Gisela Kindel

Methyl-β-carbolinium analogs of MPP+ cause nigrostriatal toxicity after substantia nigra injections in rats

Eleven beta-carbolinium compounds (beta C+s) and MPP+ were stereotaxically injected (40-200 nmol in 5 microliter of vehicle) unilaterally into the substantia nigra of anesthetized adult male Sprague-Dawley rats. The rats were sacrificed after three weeks. The ipsilateral striatum was analyzed for dopamine and DOPAC levels with HPLC. The brainstem injection site was fixed and cut coronally. The largest lesion area in each animal was measured using NIH IMAGE. Three beta C+s produced lesions whose mean areas were nearly as large as that produced by MPP+ (defined as 100%): 2,9-Me2-harman (94%), 2-Me-harmol (74%), and 2,9-Me2-norharman (57%). Three other compounds produced somewhat smaller lesions: 2-Me-harmaline (34%), 6-MeO-2-Me-harman (29%), and 2-Me-harmine (25%). The remaining compounds were ineffective (< or = 12%): norharman, 2-Me-norharman, 2-Me-harman, harmine, and 2-Me-6-MeO-harmalan. A 40 nmol dose of MPP+ reduced ipsilateral striatal dopamine to 0.6% of control. None of the beta C+s approached this, although several did significantly reduce striatal dopamine at doses of either 40 nmol (2,9-Me2-harman (37%), 2,9-Me2-norharman (42%), and 2-Me-harman (63%)) or 200 nmol (2-Me-harmaline (23%), norharman (63%), and 2-Me-norharman (64%)). There was a moderate negative correlation between lesion size and dopamine level (r = -0.65). There were also moderately strong correlation between lesion size and dopamine level (r = -0.65). There were also moderately strong correlations (r = 0.39-0.78) between the beta C+ nigral lesion area or striatal dopamine level potencies and their previously described IC50 values for inhibiting mitochondrial respiration or their toxicity to PC12 cells in culture. Interestingly, our correlation analysis revealed a remarkably strong correlation between beta C+ Ki MAO-A values and their toxicity to PC12 LDH release (r = -0.84) or PC12 protein loss (r = 0.79). Although beta C+s appear to be less specific toxins than MPP+, their levels in human substantia nigra are 8-20-fold higher than in cortex, making their role as relatively selective nigral toxins in Parkinsons disease plausible.

Sex differences and estrous cycle-variations in the AF64A-induced cholinergic deficit in the rat hippocampus

The influence of gender and stage of the estrous cycle on the levels of acetylcholine, serotonin, and noradrenaline in the hippocampus and on the susceptibility of the cholinergic septo-hippocampal pathway to the neurotoxic effect of ethylcholine aziridinium (AF64A) was investigated in the rat. Levels of acetylcholine and serotonin were consistently higher in female rats during the stage of diestrus and proestrus than in age-matched male rats (p < 0.05). Across the estrous cycle the highest levels of acetylcholine and serotonin, coinciding with the lowest levels of noradrenaline, were measured on proestrus. Eight to 10 days after the bilateral intracerebroventricular injection of a submaximal dose of AF64A (1 nmol/ventricle) the decrease of acetylcholine in hippocampus was larger in females than in male rats. The reduction of acetylcholine was most pronounced in female rats that had received submaximal doses of AF64A on proestrus (42.7 +/- 3.4%), whereas in male rats, the corresponding decrease was 25.9 +/- 5.1% (p < 0.05). At a maximal dose of AF64A (2 nmole/ventricle), the sex-specific or cycle-dependent difference in the cholinotoxicity of AF64A vanished. The dose-dependent loss of acetylcholine was associated with a secondary dose-dependent decrease in the levels of serotonin and noradrenaline, but significant differences between male and female rats or stages of estrous cycle were not apparent. The present data provide evidence that adult female rats in general, and particularly females on proestrus, are more susceptible to the neurotoxic action of submaximal doses of AF64A than age-matched male rats.

Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A)

Ethylcholine aziridinium (AF64A) has been proposed as a specific cholinergic neurotoxin. In earlier studies, using AF64A, we reported that slow infusion of 1-2 nmol of this compound into each lateral ventricle of Sprague-Dawley rats resulted in small, and transient decreases in noradrenaline (NA) and serotonin (5-HT) levels in the hippocampus, while inducing a permanent and significant cholinergic hypofunction in the same brain region. The experiments described in this paper were designed to test the hypothesis that such noradrenergic and serotonergic changes after small doses of AF64A are secondary to the changes observed in cholinergic neurons. Levels of NA, and of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were measured concurrently with levels of acetylcholine (ACh), in various brain regions of rats in which the effect of AF64A was attenuated, and in respective control animals. The effect of AF64A was diminished by inhibiting the interaction of AF64A with the high affinity transport site for choline (HAChT). This was achieved using hemicholinium-3 (HC-3), which does not cross the blood-brain barrier, and A-4 (a bis 4-methylpiperidine analog of HC-3), which is centrally active following its peripheral administration. A-4 (20 or 40 mg/kg i.p.) or HC-3 (10 micrograms/ventricle) had no effect on ACh, NA, 5-HT or 5-HIAA levels in saline-treated rats. However, all treatments significantly attenuated the decrease in ACh content produced by AF64A pretreatment. Transient decreases in NA, 5-HT and 5-HIAA contents after AF64A treatment were prevented or reduced by prior treatment with A-4 or HC-3. These results indicate that changes in noradrenergic and serotonergic neurons following AF64A administration are not due to non-specific toxicity of AF64A, but may be the result of adaptation of these neurons to withdrawal of cholinergic input, which would normally inhibit the release of NA and 5-HT. These results also indicate that AF64A can be used to produce specific lesions of hippocampal cholinergic nerve terminals.

Septal choline acetyltransferase immunoreactive neurons: Dose-dependent effects of AF64A

Two experiments were performed. In the first, the cholinotoxin, AF64A (0.5, 1.0 or 1.5 nmol/ventricle), or vehicle (3.0 microliters) was injected (ICV) bilaterally into male rats (n = 23). Choline acetyltransferase (ChAT) immunoreactive (IR) perikarya in the four subgroups of the septal complex were visualized by immunocytochemistry (PAP method) 28 days postinjection, and counted using a microprojector (x40). The 0.5 nmol/ventricle dose of AF64A significantly reduced (31%) the number of ChAT-IR cell bodies in the intermediate subgroup (rostral extension of the nucleus basalis/substantia innominata). Higher doses did not produce additional reductions. The highest dose (1.5 nmol/ventricle) of AF64A resulted in significant decreases in ChAT-IR cell bodies in the dorsal (51%) and midline (35%) subgroups (medial septum), but did not affect the number of ventral subgroup (diagonal band of Broca) ChAT-IR neurons. In the second experiment, electrolytic lesions were placed in the corpus callosum, cingulum and overlying cingulate gyrus, in order to simulate the nonselective damage seen following the 1.5 nmol/ventricle dose of AF64A. In comparison to the surgical controls (n = 3), the electrolytic lesions (n = 6) failed to significantly affect the number of ChAT-IR perikarya in any of the septal subdivisions. Thus the distinct subgroups of septal ChAT-IR neurons are differentially sensitive to the toxic effects of ICV administered AF64A: intermediate much greater than dorsal greater than midline much greater than ventral subgroup.

Clonidine prevents transient loss of noradrenaline in response to cholinergic hypofunction induced by ethylcholine aziridinium (AF64A).

Abstract: Intracerebroventricular injection of ethylcholine aziridinium (AF64A) (2 nmol/ventricle) induced a considerable decrease in the level of acetylcholine (ACh) in hippocampus (from 21.14 ± 0.84 to 10.04 ± 0.59 pmol/mg of tissue; p < 0.001) 4 days after application. The reduction of cholinergic function was accompanied by a decrease in the level of noradrenaline (NA) (from 1.96 ± 0.08 to 1.41 ± 0.06 pmol/mg of tissue; p < 0.001). Two days after administration of AF64A (1 or 2 nmol/ventricle), the dose‐dependent decrease in NA level was associated with an increase in the level of its major metabolite, 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), resulting in a considerable increase in the MHPG/ NA molar ratio (from 0.84 ± 0.06 to 1.62 ± 0.17; p < 0.002). Chronic treatment of AF64A‐injected rats with clonidine (0.02–0.2 mg/kg, i.p., every 8–12 h) had no significant effect on the loss of ACh content, whereas the decrease in NA content in hippocampus was completely prevented. Clonidine induced aggressive behavior in the AF64A‐treated rats, in contrast to sedation in vehicle‐injected rats. The response to clonidine under these experimental conditions and the increased MHPG/NA molar ratio in response to AF64A suggest that the transient loss of NA content following AF64A administration results from increased NA release. The increased noradrenergic activity in hippocampus may be linked to the reduction of tonic inhibitory cholinergic input. These results are discussed in relation to possible implications for senile dementia of the Alzheimer type.

Long-term exposure to high levels of corticosterone aggravates AF64A-induced cholinergic hypofunction in rat hippocampus in vivo

Male Sprague-Dawley rats were bilaterally adrenalectomized and corticosterone (CORT) was substituted as subcutaneous pellets in two groups of animals: low- (L-CORT: 1 x 25 mg pellet) or high-level of CORT (H-CORT: 4 x 100 mg pellet). Between 14 and 19 days after CORT substitution, ethylcholine aziridinium (AF64A) was intracerebroventricularly (i.c.v.) injected in the CORT long-term exposed rats and the dose- and time-dependent effect of this treatment was measured on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in hippocampus and septum and on serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline (NA) levels in hippocampus. Rats were killed at 2, 4, 7 and 14 days after AF64A treatment. Starting 4 days after the i.c.v. administration of 0.5 or 1.0 nmol of AF64A, an aggravation of the reduction of ChAT activity was measured in the hippocampus of the H-CORT animals compared to the L-CORT ones. In the septum of the H-CORT rats, the activity of ChAT increased within the first week after the infusion of the toxin, while no significant effect was observed in the L-CORT group. As we observed with ChAT, AF64A induced a severe inhibition of AChE activity in the hippocampus of the H-CORT rats compared to the L-CORT ones. In the septum, an increase of AChE activity was observed in both groups of CORT-exposed animals. In the hippocampus of H-CORT animals, the exacerbation of the inhibition of ChAT and AChE activity was accompanied by a parallel decrease in the content of 5-HT and 5-HIAA starting 4 days after AF64A injections. Finally, NA content in hippocampus was not affected by the toxin in the CORT-substituted animals. These data demonstrate that: (1) long-term exposure to supraphysiological levels of CORT enhances the cholinodisruption induced in hippocampus by AF64A, at doses of 0.5 and 1.0 nmol/side; (2) high circulating plasma CORT concentrations impair hippocampal cholinergic neuronal capacity to recover from damage; and (3) the degree of inhibition of the serotoninergic system is augmented in H-CORT animals, most probably due to an adaptation of the serotoninergic neurons to the larger withdrawal of cholinergic function observed in this group.

Noradrenaline depletion protects cholinergic neurons in rat hippocampus against AF64A-induced damage

The role of the noradrenergic system in the cholinotoxicity of ethylcholine aziridinium ion (AF64A) was studied in rats. Male Sprague-Dawley rats were treated with the noradrenergic neurotoxin DSP-4 (N-(2-chloroethyl)-n-ethyl-2-bromobenzylamine; 50 mg/kg i.p.) in the presence of the serotonin uptake inhibitor fluoxetine, 14 days prior to bilateral intracerebroventricular injection of AF64A (2 nmol/lateral ventricle). In rats in which noradrenaline (NA) was depleted by 94%, the loss of acetylcholine (ACh) in hippocampus induced by AF64A was significantly attenuated (p less than 0.02). However, when there was only a partial depletion of NA (50% reduction), the AF64A-induced loss of ACh was a pronounced as in rats with intact noradrenergic function. These findings indicate that the noradrenergic lesion has to be complete before a protective effect is apparent. Moreover, they imply that noradrenergic input is involved in AF64A-induced cholinergic damage in the hippocampus.

Natural and Synthetic Huperzine A: Effect on Cholinergic Function in Vitro and in Vivoa

Huperzine A has been shown to be useful in the treatment of symptoms of dementia of the Alzheimer type. Our initial attempts to synthesize (‐)Huperzine A resulted in the racemic mixture of (−)Huperzine A. We have therefore compared the in vitro and in vivo effects of (±)Huperzine A with those of (−)Huperzine A in rats. The results indicate a similar biological mechanism of action between the two, but that the racemic mixture of (±) Huperzine A has a weaker biological activity than the natural product (−)Huperzine A, presumably due to the presence in the mixture of (+)Huperzine A, which is considerably less potent than the (−)isomer.

Cerebrospinal fluid free choline in movement disorders of paediatric onset.

We measured free choline in cerebrospinal fluid (CSF) of 78 patients with movement disorders of paediatric onset and various controls as a putative index of central phospholipid metabolism. Most of the disorders studied were myoclonic disorders, such as progressive myoclonus epilepsy, the opsoclonus-myoclonus syndrome, and essential myoclonus, but other movement disorders, interictal seizure disorders, and different neurological and nonneurological disorders were also included. There were no significant differences in CSF choline concentrations in myoclonic disorders or other movement disorders compared with controls. The CSF choline levels were lowest in children with seizure disorders including progressive myoclonus epilepsy. In progressive myoclonus epilepsy, the CSF choline values resembled other epileptic disorders rather than other myoclonic disorders. When all the data were analysed collectively, no significant relation of CSF choline was found to patient age, gender, aliquot of CSF measured, or the length of time the sample was stored at -70 degrees C. Separate analyses of data from children and adults showed a trend toward a biphasic relation between patient age and CSF choline which could be pursued in developmental studies of normal subjects. Reduced CSF choline may indicate increased choline incorporation into brain phospholipids, disturbances of choline metabolism, decreased choline release, or non-neural factors.

Mivazerol inhibits intrathecal release of glutamate evoked by halothane withdrawal in rats

Background: Mivazerol is a new and selective α2‐adrenergic receptor agonist devoid of hypotensive effects (1, 2). Previous studies have demonstrated that mivazerol prevents hemodynamic instability during emergence from halothane anesthesia in rats (3). The present study was to determine whether glutamate and aspartate are involved in this action of mivazerol, at the second to third thoracic segments (T2–T3) of the spinal cord.