Potential pleiotropic effects of Mpdz on vulnerability to seizures.

We previously mapped quantitative trait loci (QTL) responsible for approximately 26% of the genetic variance in acute alcohol and barbiturate (i.e., pentobarbital) withdrawal convulsion liability to a < 1 cM (1.8 Mb) interval of mouse chromosome 4. To date, Mpdz, which encodes the multiple PSD95/DLG/ZO-1 (PDZ) domain protein (MPDZ), is the only gene within the interval shown to have allelic variants that differ in coding sequence and/or expression, making it a strong candidate gene for the QTL. Previous work indicates that Mpdz haplotypes in standard mouse strains encode distinct protein variants (MPDZ1-3), and that MPDZ status is genetically correlated with severity of withdrawal from alcohol and pentobarbital. Here, we report that MPDZ status cosegregates with withdrawal convulsion severity in lines of mice selectively bred for phenotypic differences in severity of acute withdrawal from alcohol [i.e., High Alcohol Withdrawal (HAW) and Low Alcohol Withdrawal (LAW) lines] or pentobarbital [High Pentobarbital Withdrawal (HPW) and Low Pentobarbital Withdrawal (LPW) lines]. These analyses confirm that MPDZ status is associated with severity of alcohol and pentobarbital withdrawal convulsions. Using a panel of standard inbred strains of mice, we assessed the association between MPDZ status with seizures induced by nine chemiconvulsants. Our results show that MPDZ status is genetically correlated with seizure sensitivity to pentylenetetrazol, kainate and other chemiconvulsants. Our results provide evidence that Mpdz may have pleiotropic effects on multiple seizure phenotypes, including seizures associated with withdrawal from two classes of central nervous system (CNS) depressants and sensitivity to specific chemiconvulsants that affect glutaminergic and GABAergic neurotransmission.

Behavior-related modulation of substantia nigra pars reticulata neurons in rats performing a conditioned reinforcement task.

Motor-control models of basal ganglia function have emphasized disinhibition through reduction of tonic, inhibitory output. Although these models have shed important light on basal ganglia operations, evidence emerging from electrophysiological studies of behaving primates suggests that disinhibition alone may not adequately explain the role of the basal ganglia in movement. To assess this role in the rat, the most frequently used subject in studies of basal ganglia function, we recorded neuronal activity in the primary output nucleus, the substantia nigra pars reticulata, during an operant task. After rats were trained to nosepoke into an illuminated hole for access to a 10% sucrose solution delivered through a spout, single- and multiple-unit activity was recorded during 60-120 nosepoke trials. Compared to the period 60 s before the start of the first trial in the task, 110 of 225 reticulata units increased firing >200% while 17 of 225 decreased to 40% of baseline. Of these 225 units, >60% responded coincident with specific task events such as nosepokes and spout licking. Most nosepoke-responsive units showed either excitation (>50%) or a combination of excitation and inhibition (>25%) rather than inhibition alone (>20%). Increases in firing were also common during approach and licking at the spout, with inhibitions alone comprising 30% of responses. In some units, there was evidence of reward-related responding, with changes occurring in anticipation of reward delivery or during the delivery of sucrose, but not the persistent licking that continued for several seconds after its offset. While 70% of units responded during both nosepokes and spout licking, changes in firing were typically unique depending on the motor behavior required (i.e. nosepoking vs. licking). Our results, which indicate a prominent role for increases in nigra reticulata activity during movement, add to growing evidence that although inhibitions may allow desired motor responses to emerge, excitations may help shape behavioral output by suppressing competing motor programs.

Long T methamphetamine schedules produce circadian ensuing drug activity in rats.

Eight female Sprague-Dawley rats were housed in isolated continuous 24-h/day environments under conditions of constant dim light and a rate-limited feeding schedule. Following 2 months of free-running activity, all animals were administered methamphetamine (MA) i.p. (2 mg/kg) once every 31 h for 24 injection cycles. Average wave forms of wheel-running activity showed that animals did not anticipate the 31-h schedule of MA injections, but rather displayed circadian ensuing drug activity (CEDA) between 24 and 28 h following the injections. Post-injection meals failed to meet reliably the threshold necessary to achieve food-engendered anticipatory or ensuing activity. Cosinor analysis showed that the intensity of CEDA was strongly influenced by the relative phase of the free-running rhythm. CEDA was moderately influenced by the size of the post-injection bout of activity. Because injection times rotated daily throughout local time without repeating a time of day, CEDA resulting from a long T schedule of MA administration appeared to be based on one-trial resetting of a circadian-related mechanism by a major drug of abuse.

Effects of ethanol on rat somatosensory cortical neurons.

In this study, we characterized the local effects of ethanol (EtOH) on postsynaptic potentials (PSPs) and membrane properties of layer II-III (L2-3) and layer V (L5) somatosensory cortical neurons. Intracellular recordings were done using the in vitro slice preparation of rat somatosensory cortex. Our results show that EtOH exerts local effects on cortical cell membrane at physiologically relevant concentrations. A predominant effect of EtOH was to reduce excitability of L2-3 and L5 neurons by increasing the rheobase, decreasing input resistance and repetitive firing, reducing PSPs amplitude and the probability of evoking action potentials. Early (6 ms) and late (18 ms) PSP components were affected differentially by EtOH, the late components being more suppressed. Overall, EtOH-mediated suppression of PSPs was stronger in L5 neurons. Cortical neurons were divided into three subtypes: regular spiking adapting (RS-A), regular spiking non-adapting (RS-NA) and bursting (D-IB) neurons. PSPs evoked in RS-A neurons were more sensitive to EtOH suppressant effects. EtOH effects on input resistance were distributed differentially among the three groups of neurons. These results support the notion that EtOH disrupts higher processing of somatosensory information via a differential alteration of cortical neuron's membrane properties and synaptic transmission.

Circadian activity precedes daily methamphetamine injections in the rat.

Scheduled daily injections of methamphetamine (MA) produced locomotor activity that preceded and followed the usual time of injection in rats housed under conditions of constant, moderately dim light and temporally distributed feeding. A circadian basis for pre-injection time activity was supported by its anticipatory timing in the apparent absence of reliable preceding external cues and by its persistence on a test day on which the rats remained undisturbed. Post-injection time locomotor activity also persisted on the test day, occurring from 24 to 29 h after the final MA injection. These results indicate that MA injections engage circadian processes underlying locomotor activity, and they raise the possibility that intake of drugs of abuse by humans may facilitate drug taking or relapse at times of day related to previous drug use.

Behavioral associations of neuronal activity in the ventral tegmental area of the rat.

The ventral tegmental area (VTA) is a central element in a system that mediates the reinforcing properties of natural stimuli (such as food), brain stimulation, and drugs of abuse. Although considerable effort has been applied to understanding how drugs of abuse influence this system, relatively little work has examined its function during conditioned reinforcement tasks in awake, behaving animals. In the present studies, bundles of four to eight microwire electrodes were chronically implanted in the VTA or prefrontal cortex (PFC) of male Wistar rats. Following recovery from surgery, simultaneous recordings from multiple single neurons and unit clusters were obtained in rats pressing a lever for a sucrose solution on a fixed-ratio schedule of reinforcement. Consistent with the hypothesis that these neurons encode information related to motivation, most of the neurons in both VTA and PFC showed significant modulation of firing rate associated with one or more events occurring within the response/reinforcement cycle. These events included lever presses, onset and end of a tone signaling sucrose delivery, and onset and end of sucrose consumption. Significant decreases in firing rate were observed, coincident with onset of the tone and sucrose delivery, or with consumption. These decreases were sustained through the end of sucrose consumption. A number of neurons also discharged bursts of activity associated with individual lever presses. These findings provide a clear demonstration that VTA neuronal activity is modulated during motivated behavior. Similar information about events within the ongoing response/reinforcement cycle appears to be distributed through many neurons within the VTA, and may be mirrored in target structures such as PFC.

Sensitivity to N-methyl-D-aspartic acid-induced convulsions is genetically associated with resistance to ethanol withdrawal seizures.

Mice genetically selected to be resistant (withdrawal-seizure resistant, WSR) or prone (withdrawal-seizure prone, WSP) to handling-induced convulsions during ethanol withdrawal were tested for sensitivity to convulsions induced by timed intravenous (i.v.) infusion of N-methyl-D-aspartic acid (NMDA). WSR mice displayed convulsions at infused doses of NMDA that averaged 20% lower than WSP mice. This result was present in both genetically independent replicates of the WSR and WSP mice and provides strong evidence for an involvement of the NMDA system in the difference in withdrawal seizures present in these lines.

Neural sensitivity to pentylenetetrazol convulsions in inbred and selectively bred mice.

In these experiments, sensitivity to the convulsant drug pentylenetetrazol (PTZ) was measured in 10 inbred mouse strains, and in 4 mouse lines selectively bred for severe (WSP1, WSP2) or minimal (WSR1, WSR2) ethanol withdrawal convulsions. Using a timed infusion procedure, sensitivity to convulsions was assessed by measures of latency to convulsion, effective dose (ED) infused at time of convulsion, and brain concentration (BC) of PTZ at time of convulsion. In addition, ED and latency to convulsion were measured in WSP and WSR mice at 5 different concentrations of PTZ. Higher concentrations, which increased rate of drug infusion, reduced latency but had little effect on ED. WSP1 mice were slightly more sensitive to PTZ than WSR1 mice, but WSP2 mice were equally or less sensitive than WSR2 mice. Among the inbred strains, latency, ED and brain PTZ concentration were found to be highly correlated, suggesting that pharmacokinetic factors do not significantly influence access of PTZ to sites of action in the central nervous system. The C57BL/6J strain was least sensitive by all measures, while DBA/2J mice were highly sensitive. The BALB/cJ strain was the most sensitive strain as assessed by ED and latency, but BC indicated relatively average sensitivity. Apparently, pharmacokinetic factors in this strain result in a relatively rapid accumulation of drug in brain, making it appear to be more sensitive. Thus, although ED provides a reliable estimate of neural sensitivity in general, genetic factors exist which, in some strains, modify access of PTZ and possibly other drugs to brain, potentially affecting determination of sensitivity in the absence of a measure of brain drug concentration.

Genetic correlations among inbred strain sensitivities to convulsions induced by 9 convulsant drugs.

Inbred mouse strains differed significantly in sensitivity to convulsions induced by 9 convulsant drugs administered using a timed infusion procedure. Some strains (e.g. BALB/cJ, A/J) were generally seizure-susceptible, while some were generally seizure resistant (e.g. C57BL/6J, SWR/J). However, the overall pattern of strain sensitivities was complex, and depended upon drug and convulsant sign. Five of the drugs (bicuculline, DMCM, picrotoxin, TBPS and pentylenetetrazol (PTZ] produce convulsions, at least in part, through their interactions with the GABA receptor, while the other 4 (strychnine, CHEB, 4-aminopyridine and kainic acid) act through independent mechanisms. We predicted that responses to drugs with similar mechanisms of action would be genetically correlated. However, strains sensitive to picrotoxin-induced convulsions were not necessarily sensitive to convulsions elicited by PTZ or TBPS. Furthermore, different convulsant signs produced by a single drug were not always strongly correlated. Instead, genetic correlations were found among inbred strains for sensitivity to similar convulsant signs produced by different drugs. This suggests that genetic variation in sensitivity to these convulsant drugs arises primarily from variation in systems important for the expression of the convulsion.

Estimation of genetic correlation: interpretation of experiments using selectively bred and inbred animals.

There is increasing interest in determining the extent to which multiple characters related to drug sensitivity are influenced by common genes. The principal method for testing for the existence of such genetic correlations has been examination of pairs of mouse or rat lines selectively bred for sensitivity or resistance to a single behavioral effect of a drug. When a pair of selected lines is found to differ significantly on some trait other than the one on which they were selected, it is commonly concluded that significant genetic correlation between the traits exists, implying the action of a common set of genes on the two responses. In addition, results from comparisons of lines of animals selected for trait X and tested for trait Y may be compared with results from lines selected for trait Y and tested for trait X. As the number of correlated responses in selected lines increases, it becomes more important to adhere to sensible, consensual guidelines for interpreting such line differences. The principles underlying phenotypic and genotypic correlational analyses with selected lines are discussed. A scheme is presented to allow standardization across laboratories of inferences about the relative strength of genetic association from experiments with selected lines. Statistical and practical experimental issues are addressed. Estimates of genetic correlations may also be derived from the correlation of mean trait values across a panel of inbred strains. Existing data have sometimes found estimates of genetic correlations made with one approach to be inconsistent with those estimated in other ways. Possible reasons for this are discussed. Finally, the relationship between phenotypic correlations and genetic correlations is discussed. Phenotypic and genetic correlations for a pair of traits may differ widely, and may even be opposite in sign. Both are characteristic of the population from which they are sampled. Phenotypic correlations estimated within selected lines may change over time, as the additive genetic variance in the selected trait is exhausted. A specific example of this phenomenon is given.

The effect of ethanol on behavioral temperature regulation in mice.

Mice were injected with 20% ethanol in 0.9% NaCl, or with 0.9% NaCl without ethanol during sessions of behavioral thermoregulation in a tubular temperature gradient (ambient temperature range approximately 9-38 degrees C). Internal temperature was monitored with an implanted telemetry device. An imaging system recorded the position (selected temperature) of the mouse within the gradient every 5 sec. A dose of either 2.25 or 2.60 g ethanol/kg body wt. produced significantly lower body temperatures than control (NaCl) injections. The 2.60 g/kg dose produced significantly lower selected temperatures than either the NaCl or 2.25 g/kg injections. Doses of 2.75 g ethanol/kg and above incapacitated the mice, precluding accurate behavioral thermoregulation. Utilizing a thermoregulatory index to compare the responses following experimental and control injections indicated that 2.25 or 2.60 g ethanol/kg leads to a decrease in the regulated temperature of mice.

Voluntary consumption of ethanol in WSP, WSC and WSR selectively bred mouse lines.

The genetic correlation between voluntary consumption of ethanol solutions and severity of withdrawal seizures after chronic ethanol exposure was assessed using the selectively bred Withdrawal Seizure Prone (WSP) and Resistant (WSR) mouse lines. WSP mice have at least ten-fold more severe withdrawal than WSR mice after equal chronic ethanol exposure, and withdrawal in a nonselected control line (WSC) is intermediate to withdrawal in the WSP and WSR lines. In the first experiment, mice from the WSP, WSC and WSR lines were offered a choice between 2.2, 4.6 and 10.0% ethanol solutions and water in three consecutive eight-day sessions. WSR mice consumed more ethanol than WSP mice, and WSC mice were intermediate. In a second experiment, WSP and WSR mice were offered ethanol solutions in concentrations that were adjusted up or down every two days depending upon the amount of ethanol consumed. WSP and WSR mice displayed very different patterns of drinking, with WSP mice drinking more ethanol in early stages of the experiment, and WSR mice drinking more ethanol later. Results of these experiments suggest that some genes influencing severity of withdrawal from ethanol also influence voluntary ethanol drinking.

Mice genetically selected for differences in open-field activity after ethanol.

Starting from a population of genetically heterogeneous mice, selective breeding is being used to develop lines differing in sensitivity to ethanol-induced open-field activity. Mice are tested twice for 4 min in an open field. The first test is between min 2-6 after injection of saline. Twenty-four hr later, a similar test is performed after injection of ethanol (1.5 g/kg). Two independent FAST lines are being selected for ethanol-induced increases in activity, and two independent SLOW lines are being selected for ethanol-induced decreases. After four generations of selection, the lines have diverged significantly. These lines should be useful for exploring the neuropharmacological basis for the activating and rewarding properties of ethanol.

Genetic selection of mouse lines sensitive (cold) and resistant (hot) to acute ethanol hypothermia.

Using the technique of within-family selective breeding, we have generated mouse lines that differ genetically in sensitivity to the acute hypothermia induced by injection of 3 g/kg ethanol (EtOH). After 5 generations of selection, the difference in maximal hypothermic response between COLD and HOT lines was 1.6 degrees C in the first replicate and 1.2 degrees C in the second replicate. Estimates of realized heritability were h2 = .17 in each replicate. No differences in EtOH metabolism have developed, so the differences between HOT and COLD mice are presumably in neurosensitivity. These lines of animals should be useful for studying the biological mechanisms underlying neurosensitivity to EtOH. In conjunction with other selectively bred lines, they should improve our understanding of the genetic relationships among EtOH neurosensitivity, tolerance and physical dependence.

Sensitivity and tolerance to ethanol in mice bred to be genetically prone or resistant to ethanol withdrawal seizures.

Mice genetically susceptible (withdrawal seizure prone; WSP) and resistant (withdrawal seizure resistant; WSR) to ethanol (EtOH) withdrawal convulsions have been developed by selective breeding. WSP mice show much more severe EtOH withdrawal than WSR mice after equal intensities of exposure to EtOH. The present experiments report a systematic comparison between WSP and WSR mice with respect to their neurosensitivity to two effects of EtOH, EtOH-induced hypothermia (HT) and loss of righting reflex (RR). The degree of tolerance developed to these effects was also compared between the lines. WSP and WSR mice did not differ in sensitivity to EtOH-induced HT. When EtOH was administered daily for 3 days, both lines developed tolerance as evidenced by attenuated HT, but there was no line difference. Because blood EtOH concentrations did not change, the tolerance was functional rather than pharmacokinetic. When twice-daily injections were given for 4 days before testing on the 5th day in an effort to increase the degree of tolerance achieved, functional tolerance was slightly greater in the WSR line than in the WSP line 90 to 120 min, but not 30 to 60 min, after EtOH. In similar experiments, WSP and WSR mice were found to have the same ED50 to EtOH-induced loss of RR. The brain EtOH concentrations of WSP and WSR mice were the same at the time RR was lost and at the time RR was regained. Thus, neither line developed acute functional tolerance to this effect of EtOH. WSR mice lost RR more quickly than WSP mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic differences in anticonvulsant sensitivity in mouse lines selectively bred for ethanol withdrawal severity.

WSP (withdrawal seizure-prone) mice exhibit approximately 10-fold more severe withdrawal convulsions than WSR (withdrawal seizure-resistant) mice after identical chronic ethanol exposure. Although WSP and WSR mice do not differ in threshold for seizures elicited by electroconvulsive shock (ECS), WSR mice are more sensitive to ethanol-induced elevation of ECS seizure thresholds. The current experiments demonstrated that WSR mice showed more ECS-induced seizure threshold elevation than WSP mice when tested after the administration of C1-C5 straight-chain alcohols. Whereas the brain concentrations of the C1 and C2 alcohols did not differ between the lines, WSP mice tended to have higher brain concentrations than WSR mice of the C3-C5 alcohols, even though they exhibited the smaller behavioral response in all cases. Thus, the difference between WSP and WSR mice was one of neurosensitivity and could not be attributed to pharmacokinetic differences. The WSR line was also more sensitive to ethchlorvynol, methyprylon, barbital, phenobarbital, pentobarbital, diazepam, valproic acid and phenytoin in this test. Examining loss of righting reflex (RR), we found that WSP and WSR mice did not differ in ED50, latency to lose RR or duration of loss of RR. Thus, the genetic anticonvulsant sensitivity difference is not simply a genetic difference in sensitivity to central nervous system depression between the lines. In summary, WSR mice were more sensitive to the anticonvulsant effects of a variety of compounds than WSP mice, suggesting that some genes influence both ethanol withdrawal seizures and ethanol's anticonvulsant effects.

Ethanol withdrawal in mice bred to be genetically prone or resistant to ethanol withdrawal seizures.

We are engaged in a selective breeding program developing lines of mice which differ in severity of withdrawal convulsions after ethanol treatment. Withdrawal seizure prone (WSP) mice show greater handling-induced convulsion scores than withdrawal seizure resistant (WSR) mice after 3 days of ethanol intoxication. In the present experiments, we sought to characterize these mice further as a model of genetic susceptibility to ethanol dependence and withdrawal. During withdrawal after chronic treatment with ethanol, WSP mice displayed more severe handling-induced convulsions and tremor than WSR mice, and tended to show greater reduction of exploratory activity. WSP and WSR mice did not differ in ethanol metabolism after acute treatment with ethanol alone or after chronic treatment with ethanol and pyrazole, an alcohol dehydrogenase inhibitor. Six to 10 hr after an acute injection of ethanol, WSP and WSR mice showed elevated handling-induced convulsions. This elevation was more pronounced in WSP mice than in WSR mice. WSP mice also showed slightly more severe convulsions than WSR mice when treated with saline or pyrazole alone. In summary, WSP and WSR mice treated with identical doses of ethanol differ in several symptoms of withdrawal, whereas not differing in ethanol metabolism. These mice constitute a useful population in which to study the molecular mechanisms of ethanol dependence and withdrawal.