The gonadotrophin-releasing hormone (GnRH) neurones represent the final output neurones of a complex neuronal network that controls fertility. It is now appreciated that GABAergic neurones within this network provide an important regulatory influence on GnRH neurones. However, the consequences of direct GABA(A) receptor activation on adult GnRH neurones have been controversial for nearly a decade now, with both hyperpolarising and depolarising effects being reported. This review provides: (i) an overview of GABA(A) receptor function and its investigation using electrophysiological approaches and (ii) re-examines the past and present results relating to GABAergic regulation of the GnRH neurone, with a focus on mouse brain slice data. Although it remains difficult to reconcile the results of the early studies, there is a growing consensus that GABA can act through the GABA(A) receptor to exert both depolarising and hyperpolarising effects on GnRH neurones. The most recent studies examining the effects of endogenous GABA release on GnRH neurones indicate that the predominant action is that of excitation. However, we are still far from a complete understanding of the effects of GABA(A) receptor activation upon GnRH neurones. We argue that this will require not only a better understanding of chloride ion homeostasis in individual GnRH neurones, and within subcellular compartments of the GnRH neurone, but also a more integrative view of how multiple neurotransmitters, neuromodulators and intrinsic conductances act together to regulate the activity of these important cells.

We previously defined a cholesterol recognition/interaction amino acid consensus (CRAC; ATVLNYYVWRDNS) in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), an outer mitochondrial membrane protein involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. We examined (i) the PBR-cholesterol interaction by UV crosslinking of the C17 side-chain containing progestin, promegestone, and (ii) the role of the CRAC domain of PBR in Leydig cell steroidogenesis by using a transducible peptide composed of the TAT domain of HIV and the CRAC domain of PBR. [(3)H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [(3)H]Promegestone also photoincorporated into the TAT-CRAC peptide. [(3)H]Promegestone crosslinking to TAT-CRAC could be displaced by cholesterol and promegestone, with IC50 values of 1 and 200 microM, respectively. TAT-CRAC efficiently transduced into MA-10 Leydig cells and inhibited the hCG- and cAMP-stimulated steroid production in a dose-dependent manner. TAT-CRAC did not affect the hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported steroidogenesis. Mutated TAT-CRAC lost its ability to bind [(3)H]promegestone and to inhibit the hCG-stimulated steroidogenesis. These results show that TAT-CRAC binds cholesterol and competes for cholesterol interaction with endogenous PBR, suggesting that the cytosolic carboxyl-terminal domain of PBR is responsible for taking up and bringing steroidogenic cholesterol into the mitochondria.

In steroid-synthesizing cells, like the MA-10 mouse tumor Leydig cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Expression of PBR in Escherichia coli DE3 cells, which have no PBR, no cholesterol, and do not make steroids, induced the ability to take up cholesterol in a time-dependent, temperature-sensitive, and energy-independent manner. These cells took up no other steroids tested. Addition of the high affinity PBR ligand PK 11195 to cholesterol-loaded membranes, obtained from cells transfected with PBR, resulted in the release of the uptaken cholesterol. Expression in DE3 cells of mutant PBRs demonstrated that deletions in the cytoplasmic carboxy-terminus dramatically reduced the cholesterol uptake function of PBR, although it retained full capacity to bind PK 11195. Site-directed mutagenesis in the carboxy-terminal region of PBR demonstrated that bacteria expressing the mutant PBR proteins PBR(Y153S) and PBR(R156L) do not accumulate cholesterol, suggesting that amino acids Y153 and R156 are involved in the interaction of the receptor with cholesterol. Considering these results, we postulate the existence of a common cholesterol recognition/interaction amino acid consensus pattern (-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-). Indeed, we found this amino acid consensus pattern in all proteins shown to interact with cholesterol. In conclusion, these data suggest that the expression of PBR confers the ability to take up and release, upon ligand activation, cholesterol. Considering the widespread occurrence of this protein and its tissue and cell specific subcellular localization, these results suggest a more general role of PBR in intracellular cholesterol transport and compartmentalization.

The effects of hypnotics on descriptive and functional aspects of electrophysiological sleep parameters are assessed in this report. Because of the arbitrary definition of some of the criteria underlying the conventional sleep stage scoring procedure, computer-aided methods of EEG analysis have become increasingly important for recording and interpreting pharmacological effects on sleep. Of particular interest are the changes of EEG slow-wave activity, since this parameter varies as a function of prior sleep and waking. Several types of interaction between hypnotics and sleep regulation are discussed, some recent pharmacological developments are highlighted, and some common problems in clinical trials are specified.