A role for Sv2c in basal ganglia functions.

SV2C is an isoform of the synaptic vesicle 2 protein family that exhibits a particular pattern of brain expression with enriched expression in several basal ganglia nuclei. In the present study, we have investigated SV2C implication in both normal and pathological basal ganglia functioning with a peculiar attention to dopamine neuron containing regions. In SV2C-/- mice, the expression of tyrosine hydroxylase mRNA in midbrain dopaminergic neurons was largely and significantly increased and enkephalin mRNA expression was significantly decreased in the caudate-putamen and accumbens nucleus. The expression of SV2C was studied in two models of dopaminergic denervation (6-OHDA- and MPTP-induced lesions). In dopamine-depleted animals, SV2C mRNA expression was significant increased in the striatum. In order to further understand the role of SV2C, we performed behavioral experiments on SV2C-/- mice and on knock-down mice receiving an injection of adeno-associated virus expressing SV2C miRNA specifically in the ventral midbrain. These modifications of SV2C expression had little or no impact on behavior in open field and elevated plus maze. However, even if complete loss of SV2C had no impact on conditioned place preference induced by cocaine, the specific knock-down of SV2C expression in the dopaminergic neurons completely abolished the development of a CPP while the reaction to an acute drug injection remains similar in these mice compared to control mice. These results showed that SV2C, a poorly functionally characterized protein is strongly involved in normal operation of the basal ganglia network and could be also involved in system adaptation in basal ganglia pathological conditions.

Distribution of SV2C mRNA and protein expression in the mouse brain with a particular emphasis on the basal ganglia system.

Synaptic vesicle 2 proteins (SV2), SV2A, SV2B and SV2C, are integral proteins localized on the surface of synaptic vesicles in all neurons. SV2 proteins appear to play an important, but not yet fully understood role in synaptic vesicle exocytosis and neurotransmitter release. Moreover, SV2 seems to be the receptor of the botulinum neurotoxin A. In the present study, using single and double-labeling fluorescent immunohistochemistry and in situ hybridization we have identified the brain pattern of SV2C mRNA and protein expression in mice. Our results indicated that SV2C protein was expressed in a small subset of brain regions including the olfactory bulb, olfactory tubercle, nucleus accumbens, caudate-putamen, ventral pallidum, globus pallidus, substantia nigra and the ventral tegmental area. These results were confirmed by means of in situ hybridization, except for the globus pallidus and the substantia nigra pars reticulata, in which no labeling was found, suggesting that SV2C-positive fibers in these areas are terminals of striatal projecting neurons. In the striatum, we found that, in addition to its presence in the projection neurons, SV2C was densely expressed in a fraction (around 45%) of cholinergic interneurons. In addition, our data also showed that SV2C was densely expressed in most dopaminergic neurons in the substantia nigra pars compacta and the ventral tegmental area (more than 70% of the dopaminergic neurons analyzed were SV2C-positive). Altogether, our results suggest that SV2C may contribute to the regulation of neurotransmitter release and synaptic transmission in the basal ganglia including cholinergic striatal interneurons and nigro-striatal/mesolimbic dopamine neurons.

Arylsulfonamides as a new class of cannabinoid CB1 receptor ligands: identification of a lead and initial SAR studies.

High-throughput screening of the UCB sample collection identified the piperidinyl-sulfonyl benzoic ester 1 as a novel agonist for CB(1) receptor with nanomolar affinity. We report here the pharmacological profile of compound 1 as well as preliminary biological activities in pain model. Diverse close analogs of 1 were purchased and the structure-affinity relationships among this novel class are discussed.

Diffusion-weighted MRI of infarct growth in a rat photochemical stroke model: effect of lubeluzole.

We studied the neuroprotective effect of lubeluzole, a NOS (nitric oxide synthase) pathway modulator, on the development of ischemic damage within the first six hours after a photochemically induced neocortical infarct in rats using diffusion-weighted MRI and Apparent Diffusion Coefficient (ADC) maps. A unilateral photochemical infarct was induced in the hindlimb sensorimotor neocortex of Wistar rats. One hour after infarction, rats received either vehicle (n=10) or lubeluzole (n=11; a 0.31 mg/kg i.v. bolus followed by a one-hour 0.31 mg/kg i.v. infusion). During the first six hours after infarct induction, multislice T2- and Diffusion-Weighted magnetic resonance images (MRI) were obtained to measure percent change of volume of ischemic damage, whereas regional ADC maps were used to measure time-dependent density of ischemic damage. Lubeluzole reduced the percent increase of volume of ischemic damage relative to baseline (at 1 h after infarct induction just before drug treatment), by 18% at 5 and 6 hrs after infarct induction. Lubeluzole attenuated the ADC decreases in the peripheral rim of the infarct, but left the ADC values in the core unaffected. In conclusion, the neuroprotectant lubeluzole attenuates growth of ischemic damage as well as its density in the periphery of a photochemically induced neocortical infarct in rats.

Extracellular changes of taurine in the peri-infarct zone: effect of lubeluzole.

Lubeluzole is a neuroprotective compound that has been shown to stereoselectively rescue sensorimotor function and reduce infarct size in a photochemical stroke model in rats. Tissue swelling, which occurs in the peri-infarct zone, is accompanied by a compensatory taurine release. Therefore, using a microdialysis technique, we aimed at measuring changes of extracellular concentrations of taurine in the peri-infarct zone and the effects of lubeluzole and its R-isomer. Lubeluzole blocked the increase of taurine in tissue immediately surrounding a photochemically induced thrombotic neocortical infarct. By contrast, the R-isomer was completely inactive. We hypothesize that lubeluzole may reduce osmoregulatory stress in peri-infarct tissue.

Lubeluzole blocks increases in extracellular glutamate and taurine in the peri-infarct zone in rats.

A microdialysis probe was positioned inside the peri-infarct zone of a photochemically induced neocortical infarct in rats. Extracellular glutamate rose within 20 min after the start of infarct induction and continued to increase during the 5 h observation period to 5.5-fold the pre-infarct baseline value of 0.8 +/- 0.4 micromol/l. Glutamine increased only 1.4-fold. Changes in peri-infarct glutamate were preceded by steep rises in taurine (a 3.9-fold increase from the baseline value of 2.8 +/- 0.7 micromol/l), which coincided with spreading depressions during infarct induction. Post-treatment with lubeluzole ((S)-4-(2-benzothiazolylmethylamino)-alpha-[(3,4-difluoro-phenoxy) methyl]-1-piperidineethanol, 1.25 mg/kg i.v.), a new cerebroprotective drug, blocked the peri-infarct increases of glutamate and taurine, whereas the R-enantiomer was ineffective. Since lubeluzole has previously been shown to stereospecifically decrease glutamate-activated nitric oxide (NO) toxicity in vitro, the present in vivo stereospecific effect of lubeluzole may be related to modulation of the cascade of NO toxicity, thus preventing NO toxicity-mediated increases in extracellular glutamate. Blockade of the peri-infarct taurine response suggests that lubeluzole also may have reduced cellular osmotic stress in the peri-infarct zone.

Lubeluzole protects sensorimotor function and reduces infarct size in a photochemical stroke model in rats.

Posttreatment with lubeluzole, the S-isomer of a novel 3,4-difluoro benzothiazole, potently rescued tactile/proprioceptive hindlimb placing reactions contralateral to unilateral thrombotic infarcts in the hindlimb area of the parietal sensorimotor neocortex of rats. Administered at 5 min postinfarct, a single i.v. bolus of lubeluzole was three times as potent as the racemate, whereas the R-isomer was inactive. Neurological protection was near-maximal for treatment delays through 1 hr postinfarct, but declined with longer delays. However, when administered at 6 hr, 1.25 mg/kg i.v. still protected 60% of infarcted rats. An i.v. bolus followed by a 1-hr i.v. infusion produced equieffective neurologic protection at both 6- and 3-hr delays. This optimal lubeluzole regimen, started at 5 min postinfarct, reduced infarct volume by 22 to 24% at 4 hr postinfarct and by 28% at 7 days postinfarct. Again, the R-isomer was inactive. Down-regulation of the glutamate-activated nitric oxide synthase pathway leading to neurotoxicity and neuronal death may constitute a neuroprotective mechanism of action for lubeluzole.

Towards an improved survival of rat brain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer's type.

Neuronal cell survival was investigated in rat brain cortical cultures in the presence of increasing concentrations of human brain extracts or cerebrospinal fluid (CSF) from control and Senile Dementia of Alzheimer's type (SDAT) patients. Using hippocampal brain extracts, converted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was compared to the content of the neuronal marker MAP2 in foetal rat brain neuronal cultures in order to test converted MTT as a quantitative parameter for neuronal cell survival. A significant correlation was found between both parameters. SDAT frontal cortex brain extracts induced a two four-fold increase in neuronal cell survival at 25 to 125 micrograms protein extract, whereas control brain extracts induced at similar protein concentrations a decline in neuronal cell survival. The enhanced survival yielded by SDAT brain extracts was fully abolished in the presence of control brain extract. Control CSF concentration-dependently increased neuronal cell survival in postnatal rat brain neuronal cultures independent of the difference in the protein content of CSF samples and age of the patients. SDAT CSF also concentration-dependently enhanced neuronal cell survival, however, the effect was more pronounced compared to control CSF. These observations are in favour of the hypothesis that there might be a higher neurotrophic activity in SDAT brain tissue.

Neocortical localization of tactile/proprioceptive limb placing reactions in the rat.

The present study was aimed at delineating the neocortical substrate of tactile/proprioceptive limb placing reactions in rats by means of behavioral tests that excluded the participation of facial stimuli in limb function. Using a photochemical technique, we made unilateral focal lesions in the frontal and parietal neocortex. Fore- and/or hindlimb placing deficits resulted from damage to a fronto-parietal region lying between the medial agranular cortex and the primary somatosensory (whisker barrel field) cortex. When the antero-posterior coordinate was varied from 4 mm anterior to 1 mm posterior to bregma, tactile/proprioceptive forelimb dysfunction was more pronounced after damage to the parietal forelimb area, but lesions confined to the frontal lateral agranular cortex also yielded clear-cut forelimb placing deficits. Damage to either area alone allowed for partial recovery of forelimb function. However, following combined, total destruction of both frontal and parietal forelimb areas, forelimb deficits did not recover. This resembled the irreversible hindlimb deficits after near-total destruction of the parietal hindlimb area. Damage to the medial agranular cortex left limb placing intact. Likewise, for as long as the medial edge of lesions to the whisker barrel field did not come closer than 3 mm to the midline, thus remaining outside the parietal hindlimb area, limb placing remained normal. This sharp medial and lateral delineation of the cortical substrate subserving tactile/proprioceptive limb placing coincides with the borders of a thick, dense subfield of large pyramidal neurons in the deeper parts of layer V. Limb placing remained intact when medial agranular cortex lesions damaged only 30% of that subfield, whereas 70% destruction of that layer following more laterally placed lesions in the parietal hindlimb area produced irreversible hindlimb dysfunction. The severity of hindlimb placing deficits was related to the amount of incursion by whisker barrel field lesions into the subfield of deep layer V large pyramidal neurons. Finally, very large lesions of the occipital cortex did not affect tactile/proprioceptive limb placing. We discuss the neocortical areal and laminar specificity of tactile/proprioceptive limb function in the context of recent neuroanatomical and electrophysiological findings, and their relevance to normal cortical function, recovery from neocortical stroke (including diaschisis), and age-related cortical dysfunction.

Ionic channels, cholinergic mechanisms, and recovery of sensorimotor function after neocortical infarcts in rats.

Unilateral photochemical infarcts were produced in the hind limb sensorimotor neocortex of 243 rats by intravenous injection of the fluorescein derivative Rose Bengal and focal illumination of the intact skull surface. Facial contact stimuli governed the degree and recovery rate of contralateral tactile/proprioceptive forelimb placing reactions. Contralateral forelimb placing recovered, whereas hind limb placing was resistant to recovery. Infarcted rats displayed marked recovery of spontaneous limb usage (beam traversing). However, deficits in isolated tactile/proprioceptive hind limb placing reactions endured. Posttreatment with the class IV calcium antagonist flunarizine after neocortical infarction protected sensorimotor function in a dose-dependent manner. This protective effect may be due to the peculiar ionic channel blocking profile of flunarizine. Scopolamine hydrobromide reinstated contralateral placing errors in infarcted rats at a dosage that did not affect neurologically intact rats. The cognitive enhancer sabeluzole, a novel benzothiazol derivative, dose-dependently blocked the anticholinergic-induced deterioration of a sensorimotor deficit in rats.

Animal models of cerebral stroke: pharmacological protection of function.

We used a photochemical technique which induces thrombotic infarction by intravenous injection of the fluorescein derivative Rose Bengal and focal illumination of the intact skull surface. Following such photochemically induced infarcts in the sensorimotor neocortex of rats, posttreatment with flunarizine, a class IV calcium antagonist, within a critical period of the first 6 h after infarction, results in marked sparing of sensorimotor function (tactile/proprioceptive limb placing reactions), while animals remain normoglycemic and are free of drug-induced behavioral toxicity. This could reflect flunarizine-induced coping of neuronal tissue with ischemia-related ionic shifts. It is argued that photochemical thrombosis and middle cerebral artery occlusion can fruitfully complement each other as experimental stroke models.

Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats.

We produced unilateral photochemical infarcts in the hindlimb sensorimotor neocortex of 186 rats by intravenous injection of the fluorescein derivative rose bengal and focal illumination of the intact skull surface. Infarcted rats showed specific, long-lasting deficits in tactile and proprioceptive placing reactions of the contralateral limbs, mostly the hindlimb. Placing deficits were most prominent during transition to immobility and/or when independent limb movements were required. Administration of flunarizine, a Class IV calcium antagonist, 30 minutes after infarction resulted in marked sparing of sensorimotor function in 30 rats. In contrast to 20 vehicle-treated rats, which remained deficient for at least 21 days, 15 (75%) of the rats treated with 1.25 mg/kg i.v. flunarizine showed normal placing on Day 1 after infarction, whereas the remaining five (25%) recovered within 5 days. Oral treatment of 10 rats with 40 mg/kg flunarizine was also effective. Neocortical infarct volume and thalamic gliosis, assessed 21 days after infarction, did not differ between 30 flunarizine- and 30 vehicle-treated rats. However, when 4-hour-old infarcts were measured in 16 rats, posttreatment with intravenous flunarizine reduced infarct size by 31%. In combination with appropriate behavioral analyses, photochemical thrombosis may constitute a relevant stroke model, in which flunarizine preserved behavioral function during a critical period, corresponding to the spread of ischemic damage.

Relationships between quantitative EEG measures and pharmacodynamics of alfentanil in dogs.

Intravenous alfentanil was administered as a constant 1 h infusion to 6 dogs. Before, during and up to 3 h after infusion, the effects of 3 doses (0.001, 0.004 and 0.016 mg/kg/min) on 6 quantitative EEG measures (zero-crossing frequency, root mean square (rms) amplitude, spectral edge, relative delta, alpha and beta power) were assessed in relation to plasma concentrations of alfentanil. All EEG measures, except zero-crossing frequency and rms amplitude, showed statistically significant dose-dependent changes in peak effect and duration. In addition, times-to-peak effect and return-to-baseline were sensitive to dose. The EEG effects of the low dose were smaller than those of the middle and high doses, whose peak effects did not statistically differ; but the high dose produced more persistent effects, which outlasted the infusion period for a longer time. Alfentanil-induced changes in rms amplitude and relative delta power showed the widest dynamic ranges. Measurable EEG changes occurred at low plasma concentrations, but EEG responses saturated at the middle dose. Significant correlations between plasma concentration and EEG effect were obtained for only the subperiod ranging from onset of infusion to peak EEG effect, indicating very short concentration-effect equilibration delays. On the other hand, clockwise concentration-effect loops were suggestive of acute tolerance: EEG responses peaked before peak plasma levels and they returned to baseline at dose-dependently higher plasma concentrations of alfentanil.

The acute antihypertensive effect of ketanserin increases with age.

The relationship between the acute blood pressure lowering effect of ketanserin with age was investigated in 57 patients ranging from 25 to 90 years (mean 61 years). There was a highly significant correlation between the degree of reduction of systolic and diastolic blood pressure and age, independent of the starting blood pressure. The fact that the acute blood pressure-lowering effect of ketanserin increases with age may suggest a role for serotonin in blood pressure regulation, particularly in elderly patients.

Morphine catalepsy as an adaptive reflex state in rats.

These experiments demonstrate that morphine-induced catalepsy consists of two complementary, but opposite, behavioral extremes (rigid immobility and sudden locomotor bursts), each of which can be controlled by distinct classes of external stimuli. When stimuli that involve pain and/or nonnociceptive skin pressure are tonic (continuous), morphine-induced electroencephalographic (EEG) deactivation and behavioral immobility are potentiated, even to the extent that a stimulation-bound reversible coma results. In contrast, phasic (discrete) stimulation produces behavioral and/or EEG activation. EEG and behavioral rebound effects are observed following stressful (intense, prolonged) stimuli. On the basis of the observed stimulus controls, sensorimotor characteristics, and EEG reactions, it is suggested that similarities may exist between morphine-induced catalepsy and defensive reactions of immobility and escape in drug-free animals (i.e., the adaptive death- feigning reflex).

Morphine versus haloperidol catalepsy in the rat: an electromyographic analysis of postural support mechanisms.

Electromyographic recordings from antagonistic flexor and extensor muscles in the forelegs (biceps and triceps) and hind legs (tibialis and gastrocnemius-soleus) of freely moving rats demonstrated that haloperidol (5 and 10 mg/kg, i.p.) and morphine (20 and 40 mg/kg, i.p.) produced contrasting patterns of rigidity. Haloperidol catalepsy was characterized by increases in frequency, intensity, and duration of simultaneous tonic cocontractions in antagonistic flexor and extensor muscles of the limbs. Such synergistic rigidity suggested the release of an adaptive mechanism involved in static support and the maintenance of stable static equilibrium (the positive supporting reaction of Schoen and Magnus), at the expense of locomotor mechanisms. In contrast, morphine produced antagonistic/reciprocal rigidity, which was insensitive to challenges to static equilibrium, and was compatible with locomotion. Contrary to the haloperidol-induced limb postures, which were enhanced supporting reactions, those induced by morphine were "frozen" phases of the step cycle. Haloperidol-induced synergistic rigidity and morphine-induced antagonistic/reciprocal rigidity are discussed as manifestations of contrasting movement subsystems underlying these functionally opposite immobility states. In addition, we present hypotheses concerning supraspinal and spinal mechanisms underlying cataleptic rigidity states, and their relevance as models of parkinsonian rigidity and akinesia.

Estrogen and haloperidol-induced versus handling-related catalepsy in male rats.

A single injection of 17 beta-estradiol valerate produces, 6-7 days later, potentiation of neuroleptic catalepsy. Multiple behavioral measures demonstrate that this effect occurs with an acute dose of haloperidol of 0.25 mg/kg IP. An even lower dose of haloperidol (0.10 mg/kg), which fails to make control rats cataleptic, produces catalepsy in estrogen-treated animals. Thus, estrogen lowers the threshold of haloperidol-induced catalepsy. Repeated testing alone induces cataleptic reactions in control rats. Estrogen suppresses such handling-related catalepsy in animals that subsequently show potentiation of catalepsy at a dose of haloperidol (0.10 mg/kg), which has virtually no effect on control rats. Thus, in these behavioral paradigms, estrogen by itself does not produce cataleptic effects, and estrogen-induced potentiation of haloperidol catalepsy is not merely additive to an antecedent, neuroleptic-like effect of this hormone. We interpret our results in terms of (1) the relationship of cataleptic reactions in normal rats to drug-induced cataleptic states; (2) the possible relevance of our behavioral results to basal ganglia disorders; and (3) the relationship of neuroleptic catalepsy to striatal DA receptors and their modulation by estrogen.

Effects of Estrogen on Striatal Dopamine receptor function in male and female rats.

The present study compares, biochemically and behaviorally, the effect of estrogen on central dopamine (DA) function in male and female rats. Estrogen has no direct effect in vitro on DA receptors from striatal tissue of male or female rats. In vivo administration of 17 beta-estradiol valerate to male or long-term ovariectomized female rats significantly increased the density of the striatal Da receptors by about 20 percent. Behaviorally, normal female rats have more intense stereotypy produced by apomorphine (APO stereotypy), regardless of the phase of their estrous cycle, than normal male rats, while the density of striatal DA receptors is equal. Estrogen administration to male rats increases their APO stereotypy. Normal intact female rats have no changes in APO stereotypy after the administration of estrogen. However, ovariectomy of female rats increases APO stereotypy, and estrogen administration decreases APO stereotypy back to the levels observed in the normal intact female rats. In the male rat there is a good correlation between the increased striatal Da receptor density and the increased APO stereotypy, but in the female rat factors other than striatal DA receptor density density appear to be important in the regulation of APO stereotypy.

Galloping induced by pontine tegmentum damage in rats: a form of "Parkinsonian festination" not blocked by haloperidol.

Localized lesions or local applications of gamma-aminobutyric acid (GABA) in the nucleus reticularis tegmenti pontis (NRTP) of rats cause rapidly accelerating forward locomotion. Such "festination" can coexist with blockade of the dopamine system. We suggest that (i) the akinesia produced by dopamine deficiency results at least in part from release of excessive inhibition of locomotion by a neural system whose final common inhibitory path includes the region of the NRTP and (ii) when it occurs in addition to nigrostriatal damage, destruction in the region of the NRTP might be the cause of a form of festination seen in some patients suffering from Parkinsonism.

Morphine versus haloperidol catalepsy in the rat: a behavioral analysis of postural support mechanisms.

Our experiments demonstrate that morphine and haloperidol produce two distinct and contrasting behavioral states, which can be thought of as exaggerated, isolated, and simplified forms of organized adaptive behavioral states functioning as components of normal motivated behavior. Haloperidol catalepsy constitutes an organized state in which tonic reactions subserving the maintenance of stable static equilibrium prevail, at the expense of phasic locomotor reactions. In contrast, morphine produces an immobility state characterized by inhibition of the postural support subsystem, and compatible with or preparatory to locomotor rather than static postural reactions. haloperidol-treated rats (1, 2.5, 5, 10 mg/kg i.p.) display exaggeraged bracing reactions to passive displacement as well as to stimuli which do not actively challenge stable equilibrium. In contrast, rats treated with morphine sulfate (10, 20, 40, 80 mg/kg i.p.) show a dose-dependent suppression of bracing and an exaggerated tendency to run in response to stimuli which produce bracing in haloperidol-treated rats. Further evidence that haloperidol-treated rats are organized to stand still in stable equilibrium includes their typical posture during akinesia (i.e. broad-based support), bradykinesia, tonic grasping and enhanced postural components of contact- and air-righting. Under morphine, however, the postural support subsystem is dispensed with, as evidenced by the posture of akinesia (i.e. a frozen phase of the locomotor step cycle associated with loss of limb support), absence of tonic grasping, and nature of the deficits in contact- and air-righting. Furthermore, the opiate-induced immobility state is accompanied by an increased readiness to locomote. Morphine produces an alternation between two extreme behavioral states: complete immobility (inhibition of the postural support subsystem) versus locomotor paroxysms (varying degrees of 'explosive motor behavior'). We suggest that the postures or actions adopted by morphine-treated rats involve movement subsystems concerned with the adaptive behavioral state known as the 'immobility reflex' ('tonic immobility', 'animal hypnosis').