Elevated alcohol consumption in null mutant mice lacking 5-HT1B serotonin receptors.

Substantial evidence links alcohol drinking and serotonin (5-HT) functioning in animals. Lowered central 5-HT neurotransmission has been found in a subgroup of alcoholics, possibly those with more aggressive, assaultive tendencies. Several rodent studies have also suggested that intact 5-HT systems are important determinants of sensitivity and/or tolerance to ethanol-induced ataxia and hypothermia. Null mutant mice lacking the 5-HT1B receptor gene (5-HT1B-/-) have been developed that display enhanced aggression and altered 5-HT release in slice preparations from some, but not all, brain areas. We characterized these mice for sensitivity to several effects of ethanol. Mutant mice drank twice as much ethanol as wild-type mice, and voluntarily ingested solutions containing up to 20% ethanol in water. Their intake of food and water, and of sucrose, saccharin and quinine solutions, was normal. Mutants were less sensitive than wild-types on a test of ethanol-induced ataxia and, with repeated drug administration, tended to develop tolerance more slowly. In tests of ethanol withdrawal and metabolism, mutants and wild-type mice showed equivalent responses. Our results suggest that the 5-HT1B receptor participates in the regulation of ethanol drinking, and demonstrate that serotonergic manipulations lead to reduced responsiveness to certain ataxic effects of ethanol without affecting dependence.

Activation of corticostriatal pathway leads to similar morphological changes observed following haloperidol treatment.

Treatment with haloperidol, a dopamine receptor D-2 antagonist, for one month resulted in an increase in the mean percentage of asymmetric synapses containing a discontinuous, or perforated, postsynaptic density (PSD) [Meshul et al. (1994) Brain Res., 648:181-195] and a change in the density of striatal glutamate immunoreactivity within those presynaptic terminals [Meshul and Tan (1994) Synapse, 18:205-217]. We speculated that this haloperidol-induced change in glutamate density might be due to an activation of the corticostriatal pathway. To determine if activation of this pathway leads to similar morphological changes previously described following haloperidol treatment, GABA (10(-5) M, 0.5 microliters) was injected into the thalamic motor (VL/VM) nuclei daily for 3 weeks. This treatment resulted in an increase in the mean percentage of striatal asymmetric synapses containing a perforated PSD and an increase in the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated or non-perforated PSD. Subchronic injections of GABA into the thalamic somatosensory nuclei (VPM/VPL) had no effect on the mean percentage of synapses with perforated PSDs but resulted in a small, but significant, increase in density of glutamate immunoreactivity. Using in vivo microdialysis, an acute injection of GABA (10(-5) M, 15 microliters) into VL/VM resulted in a prolonged rise in the extracellular level of striatal glutamate. The increase in asymmetric synapses with perforated PSDs and in glutamate immunoreactivity within nerve terminals of the striatum following either subchronic haloperidol treatment or GABA injections into VL/VM suggest that an increase in glutamate release may be a common factor in these two experiments. It is possible that the extrapyramidal side effects associated with haloperidol treatment may be due, in part, to an increase in release of glutamate within the corticostriatal pathway.

Audiogenic seizure susceptibility in WSP and WSR mice.

Mice selectively bred for susceptibility (WSP, withdrawal seizure prone) and resistance (WSR, withdrawal seizure resistant) to ethanol (EtOH) withdrawal seizures were tested for susceptibility to audiogenic seizures (AGS). The seizure response of mice was studied at four ages: 17, 22, 28, and 71-78 days. WSR mice exhibited no response at any age, whereas WSP mice were sensitive on days 17, 22, and 28. The maximum number of WSP mice responding to audiogenic stimulation was observed on day 22. However, the frequency and severity of responses by WSP mice was less than that of DBA/2J mice tested under identical conditions (60 vs. 100% showing at least some response). Overall, these data suggest that susceptibility to AGS and handling-induced convulsions (HIC) during EtOH withdrawal may share some common genetic determinants and presumably some common neurochemical systems. Various treatments have been shown to enhance HIC more in WSP as compared with WSR mice. Acoustic stimulation did not induce AGS in adult mice, but the treatment significantly enhanced HIC in WSP but not WSR mice. These data strongly imply that some common neurochemical pathway may regulate susceptibility to HIC elicited by diverse treatments.

Mu-opiate receptor binding and function in HOT and COLD selected lines of mice.

mu-Opiate receptor binding and function were examined in mice selectively bred for sensitivity (COLD) and resistance (HOT) to ethanol-induced hypothermia. These mice also have differential hypothermic sensitivity to mu-opiates. mu-Opiate receptor density was higher in the frontal cortex of HOT mice compared with COLD mice, but was the same in other brain areas. In addition, there were no line differences in Kd values. Basal adenylate cyclase (AC) activity was similar in both lines, as was the response to forskolin (FS) stimulation. Morphine was more effective at inhibiting FS-AC activity in the hypothalamus of HOT mice compared with COLD mice but was equally effective in the frontal and parietal cortex. There were no differences between lines in basal Ca2+, Mg2+, or Ca2+/Mg(2+)-ATPase activity. Further, 30 min after treatment ATPase activities were not altered in ethanol- or levorphanol-treated mice. These results suggests that mu-opiate biochemical pathways, but not ATPase enzyme systems, may be involved in mediating differential hypothermic sensitivity observed in HOT and COLD mice.

Olfactory bulb kindling in mice susceptible and resistant to ethanol withdrawal.

The relation between kindling and susceptibility to ethanol withdrawal seizures was investigated using withdrawal seizure-prone (WSP) and withdrawal seizure-resistant (WSR) mice. These lines were developed by selective breeding to be prone and resistant, respectively, to handling-induced convulsions after chronic exposure to ethanol. Development of kindled seizures in response to electrical stimulation of the olfactory bulb was investigated in mice aged 2 and 8 months with no exposure to ethanol. Older WSP mice kindled more slowly than older WSR mice, requiring significantly more stimulations to reach the first stage 3 and the first stage 5 seizures. In younger mice, there was no significant difference between the two lines in the rate of kindling. The lower kindling rate in mature WSP mice is in contrast to their higher sensitivity to handling-induced convulsions on withdrawal from ethanol and other agents. This finding suggests that separate genetic factors underlie these two models of mouse seizures.

Antisense GAP-43 inhibits the evoked release of dopamine from PC12 cells.

To investigate the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, F1, P-57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP-43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP-43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP-43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K+ (56 mM) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP-43 RNA in PC12 cells leads to a decrease in GAP-43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP-43 in calcium-dependent neurotransmitter release.

Serotonin and genetic differences in sensitivity and tolerance to ethanol hypothermia.

Mice have been selectively bred for genetic sensitivity (COLD) or insensitivity (HOT) to acute ethanol-induced hypothermia. COLD mice readily develop tolerance to the hypothermic effects of ethanol (EtOH) when it is chronically administered, while HOT mice do not. A number of studies have implicated serotonergic systems in both sensitivity and the development of tolerance to the hypothermic and ataxic effects of EtOH. In the experiments reported here, we administered the serotonin (5HT) neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) to HOT and COLD mice before the acute and chronic administration of equipotent doses of EtOH. 5,7-DHT lesions significantly reduced (by about 65%) whole brain levels of 5HT in both selected lines. This treatment reduced sensitivity to acute EtOH hypothermia in COLD, but not in HOT mice, and blocked the development of tolerance only in COLD mice. Metabolites of 5HT, norepinephrine, and dopamine were generally increased in hypothalamic and brain stem tissue after acute EtOH injection, but HOT and COLD mice were not differentially susceptible to these effects. These results suggest that genes affecting 5HT systems may mediate some of the differences in response to the hypothermic effects of EtOH characterizing HOT and COLD mice.

Hippocampal mossy fiber zinc deficit in mice genetically selected for ethanol withdrawal seizure susceptibility.

Hippocampal mossy fiber zinc was examined in mice selectively bred for differences in susceptibility to handling-induced convulsions during ethanol withdrawal. The density of mossy fiber zinc in the CA3 stratum lucidum was significantly decreased in the duplicate lines of untreated withdrawal seizure prone (WSP) mice compared to untreated withdrawal seizure resistant (WSR) mice. Mossy fiber zinc densities in randomly bred control lines of mice (WSC) were intermediate to WSP and WSR mice. Serum, whole brain and whole hippocampal zinc were not significantly different between WSP and WSR mice, indicating that the reduction in the chelatable pool of hippocampal mossy fiber zinc was not a consequence of deficits in brain or whole body zinc nutrition. A highly significant correlation between hippocampal mossy fiber zinc density and handling-induced convulsion indices suggests that a reduction in mossy fiber zinc may be one contributing factor in the expression of seizure susceptibility in WSP mice.

Effect of alcohols and other hypnotics in mice selected for differential sensitivity to hypothermic actions of ethanol.

Mice selectively bred for resistance (HOT) and sensitivity (COLD) to the hypothermic effect of an acute dose of ethanol were tested twice during the course of genetic selection for their hypothermic response to other alcohols and sedative hypnotics. The drugs administered were ethanol, propanol, n-butanol, t-butanol, pentanol, diazepam, phenobarbital, pentobarbital, methyprylon and ethchlorvynol, all of which have sedative effects on the central nervous system, and hydralazine, a peripheral vasodilator. All drugs decreased body temperature of both HOT and COLD mice. In mice selected for seven to nine generations, COLD mice were more sensitive than HOT mice to all sedative drugs. The longer-chain alcohols were more potent than ethanol in inducing hypothermia, but the magnitude of the response difference between HOT and COLD mice appeared to be smaller. The difference between HOT and COLD mice in hypothermic sensitivity to an acute dose of ethanol was greater after 11-15 generations of selection than after seven generations. Similarly, the differential effect of the other alcohols, phenobarbital, pentobarbital, and methyprylon, on HOT and COLD mice increased with more generations of selection but to a lesser extent than ethanol. These data demonstrate that selecting for sensitivity to acute ethanol hypothermia has produced mice that are also differentially sensitive to other sedative hypnotic agents. They also support the hypothesis that the drugs used in the present study share a common mechanism of action for inducing hypothermia, which may be regulated by a common set of genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of neurotransmitter-selective drugs in mice selected for differential sensitivity to the hypothermic actions of ethanol.

Mice selectively bred for resistance (HOT) and sensitivity (COLD) to the hypothermic effect of EtOH were tested for their hypothermic response to neurotransmitter-specific drugs and for the effect of such drugs on EtOH induced hypothermia (HT). The drugs administered were the opiate drugs morphine, levorphanol and U50488H, the dopamine agonists apomorphine, LY171535 and SKF38393, the dopamine antagonist chlorpromazine, the alpha adrenergic agonist St587, the cholinergic agonist nicotine and amphetamine, which increases the release of catecholamines. All of the drugs tested, with the exception of SKF38393 and amphetamine, induced a hypothermic response in HOT and COLD mice. SKF38393 had no effect on body temperature or HT produced by EtOH. Amphetamine caused HT at low doses and hyperthermia at high doses. COLD mice were more sensitive than HOT mice to the hypothermic effect of morphine and levorphanol, mu-opiate agonists, and U50488H, a relatively specific kappa agonist. All of the other drugs tested were approximately equally potent in HOT and COLD mice. These results suggest that the differential sensitivity of HOT and COLD mice to EtOH-induced HT may be partially mediated through genetic changes in opiate mechanisms.

Behavioral and neurochemical studies in diazepam-sensitive and -resistant mice.

Benzodiazepine (BZ) effects include anxiolyis, sedation, seizure protection, and muscle relaxation; the mechanisms underlying these various effects are not understood. We have recently used the rotarod test in conjunction with selective breeding techniques to develop lines of mice which are diazepam-sensitive (DS) and diazepam-resistant (DR). We review the general methods of selective breeding, along with a description of the DS/DR selection study, and then describe a variety of behavioral and neurochemical studies which have been conducted in an attempt to characterize these mice. We have investigated the effects of other sedative drugs believed to interact with the BZ receptor, including ethanol, pentobarbital, and phenobarbital. We have also tested these mice for seizure threshold and open-field activity. DS and DR mice do not differ in diazepam-induced seizure protection, suggesting that different mechanisms underlie rotarod performance and the anti-convulsant effect. These results provide evidence to support the search for nonsedating anti-convulsants. To determine the neurochemical basis for observed differences, BZ receptor density and chloride flux have been measured. We discuss the interaction between behavioral and neurochemical approaches, and describe a conceptual framework to guide future studies with these unique new animals.

Genetic components of ethanol responses.

A powerful technique for determining the role of a particular neurotransmitter in mediating a response to ethanol (EtOH) is the analysis of selectively bred lines of animals. Lines selected for sensitivity and resistance to an EtOH effect differ principally in gene frequencies for genes affecting the selected response. Hence, other differences between the lines are likely due to pleiotropic actions of those genes. We discuss behavioral pharmacological experiments in two sets of selected lines. Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mouse lines were selected for severe and minimal handling-induced convulsions (HIC), respectively, after withdrawal from chronic EtOH inhalation. The HIC is also elevated after acute administration of low doses of convulsant drugs. WSP mice were found to be more sensitive than WSR mice to many such drugs. There was no apparent specificity of such effects to any particular neurotransmitter system. Thus, genetic determination of a behavioral response to EtOH in this case cannot be traced to the influence of a single neurotransmitter system. COLD and HOT mice were selectively bred to show severe and mild hypothermia, respectively, after acute EtOH administration. COLD mice are also more sensitive to a number of other alcohols, barbiturates, and other general central nervous system depressants. When tested for sensitivity to a number of drugs with specific effects on neurotransmitter systems, COLD and HOT mice did not differ in sensitivity to drugs affecting dopaminergic, alpha-adrenergic, or nicotinic acetylcholinergic systems. COLD mice were more sensitive, however, to opioid and serotonergic drugs. Thus, analysis of these selected lines was successful in identifying particular neurotransmitters which may be important in EtOH-induced hypothermia.

Effect of chronic ivermectin treatment on GABA receptor function in ethanol withdrawal-seizure prone and resistant mice.

Ivermectin, a potent, effective anthelmintic, is easy to administer, has a broad spectrum of action and a wide safety margin. However, no testing has been done in hosts genetically selected for seizure susceptibility which may be more sensitive to the effects of ivermectin than other animals. This was done in the present experiments with seizure prone and seizure resistant mice infested with Syphacia obvelata (pinworm). These subjects were treated daily with oral ivermectin in their drinking water every other week for six weeks, for a total of 21 days. The treatment cleared the mice of the pinworm infestation, but did not alter the seizure susceptibility or binding parameters of [3H]flunitrazepam in either of the selected lines.

Selected mouse lines, alcohol and behavior.

The technique of selective breeding has been employed to develop a number of mouse lines differing in genetic sensitivity to specific effects of ethanol. Genetic animal models for sensitivity to the hypnotic, thermoregulatory, excitatory, and dependence-producing effects of alcohol have been developed. These genetic animal models have been utilized in numerous studies to assess the bases for those genetic differences, and to determine the specific neurochemical and neurophysiological bases for ethanol's actions. Work with these lines has challenged some long-held beliefs about ethanol's mechanisms of action. For example, lines genetically sensitive to one effect of ethanol are not necessarily sensitive to others, which demonstrates that no single set of genes modulates all ethanol effects. LS mice, selected for sensitivity to ethanol anesthesia, are not similarly sensitive to all anesthetic drugs, which demonstrates that all such drugs cannot have a common mechanism of action. On the other hand, WSP mice, genetically susceptible to the development of severe ethanol withdrawal, show a similar predisposition to diazepam and phenobarbital withdrawal, which suggests that there may be a common set of genes underlying drug dependencies. Studies with these models have also revealed important new directions for future mechanism-oriented research. Several studies implicate brain gamma-aminobutyric acid and dopamine systems as potentially important mediators of susceptibility to alcohol intoxication. The stability of the genetic animal models across laboratories and generations will continue to increase their power as analytic tools.

Sensitivity and tolerance to ethanol-induced hypothermia in genetically selected mice.

COLD mice have been genetically selected for pronounced hypothermia (HT) after acute EtOH administration, whereas HOT mice have been selected for attenuated HT. In the current experiments, HOT and COLD mice were found to differ significantly in sensitivity to EtOH-induced HT across a range of doses: the difference was greater at higher doses. After 3 g/kg of EtOH, HOT mice displayed a 1.8 degrees C HT, whereas COLD mice had a 3.6 degrees C HT. Male mice had greater HT responses than female mice regardless of genotype. Nonselected control mice were intermediate to the HOT and COLD mice in responsiveness to EtOH. After an acute EtOH dose, HOT mice were found to have slightly lower brain EtOH concentrations than COLD mice 3 and 4 (but not 1 and 2) hr after administration of EtOH, and may have eliminated EtOH slightly more rapidly than COLD mice. When tested repeatedly in a cool ambient environment (18 degrees C), COLD mice developed tolerance to EtOH hypothermia, whereas HOT mice did not. These results confirm that sensitivity to the hypothermic effects of EtOH is influenced markedly by genotype. Furthermore, selection for neurosensitivity to EtOH has produced a correlated difference in rate or magnitude of tolerance development, which is consistent with an hypothesis of the influence of common genes determining these responses to EtOH. The difference in tolerance could not be accounted for by initial HT sensitivity differences between the lines. The HOT and COLD lines should be useful for studies of the neurobiological mechanisms of EtOH-induced HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in GABA activity between ethanol withdrawal seizure prone and resistant mice.

Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) lines of mice have been genetically selected based on the severity of handling-induced convulsions after identical chronic ethanol exposure. The present experiments showed that naive WSP mice were more sensitive than WSR mice to a subconvulsant dose of picrotoxin, bicuculline or pentylenetetrazole as measured by the ability of these drugs to exacerbate handling-induced convulsions. This may reflect a difference between lines in the GABA-chloride channel. The density and affinity of [35S]t-butylbicyclophosphorothionate (TBPS) binding sites, a cage convulsant which binds to the picrotoxin site on the GABA-chloride channel, was measured in the frontal cortex, remainder of the cortex, cerebellum and hippocampus. The binding properties of [3H]flunitrazepam and the potency of gamma-aminobutyric acid (GABA) to enhance flunitrazepam binding was characterized in whole brain samples. There were no differences between lines. The behavioral results suggest a role for the GABA-chloride channel in the differential ethanol withdrawal seizure behavior of WSR and WSP mice, but this is not due to changes in receptor densities or affinities.

Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit.

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.

The sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition.

Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated 22Na+ influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Binding of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which [Na+]out/[Na+]in is varied by changing [Na+]in or [Na+]out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.

Comparison of alpha-2 adrenergic receptors and their regulation in rodent and porcine species.

The alpha-2 adrenergic antagonist [3H]yohimbine (YOH) and the alpha-2 agonist [3H]p-aminoclonidine (PAC) saturably label high-affinity binding sites in the submandibular gland from 3-week-old rats and 5-week-old pigs and in the lung from neonatal rats and 5-week-old pigs. [3H]YOH had KD values of 5.5, 1.8, 0.45 and 0.22 nM in the rat gland and lung and porcine gland and lung, respectively. KD values of 2.4, 5.3 and 1.3 nM were found for [3H]PAC in rodent and pig submandibular gland and pig lung, respectively. Both 3H-ligands labeled approximately the same density of sites within each tissue except in the rat lung in which [3H]PAC binding was too low to reliably estimate. In all cases the pharmacologic profile was indicative of an alpha-2 adrenergic receptor site. However, the Ki of yohimbine vs. [3H]PAC was 30- to 140-fold higher for the rodent relative to the porcine species. GTP decreased the affinity of (-)-epinephrine and PAC at [3H]YOH-labeled sites in the pig gland and lung, but did not shift the affinity of epinephrine in the rat gland. These results suggest the possibility of subtype or species differences for the alpha-2 receptor. The Ki values of the antagonists YOH and phentolamine were different at [3H]PAC and [3H]YOH sites. GTP caused a dose-dependent reduction in [3H]PAC binding in the porcine submandibular gland and lung. At 10 microM GTP, this loss was due to a decrease in 3H-agonist affinity, but not density.(ABSTRACT TRUNCATED AT 250 WORDS)