Joseph V. Martin

RELATIONSHIP OF NEGATIVE CONTRAST TO ANIMAL MODELS OF FEAR AND ANXIETY

The relatedness of behavior elicited by reward reduction (successive negative contrast procedure) and behaviors produced by three animal models of anxiety (open-field emergence, elevated plus-maze, and context-shock fear conditioning) was examined by correlational and factor analytic procedures. Factor analysis (oblique rotation) indicated substantial independence among the tests: Trials 1 and 2 of the plus-maze loaded on two different factors unaccompanied by any other test; open-field emergence and context-shock fear loaded on the same factor; and negative contrast loaded on a fourth factor. However, negative contrast proved to be a dynamic process, with factor loadings changing across a 4-day postshift period—moving from an independent loading on the 1st postshift day to being clustered with context-shock fear and open-field emergence on the 2nd and 3rd postshift days to being clustered with just context-shock fear on the last postshift day. These latter data support a multistage theory of successive negative contrast.

Monoamine receptors in an animal model of affective disorder.

Abstract: After a relatively mild course of uncontrollable shocks, two distinct groups of rats can be defined in terms of their performance in learning to escape from a controllable stressor. Response‐deficient (RD) rats do not learn to terminate the controllable stressor, whereas nondeficient (ND) rats learn this response as readily as do untreated control rats. The current studies were designed to determine the neu‐rochemical correlates of the behavioral differences between these groups of rats. The major findings concerned postsyn‐aptic β‐adrenergic effects in the hippocampus of RD rats. These included an up‐regulation of β‐adrenergic receptors and, in parallel experiments, an increase in the sensitivity of adenylyl cyclase to stimulation by norepinephrine. There was no difference in brain levels of catecholamines between the three groups of rats. A statistically significant increase in levels of 5‐hydroxytryptamine was noted in the hippocampus and hypothalamus of RD rats as compared to levels in ND rats, but no significant differences were measured between groups of rats in terms of S1 or S2 serotonergic receptor binding. These results implicate both β‐adrenergic and serotonergic mechanisms in the behavioral deficit caused by uncontrollable shock.

Characterization of the hypnotic effects of triazolam microinjections into the medial preoptic area

We have previously reported that microinjections of the benzodiazepine hypnotic triazolam into the medial preoptic area (MPA) of the hypothalamus enhance sleep in rats. The present study further characterizes this effect, by examining its anatomical specificity, determining whether it is mediated by interaction with central benzodiazepine receptors, and assessing whether sleep induction is associated with changes in core temperature. It was found that microinjections of 0.25 and 0.5 micrograms triazolam into two nearby structures, the lateral preoptic area and diagonal band of Broca, failed to alter sleep. Total sleep time was enhanced by microinjection of triazolam into the MPA, and this effect was blocked by co-administration of the benzodiazepine receptor blocker RO 15-1788. Sleep enhancement by triazolam was not associated with significant alterations in core body temperature. These observations continue to suggest that the MPA may be a site which mediates the hypnotic effect of triazolam, and add to the growing body of data emphasizing the importance of hypothalamic function in the regulation of sleep and waking.

Direct channel-gating and modulatory effects of triiodothyronine on recombinant GABAA receptors

We have previously shown that triiodothyronine (T3) inhibits gamma-aminobutyric acid type A (GABA(A)) receptors in synaptoneurosomes and transfected cells. To further characterize this phenomenon, the effect of T3 on recombinant GABA(A) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp method. T3 inhibited GABA-gated chloride currents in a non-competitive manner and yielded an IC50 of 7.3 +/- 0.8 microM in oocytes coexpressing alpha1beta2gamma2L receptor subunits. T3 had no inhibitory effect on alpha6beta2gamma2L or beta2gamma2L receptor constructs, indicating that the alpha1 subunit imparts T3 sensitivity to the receptor. In addition to the inhibitory effect of T3 on GABA responses, T3 alone induced a current in oocytes expressing alpha1beta2gamma2L, alpha6beta2gamma2L and beta2gamma2L constructs. This current displayed a reversal potential identical to that of GABA-gated chloride currents, and was blocked by picrotoxin (10 microM), but not by bicuculline (50 microM), indicating that T3 gates the chloride channel by binding to a site other than the GABA-binding site. The direct channel-gating action of T3 was concentration-dependent, with an EC50 of 23 +/- 5 microM. The actions of T3 are unique in that T3 acts as a noncompetitive antagonist in the presence of GABA but can directly gate the chloride channel in the absence of GABA.

Inhibition of sleep and benzodiazepine receptor binding by a β-carboline derivative

The effects of systemic injections of beta-carboline-3-carboxylate-t-butyl ester (beta-CCtB) were investigated with regard to normally occurring sleep and several measures of benzodiazepine receptor occupancy in rats. A dose of 30 mg/kg of beta-CCtB was found to have a long time-course of action as measured by an in vivo assay for benzodiazepine binding, with an 84% depletion of [3H]diazepam binding at one hour after the intraperitoneal injection. This dose of beta-CCtB was shown to delay sleep onset, decrease non-REM and total sleep in the first two hours after the injection, and to delay the appearance of REM sleep after the sleep onset. The dose- and time-dependence of the effects on sleep approximated the dose- and time-dependence of inhibitory effects of an IP injection of beta-CCtB on in vitro measures of benzodiazepine receptor affinity and number.

Behavioral and electroencephalographic effects of the adenosine1 agonist, L-PIA

The effects of N6-(L-2-phenylisopropyl)-adenosine (L-PIA), an A1 agonist, were measured on both spontaneous locomotor activity and electroencephalographic (EEG) measures of sleep in rats. L-PIA strongly inhibited motor activity at 100 micrograms/kg intraperitoneally (IP), a dose which had no statistically significant effects on EEG-defined sleep. A higher dose of L-PIA (200 micrograms/kg) increased the latency to sleep initiation and inhibited later REM sleep. These results demonstrate that L-PIA can produce a state of apparent behavioral quiescence in the presence of EEG-defined arousal.

Influence of oxygenated fuel additives and their metabolites on the binding of a convulsant ligand of the γ-aminobutyric acidA (GABAA) receptor in rat brain membrane preparations

As a foundation for evaluating potential mechanisms of the neurological effects (e.g. headache, nausea, dizziness) of some octane boosters, we studied the gamma-aminobutyric acid(A) (GABA(A)) receptor in a series of binding assays in membranes from rat brain. The GABA(A) receptor was probed using the radioligand [3H]t-butylbicycloorthobenzoate ([3H]TBOB) which binds to the convulsant recognition site of the receptor. The results demonstrated that the short-chain t-ethers and their t-alcohol metabolites inhibit binding at the convulsant site of the GABA(A) receptor. The potency of the inhibition tended to correlate with carbon chain length. For agents having an equal number of carbon atoms, potency of inhibition of [3H]TBOB binding was greater in magnitude for the alcohols than for the ethers. The descending rank order of potency for the ethers and alcohols were as follows, t-amyl alcohol (TAA); t-amyl-methyl ether (TAME); ethyl-t-butyl ether (ETBE)>t-butyl alcohol (TBA)>methyl-t-butyl ether (MTBE)>ethanol. In additional saturation binding assays, MTBE reduced apparent density of convulsant binding (B(max)).

Benzodiazepine binding varies with stage of estrous cycle in unwashed membranes from mouse brain

The influence of the stage of the estrous cycle on binding of [3H]diazepam was examined in membranes from brains of female mice. In order to conserve endogenous factors such as progesterone, other steroids, or GABA, the assay was performed without the extensive washing procedures typically employed in measurements of benzodiazepine binding. Significant variations in the apparent maximal numbers of binding sites (Bmax) were noted during the estrous cycle in both hypothalamus and cortex. The Bmax measured in membranes from proestrus female mice was significantly higher than in membranes from mice at other stages in the estrous cycle. Variations in apparent equilibrium binding dissociation constants (Kd) were not statistically significant by stage of the estrous cycle. The demonstrated variations in binding suggest the existence of a factor which varies with the estrous cycle in female mice and modulates the activity of the GABAA receptor complex.

Diazepam enhances intrasynaptosomal free calcium concentrations.

Synaptosomes prepared from brains of rats were incubated in different concentrations of diazepam under conditions designed to reduce the action of a reversed Na+/Ca2+ exchanger. In synaptosomes depolarized in the presence of added Ca2+, doses of diazepam ranging from 0.1 to 100 microM were found to significantly enhance Ca2+ levels measured with the fluorescent dye fura-2, compared to control incubations without drug. Furthermore, doses of diazepam as low as 1 microM significantly increased the concentration of Ca2+ in non-depolarized synaptosomes without added Ca2+ in the medium. The effects of depolarization and diazepam treatment were synergistic in increasing the levels of intrasynaptosomal Ca2+.

Effects of acute microinjections of thyroid hormone to the preoptic region of euthyroid adult male rats on sleep and motor activity

In adult brain tissue, thyroid hormones are known to have multiple effects which are not mediated by chronic influences of the hormones on heterodimeric thyroid hormone nuclear receptors. Previous work has shown that acute microinjections of l-triiodothyronine (T3) to the preoptic region significantly influence EEG-defined sleep in hypothyroid rats. The current study examined the effects of similar microinjections in euthyroid rats. In 7 rats with histologically confirmed microinjection sites bilaterally placed in the preoptic region, slow-wave sleep time was significantly decreased, but REM and waking were increased as compared to vehicle-injected controls. The EEG-defined parameters were significantly influenced by the microinjections in a biphasic dose-response relationship; the lowest (0.3μg) and highest (10μg) doses tested were without significant effect while intermediate doses (1 and 3μg) induced significant differences from controls. There were significant diurnal variations in the measures, yet no significant interactions between the effect of hormone and time of day were demonstrated. Core body temperature was not significantly altered in the current study. The demonstration of effects of T3 within hours instead of days is consistent with a rapid mechanism of action such as a direct influence on neurotransmission. Since the T3-mediated effects were robust in the current work, euthyroid rats retain thyroid hormone sensitivity which would be needed if sleep-regulatory mechanisms in the preoptic region are continuously modulated by the hormones. This article is part of a Special Issue entitled LInked: BRES-D-12-01552 & BRES-D-12-01363R2.