Transgenic mouse lines subdivide medial vestibular nucleus neurons into discrete, neurochemically distinct populations.

The identification of neuron types within circuits is fundamental to understanding their relevance to behavior. In the vestibular nuclei, several classes of neurons have been defined in vivo on the basis of their activity during behavior, but it is unclear how those types correspond to neurons identified in slice preparations. By targeting recordings to neurons labeled in transgenic mouse lines, this study reveals that the continuous distribution of intrinsic parameters observed in medial vestibular nucleus (MVN) neurons can be neatly subdivided into two populations of neurons, one of which is GABAergic and the other of which is exclusively glycinergic or glutamatergic. In slice recordings, GABAergic neurons labeled in the EGFP (enhanced green fluorescent protein)-expressing inhibitory neuron (GIN) line displayed lower maximum firing rates (<250 Hz) than glycinergic and glutamatergic neurons labeled in the yellow fluorescent protein-16 (YFP-16) line (up to 500 Hz). In contrast to cortical and hippocampal interneurons, GABAergic MVN neurons exhibited wider action potentials than glutamatergic (and glycinergic) neurons. Responses to current injection differed between the neurons labeled in the two lines, with GIN neurons modulating their firing rates over a smaller input range, adapting less during steady depolarization, and exhibiting less rebound firing than YFP-16 neurons. These results provide a scheme for robust classification of unidentified MVN neurons by their physiological properties. Finally, dye labeling in slices shows that both GABAergic and glycinergic neurons project to the contralateral vestibular nuclei, indicating that commissural inhibition is accomplished through at least two processing streams with differential input and output properties.

Subunit-dependent block by isoflurane of wild-type and mutant alpha(1)S270H GABA(A) receptor currents in Xenopus oocytes.

The volatile anesthetic isoflurane both prolongs and reduces the amplitude of GABA-mediated inhibitory postsynaptic currents (IPSCs) recorded in neurons. To explore the latter effect, we investigated isoflurane-induced inhibition of steady-state desensitized GABA currents in Xenopus oocytes expressing wild-type alpha(1)beta(2), alpha(1)beta(2)gamma(2s), mutant alpha(1)(S270H)beta(2) (serine to histidine at residue 270) or alpha(1)(S270H)beta(2)gamma(2s) receptors. The alpha(1) serine 270 site in TM2 (second transmembrane domain of the subunit) is postulated as a binding site for some volatile agents and is critical for positive modulation of sub-maximal GABA responses by isoflurane. For all receptor combinations, at < or =0.6 mM isoflurane (< or =2 minimum alveolar concentration (MAC)) current inhibitions were not pronounced ( approximately 10%) with block reaching half-maximal levels at supraclinical concentrations ( approximately 2 mM isoflurane, 6 MAC). Comparisons with other GABA(A) receptor blockers indicated that isoflurane blocks in a similar manner to picrotoxin, possibly via the pore of the receptor. The extent of isoflurane-induced inhibition was significantly attenuated by inclusion of the gamma(2s)-subunit but was unaffected by introduction of the S270H mutation in the alpha(1)-subunit. In conclusion, isoflurane binds with low affinity and with subunit-specificity to an inhibitory site on the GABA(A) receptor that is distinct from the site that facilitates positive modulation at the extracellular end of the pore.

General anesthetic-induced channel gating enhancement of 5-hydroxytryptamine type 3 receptors depends on receptor subunit composition.

5-Hydroxytryptamine (serotonin) (5-HT) type 3 (5-HT(3)) receptors are members of an anesthetic-sensitive superfamily of Cys-loop ligand-gated ion channels that can be formed as homomeric 5-HT(3A) or heteromeric 5-HT(3AB) receptors. When the efficacious agonist 5-HT is used, the inhaled anesthetics halothane and chloroform (at clinically relevant concentrations) significantly reduce the agonist EC(50) for 5-HT(3A) receptors but not for 5-HT(3AB) receptors. In the present study, we used dopamine (DA), a highly inefficacious agonist for 5-HT(3) receptors, to determine whether the difference in sensitivity between 5-HT(3A) and 5-HT(3AB) receptors to the potentiating effects of halothane and chloroform is due to differential modulation of agonist affinity, channel gating, or both. Using the two-electrode voltage-clamp technique with 5-HT(3A) and 5-HT(3AB) receptors expressed in Xenopus oocytes, we found that chloroform and halothane enhanced currents evoked by receptor-saturating concentrations of DA for both receptor subtypes in a concentration-dependent manner but that the magnitude of enhancement was substantially greater for 5-HT(3A) receptors than for 5-HT(3AB) receptors. Isoflurane induced only a small enhancement of currents evoked by receptor-saturating concentrations of DA for 5-HT(3A) receptors and no enhancement for 5-HT(3AB) receptors. For both receptor subtypes, none of the three test anesthetics significantly altered the agonist EC(50) for DA, implying that these anesthetics do not affect agonist binding affinity. Our results show that chloroform, halothane, and (to a much lesser degree) isoflurane enhance channel gating for 5-HT(3A) receptors and that the incorporation of 5-HT(3B) subunits to produce heteromeric 5-HT(3AB) receptors markedly attenuates the ability of these anesthetics to enhance channel gating.

Molecular properties important for inhaled anesthetic action on human 5-HT3A receptors.

Although inhaled anesthetics have diverse effects on 5-hydroxytryptamine type 3 (5-HT3A) receptors, the mechanism accounting for this diversity is not understood. Studies have shown that modulation of 5-HT3A receptor currents by n-alcohols depends on molecular volume, suggesting that steric interactions between n-alcohols and their binding sites define their action on this receptor. Electrostatic interactions also play an important role in anesthetic action on other ligand-gated receptors. We aimed to determine the contribution of molecular volume and electrostatics in defining volatile anesthetic actions on 5-HT3A receptors. Human 5-HT3A receptors were expressed in, and recorded from, Xenopus oocytes using the two-electrode voltage-clamp technique. The effects of a range of volatile anesthetics, n-alcohols, and nonhalogenated alkanes on submaximal serotonin-evoked peak currents, and full serotonin concentration-response curves were defined. Volatile anesthetics and n-alcohols, but not alkanes, smaller than 0.120 nm3 enhanced submaximal serotonin-evoked peak currents whereas all larger agents reduced currents. Most compounds tested inhibited maximal serotonin-evoked peak currents to varying degrees. However, only agents smaller than 0.120 nm3 shifted the 5-HT3A receptor's serotonin concentration-response curve to the left, whereas larger anesthetics shifted them to the right. Modulation of human 5-HT3A-mediated currents by volatile anesthetics exhibits a dependence on molecular volume consistent with the n-alcohols, suggesting that both classes of agents may enhance 5-HT3A receptor function via the same mechanism. Furthermore, the enhancing but not inhibiting effects of anesthetic compounds on 5-HT3A receptor currents are modulated by electrostatic interactions.

The N-methyl-D-aspartate receptor inhibitory potencies of aromatic inhaled drugs of abuse: evidence for modulation by cation-pi interactions.

Benzene and several close structural analogs are inhaled drugs of abuse with general anesthetic activity. By virtue of their pi electron clouds, they may engage in attractive electrostatic interactions with cationic atomic charges on protein targets. In this study, we tested the hypothesis that inhaled drugs of abuse inhibit human N-methyl-D-aspartate (NMDA) receptors with potencies that correlate with their abilities to engage in cation-pi interactions. Electrophysiological techniques were used to define the NR1/NR2B NMDA receptor inhibitory concentrations of volatile benzene analogs, and computer modeling was used to quantify their abilities to engage in cation-pi interactions and their molecular volumes. In addition, each compound's octanol/gas partition coefficient (a measure of hydrophobicity) was quantified. All 18 compounds inhibited human NR1/NR2B NMDA receptors reversibly and in a concentration-dependent manner. NMDA receptor inhibitory potency correlated strongly with the ability to engage in cation-pi interactions, weakly with hydrophobicity, and was independent of molecular volume. This is consistent with the hypothesis that cation-pi interactions enhance the binding of inhaled drugs of abuse to the NMDA receptor and suggests that the receptor binding site(s) for these drugs possesses significant cationic character.

The effects of isoflurane on desensitized wild-type and alpha 1(S270H) gamma-aminobutyric acid type A receptors.

UNLABELLED: gamma-aminobutyric acid type A receptors (GABA(A)-R) mediate synaptic inhibition and meet many pharmacological criteria required of important general anesthetic targets. During synaptic transmission GABA release is sufficient to saturate, maximally activate, and transiently desensitize postsynaptic GABA(A)-Rs. The resulting inhibitory postsynaptic currents (IPSCs) are prolonged by volatile anesthetics like isoflurane. We investigated the effects of isoflurane on maximally activated and desensitized GABA(A)-R currents expressed in Xenopus oocytes. Wild-type alpha(1)beta(2) and alpha(1)beta(2)gamma(2s) receptors were exposed to 600 microM GABA until currents reached a steady-state desensitized level. At clinical concentrations (0.02-0.3 mM), isoflurane produced a dose-dependent enhancement of steady-state desensitized current in alpha(1)beta(2) receptors, an effect that was less apparent in receptors including a gamma(2s)-subunit. When serine at position 270 is mutated to histidine (alpha(1)(S270H)) in the second transmembrane segment of the alpha(1)-subunit, the currents evoked by sub-saturating concentrations of GABA became less sensitive to isoflurane enhancement. In addition, isoflurane enhancements of desensitized currents were greatly attenuated by this mutation and were undetectable in alpha(1)(S270H)beta(2)gamma(2s) receptors. In conclusion, isoflurane enhancement of GABA(A)-R currents evoked by saturating concentrations of agonist is subunit-dependent. The effects of isoflurane on desensitized receptors may be partly responsible for the prolongation of IPSCs during anesthesia. IMPLICATIONS: Isoflurane enhances desensitized gamma-aminobutyric acid type A receptor (GABA(A)-R) currents, an effect that is subunit-dependent and attenuated by a mutation in an alpha(1)-subunit pore residue of the GABA(A)-R. As GABA release at inhibitory synapses is typically saturating, isoflurane modulation of desensitized receptors may be partly responsible for prolongation of inhibitory postsynaptic currents during anesthesia.