Alcohol withdrawal kindling: mechanisms and implications for treatment.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Larry P. Gonzalez. The presentations were (1) EEG indices of sensitization in a murine model of repeated ethanol withdrawals, by Lynn M. Veatch; (2) Long-term changes in central nervous system function after repeated alcohol withdrawals: Recommendations for the treatment of acute withdrawal, by Larry P. Gonzalez; (3) Differential regulation of GABAA and NMDA receptors by repeated ethanol treatment in cultured mammalian neurons, by Maharaj K. Ticku; and (4) Involvement of GABAA and NMDA receptors in alcohol withdrawal kindling: Implications for treatment, by Howard C. Becker.

Diazepam during prior ethanol withdrawals does not alter seizure susceptibility during a subsequent withdrawal.

The number of cycles of alcohol detoxification is suggested to be an important variable in the predisposition to severe withdrawal seizures in alcohol-dependent individuals. Several clinical studies have suggested that exposure to repeated alcohol withdrawals may lead to increased severity of subsequent withdrawal episodes. Consistent with these observations, exposure to multiple cycles of ethanol withdrawal in our previous study significantly increased sensitivity to the convulsive effects of the GABA(A) receptor inverse agonist, Ro15-4513, in comparison to continuous ethanol exposure with no intermittent withdrawals. There was also a selective increase in the occurrence of spontaneous spike and sharp wave (SSW) activity in the EEG recorded from hippocampal area CA(3) in proportion to the number of withdrawal episodes experienced. It is hypothesized that during such repeated episodes of ethanol intoxication and withdrawal, changes in neuronal excitation during prior withdrawals could serve as initially subconvulsive kindling stimuli that might eventually result in the increased severity of the withdrawal syndrome. There is some evidence of the successful suppression of such neuronal excitation during acute ethanol withdrawal by positive modulators of the GABA(A) receptor. In the present study, the benzodiazepine agonist, diazepam, at a dose (4.0 mg/kg) that suppresses acute withdrawal symptoms, when administered during intermittent withdrawals, did not alter seizure sensitivity during a subsequent nonmedicated withdrawal. Diazepam treatment during prior withdrawals also did not have any effect on the multiple withdrawal-associated increase in SSW activity in hippocampal area CA(3) during an untreated withdrawal. This finding suggests that suppression of acute withdrawal symptoms by diazepam does not prevent long-lasting changes in CNS function resulting from repeated exposures to ethanol withdrawal.

Influence of salting procedure on the composition of Muenster-type cheese.

Muenster-type cheeses were salted with a traditional saturated brine solution or by direct addition of salt to the curd. Cheeses were evaluated at 0, 30, 60, 90, 120, and 180 d of age for numbers and type of microflora, casein hydrolysis, and amounts of free fatty acids. No significant differences were found in the populations of starter, lactobacilli, or yeast for the brine- and direct-salted cheeses. The amounts of free fatty acids liberated were similar for both cheeses. The hydrolysis of alpha s1-casein was complete at 90 d of age, whereas only 40% of the beta-casein was hydrolyzed at 180 d of age. The inner layer of the brine-salted cheeses had the highest number of starter microorganisms, followed by the middle and outer layers, respectively. The salt concentrations were similar in the three layers after 4 mo of age. Results of this study showed that comparable Muenster-type cheese could be produced with either of the salting procedures. With direct salt addition to curd, a 59% reduction was observed in salt emissions from the Muenster manufacturing process.

Nifedipine alleviates alterations in hippocampal kindling after repeated ethanol withdrawal.

BACKGROUND: Current clinical treatment of alcohol detoxification commonly includes pharmacotherapy to lessen the potential for seizures, especially in those patients undergoing repeated treatment. Basic research continues to study the alcohol withdrawal-related "kindling" of seizures both to understand the mechanisms involved and to identify alternative treatments. Ethanol withdrawal has been shown to result in the delay of electrical kindling at several brain sites, which suggests a long-lasting disruption of neuronal function. METHODS: This study focused on the participation of the L-type voltage-gated calcium channels in this process by the treatment of animals during withdrawal with nifedipine, an agent that blocks these channels. Animals were randomly assigned to ethanol (ethanol-exposed/ethanol-naive) and drug treatment (nifedipine/vehicle) groups. Subjects receiving ethanol were exposed to five cycles of 3 days' ethanol exposure, with each exposure cycle separated by a 1-day withdrawal period. Drug treatment was administered twice during each withdrawal period. Twenty days after completion of ethanol exposure, animals received daily kindling stimulations to hippocampal area CA3 until the kindling criterion was attained. RESULTS: Ethanol-exposed animals that received vehicle treatment during ethanol withdrawal required more daily stimulations to become fully kindled than did ethanol-naive controls. This delay in seizure development was most pronounced in the progression from focal seizure behaviors to more generalized seizures. Animals that received the same ethanol exposure but that were treated with nifedipine required significantly fewer stimulations than did ethanol-exposed animals that received vehicle. Ethanol-exposed/ nifedipine-treated animals did not differ from ethanol-naive controls that received vehicle or nifedipine. CONCLUSIONS: Alcohol withdrawal-related alterations in seizure-sensitive neural circuitry such as the hippocampus persist long after cessation of ethanol exposure. Furthermore, the L-type voltage-gated calcium channels are involved in this effect in that blockade of these channels during acute withdrawal alleviates alterations in seizure mechanisms on a long-term basis.

Repeated ethanol withdrawal delays development of focal seizures in hippocampal kindling.

Clinical studies report an increase in the prevalence of alcohol withdrawal-related seizures in patients with a history of multiple detoxifications. In order to investigate the alcohol withdrawal-related alterations in neural activity that lead to this increase in seizure propensity, basic researchers have examined both spontaneous and elicited seizures in animals undergoing withdrawal from chronic ethanol. This study was designed to further examine alcohol withdrawal-related seizure activity in a rodent model by assessing the development of electrical kindling after chronic ethanol exposure administered in multiple or single treatment episodes. Laboratory rats were exposed to either five periods of 3 days of ethanol, one 15-day period of continuous ethanol, or a period of control handling with no ethanol exposure. Ten days after a final withdrawal episode, all animals were surgically prepared with recording and stimulating electrodes. Twenty days after final withdrawal from ethanol or an equivalent period of similar handling, daily electrical stimulation of hippocampal area CA3 was initiated. Animals exposed to ethanol required more daily stimulations to become fully kindled than did ethanol-naive controls, with those animals experiencing five withdrawals requiring the most stimulations overall and more stimulations to progress from focal to generalized seizure behaviors. These results indicate that chronic ethanol exposure and withdrawal alter neuronal mechanisms important for hippocampal kindling in a manner that persists long after cessation of ethanol exposure, and they indicate that this effect is increased by exposure to repeated withdrawal episodes.

Increased Ro15-4513-induced seizures following multiple ethanol withdrawals.

Clinical research into the etiology of ethanol withdrawal seizures has shown an increase in the number and severity of seizures with increasing numbers of withdrawal episodes. The aim of the present study was to determine the effects of multiple ethanol withdrawals on the seizure sensitivity to the GABA(A) receptor inverse agonist Ro15-4513. In this study, three groups of laboratory rats received varying amounts of either continuous or intermittent ethanol exposure. A fourth group (Naive) received no ethanol exposure. Eight hours following the last withdrawal from chronic ethanol exposure, animals were tested for sensitivity to Ro15-4513-induced motor convulsions. Seizure sensitivity was significantly increased in all ethanol-treated groups compared to ethanol-naive controls, which did not exhibit any convulsive responses to this dose of Ro15-4513. Furthermore, rats exposed to multiple ethanol withdrawals exhibited significantly higher sensitivity to drug-induced seizures than did animals experiencing only a single ethanol withdrawal. Although the specific mechanism of this enhanced convulsant effect of Ro15-4513 following multiple ethanol withdrawals remains to be determined, these results suggest an involvement of GABA(A)-benzodiazepine receptors in this multiple withdrawal phenomenon.

Electrophysiological changes after repeated alcohol withdrawal.

Studies of spontaneous and kindled electrophysiological brain activity following AW emphasize the important contribution of several variables to the severity of AW and to the associated long-lasting changes in brain function. Thus, withdrawal severity depends on the pattern of alcohol intake (e.g., withdrawal history) as well as on the total amount of alcohol exposure. Moreover, different brain regions respond differently to various patterns of alcohol exposure.

Chronic ethanol retards kindling of hippocampal area CA3.

To investigate the effects of chronic alcohol which persist long after cessation of ethanol exposure, electrical kindling of the hippocampus was studied in male Sprague-Dawley rats chronically exposed to ethanol. Kindling was initiated 14 days after either one continuous period of ethanol exposure, repeated periods of exposure and withdrawal, or control handling. Animals receiving ethanol exposure required significantly more stimulations to develop behavioral signs of kindling and to attain final kindling criterion than did ethanol-naive controls. Neither the blood ethanol level at the time of withdrawal nor the behavioral signs of acute withdrawal severity correlated with kindling measures. This study indicates that chronic ethanol exposure results in brain alterations which may be assessed long after the cessation of ethanol exposure.

Repeated ethanol withdrawal produces site-dependent increases in EEG spiking.

Recent studies have suggested an important kindling-like exacerbation of ethanol withdrawal symptoms after repeated cycles of ethanol intoxication and withdrawal. Few studies, however, have evaluated the effect of multiple episodes of intoxication and withdrawal on spontaneous EEG activity after cessation of ethanol intake. In this study, electrographic activity in cortical and subcortical structures male Sprague-Dawley rats was examined after multiple cycles of ethanol intoxication and withdrawal. After surgical implantation of electrodes, animals received repeated cycles of chronic ethanol exposure in vapor inhalation chambers for 10 or 20 days, with 4-day withdrawal periods between each. Upon removal from the inhalation chamber, spontaneous EEG activity was recorded intermittently for 72 hr. These data were then examined for the presence of spikes and sharp waves. Results indicate that the levels of spike and sharp wave activity observed vary with both length of ethanol exposure and with the number of withdrawal cycles, and that these effects varied with neuronal site. Changes in spike and sharp wave activity were first observed within hippocampal areas, with other subcortical and cortical sites showing increased activity after additional ethanol exposure or additional cycles of intoxication and withdrawal. Hippocampal areas CA1 and CA3 differed significantly from one another in their response to chronic ethanol exposure, with area CA1 most affected by changes in amount of ethanol exposure and are CA3 most affected by number of withdrawal cycles. These results indicate an increased severity of the ethanol withdrawal syndrome after repeated ethanol withdrawal episodes and suggest differential, site-specific changes in neuronal excitability.

Sensitivity to strychnine seizures is unaltered during ethanol withdrawal.

Previous research from this laboratory has indicated that animals chronically exposed to ethanol and then withdrawn exhibit a variety of symptoms of central nervous system hyperexcitability that occur in unique clusters. These clusters of symptoms were observed to differ in their duration and in the time of onset relative to the time of ethanol withdrawal. In addition, the observed clusters of spontaneous seizure events in these animals were seen to correlate with differences in sensitivity to seizure-inducing treatments. These results suggest that seizure sensitivity during ethanol withdrawal may indicate the involvement of multiple, independent, neuronal mechanisms. To investigate this possibility further, the following study examined the sensitivity of ethanol-withdrawn animals to seizures induced by the glycine antagonist strychnine. Seizure sensitivity to strychnine was evaluated at the same times following ethanol withdrawal when animals were previously seen to show the differential occurrence of spontaneous seizure events and also differences in sensitivity to picrotoxin-induced seizures. Ethanol-withdrawn animals did not differ in their responses to strychnine compared with ethanol-naive controls at any of the times tested. This lack of change in seizures induced by antagonism of glycine inhibition occurred in spite of the increased sensitivity of similarly treated animals to picrotoxin-induced seizures at the same test times. These data suggest that chronic ethanol exposure and withdrawal may not significantly after the function of glycinergic inhibitory neurotransmission and that the ethanol withdrawal syndrome is indicative of alterations in specific neuronal mechanisms rather than of a generalized state of central nervous system hyperexcitability.

Classification of drug-induced behaviors using a multi-layer feed-forward neural network.

Measurement of laboratory animal motor behavior is an important part of many studies of experimental manipulations of the central nervous system. Current automated data collection and analysis systems are limited in the number of behaviors that can be monitored and quantified simultaneously. This paper describes a signal analysis technique that when used to analyze the data from a modified Stoelting electronic activity monitor is capable of classifying multiple behavior categories automatically. In this technique, the output signal from the motility monitor is fixed-length segmented and feature extraction is performed, calculating the Fourier transform and power spectrum of each data segment. An error back-propagation neural network, implemented on a microcomputer, is used to perform behavior classification of the segment power spectra. The technique provides a high degree of accuracy in automatic behavior classification and should prove useful in the quantitative assessment of behavior.

Cocaine alters body temperature and behavioral thermoregulatory responses.

This study examined the effects of cocaine HCl and ambient temperature on the core body temperature of the rat. To further examine the mechanisms of cocaine-induced changes in temperature regulation, the effects of cocaine on behavioral thermoregulatory responses were also observed. At normal room temperature (20 degrees C) cocaine produced a reduction in core body temperature. At a higher ambient temperature (27 degrees C), however, cocaine produced hyperthermia. When given a choice between normal and warm environments, cocaine-injected animals spent significantly less time in the warm environment than did control animals, but had significantly lower core body temperature. These results suggest that cocaine treatment may lower the thermoregulatory set point.

Acute and chronic ethanol alter somatosensory-evoked potentials in conscious rats.

This study examined the effects of acute and chronic ethanol on cortical somatosensory-evoked potentials (SEPs) of laboratory rats. Evoked potentials were recorded following stimulation of the left hindpaw before and after injection of either saline or ethanol. Animals were then chronically exposed to ethanol in vapor inhalation chambers for five weeks. Recordings of SEPs before and after acute ethanol injection were then obtained 24 h and again seven days after withdrawal from ethanol exposure. The results indicate that acute ethanol produced a dose-dependent reduction in SEP amplitude, but did not alter peak latencies. Chronic ethanol exposure and withdrawal resulted in a significant increase in preinjection baseline response amplitudes when measured at 24 h after withdrawal, but not at seven days, and this treatment did not alter response latency or the effects of acute ethanol administration.

Antioxidant protection systems of rat lung after chronic ethanol inhalation.

The effect of chronic ethanol administration on pulmonary antioxidant protection systems was investigated in male Sprague-Dawley rats exposed to room air or room air containing ethanol vapors for 5 weeks. Blood ethanol concentrations in ethanol-exposed rats were usually between 200 and 300 mg/dl. Glutathione, vitamin E, and malondialdehyde concentrations were measured in lung homogenates, and antioxidant enzyme activities (catalase, glutathione peroxidase, Cu/Zn-superoxide dismutase, glutathione reductase) were determined in the supernatant fractions. For comparison, the measurements were also made using liver fractions. Ethanol treatment increased the activities of catalase (117%) and Cu/Zn-superoxide dismutase (25%) in lung but not in liver. Although chronic ethanol inhalation lowered hepatic glutathione (19%) and hepatic vitamin E (33%), there was no increase in malondialdehyde content in either liver or lung of ethanol-exposed rats. The elevation of pulmonary antioxidant enzyme activities could be interpreted to mean that lung is a target for ethanol-induced oxidative stress. However, as there was no loss of pulmonary GSH or vitamin E and no increase in malondialdehyde formation, it appears that long-term ethanol exposure did not produce a significant degree of oxidative stress in rat lung.

Spontaneous versus elicited seizures following ethanol withdrawal: differential time course.

Ethanol withdrawal symptoms in clinical populations are observed to occur in unique clusters which differ in time of onset relative to the time of withdrawal and in their duration. Since periods of mild symptoms are sometimes observed between these clusters of more severe symptoms, the symptom clusters may reflect separate periods of hyperexcitability during which times different neuronal mechanisms are involved. To investigate this possibility in an animal model of ethanol withdrawal, rats were chronically exposed to ethanol in vapor inhalation chambers. Upon withdrawal from this exposure regimen, the time course of spontaneous seizure activity was observed for a period of 84 hr and compared to sensitivity to seizures elicited by audiogenic stimuli or by the convulsant drug picrotoxin. Spontaneous seizure events were observed to occur in clusters, and these clusters were differentially correlated with periods of increased sensitivity to induced seizure activity. These results further support the suggestion that seizure sensitivity during ethanol withdrawal may indicate the involvement of multiple, independent, neuronal mechanisms.

Reduced behavioral responses to intranigral muscimol following chronic ethanol.

Increased biochemical measures of GABA activity are observed after acute administration of ethanol and decreased activity has sometimes been observed after chronic ethanol exposure. Since chronic alterations in neurotransmitter activity may result in changes in receptor function, it is possible that changes in GABA-receptive neurons may accompany chronic ethanol treatment. In the present study we examined the incidence of muscimol-induced motor behaviors in ethanol-naive and chronic ethanol-treated animals. Male Sprague-Dawley rats received bilateral cannula implants into substantia nigra pars reticulata for subsequent administration of muscimol or saline. After recovery from surgery, rats received chronic treatment in ethanol-vapor inhalation chambers for 15 days. Animals were then removed from the chambers and examined 10 hours after removal. Muscimol resulted in a general increase in motility in both control and ethanol-treated animals. Animals withdrawn from chronic ethanol exposure, however, exhibited significantly less muscimol-stimulated, repetitive 9 Hz movements. These results suggest that GABA receptive cells within the substantia nigra or its vicinity may be functionally less responsive to GABAergic stimulation after chronic ethanol administration.

Ethanol withdrawal alters apomorphine-induced motility.

Acute administration of ethanol is accompanied by alterations in dopamine turnover and release, and chronic ethanol exposure is associated with changes in biochemical measures of dopamine receptor function. This paper presents data examining the effects of chronic ethanol exposure on behavioral responses to the dopamine receptor agonist apomorphine. Measurements of behavior were obtained through the use of an electronic motility monitor which permitted the quantification of movements in terms of their characteristic frequency components. Results are presented which indicate that apomorphine-induced movements with modal frequencies of 2 Hz and of 8-9 Hz are significantly increased during the 12 to 24 hr following ethanol withdrawal, suggesting an increase in the functional responsiveness of central dopaminergic systems.

Apomorphine-induced hypothermia increases during ethanol withdrawal.

Changes in several measures of dopamine function have been observed following acute or chronic ethanol exposure. The present study examined the effects of chronic ethanol exposure on the hypothermia following acute administration of the dopamine agonist apomorphine. Animals withdrawn from chronic ethanol exposure showed a significantly greater decrease in body temperature following apomorphine than did ethanol-naive controls, suggesting an increase in sensitivity to dopaminergic stimulation during ethanol withdrawal.