Jason E. Kralic

Genetic essential tremor in γ-aminobutyric acidA receptor α1 subunit knockout mice

Essential tremor is the most common movement disorder and has an unknown etiology. Here we report that γ-aminobutyric acidA (GABAA) receptor α1–/– mice exhibit postural and kinetic tremor and motor incoordination that is characteristic of essential tremor disease. We tested mice with essential-like tremor using current drug therapies that alleviate symptoms in essential tremor patients (primidone, propranolol, and gabapentin) and several candidates hypothesized to reduce tremor, including ethanol; the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801; the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA); the GABAA receptor modulators diazepam, allopregnanolone, and Ro15-4513; and the L-type Ca2+ channel antagonist nitrendipine. Primidone, propranolol, and gabapentin reduced the amplitude (power) of the pathologic tremor. Nonsedative doses of ethanol eliminated tremor in mice. Diazepam, allopregnanolone, Ro15-4513, and nitrendipine had no effect or enhanced tremor, whereas MK-801 and CCPA reduced tremor. To understand the etiology of tremor in these mice, we studied the electrophysiological properties of cerebellar Purkinje cells. Cerebellar Purkinje cells in GABAA receptor α1–/– mice exhibited a profound loss of all responses to synaptic or exogenous GABA, but no differences in abundance, gross morphology, or spontaneous synaptic activity were observed. This genetic animal model elucidates a mechanism of GABAergic dysfunction in the major motor pathway and potential targets for pharmacotherapy of essential tremor.

Chronic ethanol consumption enhances internalization of α1 subunit‐containing GABAA receptors in cerebral cortex

The molecular mechanisms that underlie ethanol dependence involve alterations in the functional properties and subunit expression of GABAA receptors. Chronic ethanol exposure decreases GABAA receptor α1 subunits and increases α4 subunit levels in cerebral cortical membranes. This study explored the effect of chronic ethanol exposure on internalization of GABAA/benzodiazepine receptors. Chronic ethanol exposure increased α1 subunit levels by 46 ± 12% and [3H]flunitrazepam binding by 35 ± 9% in the clathrin‐coated vesicle (CCV) fraction. There was a corresponding 34 ± 8% decrease in α1 peptide expression and 37 ± 6% decrease in [3H]flunitrazepam binding in the synaptic fraction. Chronic ethanol consumption also increased the α1 subunit immunoprecipitate in the cytosolic fraction (77 ± 22%), measured by western blot analysis. Moreover, co‐immunoprecipitation of both clathrin and adaptin‐α with α1 subunits was increased in the cytosolic fraction, suggesting that α1 subunit endocytosis is enhanced by chronic ethanol consumption. In contrast, α4 subunit peptide levels were not altered in the CCV fraction despite a 39 ± 13% increase in peptide levels in the synaptic fraction of cortex. Moreover, acute ethanol exposure did not alter α1 subunit peptide expression or [3H]flunitrazepam binding in the synaptic or CCV fractions. These results suggest that chronic ethanol consumption selectively increases internalization of α1 subunit‐containing GABAA receptors in cerebral cortex.

Differential Dependence of Axo-Dendritic and Axo-Somatic GABAergic Synapses on GABAA Receptors Containing the α1 Subunit in Purkinje Cells

Synapse formation and maintenance require extensive transsynaptic interactions involving multiple signal transduction pathways. In the cerebellum, Purkinje cells (PCs) receive GABAergic, axo-dendritic synapses from stellate cells and axo-somatic synapses from basket cells, both with GABAA receptors containing the α1 subunit. Here, we investigated the effects of a targeted deletion of the α1 subunit gene on GABAergic synaptogenesis in PCs, using electrophysiology and immunoelectron microscopy. Whole-cell patch-clamp recordings in acute slices revealed that PCs from α10/0 mice lack spontaneous and evoked IPSCs, demonstrating that assembly of functional GABAA receptors requires the α1 subunit. Ultrastructurally, stellate cell synapses on PC dendrites were reduced by 75%, whereas basket cell synapses on the soma were not affected, despite the lack of GABAA-mediated synaptic transmission. Most strikingly, GABAergic terminals were retained in the molecular layer of adult α10/0 mice and formed heterologous synapses with PC spines characterized by a well differentiated asymmetric postsynaptic density. These synapses lacked presynaptic glutamatergic markers and postsynaptic AMPA-type glutamate receptors but contained δ2-glutamate receptors. During postnatal development, initial steps of GABAergic synapse formation were qualitatively normal, and heterologous synapses appeared in parallel with maturation of dendritic spines. These results suggest that synapse formation in the cerebellum is governed by neurotransmitter-independent mechanisms. However, in the absence of GABAA-mediated transmission, GABAergic terminals in the molecular layer apparently become responsive to synaptogenic signals from PC spines and form stable heterologous synapses. In contrast, maintenance of axo-somatic GABAergic synapses does not depend on functional GABAA receptors, suggesting differential regulation in distinct subcellular compartments.

NPI-031G (puerarin) reduces anxiogenic effects of alcohol withdrawal or benzodiazepine inverse or 5-HT2C agonists

Because extracts of kudzu have been used as a hangover remedy in China for many centuries, we tested the ability of NPI-031G (puerarin), an isoflavone isolated from kudzu, to counteract anxiogenic effects associated with withdrawal from chronic alcohol exposure. NPI-O31G (50 and 150 mg/kg ip) significantly increased the social interaction and locomotor activity reduced by withdrawal from 17 days of alcohol (7%) diet. The effects of NPI-031G resembled those of the benzodiazepine antagonist, flumazenil (5 mg/kg), and the 5-HT(2C) antagonist, SB 242084 (1 mg/kg). In a separate study, control rats were pretreated with NPI-031G (30 min) and then given the anxiogenic compounds DMCM, a benzodiazepine inverse agonist, or Ro 600175, a 5-HT(2C) agonist. NPI-031G significantly counteracted the reduction in social interaction induced by either compound. To identify a potential mechanism of action of NPI-031G, synaptoneurosomes were isolated from the cerebral cortex of untreated rats and chloride uptake assays were carried out. NPI-031G did not have any effect on the stimulation of chloride uptake by muscimol, a GABA(A) agonist. However, it reduced the potentiation of muscimol-stimulated chloride uptake by flunitrazepam, a benzodiazepine agonist, at a concentration of 100 microM. A reduction in [3H]flunitrazepam binding was also seen at this concentration. These findings are consistent with NPI-031G being a weak benzodiazepine site antagonist.

Chronic blockade of N-methyl-D-aspartate receptors alters γ- aminobutyric acid type A receptor peptide expression and function in the rat

Abstract: Chronic in vivo or in vitro application of GABAA receptor agonists alters GABAA receptor peptide expression and function. Furthermore, chronic in vitro application of N‐methyl‐D‐aspartate (NMDA) agonists and antagonists alters GABAA receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABAA receptor function and peptide expression in brain. Male Sprague‐Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK‐801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABAA receptor α4 and γ2 subunit expression while significantly decreasing hippocampal GABAA receptor α2 and β2/3 subunit expression. Hippocampal GABAA receptor α1 subunit peptide expression was not altered. In contrast, no significant alterations in GABAA receptor subunit expression were found in cerebral cortex. Chronic MK‐801 administration also significantly decreased GABAA receptor‐mediated hippocampal Cl‐ uptake, whereas no change was found in GABAA receptor‐mediated cerebral cortical Cl‐ uptake. Finally, chronic MK‐801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABAA receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABAA receptor regulation in brain.

GABAA receptor a1 subunit knockout mice: A novel model of essential tremor

Deletion of GABA A receptor α1 subunits in mice results in a phenotype of kinetic tremor that mimics certain features of essential tremor disease. The GABA A benzodiazepine receptor agonist diazepam and the GABAergic neuroactive steroid allopregnanolone exacerbate the tremor, whereas ethanol completely inhibits the tremor. Since the etiology of essential tremor is unknown, this animal model of genetic essential tremor may lead to an understanding of the pathophysiology of the disease and provide a valuable model system to develop therapeutic interventions. Although tremor is often associated with neurological conditions, such as Parkinsons disease, it is capable of manifesting itself in many other ways. Essential tremor, the most common of the tremor disorders, is a primary condition independent of any overlying disorder. Tremor is also commonly associated with substance withdrawal, and it is often a side effect of various drug treatments. The current studies investigating the ability of GABAergic drugs to modulate pathological tremor have provided direction for future experimental procedures investigating the pathology of the knockout tremor. Future studies may include drugs that mediate their actions through other sites of ethanol action. With continuing research on the mechanisms for genetic essential tremor and possible experimental treatments, a cure for essential tremor and other tremor behaviors may be realized in the near future.

Chapter E4 - GABA A receptor α1 Subunit Knockout Mice: A Novel Model of Essential Tremor

Deletion of GABA A receptor α1 subunits in mice results in a phenotype of kinetic tremor that mimics certain features of essential tremor disease. The GABA A benzodiazepine receptor agonist diazepam and the GABAergic neuroactive steroid allopregnanolone exacerbate the tremor, whereas ethanol completely inhibits the tremor. Since the etiology of essential tremor is unknown, this animal model of genetic essential tremor may lead to an understanding of the pathophysiology of the disease and provide a valuable model system to develop therapeutic interventions. Although tremor is often associated with neurological conditions, such as Parkinsons disease, it is capable of manifesting itself in many other ways. Essential tremor, the most common of the tremor disorders, is a primary condition independent of any overlying disorder. Tremor is also commonly associated with substance withdrawal, and it is often a side effect of various drug treatments. The current studies investigating the ability of GABAergic drugs to modulate pathological tremor have provided direction for future experimental procedures investigating the pathology of the knockout tremor. Future studies may include drugs that mediate their actions through other sites of ethanol action. With continuing research on the mechanisms for genetic essential tremor and possible experimental treatments, a cure for essential tremor and other tremor behaviors may be realized in the near future.

CHAPTER E4 – GABAA Receptor α1 Subunit Knockout Mice: A Novel Model of Essential Tremor

Deletion of GABA A receptor α1 subunits in mice results in a phenotype of kinetic tremor that mimics certain features of essential tremor disease. The GABA A benzodiazepine receptor agonist diazepam and the GABAergic neuroactive steroid allopregnanolone exacerbate the tremor, whereas ethanol completely inhibits the tremor. Since the etiology of essential tremor is unknown, this animal model of genetic essential tremor may lead to an understanding of the pathophysiology of the disease and provide a valuable model system to develop therapeutic interventions. Although tremor is often associated with neurological conditions, such as Parkinsons disease, it is capable of manifesting itself in many other ways. Essential tremor, the most common of the tremor disorders, is a primary condition independent of any overlying disorder. Tremor is also commonly associated with substance withdrawal, and it is often a side effect of various drug treatments. The current studies investigating the ability of GABAergic drugs to modulate pathological tremor have provided direction for future experimental procedures investigating the pathology of the knockout tremor. Future studies may include drugs that mediate their actions through other sites of ethanol action. With continuing research on the mechanisms for genetic essential tremor and possible experimental treatments, a cure for essential tremor and other tremor behaviors may be realized in the near future.