Guy Ebinger

Histological, behavioural and neurochemical evaluation of medial forebrain bundle and striatal 6-OHDA lesions as rat models of Parkinson's disease.

We compared the effect of an injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) and into the striatum on different parameters for evaluation of motor dysfunction and dopamine denervation in rats, as a function of time. A combination of behavioural, neurochemical and histological techniques was employed. Amphetamine-induced rotation is shown to provide a first rough estimation of motor impairment. Indeed, the number of rotations observed after amphetamine administration can distinguish between a partial and a near complete (>90%) denervation in the substantia nigra. However, lesion sizes of 50-80% resulted in similar rotational behaviour. Similarly, the elevated body swing test (EBST) can determine severe lesions, but is not sensitive enough in the partial model. In both models, determination of the dopamine tissue content with HPLC is a more precise measure of striatal dopamine innervation than striatal TH-immunostaining. The number of cells estimated by TH- and Nissl-staining correlated well in the striatal model, but there was a discrepancy between both measures in the MFB-lesioned animals. Therefore, additional Nissl-staining is necessary for better estimation of the size of the lesion at the level of the substantia nigra or ventral tegmental area in the severely lesioned animals. The MFB lesion model mimics end-stage Parkinsons disease. The striatal injection of 6-OHDA described here cannot be considered a progressive model, since there was no change in the number of TH-immunoreactive cells in the substantia nigra up to 8 weeks post-lesioning. However, the partial denervation renders its quite suitable for mimicking early stage Parkinsons disease, and is thus suitable for testing possible neuroprotective and neurotrophic drugs.

Antagonists of GLUK5-containing kainate receptors prevent pilocarpine-induced limbic seizures

Developments in the molecular biology and pharmacology of GLUK5, a subtype of the kainate class of ionotropic glutamate receptors, have enabled insights into the roles of this subunit in synaptic transmission and plasticity. However, little is known about the possible functions of GLUK5-containing kainate receptors in pathological conditions. We report here that, in hippocampal slices, selective antagonists of GLUK5-containing kainate receptors prevented development of epileptiform activity—evoked by the muscarinic agonist, pilocarpine—and inhibited the activity when it was pre-established. In conscious rats, these GLUK5 antagonists prevented and interrupted limbic seizures induced by intra-hippocampal pilocarpine perfusion, and attenuated accompanying rises in extracellular L-glutamate and GABA. This anticonvulsant activity occurred without overt side effects. GLUK5 antagonism also prevented epileptiform activity induced by electrical stimulation, both in vitro and in vivo. Therefore, we propose that subtype-selective GLUK5 kainate receptor antagonists offer a potential new therapy for epilepsy.

Effects of tryptophan and/or acute running on extracellular 5-HT and 5-HIAA levels in the hippocampus of food-deprived rats

The present microdialysis study has examined whether exercise-elicited increases in brain tryptophan availability (and in turn 5-HT synthesis) alter 5-HT release in the hippocampus of food-deprived rats. To this end, we compared the respective effects of acute exercise, administration of tryptophan, and the combination of both treatments, upon extracellular 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels. All rats were trained to run on a treadmill before implantation of the microdialysis probe and 24 h of food deprivation. Acute exercise (12 m/min for 1 h) increased in a time-dependent manner extracellular 5-HT levels (maximal increase: 47%), these levels returning to their baseline levels within the first hour of the recovery period. Besides, exercise-induced increases in extracellular 5-HIAA levels did not reach significance. Acute administration of a tryptophan dose (50 mg/kg i.p.) that increased extracellular 5-HIAA (but not 5-HT) levels in fed rats, increased within 60 min extracellular 5-HT levels (maximal increase: 55%) in food-deprived rats. Whereas 5-HT levels returned toward their baseline levels within the 160 min that followed tryptophan administration, extracellular 5-HIAA levels rose throughout the experiment (maximal increase: 75%). Lastly, treatment with tryptophan (60 min beforehand) before acute exercise led to marked increases in extracellular 5-HT and 5-HIAA levels (maximal increases: 100% and 83%, respectively) throughout the 240 min that followed tryptophan administration. This study indicates that exercise stimulates 5-HT release in the hippocampus of fasted rats, and that a pretreatment with tryptophan (at a dose increasing extracellular 5-HT levels) amplifies exercise-induced 5-HT release.

NMDA receptor-mediated pilocarpine-induced seizures: characterization in freely moving rats by microdialysis.

Pilocarpine administration has been used as an animal model for temporal lobe epilepsy since it produces several morphological and synaptic features in common with human complex partial seizures. Little is known about changes in extracellular neurotransmitter concentrations during the seizures provoked by pilocarpine, a non‐selective muscarinic agonist. Focally evoked pilocarpine‐induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10mm for 40min at a flow rate of 2μl min−1) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, γ‐aminobutyric acid (GABA) and dopamine levels were monitored and simultaneous electrocorticography was performed. The animal model was characterized by intrahippocampal perfusion with the muscarinic receptor antagonist atropine (20mm), the sodium channel blocker tetrodotoxin (1μm) and the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801 (dizocilpine maleate, 100μm). The effectiveness of locally (600μm) or systemically (10mg kg−1 day−1) applied lamotrigine against the pilocarpine‐induced convulsions was evaluated. Pilocarpine initially decreased extracellular hippocampal glutamate and GABA levels. During the subsequent pilocarpine‐induced limbic convulsions extracellular glutamate, GABA and dopamine concentrations in hippocampus were significantly increased. Atropine blocked all changes in extracellular transmitter levels during and after co‐administration of pilocarpine. All pilocarpine‐induced increases were completely prevented by simultaneous tetrodotoxin perfusion. Intrahippocampal administration of MK‐801 and lamotrigine resulted in an elevation of hippocampal dopamine levels and protected the rats from the pilocarpine‐induced seizures. Pilocarpine‐induced convulsions developed in the rats which received lamotrigine perorally. Pilocarpine‐induced seizures are initiated via muscarinic receptors and further mediated via NMDA receptors. Sustained increases in extracellular glutamate levels after pilocarpine perfusion are related to the limbic seizures. These are arguments in favour of earlier described NMDA receptor‐mediated excitotoxicity. Hippocampal dopamine release may be functionally important in epileptogenesis and may participate in the anticonvulsant effects of MK‐801 and lamotrigine. The pilocarpine‐stimulated hippocampal GABA, glutamate and dopamine levels reflect neuronal vesicular release.

Tonic GABA-ergic modulation of striatal dopamine release studied by in vivo microdialysis in the freely moving rat.

GABAA and GABAB receptor agonists and antagonists were administered locally in the striatum of intact and kainic acid lesioned rats. (+/-)-Baclofen, a GABAB receptor agonist, significantly decreased the level of extracellular dopamine in the striatum of intact rats. (+/-)-Phaclofen, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats and to a lesser extent in the striatum after kainic acid lesion. Pregnanolone (5 beta-pregnan-3 alpha-ol-20-one), a positive allosteric modulator of the GABAA receptor, significantly decreased the level of extracellular dopamine in intact rats. (-)-Bicuculline, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats, but failed to increase the level of extracellular dopamine after kainic acid lesion. The release of extracellular dopamine, due to infusion of phaclofen or bicuculline, was totally suppressed by tetrodotoxin. These results support a direct influence of GABA on the dopaminergic terminals via presynaptic GABAB receptors, while the effects via the GABAA receptor seem to be postsynaptic and mediated by striatal interneurons or the striatonigral feedback loop.

Autoradiographic localization of D1 and D2 dopamine receptors in the human brain

The distribution of dopamine D1 and D2 receptors in several human brain regions was investigated using autoradiography with the radioligands [3H]SCH 23390 and [3H]spiroperidol. The highest densities of both dopamine receptor types are seen in the nucleus caudatus, putamen and nucleus accumbens. Whereas the density of the D2 receptors is similar in the two segments of the globus pallidus, the pars medialis of the globus pallidus contains a three-fold higher concentration of D1 receptors than the pars lateralis. D1 and D2 receptors are present in the amygdala and substantia nigra. Both receptor types are absent in the cerebellum. The thalamus contains low densities of D1 receptors but no D2 receptors. Only D2 receptors are seen in the anterior lobe of the pituitary gland. The whole cerebral cortex is rich in D1 receptors, while D2 receptors, in low concentrations, are confined to the entorhinal area and cingulate cortex.

Anticonvulsant action of hippocampal dopamine and serotonin is independently mediated by D2 and 5-HT1A receptors

The present microdialysis study evaluated the anticonvulsant activity of extracellular hippocampal dopamine (DA) and serotonin (5‐HT) with concomitant assessment of the possible mutual interactions between these monoamines. The anticonvulsant effects of intrahippocampally applied DA and 5‐HT concentrations were evaluated against pilocarpine‐induced seizures in conscious rats. DA or 5‐HT perfusions protected the rats from limbic seizures as long as extracellular DA or 5‐HT concentrations ranged, respectively, between 70–400% and 80–350% increases compared with the baseline levels. Co‐perfusion with the selective D2 blocker remoxipride or the selective 5‐HT1A blocker WAY‐100635 clearly abolished all anticonvulsant effects. These anticonvulsant effects were mediated independently since no mutual 5‐HT and DA interactions were observed as long as extracellular DA and 5‐HT levels remained within these protective ranges. Simultaneous D2 and 5‐HT1A receptor blockade significantly aggravated pilocarpine‐induced seizures. High extracellular DA (> 1000% increases) or 5‐HT (> 900% increases) concentrations also worsened seizure outcome. The latter proconvulsive effects were associated with significant increases in extracellular glutamate (Glu) and mutual increases in extracellular monoamines. Our results suggest that, within a certain concentration range, DA and 5‐HT contribute independently to the prevention of hippocampal epileptogenesis via, respectively, D2 and 5‐HT1A receptor activation.

The analysis of excitatory, inhibitory and other amino acids in rat brain microdialysates using microbore liquid chromatography

Three microbore liquid chromatography (LC) assays for determination of amino acids in rat brain dialysates are described: one for separation of amino acids by gradient elution and electrochemical detection, one for analysis of GABA by isocratic elution and electrochemical detection, and one for fast measurement of glutamate and aspartate by gradient elution and fluorescence detection. The assays are reliable, reproducible and sensitive. In comparison with conventional LC, a 5-fold increase in sensitivity was obtained for GABA. Optimization of the derivatization chemistry and the microbore LC system are discussed, as well as important practical aspects.

Muscarinic Modulation of Striatal Dopamine, Glutamate, and GABA Release, as Measured with In Vivo Microdialysis

Abstract: Intrastriatal microdialysis was used to administer muscarinic drugs in freely moving rats for 40 min at a flow rate of 2 µl/min. Administration of the nonselective agonist pilocarpine at 10 mM increased striatal dopamine release and decreased extracellular GABA and glutamate overflow. Perfusion with the muscarinic M2 antagonist methoctramine at 75 µM increased extracellular dopamine and glutamate concentrations but exerted no changes on extracellular GABA levels. Intrastriatal administration of the M1 antagonist pirenzepine at 0.05 µM decreased extracellular dopamine overflow. Application of pirenzepine (0.05 and 5 µM) exerted no effects on the measured GABA or glutamate levels. There are thus important differences in applied doses of muscarinic drugs needed to obtain modulatory effects. High doses of agonists are probably needed to superimpose on the background of tonic influences of striatal acetylcholine, whereas antagonists can block the receptors in small doses. We further suggest that M1 receptors might tonically facilitate striatal dopamine release, that M2 receptors might tonically inhibit striatal glutamate efflux, and that acetylcholine does not exert tonic effects on striatal GABA release. The link with the pilocarpine animal model for temporal lobe epilepsy will be discussed.

The effect of aging on the D1 dopamine receptors in human frontal cortex.

Dopaminergic pathways to the cerebral cortex may be involved in cognitive function. We examined the effect of aging on the D1 dopamine receptors, and their high-agonist affinity (RH) sites, in postmortem human frontal cortex (n = 32; age range, 19-88 years). With aging, there was a significant decrease in the densities of the D1 dopamine receptors, and their RH sites, in human frontal cortex. The age-related reduction of cortical dopaminergic neurotransmission might contribute to the decline in cognitive abilities of elderly persons.