops in mediating consciousness, the shunted firing activities of thalamocortical neurons would be instrumental for the production of anesthesia in vivo.

Ionic mechanism of isoflurane's actions on thalamocortical neurons.

We studied the actions of isoflurane (IFL) applied in aqueous solutions on ventrobasal neurons from thalamic brain slices of juvenile rats. By using the whole cell, patch-clamp method with current- and voltage-clamp recording techniques, we found that IFL increased a noninactivating membrane conductance in a concentration-dependent reversible manner. In an eightfold concentration range that extended into equivalent in vivo lethal concentrations, IFL did not produce a maximal effect on the conductance; this is consistent with a nonreceptor-mediated mechanism of action. TTX eliminated action potential activity but did not alter IFL effects. The effects on the membrane potential and current induced by IFL were voltage independent but depended on the external [K+], reversing near the equilibrium potential for K+. External Ba2+ or internal Cs+ applications, which block K+ channels, suppressed the conductance increase caused by IFL. External applications of the Ca2+ channel blockers Co2+ or Cd2+ or internal application of the Ca2+ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid did not prevent the effects of IFL, implying little involvement of Ca2+-dependent K+ currents. A contribution of inwardly rectifying K+ channels to the increased steady-state conductance seemed unlikely because IFL decreased inward rectification. An involvement of ATP-mediated K+ channels also was unlikely because application of the ATP-mediated K+ channel blocker glibenclamide (1-80 microM) did not prevent IFL's actions. In contrast to spiking cells, IFL depolarized presumed glial cells, consistent with an efflux of K+ from thalamocortical neurons. The results imply that a leak K+ channel mediated the IFL-induced increase in postsynaptic membrane conductance in thalamic relay neurons. Thus a single nonreceptor-mediated mechanism of IFL action was responsible for the hyperpolarization and conductance shunt of voltage-dependent Na+ and Ca2+ spikes, as reported in the preceding paper. Although anesthetics influence various neurological systems, an enhanced K+ leak generalized in thalamocortical neurons alone could account for anesthesia in vivo.

Modulation of frequency selectivity by Na+- and K+-conductances in neurons of auditory thalamus.

In thalamic neurons, frequency-filter properties arise from intrinsic membrane properties which transform sensory inputs to thalamocortical signals. They also contribute to the tendency for the membrane to generate synchronized oscillations. We studied the frequency selectivities of thalamocortical neurons in the rat ventral medial geniculate body (MGBv) in vitro, using whole-cell recording techniques, sinewave (swept 'ZAP' or single) current inputs and pharmacological blockade of membrane currents. In a voltage range that was subthreshold to spike genesis, the frequency responses below 20 Hz were voltage-dependent, they exhibited lowpass characteristics at depolarized potentials and bandpass resonance (near 1 Hz) in the activation range (approximately -65 to -50 mV) of the low-threshold Ca2+-current (I(T)). A temperature increase of > 10 degrees C in 3 neurons did not change this voltage-dependence and increased the frequency of maximum resonance to 2 Hz. The removal of extracellular Ca2+, its equimolar substitution with Mg2+ or blockade of I(T) with Ni2+ (0.5 mM) completely blocked the resonance at hyperpolarized potentials or rest, as well as the low-threshold Ca2+-spike (LTS). Blockade of high threshold Ca2+-currents with Cd2+ (50 microM) did not affect the resonance. These data implied that, like the LTS, an activation of I(T) produced the membrane resonance. An increased ZAP-current input evoked action potentials near the resonant frequency as well as Cd2+-sensitive high-threshold Ca2+-spikes at depolarized membrane potentials and very low frequencies. By blocking a persistent Na+-current (I(NaP)), tetrodotoxin (300 nM) reduced the magnitude of the frequency response without affecting the frequency preference. The response was larger in amplitude, especially at frequencies lower than the maximum resonant frequency, when we used 4-aminopyridine (0.05-0.1 and 1-2 mM), Ba2+ (0.2 mM) or Cs+ (3 mM) to block voltage-dependent K+-currents. From these data, we suggest that A-type (I(A) and I(As)) and inwardly rectifying (I(KIR)) K+-currents modulate resonance, changing the quality of the lowpass filter function. We conclude that the generation of membrane resonance in MGBv neurons depends critically on I(T)-activation while the quality of the frequency response is subject to modulation by voltage-dependent conductances. The frequency selectivities in MGBv may contribute to lowpass filter functions for auditory transmission during wakefulness and oscillations observed during sleep.

Lidocaine produces a shunt in rat [correction of rats] thalamocortical neurons, unaffected by GABA(A) receptor blockade.

Low concentrations of lidocaine reversibly decrease input resistance and shunt action potentials in neurons of the ventral posterolateral thalamic nucleus (VPL) in vitro. Using differential interference contrast infrared videomicroscopy and whole-cell patch-clamp techniques in rat brain slices, we studied the effects of bicuculline methobromide to test whether the lidocaine-induced shunt in VPL neurons is mediated by GABA(A) receptors. Bicuculline (50 microM) restored tonic firing rates to control values following reduction by lidocaine (10 microM). However, bicuculline did not influence lidocaine's actions to decrease slope resistance over the voltage range from approximately -90 mV to spike threshold. Likewise, bicuculline did not affect the shunt-induced reductions of spike-afterhyperpolarizations produced by lidocaine. The results of this study imply that the effects of low lidocaine concentrations in thalamocortical neurons are not mediated by GABA(A) receptors.

Membrane properties that shape the auditory code in three nuclei of the central nervous system.

OBJECTIVE: We investigated if auditory neurons have an intrinsic ability to radically transform auditory signals. METHOD: We surveyed membrane properties that control coding by neurons, identified with intracellular staining or infrared-DIC videomicroscopy, in three stations of the auditory pathway. We used intracellular and patch-clamp techniques in slices, to study the voltage responses to current pulse injections and distinguished voltage-gated conductances with selective blockers. RESULTS: First order spherical bushy cells in the anteroventral cochlear nucleus responded at a short, stable latency with single spikes, due to a perithreshold interaction of Na+ and Ca2+ conductances. Two K+ conductances suppressed firing after this onset-spike. Second-order principal neurons of the lateral superior olive use unspecified mechanisms to secure stable onset latencies but maintained a very regular tonic firing, resulting in a chopper pattern. Other intrinsic properties induced a marked accommodation in spike rate. When depolarized as during alert states, neurons in the medial geniculate body (MGB) of the thalamus fired with variable latencies in a tonic mode. At negative resting potentials characteristic of sleep states, they responded at the onset of a depolarization and the offset of a hyperpolarization with phasic bursts due to a transient low threshold Ca2+ current. In the phasic, but not tonic mode, MGB neurons produced high-threshold Ca2+ spikes that may couple signal transmission to the neuron's metabolism. The three neuron types exhibit analogue computing abilities that transform the same input into entirely different output patterns. Isoflurane anaesthesia induces a current shunt in MGB neurons, radically changing the properties and preventing normal responses. Thus, thalamocortical auditory codes are compromised under anaesthesia. CONCLUSION: At all investigated stations of the auditory pathway, input signals are transformed by activation of voltage-controlled conductances and other intrinsic membrane properties.

Analgesic and sedative concentrations of lignocaine shunt tonic and burst firing in thalamocortical neurones.

The effects of lignocaine [lidocaine] HCl (0.6 microM(-1) mM) on the membrane electrical properties and action potential firing of neurones of the ventral posterolateral (VPL) nucleus of the thalamus were investigated using whole cell recording techniques in rat brain slices in vitro. Bath application of lignocaine reversibly decreased the input resistance (Ri) of VPL neurones. This effect was observed at low, clinically sedative and analgesic concentrations (i.e., maximal amplitude at 10 microM) whereas higher concentrations (300 microM(-1) mM) had no effect on Ri. Lignocaine (10-100 microM) depolarized VPL neurones up to 14 mV in a reversible manner. Consistent with a decreased Ri, low concentrations of lignocaine shunted the current required for spike generation in the tonic pattern. Lignocaine increased the threshold amplitude of current required for firing and decreased the tonic firing frequency, without concomitant elevation of the voltage threshold for firing or reduction in the maximal rate of rise (dV/dt(max)) of spikes. Low concentrations of lignocaine shunted low threshold spike (LTS) burst firing evoked either from hyperpolarized potentials or as rebound bursts on depolarization from prepulse-conditioned potentials. Higher concentrations of lignocaine (300 microM - 1 mM), not associated with a decrease in Ri, elevated the voltage threshold for firing and reduced the dV/dt(max) of spikes in a concentration-dependent fashion. In conclusion, low concentrations of lignocaine shunted tonic and burst firing in VPL neurones by decreasing Ri, a mechanism not previously described for local anaesthetics in the CNS. We suggest that a decreased resistance in thalamocortical neurones contributes to the sedative, analgesic, and anaesthetic properties of systemic lignocaine in vivo.

Postnatal development of signal generation in auditory thalamic neurons.

Using whole cell recording techniques, we distinguished immature from mature stages of development in auditory thalamic neurons of rats at ages P5 to P21. We compared voltage responses to injected currents and firing patterns of neurons in ventral partition of medial geniculate body (MGBv) in slices. Resting potential, input resistance and membrane time constant diminished to mature values between P5 and P14. Responses of young neurons to hyperpolarizing pulses showed delayed inward rectification; after P13, this was obscured by a rapid onset of another inward rectifier. All neurons possessed tetrodotoxin (TTX)-sensitive, depolarization-activated rectification, implying persistent Na+-current involvement. Despite a slightly higher voltage threshold for spiking, the current threshold was lower in younger neurons. Young neurons fired a short latency spike with afterhyperpolarization whereas older neurons exhibited a slow ramplike depolarization before tonic firing. Large currents caused continuous firing in all neurons. Before day P13, a high threshold Ca2+ spike (HTS) often was appended to action potentials. The low threshold Ca2+-spike (LTS) was too small in amplitude to evoke action potentials before P11 but produced a single spike at P12 and P13 and burst firing with HTS after P13. MGBv neurons have mature properties after P14, relevant for reactions to sound and the oscillations of slow-wave sleep.

GABA(B) receptor activation changes membrane and filter properties of auditory thalamic neurons.

Inhibitory inputs from nucleus reticularis thalami and the inferior colliculus activate gamma-aminobutyric acid B (GABA(B)) receptors in auditory thalamic neurons. These metabotropic receptors have been implicated in the oscillatory behavior of thalamic neurons. We studied the effects of the GABA(B) receptor agonist, baclofen, on membrane and filter properties of neurons in the ventral partition of the medial geniculate body (MGBv) of the rat, using whole-cell patch-clamp recording techniques in a slice preparation. Application of baclofen caused a concentration-dependent and reversible hyperpolarization of MGBv neurons. An increase in membrane conductance shunted voltage signals. The shunt suppressed firing in both tonic and burst modes which normally characterize the neuronal excitation from depolarized and hyperpolarized potentials, respectively. The GABA(B) receptor antagonist, CGP 35348 (0.5 mM), completely and reversibly blocked the baclofen-evoked hyperpolarization and increase in conductance. In voltage-clamp and during blockade of synaptic transmission with tetrodotoxin and Cd2+, baclofen activated an inwardly rectifying outward K+ current, that was sensitive to blockade with Ba2+ (0.5 mM). Intracellular applications of GTPgammaS occluded the baclofen current whereas similar applications of GDPbetaS prevented it, suggesting that G-proteins mediated the baclofen current. We measured the impedance amplitude profile in the frequency domain with swept sinusoidal current injection. MGBv neurons normally have lowpass filter characteristics at depolarized potentials and resonance at approximately 1 Hz at hyperpolarized potentials. Baclofen application reduced the impedance below 20 Hz which lowered the membrane filter quality and abolished the resonance. Despite its hyperpolarizing effect, therefore, baclofen eliminated an intrinsic tendency to oscillate as well as the intrinsic frequency selectivity of MGBv neurons.

Intrinsic response properties of bursting neurons in the nucleus principalis trigemini of the gerbil.

In trigeminal neurons, the spike rate, modulated by input parameters, may serve as a code for sensory information. We investigated intrinsic response properties that affect rate coding in neurons of nucleus principalis trigemini (young gerbils). Using the whole-cell recording technique and neurobiotin staining in slices, we found bursting behaviour in approximately 50% of the neurons. These neurons fired spike bursts, spontaneously, as well as at the onset of depolarizing, and offset of hyperpolarizing, current pulses. The spike rate within an initial burst was independent of stimulus strength, in contrast to single spike firing that occurred later in the response to current pulse injection. The spikes within a burst were superimposed on slow depolarizing humps. Under favourable conditions, these led to "plateau potentials", that lasted for hundreds of milliseconds at membrane potentials near approximately -20 mV. Occasionally, plateau potentials were spontaneous or evoked under control conditions: usually, they were evoked by current pulse injection during blockade of Ca2+ influx with Co2+ or Cd2+ in Ca(2+)-free extracellular media, or during blockade of K+ currents with tetraethylammonium. The plateau potentials recorded during internal Cs+ (132.5 mM) substitution of K+ had more positive amplitudes (near +20 mV). Despite relatively stable depolarization levels, the plateau potentials decreased in duration and decayed in amplitude during application of tetrodotoxin (0.6-1.8 nM). Higher tetrodotoxin concentrations (5-60 nM) eliminated the plateau potentials despite well-maintained, fast action potentials. A reduction of external [Na+] reduced the amplitudes of the spikes and plateau potentials. A hyperpolarization of long duration (> 3 s) followed a plateau potential, or a depolarizing response that was subthreshold for plateau generation. Tetrodotoxin application blocked this after-effect. We suggest that a persistent Na+ influx is a major contributor to the bursts and plateau potentials and that it mediates the hyperpolarization. Depending on Ca2+ influx, K+ conductances may regulate the amplitudes of these long-lasting depolarizations. A Ca2+ conductance, blockable by Ni2+, may support burst initiation in these neurons. In very young animals (P2-P9), we found only non-bursting neurons. Both bursting and non-bursting neurons with elongated dendritic fields showed inward rectification on hyperpolarization. The bursts in nucleus principalis trigemini neurons emphasize the onsets of stimulus transients, at the expense of using firing rate as a sensory code. Our studies describe neurons with a surprising ability to distort sensory signals, transforming depolarizing inputs into bursts of spikes by virtue of a Na(+)-conductance activation. The principal trigeminal nucleus also contains neurons with tonic firing ability, compatible with simple rate coding.