A rat model of endothelin-3-induced middle cerebral artery occlusion with controlled reperfusion.

Surge hyperemia and mechanical damage to the cerebrovascular endothelium may serve to exacerbate the neuropathological outcome in animal models of focal cerebral ischemia. We have modified an existing model of endothelin-1-induced middle cerebral artery (MCA) occlusion to enable controlled reperfusion without damage to the cerebral vasculature. Endothelin-1 (ET-1) and endothelin-3 (ET-3) were injected via a double-injection cannula into brain parenchyma adjacent to the MCA of anesthetized rats to produce focal cerebral ischemia. ET-1 and ET-3 produced large ischemic lesions that were restricted to those cortical and subcortical structures supplied by the MCA. The volume of ischemic damage produced by 100 pmol of ET-1 and ET-3 was similar. The endothelin-A (ET(A)) receptor antagonist FR139317 (3 or 30 nmol) injected 10 min after ET-1 did not significantly alter the volume of damage. By contrast, the lesion produced by ET-3 was completely inhibited by FR139317 at the 10 min time-point. FR139317 partially attenuated the ET-3-induced lesion when administered 30 min post-occlusion, but injection 90 min following ET-3 produced a lesion not different to that produced by ET-3 alone. These findings were supported by laser Doppler flowmetry which determined FR139317 induces reperfusion when injected 10 or 90 min following ET-3. ET-3-induced MCA occlusion is therefore amenable to reversal by the ET(A) receptor antagonist FR139317, and this model may offer a means to investigate the neuropathology of reperfusion without the procedure-related artifacts associated with some reperfusion models.

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

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.

Pyrazolopyridine derivatives act as competitive antagonists of brain adenosine A1 receptors: [35S]GTPgammaS binding studies.

The effects of adenosine receptor ligands and three novel pyrazolopyridine derivatives on guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to rat cerebral cortical membranes were examined. [35S]GTPgammaS binding was stimulated in a concentration dependent manner by several adenosine receptor agonists. The adenosine A2a receptor selective agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680), was ineffective confirming specificity for adenosine A1 receptor activation. 2-Chloro-N6-cyclopentyladenosine (CCPA; 10(-7) M)-stimulated [35S]GTPgammaS binding was inhibited by xanthine and pyrazolopyridine based adenosine receptor antagonists. The concentration-response curve for CCPA-stimulated [35S]GTPgammaS binding was shifted to the right with increasing concentrations of antagonist without significant changes in maximal response. Schild analyses determined pK(B) values of 8.97, 8.88, 8.21, 8.16, 7.79 and 7.65 for 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), 6-oxo-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl)-1(6H)-pyridazinebutyric+ ++ acid (FK838), 9-chloro-2-(2-furyl)[1,2,4]triazolo-[1,5c]quinazolin-5-amine (CGS 15943), 8-cyclopentyl-1,3-methylxanthine (CPT) and (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352), respectively. Schild slopes were close to unity, confirming that these novel pyrazolopyridine derivatives act as competitive antagonists at rat brain adenosine A1 receptors.

Pharmacological characterization of a simple behavioral response mediated selectively by central adenosine A1 receptors, using in vivo and in vitro techniques.

The behavioral profile of a range of adenosine receptor ligands was examined in rats using a locomotor activity model. Adenosine receptor agonists, including the selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) and the A2A agonist, 2-[(2-aminoethylamino)carbonylethyl-phenylethylamino]- 5'-ethylcarboxa midoadenosine (APEC), reduced spontaneous motor activity in a dose-dependent manner. CPA-induced locomotor depression was attenuated by adenosine A1 receptor selective antagonists, such as 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (R)-1-[(E)-3-(2-phenylpyrazolo[1, 5-a]pyridin-3-yl)-acryloyl]-2-piperidine ethanol (FK453), and (R)-1-[(E)-3-(2-phenylpyrazolo[1, 5-a]pyridin-3-yl)-acryloyl]-piperidin-2-yl acetic acid (FK352), but not by the A2A receptor antagonist, (E)-1,3-dipropyl-8-(3, 4-dimethoxystyryl)-7-methylxanthine (KF17837). By contrast, APEC-induced hypolocomotion was attenuated by KF17837 but not by DPCPX, confirming that adenosine A1 and A2A receptor activation mediates locomotor output independently. It was found that two peripheral adenosine receptor antagonists, 8-(p-sulphophenyl)-1, 3-dipropylxanthine (DPSPX) and 8-(p-sulphophenyl)-1, 3-dimethylxanthine (8-PST), did not alter CPA-induced hypolocomotion. This confirmed that pharmacological reversal of the adenosine A1 receptor-mediated response involved a central site of drug action. The relationship between occupancy of central adenosine A1 receptors and behavioral effect was therefore assessed. Regression analysis on log transformed data confirmed associations between antagonist affinity for brain [3H]DPCPX binding sites and, in order of increasing significance, the equivalent behavioral dose (EBD) for reversal of CPA-induced hypolocomotion (r2 = 0.32), the serum concentration of drug (r2 = 0.65), and most significantly with the brain concentration of drug detected 20 min after administration of the (EBD) (r2 = 0.95). These data suggest that competition between agonists and antagonists, for occupancy of central adenosine A1 receptors, is intrinsic to the pharmacological reversal of CPA-induced hypolocomotion. The validity of the model as a simple predictive screen for the blood/brain barrier permeability of adenosine A1 receptor antagonists was thereby confirmed.

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists.

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.

Intracerebroventricular infusion of the NMDA receptor-associated glycine site antagonist 7-chlorokynurenate impairs water maze performance but fails to block hippocampal long-term potentiation in vivo.

Most previous studies investigating the relationship between N-methyl-D-aspartate receptor-dependent synaptic plasticity and learning have employed drugs that either compete with glutamate for access to the primary agonist binding site (e.g., D-2-amino-5-phosphopentanoic acid) or block the associated ion channel (e.g., dizocilpine). This study targeted the glycine receptor site located on the NMDA receptor complex. Chronic intracerebroventricular infusion of the glycine site antagonist 7-chlorokynurenate (7CK; 75 mM, 0.5 microliter/h, icv, for up to 14 days) impaired performance of male Lister hooded rats during acquisition of a spatial reference memory task in the water maze. In addition, however, these animals showed sensorimotor deficits, including a prolonged righting reflex, ataxia, and difficulty in staying on the escape platform. On completion of behavioral testing, the rats were anesthetized with urethane and an attempt was made to induce LTP in the hippocampus ipsilateral to the infusion cannula. Both control and 7CK-infused animals displayed equivalent long-term potentiation (LTP) 60 min posttetanus. A novel analytical technique for assaying drug tissue levels involving high-performance liquid chromotography with fluorescence detection revealed that tissue levels of 7CK in hippocampus were extremely low and unlikely to be sufficient to affect LTP, as observed. These findings neither support nor compromise the LTP/learning hypothesis, but they illustrate some of the problems of using drugs to elucidate the neurobiological mechanisms of learning and memory and the importance of a within-subjects design incorporating behavioral, physiological, and biochemical measures.

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.

Detection of adenosine receptor antagonists in rat brain using a modified radioreceptor assay.

The present study describes a modified radioreceptor binding assay using brain homogenate or serum from drug treated animals as the 'competing drug' in a conventional in vitro radioligand binding assay. Method validation involved measurement of the brain and serum concentration of three adenosine receptor antagonists following systemic administration, using a [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) binding assay. The intrinsic [3H]DPCPX binding capacity of test samples was abolished by protein denaturation (80 degrees C, 15 min) and, endogenous ligand was depleted enzymatically, prior to determination of drug concentration. Brain and serum concentrations of the adenosine A1 receptor antagonist, DPCPX increased in a dose related manner when measured 20 min after intraperitoneal injection. Estimated brain concentrations were 13.8, 87.7 and 288 nM following injection of 0.01, 0.1 and 1.0 mg/kg DPCPX, and serum concentrations were 26.5, 195 and 1370 nM respectively. A time dependent decrease in both brain and serum concentration was noted 20-180 min following injection of 1.0 mg/kg DPCPX. The peripheral adenosine receptor antagonists, 1,3-dipropyl-8-p-sulphophenylxanthine (DPSPX; 5.6 mg/kg) and 8-(p-sulphophenyl)theophylline (8-PST; 20 mg/kg), were not detected in brain tissue 20 min after intraperitoneal injection, despite serum concentrations of 56 and 52 microM respectively. This assay provides a useful and versatile method for determining the central penetration of neuroactive drugs.

Tacrolimus (FK506) ameliorates skilled motor deficits produced by middle cerebral artery occlusion in rats.

BACKGROUND AND PURPOSE: Tacrolimus (FK506) is a potent immunosuppressant that is presently in clinical use for prevention of allograft rejection. Recently, animal studies reporting significant reductions in the volume of tissue damage associated with cardiac, hepatic, and cerebral ischemia suggest that tacrolimus may also be of use in the clinical management of stroke. In the present study, we examine whether the neuroprotective effects of tacrolimus, as assessed by histological outcome, are accompanied by an amelioration of the skilled motor deficits induced in the rat by middle cerebral artery occlusion (MCAO). METHODS: Animals were trained to perform a skilled paw-reaching task before MCAO by perivascular microinjections of endothelin-1. Tacrolimus (1 mg/kg, n = 6) or vehicle (n = 6) was administered by intravenous infusion 1 minute after MCAO. After a 5-day postoperative recovery period, the rats were retested for skilled paw-reaching ability for an additional 9 days. RESULTS: In vehicle-treated rats, MCAO resulted in a profound bilateral impairment in skilled paw use. Rats treated with tacrolimus, although still impaired, performed significantly better than those treated with vehicle alone (P < .01). Histological analysis, 14 days after occlusion, confirmed the neuroprotective efficacy of tacrolimus with a 66% reduction in the volume of hemispheric brain damage produced by MCAO (P < .01). CONCLUSIONS: The present studies show that tacrolimus not only protects neural tissue from focal cerebral ischemia but also significantly ameliorates the deficits in skilled motor ability produced by this lesion. These data provide further support for the view that tacrolimus may be of use in the treatment of stroke.

Comparison of the patterns of altered cerebral glucose utilisation produced by competitive and non-competitive NMDA receptor antagonists.

Recent studies indicate that competitive and non-competitive NMDA receptor antagonists can be readily distinguished by their effects on local cerebral glucose utilisation (1CGU). In the present study we compare the effects of the novel NMDA antagonist, (+)-1-methyl-1phenyl-1,2,3,4-tetrahydroisoquinoline (FR115427) on 1CGU, comparing its metabolic profile with that of the non-competitive NMDA receptor antagonist, dizocilpine (MK801) and of the competitive NMDA receptor antagonist CGS19755, using the 2-deoxyglucose metabolic mapping approach. Local cerebral glucose utilisation was measured in 80 anatomically discrete regions of the conscious rat brain using [14C]2-deoxyglucose quantitative autoradiography. Studies were initiated 10 min after the administration of FR115427 (0.1-3 mg/kg i.v.; n = 20), dizocilpine (0.03-0.3 mg/kg; n = 15), CGS19755 (1-30 mg/kg; n = 15) or saline (2 ml/kg; n = 5). Dizocilpine produced characteristic alterations in 1CGU with widespread increases in 1CGU in primary olfactory and limbic areas while reducing 1CGU in somatosensory and motor cortex. FR115427 produced a pattern of altered 1CGU which was broadly similar to that elicited by dizocilpine with increases in 1CGU in the pontine nuclei, presubiculum and hippocampus and reductions in somatosensory and motor cortex and within components of the auditory system. However, FR115427 was approximately 30-fold less potent than dizocilpine in this regard. In limbic structures, the effects of FR115427 were less pronounced than those produced by dizocilpine. Increases in 1CGU of 62-98% were found in retrosplenial, piriform and entorhinal cortex of dizocilpine-treated rats whereas these areas appeared relatively unaffected following FR115427 administration. A comparison of the pattern of metabolic response produced by each of these agents was performed by constructing a hierarchy of regional responsiveness using the f statistic: while focal differences in the metabolic profiles of dizocilpine and FR115427 were evident, a plot of the regional f values for dizocilpine and FR115427 revealed a strong overall relationship between the metabolic responses with a Pearson's product moment correlation of 0.78. In contrast, the correlation between the patterns produced by CGS19755 and that for dizocilpine or FR115427 was poor (r = 0.28 and 0.5 respectively).

Acquisition of conditioned inhibition in rats is impaired by ablation of serotoninergic pathways.

The effects of chronically ablating the serotoninergic inputs to various regions of the rat brain on the ability to solve a feature-negative discrimination was measured. After intracerebroventricular administration of the specific neurotoxin 5,7-dihydroxytryptamine, the rats exhibited an impaired capacity to solve such a discrimination, irrespective of whether auditory or visual stimuli were used. Further behavioural analysis revealed that this effect was not due to a reduced capacity to form excitatory associations, since both groups responded equally to reinforced stimuli. By contrast, the lesion more likely resulted in a failure to endow the non-reinforced stimuli with inhibitory properties. This suggestion was supported by the observation that, in a retardation test, the conditioned inhibitor aroused less inhibition in the lesioned group than in vehicle-injected controls. Furthermore, the conditioned inhibitor failed to pass a summation test in lesioned animals, again indicating that their hampered ability to master the discrimination was the result of an impairment in the formation of inhibitory associations. It is concluded that destruction of central 5-hydroxytryptamine-containing pathways impairs the functioning of brain areas underlying inhibitory associative learning.

Distinct components of spatial learning revealed by prior training and NMDA receptor blockade.

Synaptic plasticity dependent on N-methyl-D-aspartate (NMDA) receptors is thought to underlie certain types of learning and memory. In support of this, both hippocampal long-term potentiation and spatial learning in a watermaze are impaired by blocking NMDA receptors with a selective antagonist D(-)-2-amino-5-phosphonovaleric acid (AP5) or by a mutation in one of the receptor subunits. Here we report, however, that the AP5-induced learning deficit can be almost completely prevented if rats are pretrained in a different watermaze before administration of the drug. This is not because of stimulus generalization, and occurs despite learning of the second task remaining hippocampus dependent. An AP5-induced learning deficit is, however, still seen if the animals are pretrained using a non-spatial task. Thus, despite its procedural simplicity, the watermaze may involve multiple cognitive processes with distinct pharmacological properties; although required for some component of spatial learning, NMDA receptors may not be required for encoding the spatial representation of a specific environment.

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.

Behavioural assessment of endothelin-1 induced middle cerebral artery occlusion in the rat.

The behavioural effects of unilateral middle cerebral artery occlusion (MCAO) induced by perivascular injection of endothelin, and a unilateral excitotoxic lesion of the striatum, were explored using the staircase test of skilled paw-reaching in the rat. A profound bilateral impairment in pellet recovery, with a concomitant increase in pellet displacement, was observed in the MCAO group. By contrast the striatal lesion group exhibited a primarily contralateral impairment. The findings provide both further insight into the control of unilateral motor function and a reliable behavioural endpoint for the assessment of experimental stroke.

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.

Inhibition of nitric oxide synthase does not prevent the induction of long-term potentiation in vivo.

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 whether an inhibitor of NO synthase, N omega-nitro-L-arginine methyl ester (L-NAME), affects long-term potentiation (LTP) in vivo, as the results of recent in vitro experiments would predict. L-NAME, given as an acute injection at a dose sufficient to inhibit hippocampal NO synthase (> 90%), had no effect on perforant path-dentate gyrus LTP induced by a strongly suprathreshold tetanus, but appeared to impair LTP induced by a weak near-threshold tetanus that may be more physiologically relevant. However, subsequent studies revealed that chronic L-NAME treatment (> 95% inhibition of NO synthase) had no effect upon LTP induction, and that acute (but not chronic) treatment resulted in a gradual but significant reduction in nontetanized baseline field potentials. The baseline shift appeared to be of a magnitude sufficient to account for the apparent impairment of weak tetanus-induced LTP. This possibility was further examined in a two-hemisphere experiment in which the time course of changes in the field EPSP of the nontetanized pathway served as the within-subject control for the tetanized pathway. No impairment of LTP induction was observed; indeed, if anything, there was a trend for greater potentiation with L-NAME. Because NO has also been implicated in the control of vasodilation, the effect of L-NAME on cerebrovascular function was also investigated. Peripheral blood pressure was significantly increased by L-NAME at the same dose that affected the field EPSP. Local cerebral glucose utilization was unchanged, while local cerebral blood flow decreased significantly in various brain regions, including the hippocampus, indicating an uncoupling of cerebral metabolism and blood flow. Thus, while NO synthase inhibition does not appear to limit the induction of LTP in vivo, it does reduce the size of baseline field EPSPs and affect local cerebrovascular function.

Intracerebroventricular injection of a nitric oxide synthase inhibitor does not affect long-term slope potentiation in vivo.

Although there is evidence from in vitro studies to suggest that NO synthesis may be involved in the induction of hippocampal LTP, other in vitro studies and experiments conducted in vivo have provided conflicting results. In agreement with previous work conducted in this laboratory using an i.p. route of administration, this paper reports that i.c.v. injections of the NO synthase inhibitor, N omego-nitro-L-arginine methyl ester (L-NAME), at a dose sufficient to inhibit hippocampal NO synthase by 90-95%, failed to block the induction of LTP in the dentate gyrus in vivo (as measured by the change in the slope of the early rising phase of the field EPSP). The failure to block LTP occurred following both a strong and a weak tetanus. L-NAME injections did, however, result in a small but transient increase in the baseline slope of the field EPSP, a more prolonged enhancement of the baseline population spike, and a significant attenuation of spike potentiation induced by a strong tetanus. These results offer no support for the hypothesis that NO synthase is required for the induction of the synaptic component of LTP, but do suggest a role for NO in the control of cell excitability in the hippocampus.