Cerebellar α6GABAA Receptors as a Therapeutic Target for Essential Tremor: Proof-of-Concept Study with Ethanol and Pyrazoloquinolinones.

Ethanol has been shown to suppress essential tremor (ET) in patients at low-to-moderate doses, but its mechanism(s) of action remain unknown. One of the ET hypotheses attributes the ET tremorgenesis to the over-activated firing of inferior olivary neurons, causing synchronic rhythmic firings of cerebellar Purkinje cells. Purkinje cells, however, also receive excitatory inputs from granule cells where the α6 subunit-containing GABAA receptors (α6GABAARs) are abundantly expressed. Since ethanol is a positive allosteric modulator (PAM) of α6GABAARs, such action may mediate its anti-tremor effect. Employing the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of ethanol and α6GABAAR-selective pyrazoloquinolinone PAMs. The burrowing activity, an indicator of well-being in rodents, was measured concurrently. Ethanol significantly and dose-dependently attenuated action tremor at non-sedative doses (0.4-2.4 g/kg, i.p.). Propranolol and α6GABAAR-selective pyrazoloquinolinones also significantly suppressed tremor activity. Neither ethanol nor propranolol, but only pyrazoloquinolinones, restored burrowing activity in harmaline-treated mice. Importantly, intra-cerebellar micro-injection of furosemide (an α6GABAAR antagonist) had a trend of blocking the effect of pyrazoloquinolinone Compound 6 or ethanol on harmaline-induced tremor. In addition, the anti-tremor effects of Compound 6 and ethanol were synergistic. These results suggest that low doses of ethanol and α6GABAAR-selective PAMs can attenuate action tremor, at least partially by modulating cerebellar α6GABAARs. Thus, α6GABAARs are potential therapeutic targets for ET, and α6GABAAR-selective PAMs may be a potential mono- or add-on therapy.

Alcohol and Ganaxolone Suppress Tremor via Extra-Synaptic GABAA Receptors in the Harmaline Model of Essential Tremor.

Background: A long-standing question is why essential tremor often responds to non-intoxicating amounts of alcohol. Blood flow imaging and high-density electroencephalography have indicated that alcohol acts on tremor within the cerebellum. As extra-synaptic δ-subunit-containing GABAA receptors are sensitive to low alcohol levels, we wondered whether these receptors mediate alcohol's anti-tremor effect and, moreover, whether the δ-associated GABAA receptor α6 subunit, found abundantly in the cerebellum, is required. Methods: We tested the hypotheses that low-dose alcohol will suppress harmaline-induced tremor in wild-type mice, but not in littermates lacking GABAA receptor δ subunits, nor in littermates lacking α6 subunits. As the neurosteroid ganaxolone also activates extra-synaptic GABAA receptors, we similarly assessed this compound. The harmaline mouse model of essential tremor was utilized to generate tremor, measured as a percentage of motion power in the tremor bandwidth (9-16 Hz) divided by background motion power at 0.25-32 Hz. Results: Ethanol, 0.500 and 0.575 g/kg, and ganaxolone, 7 and 10 mg/kg, doses that do not impair performance in a sensitive psychomotor task, reduced harmaline tremor compared to vehicle-treated controls in wild-type mice but failed to suppress tremor in littermates lacking the δ or the α6 GABAA receptor subunit. Discussion: As cerebellar granule cells are the predominant brain site intensely expressing GABAA receptors containing both α6 and δ subunits, these findings suggest that this is where alcohol acts to suppress tremor. It is anticipated that medications designed specifically to target α6ßδ-containing GABAA receptors may be effective and well-tolerated for treating essential tremor. Highlights: How does alcohol temporarily ameliorate essential tremor? This study with a mouse model found that two specific kinds of GABA receptor subunits were needed for alcohol to work. As receptors with both these subunits are found mainly in cerebellum, this work suggests this is where alcohol acts to suppress tremor.

GABA-A Alpha 2/3 but Not Alpha 1 Receptor Subunit Ligand Inhibits Harmaline and Pimozide-Induced Tremor in Rats.

Treatment of tremors, such as in essential tremor (ET) and Parkinson's disease (PD) is mostly ineffective. Exact tremor pathomechanisms are unknown and relevant animal models are missing. GABA-A receptor is a target for tremorolytic medications, but current non-selective drugs produce side effects and have safety liabilities. The aim of this study was a search for GABA-A subunit-specific tremorolytics using different tremor-generating mechanisms. Two selective positive allosteric modulators (PAMs) were tested. Zolpidem, targeting GABA-A α1, was not effective in models of harmaline-induced ET, pimozide- or tetrabenazine-induced tremulous jaw movements (TJMs), while the novel GABA-A α2/3 selective MP-III-024 significantly reduced both the harmaline-induced ET tremor and pimozide-induced TJMs. While zolpidem decreased the locomotor activity of the rats, MP-III-024 produced small increases. These results provide important new clues into tremor suppression mechanisms initiated by the enhancement of GABA-driven inhibition in pathways controlled by α2/3 but not α1 containing GABA-A receptors. Tremor suppression by MP-III-024 provides a compelling reason to consider selective PAMs targeting α2/3-containing GABA-A receptors as novel therapeutic drug targets for ET and PD-associated tremor. The possibility of the improved tolerability and safety of this mechanism over non-selective GABA potentiation provides an additional rationale to further pursue the selective α2/3 hypothesis.

Quantitative measurement of postural sway in mouse models of human neurodegenerative disease.

Detection of motor dysfunction in genetic mouse models of neurodegenerative disease requires reproducible, standardized and sensitive behavioral assays. We have utilized a center of pressure (CoP) assay in mice to quantify postural sway produced by genetic mutations that affect motor control centers of the brain. As a positive control for postural instability, wild type mice were injected with harmaline, a tremorigenic agent, and the average areas of the 95% confidence ellipse, which measures 95% of the CoP trajectory values recorded in a single trial, were measured. Ellipse area significantly increased in mice treated with increasing doses of harmaline and returned to control values after recovery. We also examined postural sway in mice expressing mutations that mimic frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) (T-279, P301L or P301L-nitric oxide synthase 2 (NOS2)(-/-) mice) and that demonstrate motor symptoms. These mice were then compared with a mouse model of Alzheimer's disease (APPSwDI mice) that demonstrates cognitive, but not motor deficits. T-279 and P301L-NOS2(-/-) mice demonstrated a significant increase in CoP ellipse area compared with appropriate wild type control mice or to mice expressing the P301L mutation alone. In contrast, postural instability was significantly reduced in APPSwDI mice that have cognitive deficits but do not have associated motor deficits. The CoP assay provides a simple, sensitive and quantitative tool to detect motor deficits resulting from postural abnormalities in mice and may be useful in understanding the underlying mechanisms of disease.

Effects of iboga alkaloids on morphine and cocaine self-administration in rats: relationship to tremorigenic effects and to effects on dopamine release in nucleus accumbens and striatum.

Ibogaine, a naturally occurring alkaloid, has been claimed to be effective in treating addiction to opioid and stimulant drugs and has been reported to decrease morphine and cocaine self-administration in rats. The present study sought to determine if other iboga alkaloids, as well as the chemically related harmala alkaloid harmaline, would also reduce the intravenous self-administration of morphine and cocaine in rats. Because both ibogaine and harmaline induce tremors, an effect that may be causally related to neurotoxicity in the cerebellar vermis, the temorigenic activities of the other iboga alkaloids were assessed. Lastly, in view of the involvement of the dopaminergic mesolimbic system in the actions of drugs of abuse, the effects of some of the iboga alkaloids on extracellular levels of dopamine and its metabolites in the nucleus accumbens and striatum were determined. All of the tested alkaloids (i.e., ibogaine, tabernanthine, R- and S-coronaridine, R- and S-ibogamine, desethylcoronaridine, and harmaline) dose-dependently (2.5-80 mg/kg) decreased morphine and cocaine intake in the hour after treatment; decreases in morphine and cocaine intake intake were also apparent the day after administration of some but not all of these alkaloids (i.e., ibogaine, tabernanthine, desethylcoronaridine, and the R-isomers of coronaridine and ibogamine). In some rats, there were persistent decreases in morphine or cocaine intake for several days after a single injection or after two or three weekly injections of one or another of these alkaloids; R-ibogamine produced such effects more consistently than any of the other alkaloids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of riluzole on harmaline induced tremor and ataxia in rats: biochemical, histological and behavioral studies.

Essential tremor (ET) is one of the most common and most disabling movement disorders among adults. The drug treatment of essential tremor remains unsatisfactory. Additional therapies are required for patients with inadequate response or intolerable side effects. Thus, we aimed to investigate the therapeutic effects of riluzole on harmaline-induced tremor and ataxia in rat, and determining whether riluzole exerts its effect through modulation of glutamate levels in cerebellum. The study included 5 groups of Wistar rats weighing 80-100g, injected with harmaline (50mg/kg i.p.) for inducing experimental tremors and ataxia. The rats in group 1 served as control (saline induced) and group 2 received harmaline alone, whereas the animals in groups 3, 4 and 5, were also given riluzole intraperitoneally at doses of 2, 4 and 8 mg/kg 10 min after harmaline administration, respectively. The intensity and duration of tremor were recorded. Rotarod test, distance traveled and number of crossings were used to evaluate motor performance. Results of this study demonstrated that riluzole dose dependently attenuated duration and intensity of harmaline-induced tremors. Also, riluzole significantly improves time to fall, distance traveled and number of crossings in combined riluzole and harmaline treated rats. Histological analysis indicated that harmaline could cause vermis Purkinje cell (PC) loss and riluzole prevented this toxic effect. Harmaline also could increase glutamate levels in vermis and treatment with riluzole restored glutamate levels. In conclusion, riluzole has relatively protective effects on harmaline-induced tremor, probably related to its inhibitory effect on glutamatergic neurotransmission.

Expression of connexin57 in mouse development and in harmaline-tremor model.

Connexin57 (Cx57) was previously reported in retinal cells but not in brain nerve cells. This occurrence was tested in this study, by searching for the expression of Cx57 RNA and protein transcripts during the postnatal development of the mouse CNS. Both the Cx57 RNA (investigated by reverse transcriptase-polymerase chain reaction (RT-PCR)) and the protein (Western-Blot and immunohistochemistry using a polyclonal antibody generated in chicken) transcripts were firstly expressed in the late postnatal development (P12). The expression of Cx57 in adult life (studied at P28, by in situ hybridization and immunohistochemical analysis) concerned few regions of the brain stem (inferior olive, lateral reticular nucleus and motor trigeminal nucleus), the cerebellum (Purkinje cells and cerebellar nuclei) and the spinal cord (alpha-motoneurons). Double immunohistochemical studies using the Cx57 antibody and antibodies, which specifically labelled neuronal nuclei (NeuN) and astrocyte cells glial fibrillary acidic protein (GFAP), showed the expression of Cx57 segregated in neuronal cells. The study also confirmed the expression of Cx57 in the horizontal cells of the retinal outer plexiform layer, reported in previous investigations. Given the expression of Cx57 in the cerebellum and pre-cerebellar nuclei, such as olivary and lateral reticular nuclei, a possible role of Cx57 was hypothesized in the electrical coupling of the cerebellum. This hypothesis was tested by searching for the expression of the Cx57 transcripts in the mouse cerebellum of the harmaline-tremor model. The up-regulation of the Cx57 transcripts reported in this model suggested a possible involvement of Cx57 in the electrotonic coupling of the cerebellar system.

Harmaline-induced tremor as a potential preclinical screening method for essential tremor medications.

No preclinical method to evaluate potential new medications for essential tremor (ET) is available currently. Although harmaline tremor is a well known animal model of ET, it has not found utility as a preclinical drug screen and has not been validated with anti-ET medications. We measured harmaline tremor in rats (10 mg/kg s.c.) and mice (20 mg/kg s.c.) with a load sensor under the cage floor and performed spectral analysis on 20-minute epochs. The motion power over the tremor frequency bandwidth (8-12 Hz in rats; 10-16 Hz in mice) was divided by the motion power over the full motion frequency range (0-15 Hz in rats; 0-34 Hz in mice). The use of these measures greatly reduced data variability, permitting experiments with small sample sizes. Three drugs that suppress ET (propranolol, ethanol, and octanol) all significantly suppressed harmaline-induced tremor. We propose that, with this methodology, harmaline-induced tremor may be useful as a preclinical method to identify potential medications for ET.

Further studies on quantification of drug-induced tremor in mice: effects of antitremorgenic agents on tremor frequency.

The effects of diazepam, dantrolene and atropine on drug-induced tremors were investigated in mice from the point of view of the tremor frequency. The study involved a power spectral analysis of the random current induced by movement of a magnet (attached to the mouse) on a wire coil. To induce tremor, tremorine and harmaline were subcutaneously injected. The power spectral density function defined the frequency composition of the tremor and its severity was determined quantitatively in terms of the mean square value of the data in any frequency range of concern. Diazepam markedly depressed the power spectral density of the tremorine- and harmaline-induced tremor and reduced the tremor frequency. With higher doses of diazepam, the peak frequency of the tremorine-induced tremor shifted to the lower frequency side as if the tremor components were taken into the component of the spontaneous motor activity. Dantrolene and atropine suppressed the power spectral density without affecting tremor frequency. The relationship between the change of tremor frequency and the site of action of antitremorgenic agents are discussed.

[Harmaline-induced tremor and cerebral activities: labeling with 14C-2-deoxyglucose in the rat]. / Tremblement harmalinique et activités cérébrales: marquage au 14C-2-désoxyglucose chez le rat.

The brain structures specifically involved in harmaline-induced tremor have been identified in the Rat by using the 14C-2-deoxyglucose marking Method. The results obtained in an animal treated with tremogenic doses of harmaline, but immobilized with Faxedil, have been compared with those of another animal, not treated, but submitted to the same experimental procedure. The most specifically marked structure was the inferior olive. Secondarily marked structures were the posterior part of the lateral reticular formation, the caudato-putamen area and the prefrontal and frontal cortex.