Two hundred l of calibrators, controls, and samples were mixed by vortexing with 20 l of a 10 g/ml nalorphine solution (internal standard), 1.8 ml of saline, and 1 ml of 100 mM phosphate buffer (pH 6). -/-) mice are hypersensitive to the locomotor, rewarding, and aversive effects of cocaine (Schank et al., 2006). Pharmacological inhibition of DBH with disulfiram, which decreases the NE/DA ratio in the rodent brain (Karamanakos et al., 2001; Bourdlat-Parks et al., 2005), facilitates the development of behavioral sensitization to cocaine (Haile et al., 2003). Furthermore, a common polymorphism in the gene influences both DBH enzymatic activity and cocaine-induced paranoia (Zabetian et al., 2001; Kalayarisi et al., 2007). Noradrenergic transmission has been implicated in the modulation of seizure activity (examined by Weinshenker and Szot, 2002). Enhancement of noradrenergic transmission suppresses seizure activity (Lindvall, et al., 1988; Weinshenker et al., 2001; Kaminski et al., 2005), whereas norepinephrine depletion with 6-hydroxydopamine or disulfiram exacerbates seizures and facilitates seizure kindling (Corcoran, et al., 1974; Callaghan and Schwark, 1979; McIntyre, 1980; Abed, 1994; Amabeoku and Syce, 1997), and -/- mice have increased susceptibility to seizure induced by flurothyl, pentylenetetrazole, kainic acid, and sound (Szot et al., 1999). Approximately 27% of all drug-related emergency room episodes are related to cocaine abuse (SAMHSA, 1996). Cocaine-induced seizures are a manifestation of the toxicity associated with the drug, and estimates are that 8-12% of patients admitted to emergency departments with cocaine intoxication have seizures (Derlet and Albertson, 1989; Dhuna et al., 1991; Koppel et al., 1996). These seizures can be resistant to common anticonvulsant drugs, such as benzodiazepines and barbiturates, and constitute a major portion of cocaine-related deaths (Dhuna et al., 1991; Benowitz et al., 1993). In addition, there have been several reports of individuals without a history of epilepsy developing seizures following treatment with therapeutic doses of disulfiram (Liddon and Satran, 1967; Price and Silberfarb, 1976a, 1976b; McConchie et al., 1983; Daniel et al., 1987). Concurrent use of cocaine and disulfiram is now on the rise, as disulfiram is under evaluation as a pharmacotherapy for cocaine dependence. Because pharmacological or genetic inhibition of DBH increases the sensitivity to seizures and the behavioral effects of cocaine, we sought to examine the effects of DBH and disulfiram on susceptibility to cocaine-induced seizures (CIS). We measured the probability of having a seizure and the frequency of CIS following a high dose of cocaine (60 mg/kg) in both wild-type (+/+) and -/- mice. We hypothesized that (1) -/- mice would be hypersensitive to cocaine-induced seizures (CIS) and (2) disulfiram would exacerbate CIS in a genotype-dependent manner. To further examine whether disulfiram affects cocaine responses via a DBH-dependent mechanism, we also tested the selective DBH inhibitor nepicastat (Stanley et al., 1997). To determine whether the effects of these drugs could be attributed to changes in cocaine metabolism, we also measured peak serum cocaine levels. 2. Methods 2.1 Animals and housing Adult +/+ and mice maintained on a mixed 129/SvEv and C57BL6/J background were developed and generated as previously described (Thomas et al, 1995, 1998). Genotypes were confirmed by PCR. All mice were reared in a specific pathogen-free facility with a 12-h light/dark cycle (lights on at 0700 h, lights off at 1900 h); food and water were available ad libitum. Na?ve mice between 3 and 6 months of age were used for all experiments, as were both male and female mice. No sex differences were observed, and results were combined. Experimental protocols were approved by the Emory University IACUC and meet the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. 2.2 Cocaine-induced seizures Mice were given 3 injections of saline, disulfiram (100 mg/kg, i.p.), or the selective DBH inhibitor nepicastat (100 mg/kg, i.p.), with 2 hours between each injection. Two hours following the last injection, all mice were injected with a high dose of.Nepicastat pretreatment increased CIS frequency in wild-type but not -/- mice. by inhibiting DBH and increases CIS frequency in a DBH-independent manner. -/-) mice are hypersensitive to the locomotor, rewarding, and aversive effects of cocaine (Schank et al., 2006). Pharmacological inhibition of DBH with disulfiram, which decreases the NE/DA ratio in the rodent brain (Karamanakos et al., 2001; Bourdlat-Parks et al., 2005), facilitates the development of behavioral sensitization to cocaine (Haile et al., 2003). Furthermore, a common polymorphism in the gene influences both DBH enzymatic activity and cocaine-induced paranoia (Zabetian et al., 2001; Kalayarisi et al., 2007). Noradrenergic transmission has been implicated in the modulation of seizure activity (reviewed by Weinshenker and Szot, 2002). Enhancement of noradrenergic transmission suppresses seizure activity (Lindvall, et al., 1988; Weinshenker et al., 2001; Kaminski et al., 2005), whereas norepinephrine depletion with 6-hydroxydopamine or disulfiram exacerbates seizures and facilitates seizure kindling (Corcoran, et al., 1974; Callaghan and Schwark, 1979; McIntyre, 1980; Abed, 1994; Amabeoku and Syce, 1997), and -/- mice have increased susceptibility to seizure induced by flurothyl, pentylenetetrazole, kainic acid, and sound (Szot et al., 1999). Approximately 27% of all drug-related emergency room episodes are related to cocaine abuse (SAMHSA, 1996). Cocaine-induced seizures are a manifestation of the toxicity associated with the drug, and estimates are that 8-12% of patients admitted to emergency departments with cocaine intoxication have seizures (Derlet and Albertson, 1989; Dhuna et al., 1991; Koppel et al., 1996). These seizures can be resistant to common anticonvulsant drugs, such as benzodiazepines and barbiturates, and constitute a major fraction of cocaine-related deaths (Dhuna et al., 1991; Benowitz et al., 1993). In addition, there have been several reports of individuals without a history of epilepsy developing seizures following treatment with therapeutic doses of disulfiram (Liddon and Satran, 1967; Price and Silberfarb, 1976a, 1976b; McConchie et al., 1983; Daniel et al., 1987). Concurrent use of cocaine and disulfiram is now on the rise, as disulfiram is under evaluation as a pharmacotherapy for cocaine dependence. Because pharmacological or genetic inhibition of DBH increases the sensitivity to seizures and the behavioral effects of cocaine, we sought to examine the effects of DBH and disulfiram on susceptibility to cocaine-induced seizures (CIS). We measured the probability of having a seizure and the frequency of CIS following a high dose of cocaine (60 mg/kg) in both wild-type (+/+) and -/- mice. We hypothesized that (1) -/- mice would be hypersensitive to cocaine-induced seizures (CIS) and (2) disulfiram would exacerbate CIS in a genotype-dependent manner. To further examine whether disulfiram affects cocaine responses via a DBH-dependent mechanism, we also tested the selective DBH inhibitor nepicastat (Stanley et al., 1997). To determine whether the effects of these medicines could be attributed to changes in cocaine rate of metabolism, we also measured maximum serum cocaine levels. 2. Methods 2.1 Animals and housing Adult +/+ and mice taken care of on a combined 129/SvEv and C57BL6/J background were developed and generated as previously explained (Thomas et al, 1995, 1998). Genotypes were confirmed by PCR. All mice were reared in a specific pathogen-free facility having a 12-h light/dark cycle (lamps on at 0700 h, lamps off at 1900 h); food and water were available ad libitum. Na?ve mice between 3 and 6 months of age were utilized for all experiments, as were both male and female mice. No sex variations were observed, and results were combined. Experimental protocols were authorized by the Emory University or college IACUC and meet the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. 2.2 Cocaine-induced seizures Mice were given 3 injections of saline, disulfiram (100 mg/kg, i.p.), or the selective DBH inhibitor nepicastat (100 mg/kg, i.p.), with 2 hours between each injection. Two hours following a last injection, all mice were injected with a high dose of cocaine (60 mg/kg, i.p.). This dose was found to induce seizures in 50% of mice of the same strain during a pilot study. Mice were observed for 30 minutes following cocaine administration, and the latency to 1st seizure and seizure rate of recurrence were recorded. The 1st seizure and/or ataxia typically occurred within.Because pharmacological or genetic inhibition of DBH increases the level of sensitivity to seizures and the behavioral effects of cocaine, we sought to examine the effects of DBH and disulfiram on susceptibility to cocaine-induced seizures (CIS). improved CIS rate of recurrence in wild-type but not -/- mice. There were no genotype or treatment effects on serum cocaine levels, except for an increase in disulfiram-treated -/- mice at the highest dose of cocaine. These results suggest that disulfiram enhances CIS via two unique mechanisms: it both raises CIS rate of recurrence by inhibiting DBH and raises CIS rate of recurrence inside a DBH-independent manner. -/-) mice are hypersensitive to the locomotor, rewarding, and aversive effects of cocaine (Schank et al., 2006). Pharmacological inhibition of DBH with disulfiram, which decreases the NE/DA percentage in the rodent mind (Karamanakos et al., 2001; Bourdlat-Parks et al., 2005), facilitates the development of behavioral sensitization to cocaine (Haile et al., 2003). Furthermore, a common polymorphism in the gene influences both DBH enzymatic activity and cocaine-induced paranoia (Zabetian et al., 2001; Kalayarisi et al., 2007). Noradrenergic transmission has been implicated in the modulation of seizure activity (examined by Weinshenker and Szot, 2002). Enhancement of noradrenergic transmission suppresses seizure activity (Lindvall, et al., 1988; Weinshenker et al., 2001; Kaminski et al., 2005), whereas norepinephrine depletion with 6-hydroxydopamine or disulfiram exacerbates seizures and facilitates seizure kindling (Corcoran, et al., 1974; Callaghan and Schwark, 1979; McIntyre, 1980; Abed, 1994; Amabeoku and Syce, 1997), and -/- mice have improved susceptibility to seizure induced by flurothyl, pentylenetetrazole, kainic acid, and sound (Szot et al., 1999). Approximately 27% of all drug-related emergency room episodes are related to cocaine misuse (SAMHSA, 1996). Cocaine-induced seizures are a manifestation of the toxicity associated with the drug, and estimations are that 8-12% of individuals admitted to emergency departments with cocaine intoxication have seizures (Derlet and Albertson, 1989; Dhuna et al., 1991; Koppel et al., 1996). These seizures can be resistant to common anticonvulsant medicines, such as benzodiazepines and barbiturates, and constitute a major portion of cocaine-related deaths (Dhuna et al., 1991; Benowitz et Adam23 al., 1993). In addition, there have been several reports of individuals without a history of epilepsy developing seizures following treatment with restorative doses of disulfiram (Liddon and Satran, 1967; Price and Silberfarb, 1976a, 1976b; McConchie et al., 1983; Daniel et al., 1987). Concurrent use of cocaine and disulfiram is now on the rise, as disulfiram is definitely under evaluation like a pharmacotherapy for cocaine dependence. Because pharmacological or genetic inhibition of DBH increases the level of sensitivity to seizures and the behavioral effects of cocaine, we wanted to examine the effects of DBH and disulfiram on susceptibility to cocaine-induced seizures (CIS). We measured the probability of possessing a seizure and the rate of recurrence of CIS following a high dose of cocaine (60 mg/kg) in both wild-type (+/+) and -/- mice. We hypothesized that (1) -/- mice would be hypersensitive to cocaine-induced seizures (CIS) and (2) disulfiram would exacerbate CIS inside a genotype-dependent manner. To further analyze whether disulfiram affects cocaine responses via a DBH-dependent system, we also examined the selective DBH inhibitor nepicastat (Stanley et al., 1997). To determine if the ramifications of these medications could possibly be attributed to adjustments in cocaine fat burning capacity, we also assessed top serum cocaine amounts. 2. Strategies 2.1 Pets and casing Adult +/+ and mice preserved on the blended 129/SvEv and C57BL6/J background had been developed and generated as previously defined (Thomas et al, 1995, 1998). Genotypes had been verified by PCR. All mice had been reared in a particular pathogen-free facility using a 12-h light/dark routine (lighting on at 0700 h, lighting off at 1900 h); water and food were available advertisement libitum. Na?ve mice between 3 and six months old were employed for all experiments, as were both male and feminine mice. No sex distinctions were noticed, and results had been mixed. Experimental protocols had been accepted by the Emory School IACUC and meet up with the guidelines from the Association for Evaluation and Accreditation of Lab Animal Treatment. 2.2 Cocaine-induced seizures Mice received 3 injections of saline, disulfiram (100 mg/kg, i.p.), or the selective DBH inhibitor nepicastat (100.Serum cocaine amounts were measured by HPLC. boosts CIS regularity by inhibiting DBH and boosts CIS regularity within a DBH-independent way. -/-) mice are hypersensitive towards the locomotor, rewarding, and aversive ramifications of cocaine (Schank et al., 2006). Pharmacological inhibition of DBH with disulfiram, which reduces the NE/DA proportion Indoramin D5 in the rodent human brain (Karamanakos et al., 2001; Bourdlat-Parks et al., 2005), facilitates the advancement of behavioral sensitization to cocaine (Haile et al., 2003). Furthermore, a common polymorphism in the gene affects both DBH enzymatic activity and cocaine-induced paranoia (Zabetian et al., 2001; Kalayarisi et al., 2007). Noradrenergic transmitting continues to be implicated in the modulation of seizure activity (analyzed by Weinshenker and Szot, 2002). Improvement of noradrenergic transmitting suppresses seizure activity (Lindvall, et al., 1988; Weinshenker et al., 2001; Kaminski et al., 2005), whereas norepinephrine depletion with 6-hydroxydopamine or disulfiram exacerbates seizures and facilitates seizure kindling (Corcoran, et al., 1974; Callaghan and Schwark, 1979; McIntyre, 1980; Abed, 1994; Amabeoku and Syce, 1997), and -/- mice possess elevated susceptibility to seizure induced by flurothyl, pentylenetetrazole, kainic acidity, and audio (Szot et al., 1999). Around 27% of most drug-related er episodes are linked to cocaine mistreatment (SAMHSA, 1996). Cocaine-induced seizures certainly are a manifestation from the toxicity from the medication, and quotes are that 8-12% of sufferers admitted to crisis departments with cocaine intoxication possess seizures (Derlet and Albertson, 1989; Dhuna et al., 1991; Koppel et al., 1996). These seizures could be resistant to common anticonvulsant medications, such as for example benzodiazepines and barbiturates, and constitute a significant small percentage of cocaine-related fatalities (Dhuna et al., 1991; Benowitz et al., 1993). Furthermore, there were several reports of people without a background of epilepsy developing seizures pursuing treatment with healing dosages of disulfiram (Liddon and Satran, 1967; Cost and Silberfarb, 1976a, 1976b; McConchie et al., 1983; Daniel et al., 1987). Concurrent usage of cocaine and disulfiram is currently increasing, as disulfiram is certainly under evaluation being a pharmacotherapy for cocaine dependence. Because pharmacological or hereditary inhibition of DBH escalates the awareness to seizures as well as the behavioral ramifications of cocaine, we searched for to examine the consequences of DBH and disulfiram on susceptibility to cocaine-induced seizures (CIS). We assessed the likelihood of developing a seizure as well as the regularity of CIS carrying out a high dosage of cocaine (60 mg/kg) in both wild-type (+/+) and -/- mice. We hypothesized that (1) -/- mice will be hypersensitive to cocaine-induced seizures (CIS) and (2) disulfiram would exacerbate CIS within a genotype-dependent way. To further look at whether disulfiram impacts cocaine responses with a DBH-dependent system, we also examined the selective DBH inhibitor nepicastat (Stanley et al., 1997). To determine if the ramifications of these medications could possibly be attributed to adjustments in cocaine fat burning capacity, we also assessed top serum cocaine amounts. 2. Strategies 2.1 Pets and casing Adult +/+ and mice preserved on the blended 129/SvEv and C57BL6/J background had been developed and generated as previously defined (Thomas et al, 1995, 1998). Genotypes had been verified by PCR. All mice had been reared in a particular pathogen-free facility using a 12-h light/dark routine (lighting on at 0700 h, lighting off at 1900 h); water and food were available advertisement libitum. Na?ve mice between 3 and six months old were employed for all experiments, as were both male and feminine mice. No sex distinctions were noticed, and results had been mixed. Experimental protocols had been accepted by the Emory School IACUC and meet Indoramin D5 up with the guidelines from the Association for Evaluation and Accreditation of Lab Animal Treatment. 2.2 Cocaine-induced seizures Mice received 3 injections of saline, disulfiram (100 mg/kg, i.p.), or the selective DBH inhibitor nepicastat (100 mg/kg, we.p.), with 2 hours between each shot. Two hours following last shot, all mice had been injected with a higher dosage of cocaine (60 mg/kg, i.p.). This dosage was discovered to induce seizures in 50% of mice from the same stress throughout a pilot research. Mice were noticed for thirty minutes pursuing cocaine administration, as well as the latency to 1st seizure and seizure rate of recurrence were recorded. The first seizure and/or ataxia occurred within 2-4 mins postinjection typically. Seizures were thought as repeated, rapid intervals of jumping, wild-running, tonic-clonic activity, or a lack of the righting reflex. N = 9-16 for every treatment group. 2.3 Cocaine metabolism Mice received 3 injections of either saline or disulfiram (100 mg/kg, Indoramin D5 i.p.) each 2 hours apart..Serum cocaine amounts were measured by HPLC. -/- mice. There have been no genotype or treatment results on serum cocaine amounts, except for a rise in disulfiram-treated -/- mice at the best dosage of cocaine. These outcomes claim that disulfiram enhances CIS via two specific systems: it both raises CIS rate of recurrence by inhibiting DBH and raises CIS rate of recurrence inside a DBH-independent way. -/-) mice are hypersensitive towards the locomotor, rewarding, and aversive ramifications of cocaine (Schank et al., 2006). Pharmacological inhibition of DBH with disulfiram, which reduces the NE/DA percentage in the rodent mind (Karamanakos et al., 2001; Bourdlat-Parks et al., 2005), facilitates the advancement of behavioral sensitization to cocaine (Haile et al., 2003). Furthermore, a common polymorphism in the gene affects both DBH enzymatic activity and cocaine-induced paranoia (Zabetian et al., 2001; Kalayarisi et al., 2007). Noradrenergic transmitting continues to be implicated in the modulation of seizure activity (evaluated by Weinshenker and Szot, 2002). Improvement of noradrenergic transmitting suppresses seizure activity (Lindvall, et al., 1988; Weinshenker et al., 2001; Kaminski et al., 2005), whereas norepinephrine depletion with 6-hydroxydopamine or disulfiram exacerbates seizures and facilitates seizure kindling (Corcoran, et al., 1974; Callaghan and Schwark, 1979; McIntyre, 1980; Abed, 1994; Amabeoku and Syce, 1997), and -/- mice possess improved susceptibility to seizure induced by flurothyl, pentylenetetrazole, kainic acidity, and audio (Szot et al., 1999). Around 27% of most drug-related er episodes are linked to cocaine misuse (SAMHSA, 1996). Cocaine-induced seizures certainly are a manifestation from the toxicity from the medication, and estimations are that 8-12% of individuals admitted to crisis departments with cocaine intoxication possess seizures (Derlet and Albertson, 1989; Dhuna et al., 1991; Koppel et al., 1996). These seizures could be resistant to common anticonvulsant medicines, such as for example benzodiazepines and barbiturates, and constitute a significant small fraction of cocaine-related fatalities (Dhuna et al., 1991; Benowitz et al., 1993). Furthermore, there were several reports of people without a background of epilepsy developing seizures pursuing treatment with restorative dosages of disulfiram (Liddon and Satran, 1967; Cost and Silberfarb, 1976a, 1976b; McConchie et al., 1983; Daniel et al., 1987). Concurrent usage of cocaine and disulfiram is currently increasing, as disulfiram can be under evaluation like a pharmacotherapy for cocaine dependence. Because pharmacological or hereditary inhibition of DBH escalates the level of sensitivity to seizures as well as the behavioral ramifications of cocaine, we wanted to examine the consequences of DBH and disulfiram on susceptibility to cocaine-induced seizures (CIS). We assessed the likelihood of creating a seizure as well as the rate of recurrence of CIS carrying out a high dosage of cocaine (60 mg/kg) in both wild-type (+/+) and -/- mice. We hypothesized that (1) -/- mice will be hypersensitive to cocaine-induced seizures (CIS) and (2) disulfiram would exacerbate CIS inside a genotype-dependent way. To further analyze whether disulfiram impacts cocaine responses with a DBH-dependent system, we also examined the selective DBH inhibitor nepicastat (Stanley et al., 1997). To determine if the ramifications of these medicines could possibly be attributed to adjustments in cocaine rate of metabolism, we also assessed maximum serum cocaine amounts. 2. Strategies 2.1 Pets and casing Adult +/+ and mice taken care of on the combined 129/SvEv and C57BL6/J background had been developed and generated as previously referred to (Thomas et al, 1995, 1998). Genotypes had been verified by PCR. All mice had been reared in a particular pathogen-free facility having a 12-h light/dark routine (lamps on at 0700 h, lamps off at 1900 h); water and food were available advertisement libitum. Na?ve mice between 3 and six months old were useful for all experiments, as were both male and feminine mice. No sex variations were noticed, and results had been mixed. Experimental protocols had been authorized by the Emory College or university IACUC and meet up with the.