Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABA(A) receptor.

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with ß3 subunits, we tested the hypothesis that etomidate acts through ß3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR ß3-subunit (ß3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by ß3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5ß1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 µM and below), whereas α5ß2γ2L and α5ß3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5ß3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5ß2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons).

Inhibitory ligand-gated ion channels as substrates for general anesthetic actions.

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.

"Not proper mammals": immunity in monotremes and marsupials.

Immune systems and responses in Monotremata and Marsupialia are reviewed. The Monotremata (Prototheria) are egg-laying mammals. Few studies have been carried out on monotremes. The structure of the lymph nodules of Tachyglossus aculeatus is unusual, and the occurrence of IgG in this species is noteworthy: IgG has not yet been found in any non-mammal. A number of Marsupialia (Metatheria) species have been used as immunological models. Generally immune responses are somewhat slower and less accentuated than in placental (eutherian) mammals. Of interest is the presence of cervical and thoracic thymuses in several marsupials. Marsupials are born very immature and possess rather rudimentary immune responses at birth: the neonate may provide a helpful model for immune ontogenesis. Marsupials have a full repertoire of immunoglobulin classes. MHC Class II (but not Class I) gene polymorphism may be limited. Studies using molecular biology techniques are awaited to elucidate the structural organization of the immune components and to determine similarities and differences between marsupials' and other animals' immune systems.

The compatibility of Xenopus antibody and homoiothermic complement.

Adult Xenopus laevis serum containing an anti-chicken erythrocyte antibody exhibits complement (C) activity by both the classical and the alternative pathways. Lytic activity is removed by heating serum to 50 degrees C for at least 20 min; it is also removed by polysaccharides and by 7.5 mM EDTA. The optimum temperature for Xenopus C activation is 25 degrees C. The ability of Xenopus antibody against chicken erythrocytes to co-operate with homoiothermic C in in vitro by lysis of chicken erythrocytes was tested. Removal of Xenopus C by heating resulted in apparent damage to the antibody, so C was removed by absorption with antibody/antigen complex. Xenopus antibody will apparently combine with guinea-pig and rat C to effect lysis of chicken erythrocytes; low levels of haemolysis were seen with human, rabbit and chicken sera, while horse and mouse sera were non-lytic. Xenopus C was able to restore the lytic activity of decomplemented rabbit anti-chicken erythrocyte serum.

Maturation of transplantation antigens in Ambystoma mexicanum.

Both juvenile (14-16 week) and adult (18 month) Ambystoma mexicanum reject skin allografts from adult Ambystoma more speedily than they reject such grafts from juvenile axolotls. Donor-specific histocompatibility antigen, prepared from splenocytes, is more effective in inhibiting adult host splenocyte migration when the antigen is prepared from spleen cells from adult, rather than from juvenile Ambystoma. The thymus is fully developed in juvenile Ambystoma, suggesting that the delayed kinetics of rejection of juvenile allografts reflects immaturity in the expression of histocompatibility antigens to donor skin cells.

Effect of temperature on transplantation immunity in Ambystoma mexicanum.

Juvenile and mature neotenous Ambystoma mexicanum received allogeneic and xenogeneic skin transplants. Rejection times for 1st-set, and 3rd-set grafts were noted at 21 degrees C, and for axolotls placed in a cold-room at 7 degrees C 7 h post-grafting, 1 week post-grafting and 2 weeks post-grafting respectively. In all cases graft-rejection was chronic; 2nd and 3rd-set grafts were rejected progressively more rapidly than 1st-set grafts. An important temperature-sensitive event occurs during the 1st week post-grafting.

A natural heterohaemagglutinin in Xenopus laevis serum.

In most adult Xenopus laevis the serum contains a 'natural' factor capable of lysing the erythrocytes from a wide variety of amniote species. The factor has no effect on the erythrocytes of another amphibian, Ambystoma mexicanum, nor will serum from one animal lyse red cells from another Xenopus individual. No lysing factor was present in the serum of larval (tadpole) Xenopus. Heating of Xenopus serum to 56 degrees for 30 min, absorption of the serum with zymosan or inulin, or removal of calcium and magnesium ions results in loss of lytic activity, although haemagglutinating activity remains, suggesting that the factor can fix complement. The factor elutes from a gel chromatography column in the 19S peak, and is inactivated by thiol reduction and subsequent alkylation. These findings, coupled with immunoabsorption studies suggest that the haemagglutinin is an immunoglobulin of the IgM class. The significance of this suggestion is discussed in the light of previous reports of 'natural' heterohaemagglutinins in other species.

Antibody-dependent cellular cytotoxicity in poikilotherms.

51Cr-chromate labelled chicken red blood cells, treated with rabbit (anti-chicken red blood cell) serum, are lysed in vitro, in the absence of complement, by spleen cells from Xenopus laevis, Ambystoma mexicanum or Lacerta viridis. Optimal conditions for lysis by Xenopus spleen cells were determined. The phenomenon seems homologous with antibody-dependent cellular cytotoxicity (ADCC) mediated by mammalian or avian K cells. The phylogenetic significance of the finding is discussed.

Surface immunoglobulin of guinea-pig leukaemic lymphocytes.

The surface immunoglobulin of the transplantable L2C leukaemia of strain 2 guinea-pigs has been investigated. The immunoglobulin is seen to be synthesized when the cells are maintained in culture, indicating its intrinsic origin. Immunolabelling of the cell surface and immunochemical study of the Fab released by limited surface proteolysis indicate the presence of immunoglobulin of class IgM. IgG and free light chains were not detected, and there is unlikely to be an appreciable amount of immunoglobulin of any other class. The amount of immunoglobulin present, in terms of 4-chain monomers, is approximately 100,000 molecules per cell. Its half-life, calculated from the rate of reappearance in vitro of surface Fab after proteolytic clearing, is approximately 5 hours. Immunoglobulin secreted into the environment appears to arise predominantly or entirely from the cell surface: there is no evidence of an appreciable export of immunoglobulin which does not have a surface phase. Papain at 0.06 mg/ml rapidly removes the surface Fab. Residual Fcmu can then be detected by immunofluorescence, suggesting that papain cleaves surface IgM at a hinge region with the molecule in situ on the membrane. The released Fab is only moderately susceptible to degradation by papain at the enzyme: substrate ratio prevailing. It has been possible to isolate it from the papain digest by immuno-adsorption, with a notional yield of 75 mug per 10-10 cells, and then to prepare antisera against it.