4-methyl benzylamine stimulates food consumption and counteracts the hypophagic effects of amphetamine acting on brain Shaker-like Kv1.1 channels

1.--4-methyl benzylamine (4-MBZ; 28 microg, 231 nmol) elicits a hyperphagic response in starved mice in contrast to the hypophagia induced by the parent compound benzylamine (BZ; 33 microg, 231 nmol) or by amphetamine (AMPH, 2 mug). 2.--In mice starved for only 4 h, and therefore with little stimulation to eat, the maximal increase in food consumption induced by intracerebroventricular (i.c.v.)-injected 4-MBZ was 190% over that of the controls (ED(50) 8.3+/-2.7 microg mouse(-1); 68+/-22 nmol mouse(-1)), whereas after i.p. administration, these values were 160% and approximately 129 mg kg(-1), respectively. 3.--The hyperphagic effect of 4-MBZ was reduced by more than 60% in mice pretreated with antisense oligodeoxyribonucleotide (aODN(1)) previously found to selectively inhibit (over 50%) the expression of Shaker-like Kv1.1 channels. 4.--In mice highly stimulated to eat after 12-h fasting, 4-MBZ (28 microg) significantly reduced (to about 70%) the hypophagic response by AMPH (2 microg) or BZ (33 microg). Conversely, these two compounds reduced (respectively, by 69 and 44%) the hyperphagic response of 4-MBZ in 4-h fasting mice. 5.--4-MBZ (28 microg) also reduced the hypermotility and the stimulation of inspection activity elicited by AMPH in mice and the release of DA stimulated by AMPH (2 microg) from the nucleus accumbens of rats.We hypothesize that 4-MBZ elicits hyperphagic effects probably by opening Shaker-like Kv1.1 subtypes in the brain, whereas AMPH and BZ are hypophagic by blocking these channels.     

Methylamine and benzylamine induced hypophagia in mice: modulation by semicarbazide-sensitive benzylamine oxidase inhibitors and aODN towards Kv1.1 channels

1. In starved mice, the anorectic activity of methylamine (MET) and benzylamine (BZ), both substrates of semicarbazide-sensitive benzylamine oxidases (Bz-SSAO), was compared with that of the potassium channel blocking agents charybdotoxin (ChTX), tetraethylammonium (TEA), gliquidone (GLI), ammonium chloride (NH(4)(+)) and of the anoressants amphetamine (AMPH) and nicotine (NIC). After i.c.v. administration, an approximate ranking order of potency was: ChTX> or =AMPH>NIC=TEA> or =GLI> or =MET>BZ>NH(4)(+). 2. Clorgyline (2.5 mg kg(-1) i.p.) or deprenyl (10 mg kg(-1) i.p.) potentiated the anorectic effect of i.c.v.-administered BZ, NIC and AMPH. The effect of TEA was increased only by deprenyl, while MET, NH(4)(+), ChTX and GLI were not affected by either of the inhibitors. 3. The Bz-SSAO inhibitors alpha-aminoguanidine (50 mg kg(-1) i.p.), B24 (100 mg kg(-1) i.p.) and MDL 72274 (2.5 mg kg(-1) i.p.) potentiated the effect of i.p., but not of i.c.v.-administered MET. 4. Antisense oligodeoxyribonucleotides (aODN) to Kv1.1 potassium channels abolished the effect of BZ and TEA, but was ineffective in reducing the activity of MET and other compounds. 5. These results suggest that MET is endowed with peculiar hypophagic effects at dosage levels that are not able to affect gross behaviour in mice. The effect of MET, differently from BZ, seems unrelated to an increase in the central release of monoaminergic mediators, as well as to a Kv1.1 blocking activity. Through a reduction of the endogenous breakdown of MET, Bz-SSAO inhibitors enhance the central pharmacological activity of this amine.     

Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels

The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.     

Long-lasting effects of escalating doses of d-amphetamine on brain monoamines, amphetamine-induced stereotyped behavior and spontaneous nocturnal locomotion

The repeated intermittent administration of relatively low doses of amphetamine (AMPH) produces an enduring hypersensitivity to the motor stimulant effects of AMPH (behavioral sensitization), and this is accompanied by enhanced mesotelencephalic dopamine (DA) utilization/release. In contrast, chronic treatment with very high doses of AMPH does not produce sensitization, but is neurotoxic, resulting in the depletion of brain DA (and often other monoamines). However, gradually escalating doses of AMPH provide protection against the neurotoxic effects of higher doses given later. Therefore, the purpose of the present experiment was to determine if a regimen of gradually escalating doses of AMPH, culminating in much higher doses than usually used to study sensitization, would produce neural and behavioral changes associated with AMPH neurotoxicity (DA depletion) or behavioral sensitization (increased DA utilization). Female rats were given 60 injections (2/day) of increasing (1 to 10 mg/kg) doses of d-AMPH, culminating in rats receiving 20 mg/kg/day for four consecutive days. This treatment did not deplete brain DA or serotonin, but did produce a long-lasting enhancement (at least 12 days) in striatal and nucleus accumbens DOPAC concentrations, and DOPAC/DA ratios. These neurochemical changes were accompanied by an enduring hypersensitivity to the stereotypy-producing effects of a subsequent AMPH 'challenge.' In contrast to this enhanced response to a challenge, AMPH-pretreated rats showed a marked reduction in spontaneous nocturnal motor activity. It is concluded that rats can be given escalating doses of AMPH, which mimic to some extent the AMPH 'runs' common in addicts and that this produces neural and behavioral changes consistent with the development of sensitization; not neurotoxicity.     

Modulation of voltage-gated K(+) channels Kv11 and Kv1 4 by forskolin

Forskolin (FSK) affects voltage-gated K(+) (Kv) currents in different cell types, but it is not known which of the various subunits form FSK-sensitive Kv channels. We compared the effect of the compound at Kv1.1 and Kv1.4 channels ectopically expressed in HEK 293 cells. Low FSK concentrations induced a phosphorylation-dependent potentiation of Kv1.1 currents. At higher concentrations, this effect was superimposed by a fast, cAMP-independent channel block. Kv1.4 currents were inhibited with lower potency by FSK but were not modified by phosphorylation. The variable effect of the compound might help to distinguish between Kv subunits expressed by native cells.     

Activity density in the open field: a measure for differentiating the effect of psychostimulants

Traditional open-field activity measures do not provide a sharp behavioral differentiation across psychomotor stimulants such as d-amphetamine (AMPH) and cocaine (COC) in the mouse. We used Software for the Exploration of Exploration (SEE) to investigate and develop a novel behavioral endpoint to characterize the "structure" of AMPH- and COC-induced locomotor behavior in two inbred strains of mouse, C57BL/6 (B6) and DBA/2 (D2). We suggest a measure we term "activity density" as a means to differentiate the behavioral effects of COC and AMPH. Activity density is defined as the activity divided by the range over which it took place. It characterizes the restriction of behavioral repertoire that does not result merely from inactivity. In both the B6 and D2 mice, AMPH increased activity density in a dose-dependent fashion by restricting the range of activity compared with COC doses producing the same level of activity. While AMPH restricted the range in both genotypes, characterizing the geographical region in which the restriction took place further differentiated the genotypes. The newly developed activity density measure thus provides a more general measure than stereotypy of the path, and can differentiate the effects of AMPH and COC both within and across genotypes.     

Effects of Acute Oral Naltrexone on the Subjective and Physiological Effects of Oral D-Amphetamine and Smoked Cocaine in Cocaine Abusers

Despite the prevalent worldwide abuse of stimulants, such as amphetamines and cocaine, no medications are currently approved for treating this serious public health problem. Both preclinical and clinical studies suggest that the opioid antagonist naltrexone (NTX) is effective in reducing the abuse liability of amphetamine, raising the question of whether similar positive findings would be obtained for cocaine. The purpose of this study was to evaluate the ability of oral NTX to alter the cardiovascular and subjective effects of D-amphetamine (D-AMPH) and cocaine (COC). Non-treatment-seeking COC users (N=12) completed this 3-week inpatient, randomized, crossover study. Participants received 0, 12.5, or 50 mg oral NTX 60 min before active or placebo stimulant administration during 10 separate laboratory sessions. Oral AMPH (0, 10, and 20 mg; or all placebo) was administered in ascending order within a laboratory session using a 60-min interdose interval. Smoked COC (0, 12.5, 25, and 50 mg; or all placebo) was administered in ascending order within a laboratory session using a 14-min interdose interval. Active COC and AMPH produced dose-related increases in cardiovascular function that were of comparable magnitude. In contrast, COC, but not AMPH, produced dose-related increases in several subjective measures of positive drug effect (eg, high, liking, and willingness to pay for the drug). NTX did not alter the cardiovascular effects of AMPH or COC. NTX also did not alter positive subjective ratings after COC administration, but it did significantly reduce ratings of craving for COC and tobacco during COC sessions. These results show that (1) oral AMPH produces minimal abuse-related subjective responses in COC smokers, and (2) NTX reduces craving for COC and tobacco during COC sessions. Future studies should continue to evaluate NTX as a potential anti-craving medication for COC dependence.     

Barbiturate dependence and drug preference

Rats were administered chronic multiple injections of amphetamine (AMPH) using dosage regimens which produce tolerance to the AMPH facilitation of self-stimulation responding, or reverse tolerance (sensitization) to the locomotor stimulant and stereotypy-producing effects of the drug. Subsequently rats were challenged with AMPH at behaviorally relevant doses and times and striatal and mesolimbic dopamine (DA) dynamics were assessed using the conversion of ³H-tyrosine to ³H-DA, and endogenous levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as indices of dopaminergic function. Acute administration of AMPH produced dose and time related changes in all indices of DA function in both the striatal and mesolimbic brain regions. Co-administration of haloperidol during chronic AMPH pretreatment prevented the appearance of most of the behavioral changes induced by chronic AMPH, suggesting an important role for DA systems. However, following chronic AMPH treatment, no additional biochemical changes in striatal or mesolimbic DA metabolism could be detected which would parallel the development of tolerance to AMPH facilitation of self-stimulation behavior or reverse tolerance to AMPH as reflected in enhanced post-stereotypy locomotor activity or a suggested increased intensity of stereotypy. Challenge with AMPH after chronic AMPH pretreatment did accelerate the changes in striatal but not mesolimbic DA metabolism, correlating with the more rapid onset of stereotypy induced by chronic AMPH. Thus, while DA systems appear to be a critical link, not only in the acute effects of AMPH, but also in the development of tolerance and reverse tolerance, most of the behavioral differences between acutely and chronically treated animals are not reflected by comparable differences in DA synthesis and metabolism.     

宫内暴露可卡因和胎鼠发育:不同给药剂量和给药时间的比较

目的　在妊娠期可卡因总给药剂量相等的情况下 ,探讨不同的给药时间及单次给药剂量对胎鼠发育的影响。方法　实验动物分为盐水对照组 (SAL)、可卡因小剂量组(COC2 0 )和可卡因中等剂量组 (COC4 0 )。妊娠 (Embryonicday ,E) 17d取材 ,记录各组胎鼠体重、脑重、纹状体重。并采用HPLC结合电化学检测器的方法测定各组胎鼠纹状体中多巴胺 (dopamine ,DA)和 5 羟色胺 (serotonin ,5 HT)的浓度。结果　COC4 0和COC2 0组胎鼠体重、脑重、纹状体重均低于SAL组 ;然而 ,仅COC4 0组胎鼠的脑 /体重比低于SAL组 (P <0 0 1)。HPLC结果表明 ,COC4 0、COC2 0组胎鼠纹状体DA、5 HT含量均高于SAL组 ,且COC4 0组纹状体 5 HT的含量显著高于COC2 0组。结论　与妊娠中晚期接触小剂量的可卡因相比 ,妊娠中期中等剂量接触可卡因对中枢神经系统发育的影响明显大于对胎鼠整体发育的影响     

Behaviorally equivalent stressors differentially modify the monoamine altering property of d-amphetamine

We previously demonstrated that behaviorally equivalent heat and cold stressors interacted with d-amphetamine (AMPH) treatment to produce different effects in rats responding for food on a fixed ratio 15 (FR15) schedule of reinforcement [25]. The present study was carried out to determine if these stressors differentially affect the disposition of AMPH to brain and/or if the stressors alone or in combination with AMPH affect CNS monoamines in a dissimilar manner. Exposure to either heat or cold stressor produced equivalent elevations of [3H]-AMPH in brain following 3 mg AMPH/kg but not 1 mg AMPH/kg. Neither stressor alone significantly altered any of the neurochemical parameters measured in any of the brain regions studied. In forebrain, heat and cold stressors interacted with AMPH treatment in different manners. Thus, although [3H]-AMPH was equally elevated in stressed groups following the high dose, cold-induced stress was not associated with an increase in dopamine (DA) levels, which was observed in Nonstressed and Heat-Stressed subjects. Although serotonin (5-HT) levels were not changed by any manipulation, 5-hydroxyindoleacetic acid (5-HIAA) levels were lowered in Nonstressed and Cold-Stressed subjects following both doses of AMPH. This effect was not associated with heat-induced stress. The apparent attenuation of AMPH behavioral toxicity observed in Cold-Stressed and/or exacerbation in Heat-Stressed rats observed in the earlier study may involve a pharmacodynamic interaction of AMPH and stress with transmitter substances, including DA and/or 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitization to systemic amphetamine produces an enhanced locomotor response to a subsequent intra-accumbens amphetamine challenge in rats

Repeated amphetamine (AMPH) administration into the nucleus accumbens does not enhance (sensitize) the locomotor activity produced by a subsequent systemic AMPH challenge. We report here, however, that pretreatment with systemic injections of AMPH does produce a significant enhancement in the locomotor stimulant effects produced by intra-accumbens AMPH given 21 days after the last pretreatment injection of AMPH. These data support the hypothesis that neural adaptations in dopamine (DA) terminal fields are sufficient for theexpression of AMPH sensitization, although an action on DA cell bodies may be required for theinduction of AMPH sensitization.     

妊娠中晚期接触可卡因对小鼠胎儿神经系统发育的影响

目的　观察妊娠母体接触可卡因引起的胎儿神经系统发育异常 ,研究宫内暴露可卡因引起的胎儿发育迟缓是否与母体孕期营养不良有关以及胎儿神经系统发育异常与神经递质的变化之间的关系。方法　小鼠妊娠d 8(E8)到d 17(E17) ,每日 2次颈背部皮下注射盐酸可卡因 2 0mg·kg-1·d-1,同时建立饮食对照组和盐水对照组 ,每日 2次注射生理盐水 2 0ml·kg-1·d-1,在此期间记录各组母鼠、胎鼠生理指标。E17取材 ,用HPLC法检测给药组血中可卡因浓度及各组母鼠、胎鼠纹状体多巴胺 (dopamine ,DA)、5 羟色胺 (sero tonin ,5 HT)的浓度。结果　可卡因给药组 (COC)与盐水对照组 (SAL)相比 ,母体体重增长量及总摄食量均减少 ,纹状体多巴胺和 5 羟色胺含量增高 (COCn =16 ,SALn =11,P<0 0 1) ;胎鼠体重、脑重、纹状体重也存在明显差异 (SALn=76 ,COCn =92 ,P <0 0 1)。而可卡因给药组与饮食对照组 (SPF)相比 ,在母鼠摄食量相等、体重增长量无明显差异 (COCn =16 ,SPFn =12 ,P >0 0 1)的情况下 ,胎鼠的各项生理指标均下降 (COCn =92 ,SPFn =6 5 ,P <0 0 1) ,且脑纹状体多巴胺、5 羟色胺水平明显升高 ,分别为 (88± 12 )ng·g-1,(2 4 2± 18)ng·g-1。结论　宫内暴露可卡因可引起子代神经系统发育异常 ,这种发育异常?     

Amphetamine discrimination: effects of dopamine receptor agonists

Dopamine (DA) D-1 and D-2 receptor agonists and antagonists were characterized in receptor binding and adenylate cyclase assays with respect to affinity, selectivity and efficacy. The ability of the ligands to interact with the discriminative stimulus effects of d-amphetamine (AMPH) was then assessed. The D-2 agonists, quinpirole, pergolide and CH 29-717, substituted completely for AMPH while neither partial (SKF 38393 and SKF 75670) nor full D-1 receptor agonists (SKF 89626 and SKF 81297) substituted. On the other hand, the selective D-1 and D-2 antagonists all blocked AMPH. The substitution for AMPH by pergolide was blocked by raclopride but not by SCH 23390, indicating D-2 mediation. In contrast, the motor effects of pergolide were blocked by both raclopride and SCH 23390, indicating mixed D-1/D-2 receptor involvement. These results suggest that D-1 and D-2 are equally involved in the expression of functional effects in the DAergic motor systems. Conversely, D-2 receptors may play a primary role in the DA systems involved in the AMPH cue; furthermore, the D-1 and D-2 receptors in the systems are relatively uncoupled.     

Effect of potassium channel modulators in mouse forced swimming test.

1. The effect of intracerebroventricular (i.c.v.) administration of different potassium channel blockers (tetraethylammonium, apamin, charybdotoxin, gliquidone), potassium channel openers (pinacidil, minoxidil, cromakalim) and aODN to mKv1.1 on immobility time was evaluated in the mouse forced swimming test, an animal model of depression. 2. Tetraethylammonium (TEA; 5 microg per mouse i.c.v.), apamin (3 ng per mouse i.c.v.), charybdotoxin (1 microg per mouse i.c.v.) and gliquidone (6 microg per mouse i.c.v.) administered 20 min before the test produced anti-immobility comparable to that induced by the tricyclic antidepressants amitriptyline (15 mg kg(-1) s.c.) and imipramine (30 mg kg(-1) s.c.). 3. By contrast pinacidil (10-20 microg per mouse i.c.v.), minoxidil (10-20 microg per mouse i.c.v.) and cromakalim (20-30 microg per mouse i.c.v.) increased immobility time when administered in the same experimental conditions. 4. Repeated administration of an antisense oligonucleotide (aODN) to the mKv1.1 gene (1 and 3 nmol per single i.c.v. injection) produced a dose-dependent increase in immobility time of mice 72 h after the last injection. At day 7, the increasing effect produced by aODN disappeared. A degenerate mKv1.1 oligonucleotide (dODN), used as control, did not produce any effect in comparison with saline- and vector-treated mice. 5. At the highest effective dose, potassium channels modulators and the mKv1.1 aODN did not impair motor coordination, as revealed by the rota rod test, nor did they modify spontaneous motility as revealed by the Animex apparatus. 6. These results suggest that modulation of potassium channels plays an important role in the regulation of immobility time in the mouse forced swimming test.     

Antagonism of the amphetamine cue by both classical and atypical antipsychotic drugs

Rats were trained to discriminate the stimulus properties of 1 mg/kg of d-amphetamine sulphate (AMPH) from saline in a two-lever task in which correct responding was reinforced with water under a fixed ratio (FR 32) schedule. Classical antipsychotic drugs from different chemical classes were all able to block the AMPH cue. Doses (mg/kg) inhibiting the cueing effect to 50% (ID50) were 0.035 (haloperidol), 0.04 (spiroperidol), 0.09 (cis(Z)-flupenthixol), 0.12 (trifluperazine), 0.15 (perphenazine), 0.92 (chlorpromazine) and 1.40 (pimozide). The AMPH cue was also antagonized by antipsychotic drugs that are considered atypical due to their relative lack of activity in conventional animal models or inability to produce extrapyramidal symptoms in the clinic. The following ID50 values were obtained: 0.88 (molindone), 1.22 (clozapine), 5.48 (metoclopramide), 15.4 (thioridazine) and 52.8 [-)-sulpiride). In addition, the AMPH cue was blocked by the D-1 selective dopamine (DA) antagonist, SCH 23390 (ID50 = 0.014 mg/kg). The abilities of these drugs to block the AMPH cue were unrelated to the drugs' effect upon the rate of responding. For example, some drugs (e.g. haloperidol, spiroperidol and SCH 23390) blocked the AMPH cue completely without any effect on the response rate. Furthermore, the non-antipsychotic phenothiazine, promethazine (2.5-12.5 mg/kg) failed to affect the AMPH cue although the drug strongly suppressed the response rate. However, the potent DA agonists, apomorphine (0.05-0.33 mg/kg) and lisuride (0.02-0.08 mg/kg), and the DA and norepinephrine agonist, DPI (0.4 and 0.8 mg/kg), did not mimic the AMPH cue or did so only partially. These results suggest that the 1 mg/kg AMPH cue depends on (DA) systems other than those involved in the stereotyped motor behavior commonly produced by high doses of AMPH or DA agonists. Low-dose AMPH discrimination may thus serve as a new model for studying antipsychotic drug action.     

Supersensitivity to amphetamine in protein kinase-C interacting protein/HINT1 knockout mice.

Protein kinase C interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) is a member of the histidine triad protein family. Although this protein is widely expressed in the mammalian brain including mesocorticolimbic and mesostriatal regions, its physiological function in CNS remains unknown. Recent microarray studies reported decreased mRNA expression of PKCI/HINT1 in the frontal cortex of individuals with schizophrenia, suggesting the possible involvement of this protein in the pathophysiology of the disease. In view of the documented link between dopamine (DA) transmission and schizophrenia, the present study used behavioral and neurochemical approaches to examine the influence of constitutive PKCI/HINT1 deletion upon: (i) basal and amphetamine (AMPH)-evoked locomotor activity; (ii) DA dynamics in the dorsal striatum, and (iii) postsynaptic DA receptor function. PKCI/HINT1(-/-) (KO) mice displayed lower spontaneous locomotion relative to wild-type (WT) controls. Acute AMPH administration significantly increased locomotor activity in WT mice; nonetheless, the effect was enhanced in KO mice. Quantitative microdialysis studies revealed no alteration in basal DA dynamics in the striatum or nucleus accumbens of KO mice. The ability of acute AMPH to increase DA levels was unaltered indicating that function in presynaptic DA neurotransmission in these regions do not underlie the behavioral phenotype of KO mice. In contrast to WT mice, systemic administration of the direct-acting DA receptor agonist apomorphine (10 mg/kg) significantly increased locomotor activity in KO mice suggesting that postsynaptic DA function is altered in these animals. These results demonstrate an important role of PKCI/HINT1 in modulating the behavioral response to AMPH. Furthermore, they indicate that the absence of this protein may be associated with dysregulation of postsynaptic DA transmission.     

Brain excitatory amino acid transporters (EAATs) and treatment of methamphetamine toxicity.

Based on the phenomenon of the abnormally increased transport of brain excitatory amino acids induced by the increased release of dopamine (DA) in the brain, the effects of intraperitoneal L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a non-selective excitatory amino acid transporter (EAAT) inhibitor, and (+/-)-threo-3-methylglutamic acid (MG), a specific EAAT2 inhibitor, were examined against methamphetamine (MA) and cocaine (COC) toxicity in mice. The MA (5 mg/kg)-increased activity counts, which included counts of both ambulatory and stereotyped behaviors, were attenuated by 10 and 20 mg/kg of PDC, but the COC (40 mg/kg)-increased activity counts were attenuated only by 20 mg/kg PDC. PDC (20 mg/kg) significantly attenuated both the mortality rate and the seizure score in acute MA (18 mg/kg) toxicity, but attenuated only the seizure score in acute COC (75 mg/kg) toxicity. PDC and MG (repeated doses of 5 and 10 mg/kg) attenuated the mortality rate (significant attenuation in the PDC group) and seizure score against repeated MA (12 mg/kg) toxicity, but had no effect on repeated COC (60 mg/kg) toxicity. Furthermore, MA (5 mg/kg) and COC (40 mg/kg) induced stressor-like and anxiogenic effects, the former of which were attenuated by PDC only (10 and 20 mg/kg in the MA group and 20 mg/kg in the COC group), and the latter of which were attenuated by both PDC and MG (for both drugs, 10 and 20 mg/kg in the MA group and 20 mg/kg in the COC group). Therefore, it was concluded that not only EAAT2 but also the other EAATs contributed to the occurrence of the MA-induced effects and part of the COC-induced effects, and that a non-selective EAAT inhibitor notably blocked the behavioral effects accompanying the MA-induced over-release of DA.     

The effect of 6-hydroxydopamine on the antinociceptive action of oxotremorine

Mice received intraventricular 6-hydroxydopamine (6-OHDA) to deplete brain noradrenaline (NA) and dopamine (DA). 6-OHDA reduced the reaction time of mice to a nociceptive stimulus (hot plate) and attenuated the antinociceptive action of oxotremorine. The administration of 6-OHDA to desipramine treated mice prevented both the loss of cerebral NA and the antogonism of oxotremorine's antinociceptive action. The administration of 6-OHDA to pargylinetreated mice increased the depletion of cerebral DA, but the antagonism of oxotremorine's antinociceptive action persisted. Catecholamines were measured in mouse brain at intervals from 1 h to 3 days after administration of 6-OHDA. DA levels failed to correlate with either the reduction in the hot plate reaction time or the antagonism of oxotremorine analgesia, whereas these effects were usually accompanied by a loss of brain NA. Centrally acting DA agonists failed to restore oxotremorine's antinociceptive action in 6-OHDA-treated mice. Intraventricular administration of the acetylcholine synthesis inhibitor triethylcholine to mice did not effect the antinociceptive action of oxotremorine. It is concluded that the antinociceptive action of oxotremorine in mice is initiated by stimulation of muscarinic receptors and involves noradrenergic neurones.     

1,4-Diazabicyclo[2.2.2]octane derivatives: a novel class of voltage-gated potassium channel blockers

Voltage-gated potassium (Kv) channels are targets for therapeutic drugs in the treatment of pathologic conditions including cardiac arrhythmia and epilepsy. In this study, we synthesized three classes of novel polyammonium compounds incorporating the bicyclic unit 1,4-diazabicyclo[2.2.2]octane (DABCO) and tested their action on three representative mammalian Kv channels (Kv2.1, Kv3.4, and Kv4.2) expressed in Xenopus laevis oocytes. Nonsubstituted DABCO did not block the Kv channels tested. Simple DABCO monostrings and diDABCO strings inhibited Kv2.1 and Kv3.4 channels, with potency increasing with string length for both these DABCO classes. Both Kv2.1 and Kv3.4 were most sensitive to C16 monostrings, with IC50 values of 1.9 and 0.6 microM, respectively. For compounds comprising two DABCO groups separated by an aromatic ring, inhibition depended upon relative positioning of the two DABCO groups, and only the para form (JC638.2alpha) was active, blocking Kv2.1 with an IC50 of 186 microM. Kv4.2 channels were relatively insensitive to all compounds tested. Unlike the tetraethylammonium ion (TEA), neither JC638.2alpha nor C16 monostring TA279 produced block when applied intracellularly via the recording electrode to Kv2.1 channels expressed in Chinese hamster ovary cells, suggesting against an internal site of action. However, JC638.2alpha protected an introduced cysteine (K356C) in the Kv2.1 outer pore from permanent modification by methanethiosulfonate ethyltrimethylammonium (MTSET). These data suggest that JC638.2alpha occupies an external binding site similar to that of TEA in the Kv2.1 outer pore, but with much higher affinity than TEA. These DABCO salts represent a new class of Kv channel blockers, some with higher potencies than any previously described quaternary ammonium ions. The potential for synthesis of an array of modular derivatives suggests that DABCO compounds hold promise as probes of Kv channel structure and identity and as potential therapeutic agents.