Steven P. Butcher

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Abstract: Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three‐vessel occlusion combined with hypotension (50 mm Hg) in male Sprague‐Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nudeotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 μM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.

Extracellular Overflow of Neuroactive Amino Acids During Severe Insulin‐Induced Hypoglycemia: In Vivo Dialysis of the Rat Hippocampus

Hypoglycemia‐evoked changes in levels of extracellular excitatory and inhibitory amino acids were studied using the microdialysis technique. A newly designed dialysis probe was inserted stereotaxically into the rat hippocampus. Animals were then subjected to insulin‐induced hypoglycemia; then blood glucose levels were restored by glucose injections after a 30‐min period of isoelectric electroencephalography. Dialysates were collected before, during, and after the isoelectric period. Amino acids in the dialysates were analyzed by liquid chromatography and fluorescence detection following automatic precolumn derivatization with o‐phthaldialdehyde. During the isoelectric phase, the concentration of aspartate increased 15‐fold, whereas glutamate, γ‐aminobutyric acid, taurine, and phosphoethanolamine levels were elevated three‐ to sixfold. Smaller increases were observed for nonneuroactive amino acids such as asparagine, alanine, and phenylalanine. In contrast to all other amino acids, the glutamine content was reduced to <30% of preisoelectric values. The concentrations of the neuroactive amino acids were restored to normal in the postisoelectric phase. These data demonstrate that there is an extracellular overflow of neuroactive amino acids, especially aspartate, during severe hypoglycemia.

Amphetamine‐Induced Dopamine Release in the Rat Striatum: An In Vivo Microdialysis Study

The effects of a number of biochemical and pharmacological manipulations on amphetamine (AMPH)‐induced alterations in dopamine (DA) release and metabolism were examined in the rat striatum using the in vivo brain microdialysis method. Basal striatal dialysate concentrations were: DA, 7 nM; dihydroxyphenylacetic acid (DOPAC), 850 nM; homovanillic acid (HVA), 500 nM; 5‐hydroxyindoleacetic acid (5‐HIAA), 300 nM; and 3‐methoxytyramine (3‐MT), 3 nM. Intraperitoneal injection of AMPH (4 mg/kg) induced a substantial increase in DA efflux, which attained its maximum response 20–40 min after drug injection. On the other hand, DOPAC and HVA efflux declined following AMPH. The DA response, but not those of DOPAC and HVA, was dose dependent within the range of AMPH tested (2–16 mg/kg). High doses of AMPH (>8 mg/kg) also decreased 5‐HIAA and increased 3‐MT efflux. Depletion of vesicular stores of DA using reserpine did not affect significantly AMPH‐induced dopamine efflux. In contrast, prior inhibition of catecholamine synthesis, using α‐methyl‐p‐tyrosine, proved to be an effective inhibitor of AMPH‐evoked DA release (<35% of control). Moreover, the DA releasing action of AMPH was facilitated in pargyline‐pretreated animals (220% of control). These data suggest that AMPH releases preferentially a newly synthesised pool of DA. Nomifensine, a DA uptake inhibitor, was an effective inhibitor of AMPH‐induced DA efflux (18% of control). On the other hand, this action of AMPH was facilitated by veratrine and ouabain (200–210% of control). These results suggest that the membrane DA carrier may be involved in the actions of AMPH on DA efflux.

Neuroprotective Actions of FK506 in Experimental Stroke: In Vivo Evidence against an Antiexcitotoxic Mechanism

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, although in vitro studies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The present in vivo study demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56–58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.

Inhibition of nitric oxide synthase does not impair spatial learning.

Nitric oxide (NO), a putative intercellular messenger in the CNS, may be involved in certain forms of synaptic plasticity and learning. This article reports a series of experiments investigating the effects of N omega-nitro-L-arginine methyl ester (L-NAME) upon various forms of learning and memory in the watermaze. L-NAME (75 mg/kg, i.p., sufficient to bring about > 90% inhibition of NO synthesis in brain) produced an apparent impairment in spatial learning when given to naive rats during acquisition (3 d, six training trials per day). This impairment was dose related, stereoselective, and attenuated by coadministration of L-arginine. A second study showed that L-NAME did not affect the retention of a previously learned spatial task. In addition, in a visual discrimination task, the rate at which criterion levels of performance were reached was unaffected by L-NAME. Thus, inhibition of NO synthase may cause a selective impairment of spatial learning without effect upon retention. However, analysis of the early training trials of the visual discrimination task revealed significantly elevated escape latencies in the L-NAME-treated rats, suggesting that inhibition of NO synthase may have more general effects. As normal rats learn the spatial task very rapidly, the possibility arises that the apparent deficit in learning is due to a disruption of some process other than learning per se. A further series of experiments investigated this possibility. L-NAME was found not to impair the learning of a new platform position in the same spatial environment. Surprisingly, L-NAME also had no effect on spatial learning in a second watermaze located in a novel spatial environment by rats well practiced with all aspects of watermaze training. Finally, L-NAME had no effect on spatial learning in naive rats trained with just one trial per day. Thus, systemic injection of an NO synthase inhibitor impairs behavioral performance in two tasks during their initial acquisition, but the basis of this functional disruption is unlikely to be due to any direct effect upon the mechanisms of spatial learning.

Effects of Selective Monoamine Oxidase Inhibitors on the In Vivo Release and Metabolism of Dopamine in the Rat Striatum

Abstract: Brain microdialysis was used to examine the in vivo efflux and metabolism of dopamine (DA) in the rat striatum following monoamine oxidase (MAO) inhibition. Relevant catecholamines and indoleamines were quantified by HPLC coupled with a electrochemical detection system. The MAO‐B inhibitor selegiline only affected DA deamination at a dose shown to inhibit partially type A MAO. Alterations in DA and metabolite efflux were not observed when using the MAO‐B‐selective dose of 1 mg/kg of selegiline. At 10 mg/kg, selegiline reduced the efflux of DA metabolites to ∼70% of basal values without affecting DA efflux. K+‐and veratrine‐stimulated DA efflux was not affected by selegiline. Experiments using amphetamine and the DA uptake inhibitor nomifensine demonstrated that the effect of selegiline on DA metabolism was unlikely to be mediated either by inhibition of DA uptake or by an indirect effect of its metabolite amphetamine. The possibility that the effect of selegiline is mediated via a nonspecific inhibition of MAO is discussed. In contrast, the MAO‐A inhibitor clorgyline inhibited basal DA metabolism and increased basal and de‐polarisation‐induced DA efflux. A 1 mg/kg dose of clorgyline reduced basal DA metabolite efflux (40–60% of control values) without affecting DA efflux. At 10 mg/kg of clorgyline, DA efflux increased to 253 ± 19% of basal values, whereas efflux of DA metabolites was reduced to between 15 and 26% of control values. The release of DA induced by K+ and vera‐trine was not affected by 1 mg/kg of clorgyline but was increased by ∼200% following pretreatment with 10 mg/kg of clorgyline. The nonselective MAO inhibitor pargyline caused similar but more pronounced alterations in these parameters. For example, using a 75 mg/kg dose of pargyline, efflux of DA increased maximally to 310 ± 20% of basal values, and the release of DA induced by K+ and veratrine was increased by ∼300% following pargyline pretreatment. These data suggest that DA metabolism is mediated principally by MAO‐A in the rat striatum. However, under conditions of MAO‐A inhibition, a component of metabolism mediated by the type B enzyme becomes apparent.

Behavioural analysis of the acute and chronic effects of MDMA treatment in the rat.

Abstract  Rationale: A variety of animal models have shown MDMA (3,4-methylenedioxymethamphetamine) to be a selective 5-HT neurotoxin, though little is known of the long-term behavioural effects of the pathophysiology. The widespread recreational use of MDMA thus raises concerns over the long-term functional sequelae in humans. Objective: This study was designed to explore both the acute- and post-treatment consequences of a 3-day neurotoxic exposure to MDMA in the rat, using a variety of behavioural paradigms. Methods: Following training to pretreatment performance criteria, animals were treated twice daily with ascending doses of MDMA (10, 15, 20 mg/kg) over 3 days. Body temperature, locomotor activity, skilled paw-reaching ability and performance of the delayed non-match to place (DNMTP) procedure was assessed daily during this period and on an intermittent schedule over the following 16 days. Finally, post mortem biochemical analyses of [3H] citalopram binding and monoamine levels were performed. Results: During the MDMA treatment period, an acute 5-HT-like syndrome was observed which showed evidence of tolerance. Once drug treatment ceased the syndrome abated completely. During the post-treatment phase, a selective, delay-dependent, deficit in DNMTP performance developed. Post-mortem analysis confirmed reductions in markers of 5-HT function, in cortex, hippocampus and striatum. Conclusions: These results confirm that acutely MDMA exposure elicits a classical 5-HT syndrome. In the long-term, exposure results in 5-HT neurotoxicity and a lasting cognitive impairment. These results have significant implications for the prediction that use of MDMA in humans could have deleterious long-term neuropsychological/psychiatric consequences.

Characterisation of an experimental model of stroke produced by intracerebral microinjection of endothelin-1 adjacent to the rat middle cerebral artery

A novel experimental model of stroke has been developed using the powerful vasoconstrictor peptide, endothelin-1, to occlude the middle cerebral artery (MCA) of anaesthetised rats. Intracerebral microinjections of endothelin-1 were administered under stereotaxic guidance adjacent to the MCA, and after 3 days rats were perfusion fixed for histopathological determination of ischaemic brain damage. The pattern of brain damage noted using this model was similar to that reported following permanent surgical occlusion of the MCA. Brain damage was apparent in the dorsal and lateral neocortex (98 +/- 12 mm3) and striatum (32 +/- 3 mm3) ipsilateral to the insult. Rats anaesthetised with halothane and barbiturate exhibited a similar volume of brain damage. However, infarct volume increased when the duration of halothane anaesthesia was extended from 5 to 180 min post-occlusion. Neuroprotection studies demonstrated that dizocilpine (5 mg/kg, i.p.), administered 30 min prior to MCA occlusion, reduced the volume of cortical brain damage by 51% (P < 0.05) but did not alter the volume of striatal brain damage. The present results demonstrate that microinjections of endothelin-1 adjacent to the rat MCA result in a reproducible pattern of focal cerebral infarction which is sensitive to the duration of anaesthesia and can be reduced by dizocilpine.

Dopamine release and metabolism in the rat striatum: An analysis by ‘in vivo’ brain microdialysis

Brain microdialysis studies on the mechanisms underlying dopamine release in the rat striatum provide evidence that both exocytotic and carrier-dependent processes operate in vivo. While several releasers (potassium, veratrine, amphetamine, ouabain) utilize newly synthesized stores of dopamine, tyramine is uniquely sensitive to depletion of vesicular storage by reserpine. Extracellular DOPAC is closely associated with the newly synthesized pool of dopamine and experiments with selective monoamine oxidase inhibitors suggest that DOPAC is formed mainly by MAO-A. Recent work on the two dopamine receptors suggest that release by different mechanisms may selectively activate D1 or D2 receptor subtypes.

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

Abstract: The effects of corticostriatal deafferentation (de‐cortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine‐releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra‐and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. γ‐Aminobutyric acid (GABA), tau‐nne (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month postlesion. The intracellular pool of Glu and Asp was also reduced in kainate‐lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of nonneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion‐induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood‐brain bamer transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine. GABA, Tau, and PEA effluxes were also decreased in kainate‐lesioned striata. These findings are consistent with the proposed roles of an acidic amino acid as the corticostriatal transmitter, and of GABA as a transmitter in intrinsic striatal neurons. The existence of releasable pools of Tau and PEA within kainate‐sensitive striatal neurons would also appear to be likely.