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The effects of acute and chronic ethanol administration on the expression ofGABA a /BZ receptor subunit mRNA in the mouse cerebellum
Wu, Chieh-Hsi, Ph.D.
The Ohio State University, 1994
Copyright ©1994 by Wu, Chi eh-Hal. All rights reserved.
UMI 300 N. Zeeb Rd. Ann Arbor. M I 48106 THE EFFECTS OF ACUTE AND CHRONIC ETHANOL ADMINISTRATION ON THE EXPRESSION OF
GABAa /BZ RECEPTOR SUBUNIT mRNA
IN THE MOUSE CEREBELLUM
DISSERTATION
Presented In Partial Fulfillment of the Requirements for the degree Doctor of Philosophy in the Graduate School of The Ohio State University
By Chieh-Hsi Wu, B.S.
The Ohio State University 1994
Dissertation Committee: Approved by Andrej Rotter Adrienne Frostholm
Richard Fertel Thomas Boyd Department of Pharmacology ACKNOWLEDGMENTS
I would like to express my sincere appreciation to my advisor, Dr. Andrej Rotter for his guidance and instruction throughout my entire graduate career. With his patience and understanding, I was able to conquer many academic difficulties and gradually build up my confidence in designing experiments, thus accomplishing my dissertation work. Thanks also go to Dr. Adrienne Frostholm for her conceptual and technical assistance on my experiments. I thank her for letting me mature in science. Without her help, patience and enthusiasm, the writing of this dissertation would not have been possible. Like a friend, supervisor, and lecturer, Dr. Frostholm gave a lot of critical advice and her efforts on my behalf will be appreciated for ever. My appreciation is extended to my dissertation committee members, Drs. Richard Fertel and Tom Boyd, for their encouragement and valuable evaluation of this document. To my colleagues in the laboratory, Dr. Vera Luntz-Leybman, Dr. Jim Evans, and Anne Chang, thank you for giving me a great deal of help and an enjoyable time together. Gratitude and love is also extended to my family and friends, my mother Chaieshe, my siblings, Meeiling, Meeibi, Meeifang, Cheihchi, and Anning. Thank you all for the endless spiritual support. VITA
September 11, 1964 Born - Taiwan, R.O.C.
1986 B.S., Taipei Medical College Taiwan, R.O.C. Major: Pharmacy
1 989-present The Ohio State University Department of Pharmacology College of Medicine Columbus, Ohio
PUBLICATIONS
1. Varecka L., Wu C. H., Rotter A. and Frostholm A. "GABAA/Benzodiazepine receptor a6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice" J. Comp. Neurol.383 (1993) 1-12.
2. Wu C. H., Frostholm A. and Rotter A. "Effect of acute and chronic ethanol exposure on gene expression for GABAA/Benzodiazepine receptor subunits in mouse cerebellum" Soc. Neurosci. Abstr. 19 (1993) 595.
FIELDS OF STUDY
Major Field: Pharmacology Studies in: Neuroscience TABLE OF CONTENTS
Page ACKNOWLEDGMENTS...... ii VITA...... in LIST OF TABLES...... ix LIST OF FIGURES...... xi
CHAPTER I: INTRODUCTION...... 1 A. Introduction ...... 1 B. The pharmacokinetics of ethanol consumption ...... 3 C. The physiological effects of ethanol consumption ...... 5 D. Structure and function of the GABAA/BZ receptor ...... 8
E. The pharmacological properties of GABAa /BZ receptor
subunit variants ...... 17
F. The relationship between ethanol and the GABAa /BZ
receptor ...... 18 1. Acute ethanol effects ...... 21 2. Chronic ethanol effects ...... 22 3. Molecular studies ...... 24 G. Ethanol and the cerebellum ...... 25 1. An overview of cerebellar anatomy ...... 25
i v 2. The effect of ethanol on adult and developing cerebellar neurons ...... 30 H. GABAa/BZ receptor subunit localization in the
cerebellum ...... 33 I. Hypothesis ...... 36
CHAPTER II: PREPARATION OF RIBONUCLEOTIDE PROBES AND GENERAL METHODOLOGY...... 38 A. Ethanol treatment ...... 38 1. Animals...... 38 2. Acute ethanol treatment ...... 38 3. Chronic ethanol treatment ...... 39 4. Prenatal Ethanol Treatment ...... 40 5. Blood ethanol concentration measurement ...... 40 B. Riboprobe synthesis ...... 41 1. mRNA isolation by oligo (dT) chromatography ...... 41 2. Reverse transcription ...... 43 3. Polymerase chain reaction (PCR) ...... 44 4. Ligation of amplified fragments into pBluescript II SK+ phagemid vector ...... 46 5. Small-scale preparation of pBluescript
SK+ plasmid (Miniprep)...... 49 6. Large-scale plasmid isolation (Maxiprep) ...... 52 7. Linearization of the insert-containing
plasmids for transcription...... 54 C. Sequencing double-stranded plasmid template ...... 54
v 1. Alkali denaturation of supercoiled plasmid DNA ...... 54 2. Probe labeling ...... 55 3. Gel preparation and electrophoresis ...... 56 4. Autoradiography ...... 59 D. Northern blot analysis ...... 59 1. Labeling riboprobes with p2p] UTP ...... 59 2. RNA electrophoresis ...... 60 3. Northern blot ...... 61 4. Hybridization ...... 62 E. In vitro transcription of linearized template ...... 63 F. In situ hybridization ...... 64 1. Tissue preparation ...... 64 2. Prehybridization wash ...... 65 3. Hybridization ...... 66 4. Posthybridization washing ...... 67 G. Immunocytochemistry ...... 68 H. Radioligand binding ...... 70 I. Autoradiography ...... 71 J. Quantification ...... 72
CHAPTER III: RESULTS...... 75 A. Probe design and specificity...... 75 B. Blood ethanol concentration and body weight ...... 76 C. The effect of chronic ethanol treatment on Purkinje cell number ...... 78 D. Acute ethanol administration ...... 80
v i 1. In situ hybridization with GABAa /BZ receptor
a i, a6, P2> P3. and 72 subunit cRNA probes ...... 80 2. The effect of acute ethanol treatment on [3H]muscimol and (3H]flunitrazepam binding sites 82 3. The effect of acute ethanol administration on the expression of (52/3 subunit protein ...... 83
E. Chronic ethanol administration ...... 83
1. The expression of GABAa /BZ receptor subunit mRNAs
after chronic ethanol treatment ...... 83 2. The effect of chronic ethanol treatment on [3H]flunitrazepam and [3H]muscimol binding sites in mouse cerebellum ...... 85 3. The effect of chronic ethanol administration on the expression of p2/3 subunit protein ...... 85
F. The effect of chronic maternal ethanol treatment
on the expression of GABAa /BZ receptor a i, a6, P2.
P3, and 7 2 subunit mRNAs in the developing mouse
cerebellum ...... 86
CHAPTER IV: DISCUSSION...... 158
A. The effect of acute ethanol treatment on
GABAa /BZ receptor ...... 164
B. The effect of chronic ethanol treatment on
GABAa /BZ receptor ...... 169
C. The effects of maternal ethanol treatment during
gestation on the expression of GABAa /BZ receptor v i i subunit mRNAs ...... 175 D. Summary ...... 177
REFERENCES...... 179
v i i i LIST OF TABLES
Table Page
1. Species from which the sequences of the GABAa /BZ receptor subunit variants are reported and the chromosomal localization of the genes which encode these subunit variants ...... 13
2. The distribution of possible functional GABA^/BZ receptor combinations in the CNS ...... 19
3. The localization of GABAa /BZ receptor subunits in the adult cerebellum ...... 34
4. Sequence of upstream and downstream primers of GABAa /BZ receptor a i t a6, P2, P3 and 72 subunits ...... 88
5. Northern blot analysis of the number and size of the mRNA species recognized by the mouse-specific riboprobes ...... 89
6. Blood ethanol concentration in acute and chronic ethanol treated mice ...... 90
7. The direct effect of ethanol on the expression of GABAa /BZ receptor ai subunit mRNA ...... 91
8. The summary of acute and chronic effects of ethanol treatment on the expression of GABAa/BZ receptor a t , <*6, P2. P3. and 7 2 subunit mRNAs in adult mouse cerebellum ...... 92
i x The summary of the effects of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor a i, <*6, P2. P3. and 7 2 subunit mRNAs in the mouse cerebellum ...... LIST OF FIGURES
Figure Page
1. A schematic model for the topology of the GABA/\ receptor in the cell membrane, which includes the a, p, y and 8 subunits ...... 14
2. The neuronal distribution and synaptic projection in the cerebellar cortex ...... 27
3. The laminar organization of the cerebellar cortex ...... 94
4 The autoradiographic distribution of the GABAa/BZ receptor a i, a6, P2, P3 and 7 2 subunit mRNAs in mouse cerebellum ...... 96
5. The blood ethanol concentration of mice at different time intervals after receiving a 4g/Kg ethanol IP injection...... 98
6. The body weight of each mouse receiving liquid diet with either alcohol or dextrose for chronic alcohol treatment ...... 100
7. The number of Purkinje cells labeled by the Calbindin monoclonal antibody in the cerebellar vermis and hemisphere of chronic ethanol treated mice and dextrose controls...... 102
8. The number of Purkinje cells stained by cresyl fast violet in the cerebellar
x i vermis (v) and hemisphere (h) of chronic ethanol treated mice and dextrose controls ...... ,104
The effect of acute ethanol administration on the expression of the GABAa /BZ receptor « 1 . <*6. P2. P3. and Y2 subunit mRNAs within the cerebellum ...... 106
The effect of acute ethanol treatment on the expression of GABAa /BZ receptor ai subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 108
The effect of acute ethanol treatment on the expression of GABAa/BZ receptor P3 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 110
The effects of acute and chronic ethanol treatment on the expression of GABAa /BZ receptor a6 subunit mRNA in mouse cerebellar granule cells ...... 112
The effect of acute ethanol treatment on the expression of GABAa /BZ receptor P2 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN)...... 114
The effect of acute ethanol treatment on the expression of GABAa /BZ receptor Y2 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 116
Autoradiographs of [3H]flunitrazepam binding and [3H]muscimol binding sites following acute ethanol administration ...... 118
x i i 16. The effect of acute ethanol treatment on [3H]flunitrazepam binding sites in the mouse cerebellar molecular layer (ML), granule cells (GC), and deep cerebellar nuclei (DCN) ...... 120
17. The effect of acute ethanol treatment on [3H]muscimol binding in the mouse cerebellar molecular layer (ML) and granule cells (GC) ...... 122
18. The acute effect of ethanol administration on monoclonal antibody 62-3G1 immunostaining in the mouse cerebellar granule cell layer ...... 124
19. The effect of acute ethanol administration on the expression of GABAa /BZ receptor (32/3 subunits in the mouse cerebellar molecular layer (ML) and granule cells (GC) ...... 126
20. The effect of chronic ethanol administration on the expression of the GABAa /BZ receptor a i, a6, p2. P3. and Y2 subunit mRNAs within the cerebellum ...... 128
21. The effect of chronic ethanol treatment on the expression of GABAa /BZ receptor a i subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 130
22. The effect of chronic ethanol treatment on the expression of GABAa /BZ receptor Y2 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 132
XIII 23. The effect of chronic ethanol treatment on the expression of GABAa /BZ receptor (32 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 134
24. The effect of chronic ethanol treatment on the expression of GABAa /BZ receptor (33 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons(DCN) ...... 136
25. Autoradiographs of [3H]flunitrazepam binding and [3H]muscimol binding sites following chronic ethanol administration ...... 138
26. The effect of chronic ethanol treatment on the pHjflunitrazepam binding in the mouse cerebellar molecular layer (ML), granule cells (GC), and deep cerebellar nuclei (DCN) ...... 140
27. The effect of chronic ethanol treatment on the pHjmuscimol binding in the mouse cerebellar molecular layer (ML) and granule cells (GC) ...... 142
28. The effect of chronic ethanol administration on monoclonal antibody 62-3G1 immunostaining in the m?use cerebellar granule cell layer and molecular layer ...... 144
29. The effect of chronic maternal ethanol administration on the expression of the GABAa /BZ receptor a i, a6, P2. P3. and y2 subunit mRNAs in the P7 mouse cerebellar vermis (lobules 8 and 9) ...... 146
30. The effect of chronic maternal ethanol exposure on the expression of GABAa /BZ
x iv receptor a i subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 148
31. The effect of chronic maternal ethanol exposure on the expression of GABAa /BZ receptor a6 subunit mRNA in P7 mouse cerebellar granule cells ...... 150
32. The effect of chronic maternal ethanol exposure on the expression of GABAa /BZ receptor p2 subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 152
33. The effect of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor p 3 subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 154
34. The effect of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor 7 2 subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN) ...... 156
xv CHAPTER I INTRODUCTION
A.Introduction Ethyl alcohol (ethanol, C 2 H5 OH) has a major impact on society.
Although the primary use of alcohol is recreational, it has been used clinically as a sedative-hypnotic drug, and an anesthetic (Deitrich et al., 1989). Ethanol is a very small water-soluble molecule that can be completely and rapidly absorbed from the gastrointestinal tract. After being absorbed, it is distributed to almost every part of the body. Therefore, the effects of ethanol on the physiological system are quite extensive. The gastrointestinal, cardiovascular, endocrine (Lee and Becker, 1989) and neuronal systems (Treistman and Wilson, 1987) are all affected by ethanol. Among these systems, the central nervous system is most vulnerable to the effects of ethanol. The overall effects of ethanol in the central nervous system appear to be sedation (Abraham and Hunter, 1982; Abel, 1984), loss of consciousness (Little, 1991), slurred speech (Colangelo and Jones, 1982), ataxia and impaired judgment (Leonard, 1987). These adverse effects are often seen after acute ingestion of ethanol. Physical dependence on, and tolerance to, ethanol
1 2 frequently occurs after long term consumption; this may be accompanied by various problems associated with withdrawal reactions or abstinence syndromes, including motor agitation, anxiety, insomnia, and reduction of seizure threshold (Lee and Becker, 1989). Pregnancy after prolonged consumption of ethanol may result in teratogenesis. These abnormalities, which have been defined as fetal alcohol syndrome (Jones and Smith, 1973), include delays in motor development, problems with fine motor tasks, retarded body growth, microencephaly, congenital heart defects, and mental retardation . The wide availability of ethanol has led to abuse in many societies. There is an enormous cost of ethanol intoxication, which often results in human tragedy and economic loss. The appeal of ethanol may lie in its ability to reduce social stress and anxiety. Ethanol has been proposed as an anxiolytic by (Breese et al., 1979), who suggested that ethanol is useful in calming, alleviating anxiety and reducing social inhibition. Thus, a desire to reach those various low-anxiety states may become a major motivating factor in ethanol consumption. More recent work (Breese et al., 1984) has also suggested that ethanol does produce a short term anxiolytic effect; however, this effect decreases gradually, followed by a prolonged dysphoric state. The anxiolytic effect can be temporarily reached by further ethanol ingestion. Therefore, people who ingest ethanol to alleviate anxiety have to continue taking ethanol to obtain its original effects, and thus may become addicted. 3 Although alcoholism is not a novel disease, and much effort has been exerted to prevent, and possibly cure, this growing problem, there is very little understanding of the cellular and neuronal mechanisms which are responsible for ethanol intoxication and dependence. A wide spectrum of studies have been carried out to explore these ethanol-related mechanisms. These include studies on structure-activity relationships (Huidobro-Toro et al., 1987), effects on membrane properties (Harris et al., 1987), voltage or second-messenger gated ion channels (Hunt, 1985; Treistman and Wilson, 1987; Daniell and Harris, 1989), neurotransmitters, and neuromodulators and their receptors (Allan and Harris, 1987; Suzdak et al., 1986a; Suzdak et al., 1988b; Gruol,1982). The effects of ethanol have been studied extensively in the CNS, using biochemical (Mhatre and Ticku, 1992), behavioral (Rassnick et al., 1993) and electrophysiological approaches (Freund et al., 1993). In addition, anatomical and molecular approaches have been used to pinpoint the brain regions, cell types, and molecular components that appear to be particularly sensitive to the effects of ethanol.
B. The pharmacokinetics of ethanol consumption Ethanol is ingested and absorbed through the gastrointestinal tract by diffusion. After ethanol is absorbed into the body, it rapidly diffuses throughout the aqueous compartments, going wherever water goes, without any difficulty. Although ethanol is not a very hydrophobic compound (its partition coefficient between lipid and aqueous phase is about 0.1), it can easily get through the 4 blood-brain-barrier, as well as the placenta. The process of absorption is fast, especially with a high concentration of ethanol and an empty stomach (Ritchie, 1970). Diluted beverages, such as beer (3-5% V/V ethanol) or wines (about 12% V/V) are absorbed less in amounts in small intestine than distilled spirits, which contain 40-50% ethanol by volume. Ingestion with food will also slow ethanol absorption. This is due to the dual effect of food: it dilutes the ethanol in the gastrointestinal tract, and it delays stomach emptying. Consequently, ethanol is delayed in reaching the small intestine where ethanol is absorbed extremely rapidly and completely (Ritchie, 1970). The rate at which ethanol distributes into different tissues depends mainly on their blood supply. For example, because of its extensive vascular supply, concentrations in the brain rapidly reflect changes in the concentration of ethanol in arterial blood. Ethanol is almost entirely metabolized to acetaldehyde in the liver by oxidation. Although other tissues such as brain and intestines have some metabolic capacity, their ability to metabolize ethanol is barely detectable. The liver contains three different enzyme systems which are involved in ethanol metabolism: alcohol dehydrogenase, alcohol catalase and the microsomal ethanol-oxidizing system (Lee and Becker, 1989). Of the three enzymes, alcohol dehydrogenase is the most important; it has been extensively studied by biochemists because it is stable and easily purified. It is a soluble enzyme that uses nicotinamide adenine dinucleotide (NAD) as a coenzyme to oxidize ethanol to 5 acetaldehyde by the following reaction: Equation 1: c h 3c h 2o h + n a d + ^-alcohol dehydrogenase CH3CH0 , + NADH + H+
Acetaldehyde, the product of the alcohol dehydrogenase reaction, is further oxidized by acetaldehyde dehydrogenase to CO 2 and H2 O
(Algar and Holmes, 1989: Tomita et al., 1992), which are then excreted from the body. Both catalase and the microsomal ethanol-oxidizing system (MEOS) can also oxidize ethanol. However, they play smaller roles in ethanol metabolism than alcohol dehydrogenase. Catalase, which is abundant in the liver, acts as a peroxidase which uses hydrogen peroxide formed by various oxidases to oxidize ethanol. The MEOS of the liver can oxidize ethanol in the presence of molecular oxygen and NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). Under the normal oxidation process, alcohol dehydrogenase oxidizes ethanol first followed by oxidation by MEOS.
Although most of the ethanol is excreted in the form of CO 2 and
H 2 O, there is still 5-10% excreted unchanged in expired air and in urine. The excretion of this portion depends on the dose, since it follows first-order kinetics, i.e., at higher doses more ethanol is excreted.
C. The physiological effects of ethanol consumption The physiological effects of ethanol can be categorized into two classes: the effects caused by ethanol metabolites and the direct effects of ethanol itself. The metabolites of ethanol greatly affect physiological systems. As a result of oxidation by alcohol 6 dehydrogenase, MEOS, or catalase, an excess of reducing equivalents are generated in the liver. In order to get rid of these reducing equivalents, a large amount of oxygen is consumed. Thereafter, there is a possible risk of hypoxia, which may induce hepatic necrosis (Klatsky et al., 1981). Acetaldehyde, the immediate metabolite of ethanol, can cause nausea, vomiting, flushing, and hypotension. Moreover, ethanol oxidation may reduce the cofactor NAD to NADH in the liver (Lee and Becker, 1989). The increased level of NADH can decrease the amount of pyruvate in the gluconeogenesis reaction, an important step in the formation of glucose in the physiological system (Xu et al., 1993). If this reduction occurs, hypoglycemia may ensue (Klatsky et al., 1981). Some other disorders related to ethanol intoxication, such as hyperuricemia and fatty liver, may also be induced by increased amounts of reducing equivalents (Geokas, 1984). Although there are no specific ethanol receptors, ethanol can alter a variety of physiological systems directly. With its ability to dissolve in the lipid bilayer of cell membranes, ethanol may affect the viscosity of the membranes of many types of cells. This has been called the fluidization effect of ethanol (Goldstein, 1986). The molecular and cellular mechanisms underlying ethanol-induced disorders are poorly understood; however, it has been suggested that a change in the neuronal membrane is one of the most likely mechanisms related to the clinical alcohol syndromes (Chin and Glodstein, 1977a,b). Such changes in the neuronal membrane often cause alterations of membrane transduction systems, such as 7 receptor proteins and ion channels (Little, 1991). Since receptor proteins are located in the cellular membranes, there is the possibility that when ethanol fluidizes the cell membrane it can alter the normal structure of certain receptors, and physiological and biochemical functional states may be changed. For example, the influx of Cl" through the glycine receptor in synaptoneurosomes was increased by exposure to ethanol (Nordberg and Wahlstrom, 1992); supersensitivity of muscarinic receptors was induced by chronic ethanol administration (Nordberg and Wahlstrom, 1992); and dopamine receptor density was changed by ethanol treatment (Hamdi and Prasad, 1992). Ethanol also affects NMDA receptors: The NMDA-enhanced sodium efflux from striatal slices was diminished by ethanol (Teichberg et al., 1984). Inhibitory y-aminobutyric acidA/ benzodiazepine (GABAa/BZ) receptors also have been reported to be affected by ethanol administration (Ticku, 1980; Wafford et al., 1991; Morrow et al., 1992; Sigel et at., 1993). Taken together, the above ooservations indicate that ethanol extensively influence a variety of neurochemical receptor systems.
Among these receptors, GABAa /BZ receptor has been most extensively studied because there is considerable evidence showing that GABAa /BZ receptor agonists are involved in both the acute and chronic actions of ethanol (Ticku, 1980; Volicer and Biagioni, 1982; Harris et al., 1983), and these agonists and ethanol share a variety of pharmacological properties (Mihic et al., 1992), including sedation, hypnosis and anesthesia. Acute exposure of cellular 8 membranes to ethanol in vitro results in an increased ability of benzodiazepine inverse agonists to reduce muscimol-activated chloride channel function (Buck and Harris, 1990a). Benzodiazepine inverse agonists also have been reported to be ethanol antagonists (Lister and Nutt, 1987), which reverse the ethanol-induced stimulatory effect on muscimol-mediated 36CI" influx (Suzdak et al., 1986b). Once the inhibition of benzodiazepine inverse agonists on muscimol-activated chloride channel function is increased, more ethanol is needed to reach its original effect on Cl' influx. Therefore, the ability of ethanol exposure to enhance the inhibition of endogenous benzodiazepine inverse agonists on chloride channel function may be involved in the development of ethanol tolerance
(Buck et al., 1991).
P. Structure and function of the GABA^/BZ re.CePlflr
The GABA receptor is one of the major, well characterized inhibitory receptors in the CNS. There are two types of GABA receptor: GABAa and GABAb , which have been distinguished both by their selectivities for agonists and antagonists, and by their channel coupling mechanisms (Bomann, 1985). The GABAa receptor is a multisubunit ligand-gated chloride ion channel which exerts an inhibitory influence on the central nervous system, whereas GABAb receptors are associated with Ca^+ and K+ channels using a G- protein linked mechanism (Bowery et al., 1983; Kerr et al., 1987).
There appear to be less GABAb receptors than GABAa receptors, the 9 latter being present in approximately one third of all brain synapses (Bloom and Iverson, 1971), including those in the cortex, hippocampus, striatum, midbrain and cerebellum. Their presence in the midbrain and cerebellum shows that the GABAa receptors may have a major inhibitory function in the central coordination of excitability, and in motor and autonomic function (Palmer and Paul,
1990). Since the GABAa receptor is a complex containing both GABA and benzodiazepine binding sites, it is frequently denoted as the
GABAa /BZ receptor.
During the past decade, studies on the GABAa /BZ receptor have attracted increased attention because psychiatric problems, such as schizophrenia and affective psychosis, have been related to pathological changes in these receptors (Squires and Saederup,
1991; Dixon et al., 1989). The membrane-bound GABAa /BZ receptor protein binds the endogenous neurotransmitter, GABA, which opens the ion channel and allows chloride ions to pass through. The increased intracellular concentration of chloride ions then hyperpolarizes the cellular membrane and decreases its physiological activity. This response can be modified by several drugs, including benzodiazepines (Tallman et al., 1980; Schwartz, 1988; Brioni et al., 1989), barbiturates {Ticku and Olsen, 1978; Peters et al., 1988), convulsants (Ticku et al., 1978; Squires et al., 1983) and neurosterords (Majewska et al., 1988; Perez et al., 1988; Smith, 1989). Benzodiazepines (Haefely, 1989; Pritchett et al., 1989) and barbiturates (Olsen, 1982) can enhance the GABA response, the latter more strongly. Benzodiazepines exert their 10 effects on the GABAa /BZ receptor complex by increasing the probability of channel openings, whereas barbiturates do so by extending the channel open time {Olsen, 1982). Convulsants, such as picrotoxin, decrease GABAa /BZ receptor function by reducing the average channel open time (Twyman et al., 1992). The benzodiazepine receptor, an important component of the
GABAa /BZ receptor complex, has been categorized into several subtypes according to its response to various drugs, including benzodiazepine agonists and inverse agonists. Benzodiazepine inverse agonists, including several of the p-carbolines, not only block agonist effects by competing for benzodiazepine binding site, but also produce pharmacological effects opposite to those of agonists, such as flunitrazepam and diazepam (Braestrup and Nielsen, 1981). Partial inverse agonists, such as FG7142 and Ro 15-4513, reduce the seizure threshold to other convulsants (Cowen et al., 1981) and decrease the GABA-mediated enhancement of Cl- influx (Little, 1984). It has been suggested that Ro 15-4513 also reduces the ataxic and anesthetic effects of ethanol (Suzdak et al., 1986a). These agonists and antagonists have been used extensively to define GABAa /BZ receptor subtypes. According to binding studies
(Squires et al., 1979), competitive inhibition of (^HJflunitrazepam binding by the benzodiazepine agonist CL 218872, a triazolopyridazine, appears biphasic in many brain regions. The sites with higher affinity for CL 218872 are known as type I BZ receptors; they predominate over the type II receptors which have a lower affinity for CL 218872. Type I receptors are extensively 11 expressed in the cerebellum, whereas the type II receptors are present in other CNS regions, such as the hippocampus, striatum, and spinal cord (Palmer and Paul, 1990; Yakushiji et al., 1993). Beta-carbolines have also been used to define the two major benzodiazepine receptor subtypes, BZ1 and BZ2 receptors (Braestrup and Nielsen, 1981). The distributions of Type I and BZI receptors are not completely identical, nor are these of Type II and BZII receptors (Squires et al., 1979; Braestrup and Nielsen, 1981; Levitan et al., 1988a), suggesting that there are more than two benzodiazepine receptor subtypes. More recently, molecular cloning studies (Schofield et al., 1987; Pritchett et al., 1990; Bateson et al., 1991; Wilson-Shaw et al., 1991; Cutting et al., 1991) have shown that the GABA/^/BZ receptors are composed of subunits belonging to at least 5 classes: a, p, y, 8 and p. The a and p subunits were discovered first (Mohler et al., 1980; Siegel et al., 1983; Schofield et al., 1987), followed by y, 5, and p. The a-subunit (Mr 53k) has been shown to contain the BZ binding site (Casalotti et al., 1986; Vitorica et al., 1987) and the p-subunit (Mr 57k) possesses the GABA binding site (Mamalaki et al., 1987; Vitorica et al., 1987). The deduced protein sequences and hydropathy profiles reveal striking similarities in the structure and general architecture of these two GABAa receptor subunits, suggesting a common evolutionary origin. There is about 70-80% identity of amino acid sequence between variants within the same subunit family, while different subunit families show about 30-40% homology (Burt and Kamatchi, 1991). The five subunits have several 1 2 variants. The reported sequences and their chromosomal localization are shown in table 1.
Among the subunit variants, there is a common structural topology (Fig. 1). Each variant has four hydrophobic transmembrane domains, which are denoted as M1, M2, M3 and M4. These are segments of their primary amino acid sequence that have been proposed to form a-helical spans across the plasma membrane. A group of positively-charged side-chains are located on both sides of these transmembrane domains, which are presumed to form the mouth of the ion channel. It has been suggested that a proline residue in the M1 segment forms a bend which protrudes into the channel lumen; this keeps the channel closed in the absence of a neurotransmitter (Schofield et al., 1987). The M2 transmembrane domain may play a role in lining the ion-channel wall. The lining of the channel may be responsible for the conduction of ion flow due to the presence of threonines and serines in these transmembrane domains (Schofield et al., 1987). The positive charges of threonines and serines can stabilize the negative charge of chloride inside the channel. Flanking transmembrane domains M1 and M4 in the extracellular space are the N- and COOH-terminals, respectively. The binding sites for the various GABA and BZ agonists and antagonists are postulated to be located in these large N-terminal extracellular domains (Schofield et al., 1987). The structure of the binding site in the N-terminal domain is thought to be a loop which is composed of several N-linked glycosylation sites (2 in the a subunit and 3 in the (3 subunit) and nearby Cys residues (Burt and 1 3
Table 1. Species from which the sequences of the GABA^/BZ receptor subunit variants are reported and the chromosomal localization of the genes which encode these subunit variants.
Subunit Species and Chromosomal localization Variant a l rat (Khrestchatisky et al.. 1989);bovine (Schofield et al., 1987);human (Schofield et al., 1989) Chromosome 15 (human); 11 (mouse) (Keir et al., 1991) a2 rat (Khrestchatisky et al., 1991); bovine (Levitan et al., 1988b) chromosome 4 (human); 5 (mouse) (Danciaer et al.. 1993) a3 rat (Malherbe et al., 1990b); bovine (Levitan et al., 1988b) chromosome x (human or mouse) (Buckfe et al„ 1989) a4 rat (Khrestchatisky et al., 1989) chromosome 7 (mouse) (Danciaer et al., 1993) aS rat (Pritchett and Seeburg, 1990); mouse (Nakatsu et al., 1993) chromosome 15 (human) (Knoll et al., 1993) a 6 mouse (Kato. 1990); rat (Luddens et al., 1990) PI rat (Malherbe et al., 1990a); human (Schofield et al., 1989) chromosome 4 (human); 5 (mouse) (Danciaer et al., 1993) P2 rat . bovine (Ymer et al., 1989b) (J3 rat, bovine (Ymer et al., 1989b); mouse (Nakatsu et al., 1993) chromosome 15 (human); 7 (mouse) (Waastaff et al., 1991) f * chicken (Bateson et al.. 1991) Y1 rat , bovine, human (Ymer et al., 1990) chromosome 6 (human); 4 (mouse) (Wilcox et al., 1992) 72 rat (Malherbe et al., 1990b); human (Pritchett et al., 1989) chromosome 6 (human); 4 (mouse) (Wilcox et al.. 1992) Y3 mouse (Wilson-Shaw et al.. 1991; Nakatsu et al., 1993); rat (Knoflach et al., 1991) 6 rat (Shivers et al., 1989) chromosome 1 (human) (Sommer et al.. 1990) P human (Cutting et al., 1992) chromosome 6 (human) (Cutting et al., 1992) Fig. 1. A schematic model for the topology of the GABAa receptor in the cell membrane, which includes the a, p, y and 5 subunits
(modified from Schofield et al., 1987). The subunit with benzodiazepine (BZ) binding site is a common structure for both a and y subunits; the subunit with GABA binding site is a common structure for both p and S subunits 1 5
NH2 NH2 GABA binding site
COOH
COOH M l
M t
Serine phosphorylation BZ binding site site
p and& and y common common structure structure
Fig. 1 1 6 Kamatchi, 1991). When the ligand binds to the receptor complex, the conformation of this receptor changes and exposes some of the positively-charged residues at the channel mouth to attract the negatively-charged chloride ions. This conformational change can then shift the configuration of M1 domain where the proline residue is withdrawn from the channel lumen and the channel is opened (Schofield et al., 1987). Although there is considerable homology both within the subunit variants, and with other receptors (for example, the nicotinic acetylcholine receptor (Olsen and Tobin, 1990) and the strychnine-sensitive glycine receptor (Grenningloh et al., 1987)), no sequence homology among the subunit variants is apparent in the intracellular loop which connects M3 and M4 domains. In each of the subunits, this region is of different length. Recent studies suggest that the intracellular region of certain p and y subunit variants contains a unique cAMP-dependent serine phosphorylation consensus sequence (Feramisco et al., 1980; Sigel et al., 1991): The cytoplasmic loop of the P3 subunit is the substrate for cyclic AMP- dependent protein kinase (PKA). The long form of the y2 subunit contains an 8 amino acid insert in which there is a consensus sequence which is phosphorylated by Ca2+-phospholipid-dependent protein kinase C (PKC); this region appears to be essential for modulation of GABAa receptor function by ethanol (Kofuji et al.,
1991). It has been reported that desensitization of GABAa /BZ receptors may be modulated by phosphorylation at these two sites (Whiting et al., 1990; Walaas and Greengard, 1991); this reduces the 1 7 functional inhibitory effects of the GABAa /BZ receptor. For example, electrophysiological studies, using Xenopus oocytes injected with GABAa receptor subunit mRNAs, have shown that activation of PKA decreases GABAa receptor-mediated chloride conductance (Porter et al., 1990), whereas phorbol esters blocked
GABAa receptor function by activating PKC (Sigel et al., 1991).
E. The pharmacological properties ofGABA a/BZ receptor subunit valiants The different subunit variants confer highly specific pharmacological properties upon the GABAa /BZ receptor (DeLorey and Olsen, 1992); therefore, the effects of GABA and other drugs acting on GABAergic transmission are very much dependent on the subunit composition at specific sites (Baude et al., 1992). The diversity of these subunits makes the study of GABAa /BZ receptors extremely complicated, since at least 150,000 combinations of the
GABAa /BZ receptor subunit variants are theoretically possible (Burt and Kamatchi, 1991). Although not all of them may exist in vivo, there still are a huge variety of these receptors distributed in different neuronal populations, displaying distinct kinetics and modulatory properties (Seeburg et al., 1990). Therefore, extensive experimental corroboration is needed to clarify the diverse roles of GABA^/BZ receptors in specific regions of the CNS. Coexpression studies, using cloned subunit variants in mammalian cells, have shown that ai Pi subunits respond to GABA, but the response is only weakly and inconsistently modified by benzodiazepines (Malherbe 1 8 et al., 1990c). It is now known that the coexpression of the y2
subunit in the Xenopus oocytes confers a strong benzodiazepine sensitivity to the subunit composition (Malherbe et al., 1990b; Pregenzer et al., 1993). Some of the possible functional receptor combinations proposed by electrophysiological and ligand binding studies are listed in table 2.
F. The relationship between ethanol and theG A B A ^ /B Z receptor A variety of behavioral (Liljequist and Engel, 1982; Martz et al., 1983), biochemical (Buck et al., 1991; Morrow et al., 1992), electrophysiological (Benke et al., 1991; Pregenzer et al., 1993), and pharmacological (Ticku et al., 1986; Buck and Harris, 1990a, b) studies have shown that GABAa /BZ receptor function may be modified by ethanol. The precise mechanism by which this occurs is unclear. However, ethanol possesses muscle relaxant, antianxiety and sedative-hypnotic properties similar to those of the benzodiazepines and barbiturates, which exert their effects through the GABAa /BZ receptor (Burch and Ticku, 1980; Skolnick and Paul,
1981). The benzodiazepines are well known as a class of pharmacological agents which have high efficacy in the treatment of anxiety (Givens and Breese, 1987). Since ethanol was once classified as an anxiolytic (Breese et al., 1979), the similarity between the behavioral actions of ethanol and the benzodiazepines might suggest a possible mechanism for the effects of ethanol. The clinical syndromes of acute intoxication and withdrawal produced 1 9 Table 2. The distribution of possible functional GABAA/BZ receptor
combinations in the CNS
Receptor Functional Properties Regional distribution composi tion aipiy2 high affinity for BZ agonists: olfactory bulb mitral cells, sensitive to picrotoxin; higher hippocampal pyramidal cells, sensitivity to diazepam than granule cells of the dentate gyrus, al(32Y2; type I BZ receptor. cerebellum (Pritchett et al., 1989b; Malherbe et al.. 1990; Siael et al.. 1990). alp2y2 high affinity for BZ agonists Cerebellum, inferior colliculi, red insensitive to picrotoxin; the mostnucleus, olfactory bulb mitral widely distributed composition incells the brain; type I BZ receptor. (Benke et al., 1991; Wisden et al., 1992; Laurie et al., 1992). a2P3yx the most abundant receptor Nucleus accumbens, caudate composition in the hippocampus;nucleus, amygdaloid nucleus, type If BZ receptor. hypothalamus, hippocampus (Wisden et al., 1992; Pritchett et al., 1989a; Goodchild, 1993). a3pxy2 low affinity for CL 218872 and p-spinal cord, hippocampus, carboline; type II BZ receptor. cerebral cortex (Pritchett et al., 1989a; Sigel et al., 1990; Goodchild. 1993). a4f)25 high binding affinity for GABA hippocampus, thalamus, neocortex ligands, but not for BZ agonists; (Shivers et al., 1989; Wisden et highly expressed in thalamus. al.. 1991). a5pxy2 high affinity for GABA gating of thehypothalamus, hippocampus channel; induces cooperativity of(Wisden et al., 1992; Sigel et al., the gating; type II BZ receptor. 1990: Goodchild, 1993) a6p2/35 binds [3H]muscimol but not cerebellar granule cells (Laurie et benzodiazepines; exists at synaptical., 1992) sites but not the somatic membranes of cerebellar granule cells. x: any one of the subunit variants 20 by ethanol and benzodiazepines are extremely similar (Greenberg et al., 1984). It has thus been suggested that cross-tolerance exists between ethanol and benzodiazepines, and benzodiazepines have been clinically used to relieve ethanol withdrawal syndromes (Biosse and Okamoto, 1980). At the cellular level, ethanol produces an effect similar to that of benzodiazepines in that it enhances the GABA-mediated chloride flux in synaptoneurosomal preparations (Ticku et al., 1986; Allan and Harris, 1987; Suzdak et al., 1986b; Mehta and Ticku, 1989). This ethanol-induced increase in GABA- mediated chloride flux was diminished by the partial benzodiazepine-inverse agonist, Ro 15-4513 (Suzdak et al., 1986a; Kulkarni and Ticku, 1989). In addition, ethanol, GABA receptor agonists and benzodiazepines all produce similar effects on postsynaptic second messengers by decreasing cyclic GMP levels (Biggio et al., 1977; Mohler et al., 1980).
The effects of ethanol on the GABAa /BZ receptor mainly depend on the experimental conditions used, such as the length of ethanol treatment (Davis and Ticku, 1981), the dose of ethanol (Ticku, 1980; Ticku and Burch, 1980), the anatomical regions of CNS (Little, 1991), and the species (Volicer and Biagioni, 1982). Acute and chronic ethanol treatments have shown differential effects on the GABAa /BZ receptor, suggesting that intoxication mechanisms of different period of ethanol exposure are different. 21 1. Acute ethanol effects Acute ethanol administration was reported to increase the density of GABAa /BZ receptors (Ticku, 1980): GABA binding to a whole brain membrane fraction was increased 30 minutes after a 4 g/Kg dose of ethanol intraperitoneally injected into rats (Ticku, 1980) and mice (Ticku and Burch, 1980). Scatchard analysis revealed that this change was due to an increase in the number of low-affinity binding sites for GABA (Little, 1991). These changes in low affinity binding sites were not observed, however, after a 4 g/Kg oral dose (Volicer and Biagioni, 1982), suggesting that the route of administration can also influence the effect of ethanol. These differential effects may be due to different pathways of ethanol metabolism and absorption. GABA-mediated chloride flux was also increased by ethanol over the concentration range 5-100 mM in cultured spinal cord neurons (Gruol, 1982; Mehta and Ticku, 1988) and synaptoneurosomes from rat cerebral cortex (Suzdak et al., 1986b). Muscimol, a GABA agonist, induced chloride uptake into mice cortical membrane vesicles, which was also increased by ethanol at a concentration of 10-45 mM (Allan and Harris, 1987). There is no evidence that ethanol affects GABA release (Suzdak et al., 1986b; Howerton and Collins, 1984), and the synthesis and degradation of GABA is affected by ethanol to a minimal degree (Hakkinen and Kulonen, 1976), indicating that presynaptic mechanisms are involved in the action of ethanol only to a minimal extent. 22 Ethanol also has been reported to increase benzodiazepine binding. Studies using a Lubrol-solubilized fraction, a tissue preparation carrying specific binding sites for muscimol {GABA agonist), diazepam (benzodiazepine agonist), and dihydropicrotoxin (picrotoxin agonist), have shown that acute ethanol administration enhanced [3H]diazepam binding by inhibiting the picrotoxin- sensitive site on the GABAa /BZ receptor (Davis and Ticku, 1981).
This potentiation appeared at ethanol concentrations between 20 and 100 mM; at concentrations higher than 100 mM, the effect was reduced. The coupling between benzodiazepine inverse agonist sites and the chloride ion channel was also increased by acute exposure to ethanol (Buck and Harris, 1990a). The interaction between ethanol and GABAa /BZ receptors was neuroanatomically specific; the microinjection of GABA agonists or antagonists into the medial septal nucleus altered aerial righting deficits induced by acute ethanol treatment, while microinjection of such drugs into the nearby lateral septum did not have similar effects (Givens and Breese, 1987).
2. Chronic ethanol effects Chronic ethanol effects on GABAergic transmission system show a different pharmacological profile from that seen in response to acute ethanol treatment. The potentiation of ethanol on GABA- stimulated chloride uptake was no longer observed after long term ethanol administration (Morrow et al., 1988). Low affinity GABA binding, as well as benzodiazepine binding, was reduced in rodent 23 brain after chronic ethanol treatment (Freund, 1980; Ticku and Burch, 1980; Linnoila et al., 1981; Volicer and Biagioni, 1982; Ticku et al., 1983). A decrease in the Bmax for the low affinity site for muscimol after 7 days intragastric administration of ethanol suggested that the GABAa /BZ receptor number was reduced by chronic ethanol treatment (Volicer and Biagioni, 1982). This decrease in the Bmax may be due to downregulation of GABAa /BZ receptor in response to the prolonged existence of ethanol-induced CNS depression (Unwin and Taberner, 1980; Buck and Harris, 1991).
Nevertheless, the binding of a benzodiazepine-inverse agonist Ro 15-4513 (a postulated ethanol antagonist) to cerebellum and cerebral cortex was increased after chronic exposure to ethanol (Mhatre et al., 1988). The increased number of binding sites suggest that the benzodiazepine-inverse agonists may be used clinically to reverse some of the adverse effects of chronic ethanol ingestion. In contrast to pharmacological and biochemical studies, electrophysiological studies are less consistent regarding the effects of ethanol on the GABAa /BZ receptor. Takada et al. (1989) showed that the GABA-mediated inhibition of population spikes in the CA1 area of hippocampus was increased by 70 mM or greater ethanol. Voltage clamp studies showed that application of ethanol (20-50 mM) on chick spinal cord neurons was found to increase the actions of GABA (Celentano et al., 1988). However, Carlen et al. (1982) and Siggins et al. (1987) found no evidence of potentiation of GABA by ethanol. Gruol (1982) indicated that the effects of ethanol (20-80 mM) either did not change, or slightly increased, the GABA 24 responses in cultured mouse spinal cord neurons. The discrepancy between these results may have been due to the duration of ethanol administration, or the concentrations of ethanol used (Little, 1991).
3. Molecular studies At a more molecular level, the expression of several
GABAa /BZ receptor a subunit variant mRNAs of the GABAa /BZ receptor has been studied mainly after chronic ethanol treatment (Morrow, 1990; Montpied et al., 1991; Morrow et al., 1992). Northern blot analysis, using cRNA probes for these subunit variants, has shown that long term ethanol treatment affects the expression of these variant mRNAs in the cerebral cortex and cerebellum. In the cerebral cortex, chronic ethanol inhalation reduced the level of a1 subunit mRNA by 40-50% (Montpied et al., 1991), whereas the levels of both the 6Kb and 3Kb of a2 mRNA were decreased by 61 and 45%, respectively. The levels of a5 mRNA were also decreased by 51% in the cerebral cortex of rats (Mhatre and Ticku, 1992). However, a3 subunit mRNA level showed no change under prolonged ethanol exposure (Mhatre and Ticku, 1992). In the cerebellum, Northern blot studies show the level of GABAa /BZ receptor a1 subunit mRNA was reduced by 30%; however, a6 subunit mRNA level was increased by
45% following chronic ethanol ingestion (Morrow et al., 1992). The level of total RNA, poly(A)+RNA and glutamic acid decarboxylase mRNA levels remained unchanged after chronic ethanol treatment, suggesting that the decreased levels of a1, a2, and a5 mRNA, and the increased levels of a6 mRNA were not due to a generalized 25 effect of ethanol on mRNA transcription or turnover (Montpied et al., 1991). The majority of molecular studies have used Northern blots to examine the effects of ethanol on mRNA synthesis. Although this technique can show the changes in GABAa /BZ receptor subunit variant mRNAs caused by ethanol in gross brain regions, the changes in different cell types within the same brain regions can not be distinguished since they are derived from a homogenate. For example, the changes in subunit mRNA levels in response to ethanol in Purkinje cells may be greater than in deep cerebellar neurons; however, Northern blot studies would not show this difference. Moreover, if one cell type shows a reduction in the mRNA level, while another cell type in the same brain region shows an increase, the net result shown by the Northern blot technique will be an average of the two results; this may, and has, caused misleading results. In situ hybridization, a technique used to identify a particular type of mRNA in different anatomical localizations throughout the brain, can be used to correct the disadvantages of Northern blot analysis.
G. Ethanol and the cerebellum 1. An overview of cerebellar anatomy The cerebellum is an excellent CNS region in which to study the effects of ethanol, since it is involved in the control of locomotor activity which is severely disturbed in response to ethanol. The cerebellar cortex is a uniform structure that is divided 26 into distinct molecular, Purkinje cell, and granule cell layers that together contain only five types of neurons: stellate, basket, Purkinje, Golgi, and granule cells. The distribution of these neurons within the cerebral cortex is shown in fig. 2. In the adult, the deepest cerebellar layer, the granule cell layer, mainly contains densely packed granule cells and infrequent Golgi cells. Above the granule cell layer is the molecular layer, the most superficial layer, which contains the dendrites of the Purkinje and Golgi cells, and the axons of granule cells. Two interneurons, the stellate and basket cells, are also situated in this layer. Between these two regions is the Purkinje cell layer, which consists of a single layer of Purkinje cells, the major output neurons of cerebellum. Depending on the location in the cerebellum, Purkinje cells project inhibitory GABAergic axons to the deep cerebellar nuclei (DCN), which innervate the motor neurons (Ross et al., 1990) which in turn project to other brain regions: Purkinje cells in the medial zone (vermis) of cerebellar cortex project, via the medial DCN (fastigial nuclei), onto the pontine reticular formation or vestibular nucleus in the brain stem, which then influence the pontine reticulospinal tract or lateral vestibulospinal tract, respectively. Purkinje cells in the intermediate zone project, via the intermediate DCN (interposed nuclei), onto the red nucleus in the brain stem, which in turn influence the rubrospinal tract. Purkinje cells in the cerebellar hemispheres project, via the lateral DCN (dentate nuclei), onto the ventral lateral nucleus of the thalamus in the brain stem, which sends projections to the motor Fig. 2. The neuronal distribution and synaptic projection in the cerebellar cortex
27 28
Parallel fiber Stellate cell ^ Molecularlauer
Basket cell Purkinje Purkinje ceil cell Bergmann glia layer Gol ]1 cell
Axon Granular cell Granule lauer cell
Deep Cerebellar Deep Cerebellar Nuclei Nuclei
Climbing Mossy fiber fiber
Fig. 2 29 cortex influencing the lateral corticospinal tract. These influences on motorneurons make the cerebellum an important component in the control of motor coordination. Granule cell axons constitute the parallel fiber system. Each granule cell axon runs perpendicularly through a row of massive Purkinje cell dendritic arbors, contacting approximately 50 Purkinje cells (Berne and Levy, 1988). Each Purkinje celt is contacted by approximately 200,000 parallel fibers (Ghez and Fahn, 1981). In addition to innervating the Purkinje cells, the glutamatergic parallel fibers activate three types of cerebellar cortical interneurons (stellate cells, basket cells, and Golgi cells), all of which have their dendrites in the molecular layer. These interneurons, which release y-aminobutyric acid (GABA) as neurotransmitter, are all inhibitory. Basket and stellate cells both feed forward to inhibit Purkinje cells, whereas Golgi cells feed back to inhibit the granule cells. Stellate cells inhibit the more distal Purkinje cell dendrites, while basket cells inhibit the more proximal dendrites and soma of Purkinje cells. The cerebellar cortex has two major excitatory afferent systems, the mossy fibers and climbing fibers. Mossy fibers arise from spinal cord and brain stem and release glutamate to excite the granule cells (Ross et al., 1990), whereas the climbing fibers originate in the contralateral inferior olive and release aspartate to activate Purkinje cells (Ito, 1984). Each mossy fiber makes synapses with many granule cells. In contrast, an individual climbing fiber innervates only one Purkinje cell in the adult (Crepel, 30 1982; Mariani and Changeux, 1981a,b). Because of this different pattern of synaptic innervation, the physiological outcomes of these two fiber systems are also different. In the adult, the climbing fiber makes a significant number of contacts with the dendrites of one Purkinje cell; therefore, a single discharge from a climbing fiber can trigger a burst of impulses from the Purkinje cell. However, mossy fibers activate many granule cells, which in turn innervate Golgi cells, an inhibitory input to mossy fibers. The overall output of mossy fibers to activate Purkinje cells is, therefore, not as great as that of climbing fibers (Berne and Levy, 1988). Together with its various influences on the descending motor pathways to modulate ongoing movements, cerebellum has been reported to be one of the most important structures that coordinate the muscular activity and regulate the rate, range, force and direction of movements (Frye and Fincher, 1988; Berne and Levy, 1988).
2. The effect of ethanol on adult and developing cerebellar neurons The cerebellar Purkinje cells are integrally involved in the control of motor coordination, which can be altered by ethanol administration (Stinchcomb et al., 1989; Jackson and Nutt, 1992; Yoshida, 1993). Therefore, in order to explore the underlying mechanisms of ethanol intoxication, its effects on cerebellar single-unit electrical activities have been extensively studied (Deitrich et al., 1989). A number of electrophysiological studies 31 have shown that intraperitoneal injection of low doses of ethanol excite, whereas high doses tend to inhibit, the spontaneous activity of Purkinje cells (Sinclair et al., 1980; Sorensen et al., 1981). Both the rate and pattern of spontaneous activities of Purkinje cells are modified by ethanol treatment (Basile et al., 1983; George and Chin, 1984): Cells with steady firing pattern were inhibited by perfusion with 10-300 mM ethanol; cells with high frequency activity, followed by a short period of electrical silence, showed a reduced period of repetitive spiking by ethanol (George and Chin, 1984). In contrast to the firing rate, the regularity of Purkinje cell firing in cultures of cerebellar neurons was increased by ethanol treatment (Franklin and Gruol, 1987). This increased regularity of firing of Purkinje cells was not a generalized effect of ethanol on all cerebellar neurons because the pattern of granule cell firing became more irregular under the same ethanol treatment (Deitrich et al., 1989). The cerebellum of both young and adult animals is highly sensitive to ethanol (Phillips and Cragg, 1982; West, 1986). It has been reported that cerebellar size, neuronal differentiation and synapse formation are all affected by ethanol exposure in the developing rat cerebellum (Mohamed et al., 1987; Nathaniel et al., 1986; Phillips, 1985; Smith et al., 1986; Hamre and West, 1993). However, these effects are dependent on the time that the brain is exposed to ethanol during development. It has been suggested that the CNS is most vulnerable to ethanol during brain growth spurt
(Hamre and West, 1993). In humans, the brain growth spurt starts 32 from approximately the fifth prenatal month and reaches the peak at birth, whereas in rodents it occurs in the first two postnatal weeks (Dobbing, 1985; Hamre and West, 1993). This is an important period since the formation of granule cells, arborization of Purkinje cell dendrites, and synaptogensis in the cerebellum all occur during this period (Dobbing and Sands, 1979). Hamre and West (1993) have reported that cerebellar weights of rats at Postnatal day 21 are significantly reduced by exposure to ethanol at postnatal day 4-7, and the number of both granule and Purkinje cells is also significantly decreased. Because the pattern of granule cell loss was similar to that of Purkinje cells, they suggested that the reduction in the number of granule cells may be due to the loss of their targets, the Purkinje cells. This susceptibility to ethanol effects in cerebellum is region-dependent: The vermis was affected by ethanol to the maximal extent; the most severe loss of granule and Purkinje cells was in lobules l-lll and IX, whereas the least severe loss was in lobules VI and VII (Phillips and Cragg, 1982; Pierce et al., 1989). These results are consistent with an earlier assertion (Altman, 1982) that lobules maturing the earliest were maximally affected by alcohol, while those maturing later were less affected. The number of synapses between parallel fibers and dendritic spines of Purkinje cells was also decreased by ethanol treatment (Smith and Davis, 1990). However, those changes were no longer observed when alcohol was administered after postnatal day 7 (Hamre and West, 1993). 33 tL GABA^ /BZ _receplo i subunit localization in the cerebellum The mRNAs which code for many of the GABA^/BZ receptor subunit variants have been localized in specific regions of the rodent cerebellum by in situ hybridization (Wisden et al., 1992; Laurie et al., 1992; Zdilar et al., 1991; Zdilar et al., 1992; Luntz- Leybman et al., 1993). Each cerebellar neuron expresses a subset of the GABAa /BZ subunit variant mRNAs; these are summarized in table 3. The Purkinje cell contains the a1, (32, (33, and y2 variants
(Malherbe et al., 1990; Laurie et al., 1992; Zdilar et al., 1992; Luntz-Leybman et al., 1993). Among these subunits, a1 mRNA is most abundantly expressed, and its translated protein carries the binding sites for benzodiazepines (Sigel et al., 1983; Casatotti et al., 1986; Vitorica et al., 1987) and share some pharmacological properties with ethanol (Abel, 1984; Deitrich et al., 1989). The (32 and (33 subunits carry the GABA binding site (Casalotti et al., 1986;
Mamalaki et al., 1987) which controls the Cl" channel activity. These channel responses to GABA can be modulated by benzodiazepines (Olsen and Venter, 1986). However, recombinant
GABAa receptors expressed in Xenopus oocytes, composed of a and [3 subunits, show only a weak modification of GABA responses by benzodiazepines (Schofield et al., 1987; Malherbe et al., 1990). The benzodiazepine sensitivity appears to be conferred on recombinant
GAB A A-receptors only when the y2 subunit is coexpressed with a and (3 subunits (Pritchett et al., 1989), suggesting that the y subunit 34 Table 3. The localization of GABA/\/BZ receptor subunits in the adult cerebellum localization in the subunit variants Reference cerebellum Purkinje celts a l + + + + + Schofield et at., 1989; y2 + + + + Malherbe et al., 1990; |12 + + + Laurie et al., 1992; Zdilar P3 + et al., 1992; Luntz- Leybman et al., 1993 Granule cells a6 + + + + + Montpied et al., 1989; a l + + + + Shivers et al., 1989; y2 + + + + Khrestchatisky et al., p2 + + + + 1989; Ymer et al., 1989b; 5 + + + + Kato, 1990; Zhang et al., P3 + + + 1991; Pollard et al., Y3 + a4 + 1991; Laurie et al., 1992; Zdilar et al., 1992; Varecka et al., 1993 Stellate/basket cells a l + + + Montpied et al., 1989; y2 + + + Levitan et al., 1988; P2 + + Wisden et al., 1989; Ymer p3 + et al., 1989b; Zdilar et al., 1992; Laurie et al.. 1992 Deep cerebellar nuclei a l + + + + + Ymer et al., 1989b; Zhang y2 + + + + et al., 1991; Laurie et al., p2 + + + 1992; Zdilar et al., 1992; p3 + + Luntz-Leybman et al., 1993 Bergmann glia cells a2 ++ Olsen and Tobin, 1990; pi + + Luddens and Wisden, 1991; yl + + Ymer et al., 1990; Wilcox et al., 1992; Laurie et at., 1 992; + + + + + very strong signal + + + + strong signal + + + moderate signal + + weak signal + very weak signal 35 is essential for the complete benzodiazepine response. There are two different forms of y2 subunit generated by alternative splicing: the long form of y2 subunit (y2L) carries 8 more amino acids than the short form (y2S), and this 8-amino-acid encodes a serine phosphorylation site for protein kinase C (Kofuji et al., 1991). While Purkinje cells express only 4 subunits, many more of the 15 known subunits are expressed by granule cells. In addition to those subunits expressed in Purkinje cells, a6, a4, y3, and 8 are also found in the granule cells (Montpied et al., 1989; Khrestchatisky et al., 1989; Kato, 1990; Laurie et al., 1992; Zdilar et al., 1992; Varecka et al., 1993). The a6 subunit is expressed exclusively in granule cells (Kato, 1990; Varecka et al., 1993), and carries the diazepam-insensitive binding sites for the ethanol antagonist Ro 15-4513. Similarly, although expressed in a much lower level, the a4 subunit also carries Ro 15-4513 binding sites (Khrestchatisky et al., 1989). These two subunits possess different pharmacological properties from the other a subunits, and have been classified as a third class of benzodiazepine receptor subunits (other than type I and II receptor) (Luddens and Wisden, 1991). The y3 subunit has a distribution pattern similar to that of y2 subunit mRNA (Wiison-
Shaw et al., 1991) and also confers benzodiazepine sensitivity on
recombinant GABAa /BZ receptors (Knoflach et al., 1991). The 8
subunit, which is associated with muscimol binding sites and has no benzodiazepine binding affinity (Shivers et al., 1989), is also expressed in the granule cells. 36 As in Purkinje cells, the at, p2, p3, and y2 subunits are expressed in the molecular layer (stellate and basket cells) and deep cerebellar nuclei (DCN). These subunits in the DCN are expressed at approximately the same level as in Purkinje cells, suggesting the receptor compositions in both cell types are very similar. This may be related to the fact that both deep cerebellar neurons and Purkinje cells originate in the ventricular germinal zone (Miale and Sidman, 1961), and have similar size and morphology. In addition, both of them receive inhibitory GABAergic inputs: Deep cerebellar neurons being innervated by Purkinje cells, the sole inhibitory output of the cerebellum, whereas Purkinje cells receive GABAergic innervation from stellate and basket cells.
Another cell type, the Bergmann glia, also contains GABAa receptor subunits. These cells are located at the interface of the Purkinje cell and granule cell layers; they synthesize GABA neurotransmitter and mediate the GABAergic transmission (Martin and Rimvall, 1993). The a2, (31, and y1 subunit mRNAs have been found in Bergmann glia cells (Laurie et al., 1992; Zdilar et al.,
1992), and may represent the native astrocytic GABAa /BZ receptor.
L Hypothesis An important cerebellar pathway involves granule cells which activate Purkinje cells, which in turn innervate the DCN and influence a variety of neurons in the CNS. If ethanol affects cerebellar function, the cells involved in this pathway should also be affected. The GABAa /BZ receptor is widely distributed in these 37 cells; therefore, the response of the receptor to ethanol might be a compensatory response of cellular function to ethanol administration. Two hypotheses were tested in this dissertation: 1 )
GABAa /BZ subunit mRNA levels are differentially affected by ethanol treatment. 2) Different length of ethanol exposure time produces different effects on GABAa /BZ subunit mRNA expression. CHAPTER II PREPARATION OF RIBONUCLEOTIDE PROBES AND GENERAL METHODOLOGY
A, Ethanol treatment 1. Animals C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and maintained in the vivarium on a 12 hour light and 12 hour dark cycle. Mice are nocturnal and their peak activity occurs in the dark cycle. The 12 hour dark cycle was set up from 8:00 AM to 8:00 PM to match our normal experimental schedule. Before they were assigned to one of the experimental procedures, the mice were fed with mouse pellets and water ad libitum.
2. Acute ethanol treatment Twelve adult (40-60 days old) female C57BL/6J mice were injected intraperitoneally with a single hypnotic dose of ethanol (4.0 g/Kg; 0.02 ml 25% ethanol/g of body weight). Twelve control mice were given an equivalent volume of physiological saline. Five minutes after ethanol injection, the mice began to lose their motor coordination and lay down on the cage floor with fast, shallow
38 39 breathing. About 15 minutes later, the mice remained lying on the cage floor and their hind legs were extending and contracting for approximately 10 minutes. The behaviors of these ethanol treated mice gradually returned to normal, but had not completely recovered after 30 minutes of injection. Mice injected with saline behaved normally until sacrificed. Both ethanol-treated and control mice were sacrificed 30 minutes after injection.
3. Chronic ethanol treatment Twelve adult (40-60 days old) female C57BL/6J mice were given a nutritionally complete liquid diet (Dyets Co.), according to the methods of Morrow et al. (1992) with slight modifications. Mice were initially given the liquid diet ad libitum for 4 days to acclimate to the diet. They were then fed the diet containing 5% (v/v) ethanol for 7 days, and 7.5% ethanol for the following 7 days. Another 12 control mice were pair-fed an equicaloric diet with dextrose substituted for ethanol. During this period, liquid diet was the only source of food and liquid available to the mice. The body weight and dietary consumption of each mouse was measured every day to monitor the health of the animals. Both ethanol treated and control mice of acute and chronic groups were anesthetized with
CO 2 and decapitated following the end point of ethanol administration; brains were rapidly removed, frozen on dry ice and stored at -70°C. 40 4. Prenatal Ethanol Treatment The liquid diet (Dyets Co.) containing 5% (V/V) ethanol were given to six pregnant C57BL/6J mice from gestational day (GD) 12 to postnatal day (PD) 7 of the pups. Six control pregnant mice were fed with equicaloric dextrose-contained diet following the same schedule as that of ethanol-treated groups. The presence of a vaginal plug, an indication of copulation occurring, in thefemale mouse was designated as GD 1 (Smith and Davies, 1990), and PD 1 was defined as the day of birth of the pups. Mouse pups, anesthetized with metofane (Pitman-Moore Inc., NJ) were sacrificed at PD 7; brains were removed and stored at -70°C.
5. Blood ethanol concentration measurement Alcohol dehydrogenase (ADH) catalyzes the oxidation of ethanol to acetaldehyde. Nicotinamide adenine dinucleotide (NAD) is reduced to NADH by the following reaction: Equation 2: Ethanol + NAD -AQ tL> Acetaldehyde + NADH The increased absorbance at 340 nm due to the increased amount of NADH is directly related to blood ethanol concentration (BEC). After decapitation and removal of the brain, blood was obtained from the trunk. The blood sample was first deproteinized by slowly adding 0.2ml blood sample to 1.8ml of trichloroacetic acid solution (Diagnostic Alcohol Reagents, Kit No. 332-UV, Sigma). The deproteinized blood sample stood at room temperature for about 5 minutes to complete the reaction, and then was centrifuged at approximately 2000 rpm at 4°C for 5 minutes to obtain a clear 41 supernatant. The NAD-ADH Single Assay vials (Diagnostic Alcohol reagents, Sigma) were labeled as TEST and BLANK for the assay of the supernatant of deproteinized blood sample. Powdered alcohol dehydrogenase was coated on the bottom of each vial, and dissolved by addition of 2.9ml of glycine buffer (Diagnostic Alcohol Reagents, Kit No. 332-UV, Sigma). One hundred microliters of protein-free supernatant was dispensed into each TEST vial, and 10Optl of deionized water was distributed to the BLANK vial as a background control. These vials were capped tightly, mixed by gentle inversion, and incubated for 10 minutes at room temperature. The BLANK sample was first read at 340nm in the spectrophotometer (SPECTRONIC 1201, Milton Roy Co.) to compensate for the background, followed by reading the absorbance of each TEST sample. The readings were completed within 10 minutes.
B. Riboprobe synthesis 1. mRNA isolation by oligo (dT) chromatography mRNA was isolated from mouse brain using a modification of the method of Badley et al. (1988). Two whole brains (approximately 1 gram) were dissected within 30 seconds and immersed into liquid nitrogen in a sterile 50ml polypropylene centrifugation tube. The frozen brains were transferred into a mortar, which was pre chilled in dry ice, and ground by a pestle. The slurry of the ground tissue was poured into a sterile 50ml polypropylene tube, and the liquid nitrogen left to evaporate. Upon evaporation, 22.5ml of lysis buffer (0.2M NaCI; 0.2M Tris-CI, pH 7.5; 1.5mM MgCI2; 2% SDS; 42 200ng/ml proteinase K) was added to the powdered brain tissue. The suspension was pulled and pushed by a syringe through 18 gauge needle until the tissue was completely dispersed. Incubation at 4 5°C for 120 minutes led to the complete solubilization of the tissue. After incubation, additional 21 gauge needle was used to homogenize the suspension again. The NaCI concentration of lysate was adjusted to 0.5M and used for affinity chromatography on a powdered oligo (dT) cellulose carrier, prepared as follows: Sixty milligrams of oligo (dT) cellulose (Boehringer) was suspended in 10ml of elution buffer (0.01 M Tris-CI, pH 7.5) and spun down slowly by means of a hand- driven centrifuge; the pellet was resuspended twice with 10ml of binding buffer (0.5M NaCI; 0.01 M Tris-CI, pH 7.5). The lysate was incubated with the cellulose carrier for 20 minutes at room temperature (RT) on a rotary mixer. The mixture of lysate and cellulose carrier was centrifuged and the supernatant was discarded. The pellet was resuspended with 14ml of binding buffer and centrifuged as above. The pellet was then resuspended with 5ml of binding buffer and transferred to a disposable Poly-Prep chromotography column (Part No. 731-1550, Bio Rad). The column was washed with 50ml of binding buffer. The mRNA in the column was then eluted with 2.5ml of elution buffer. The elution was mixed with 6.25ml of 100% EtOH and 0.25ml of 3M NaAc, pH 5.2, and incubated overnight at -20°C to precipitate the mRNA. On the following day, the mixture was centrifuged for 15 minutes at
12,000 x g at 4°C (Beckman microcentrifuge); the pellet was 43 collected and washed with prechilled 70% ethanol, and vacuum dried at room temperature overnight. The pellet was then solubilized in
10-15pl diethylpyrocarbonate (DEPC) treated H 2 O at 65°C, and a
1pl aliquot of mRNA was taken for measuring the concentration. The concentration of the preparation was obtained by spectrophotometry. The addition of 1pl of the preparation to 1ml
DEPC-H 2 O usually results in an absorbance value between 0.02 to
0.1 O.D. unit (One O.D. unit corresponds to the concentration of approximately 40 pg/ml).
2. Reverse transcription During reverse transcription, a single strand of complementary DNA is synthesized from mRNA to provide a template for amplification of a gene segment of interest. The GeneAmp Kit (Perkin Elmer Cetus) used in the reverse transcription reaction includes master mix and random hexamers. The master mix is composed of 5mM MgC^ 1 x PCR Buffer, 1mM of dATP, dGTP, dCTP, and dTTP, 1 U/pl RNAase inhibitor, and 2.5U/pl reverse transcriptase. The master mix, together with 0.25jiM of random hexamers and 1pg mRNA, was incubated at room temperature for 10 minutes. This allowed the extension of the hexameric primers by reverse transcriptase. Upon raising the reaction temperature to 42°C, the extended hexameric primers remained annealed to the mRNA template. One additional tube, containing 15pl of master mix and 5pl of DEPC-H 2 O without mRNA, served as negative controls (-
RT). All tubes were incubated in a Perkin-Elmer Cetus DNA thermal 44 cycler at 42°C for 40 minutes to initiate reverse transcription, and at 99°C for 10 minutes to inactivate reverse transcriptase. These tubes were then stored at 4°C for the following polymerase chain reaction.
3. Polymerase chain reaction (PCR) This procedure uses the template synthesized from reverse transcription to amplify a gene segment. The reagents used for polymerase chain reaction are from GeneAmp RNA PCR Kit (Perkin Elmer Cetus). The PCR Master Mix of 59.5pl was dispensed into a tube with 10pl of reverse transcription product. PCR Master Mix was composed of 2mM MgCl2, 1 x PCR Buffer (10mM Tris, 50mM KCI, pH 8.3), and 2.5U/100jil AmpliTaq polymerase. Downstream (sense) and upstream (antisense) primers (0.2pM each) were dispensed into the tube; micropipette tips were changed between primer additions to avoid sample carryover. A tube containing all the above components, except the reverse transcription product, served as a control. Primers which include sequence for restriction enzyme Bam HI and Hind III were used to synthesize sense and antisense probes, respectively. The surface of the PCR mixture was covered by a small amount of mineral oil (Sigma) to prevent evaporation during the high temperature reaction. The tubes were centrifuged at 4°C for approximately 30 seconds to separate the water and oil phases. The temperature cycling for PCR was as follows: 1st step-cycle: 3 minutes at 94°C 2nd step-cycle: 1 minute at 94°C, and 1 minute at 60°C (a1, p2, and 45 y2) or 55°C (a6 and (33), and 1 minute at 72°C for 30 cycles 3rd step-cycle: 5 minutes at 72 °C Soak: 4 °C In the PCR cycling steps, the double stranded template was denatured at 94°C to form a single strand cDNA; the primers were annealed to a single strand at 60°C (In general, this temperature should be lower than the melting point of primer); the extension of primers along the template was initiated at 72°C. At the end of PCR, the mineral oil was extracted by 10Ojal of chloroform. The mineral oil was dissolved in the chloroform layer in the lower part of the tube, and the upper phase was carefully transferred to a clean microcentrifuge tube and briefly centrifuged at 4°C. The tube was then checked to see if there was any residual chloroform layer on the bottom. Amplificates were then stored at -20°C . The size of PCR product was checked by electrophoresis. The 2% gel was made by dissolving 0.6g of agarose in 30ml of 1 x TAE buffer (40mM Tris-acetate; 2mM EDTA), then heated until the agarose slowly boiled and dissolved. Agarose was cooled to 60°C and 30|il of 0.5mg/ml ethidium bromide (EtBr) (Boehringer) was added to the gel mix. Both ends of the gel tray of the electrophoresis apparatus (Bio Rad) were sealed with an adhesive tape, and the comb was placed in the proper position (1-2cm away from the end of the gel). The apparatus was kept in a horizontal position to prevent uneven distribution of the gel. The gel solution was poured into the gel tray, and solidified for about 30 minutes at 46 room temperature. The tapes and comb were then removed, and the tray with gel was placed in the electrophoresis tank covered with a thin layer of 1 x TAE buffer several millimeters thick. The 10ptl PCR product was mixed with 4pl of 5 x loading buffer (TAE, Ficoll, and bromophenol blue) and 6pl of H 2 O; the sample was loaded into the wells with a micropipette. Micropipette tips were changed between samples to prevent carryover. The gel was run at 60 volts until the tracking dye migrated through two thirds of the gel length. Thereafter, the gel was removed carefully, the sample size was checked under UV light, and the gel was photographed with a Polaroid instant camera.
4. Ligation of amplified fragments into pBluescript II SK+ phagemid vector The ligation procedure inserts the gene segments of interest produced by PCR into a plasmid for further amplification. Amplificates from PCR were purified through Nensorb 20 purification columns using the following procedures: The column was fixed on a stand with a clamp, and washed with 3ml of methanol and 3ml of reagent A (according to the protocol from NEN, 100mM Tris-CI, pH 7.7; 10mM triethylamine; 1mM disodium or dipotassium EDTA). The PCR product (40|il), dissolved in 0.5ml of reagent A (100mM Tris-CI, pH 7.7; 10mM triethylamine; 1mM disodium or dipotassium EDTA), was applied on the column, and washed with 2ml of reagent A and 2ml of water. The PCR product was eluted with 1ml of 50% methanol. The first 0.5ml was 47 collected and dried in a SpeedVac Savant Drier. The pellet was then dissolved in 17jil of DEPC-treated water at 65°C and stored in a 0.5ml SafeLock Eppendorf tube. Approximately 17 pi of purified amplificate was mixed with 2pl of restriction buffer B (supplied with restriction enzymes kit; 10mM Tris-HCI, 300mM KCl, 1mM EDTA, 1mM dithiothreitol, 0.015% Triton X-100, 50% glycerol, pH 7.4; Boehringer) in a 0.5 ml Eppendorf tube, and digested with 0.5pl of Bam HI {10U/pl) and Hind III (10U/pl) enzymes. A Perkin-Elmer Cetus DNA thermal cycler was used for incubation, with the following program: 120 minutes at 37°C, 10 minutes at 70°C, and infinitely at 4°C. The Hind III (5U) and Bam HI (5U) restriction enzymes were used to digest 0.5pl of Ipg/pl pBluescript II SK+ phagemid. After digestion, the restriction enzymes were heat-inactivated at 70°C for 10 minutes. The plasmid then was treated with 2pl of calf intestine alkaline phosphatase (CIAP) (1 U/pl) and 4pl of 10 x CIAP buffer (Boehringer) in 14pl of water for 45 minutes at 37° C to prevent reannealing. The dephosphorylated plasmid (vector) and restricted amplificate (insert) were purified by electrophoresis on a 1% NuSieve (or Sea Plaque) mini-agarose gel, prepared in TAE buffer. The bands corresponding to vector and insert sizes were excised under long wavelength UV light (366 nm) by razor blades; the smallest possible regions were excised so that the dilution of bands by the gel was minimal. Razor blades were changed between each dissection in order not to cross-contaminate vector and insert 48 bands. A small portion of gel which contained no sample was excised and used as a control. If necessary, the agarose gel was * cooled in order to improve handling. The gel containing vector and insert was melted at 70°C and maintained at 37°C for the ligation procedure (Kalvakolanu and Livingston, 1991). Three Eppendorf tubes were labeled as Vector, Vector+Ligase, and Vector+Ligase+lnsert. Each tube contained 5pl of molten vector and 40pl of 2 x ligase buffer. The Vector+Ligase and Vector+Ligase+lnsert tubes contained additional 3pl of 1U/pl T4 DNA ligase. The Vector+Ligase+lnsert tube contained an additional 35pl of molten insert. The components in the Vector and Vector+Ligase tubes served as controls for restriction efficiency and dephosphorylation efficiency, respectively. All tubes were incubated overnight at 17°C. After incubation, the ligation mixtures in each of the three tubes were melted at 70°C for 10 minutes, and supplemented with 80pl of 2 x ice cold TCM buffer (20mM Tris, pH7.5, 20mM CaCl2, and 20mM MgCl 2 -stock) and 160pl of competent
E. coli cells {Transfinity/Subcloning Efficiency DH5 alpha Competent Cells, BRL). These components were vortexed briefly, and kept on ice for 1 hour. The suspension was then heat-shocked for 2 minutes at 45°C, and diluted with 1ml sterile Luria-Bertaini medium (LB medium) without ampicillin. The diluted suspension was incubated in a shaking water-bath for 60 minutes at 37°C and centrifuged at 12,000 x g at 4°C for 12 seconds, and the supernatant was removed. The pellet from each of the three tubes was resuspended with 100pl of LB medium without ampicillin and 49 smeared on a sterile LB plate containing 75pg/ml ampicillin. LB plates and medium were prepared as follows: A mixture of 10g Bacto/Tryptone (Difco), 5g NaCI, 5g Bactoyeast extract {Difco), and 15g agar (Difco) was dissolved in 1000 ml water and adjusted to pH 7.2-7.4. It was then autoclaved for 15 minutes at 125°C, and poured onto Petri dishes. For making plates with ampicillin, 75mg of ampicillin was added to the above preparation at the point that the temperature of the medium decreased to 50°C. These LB plates were stored at 4°C. LB medium was prepared as above without the addition of agar. Three LB plates labeled as Vector, Vector+Ligase, and Vector+Ligase+lnsert were incubated overnight at 37°C. In plates with vector alone, or vector + ligase, there were no colonies, whereas between 1 to 10 colonies grew in the Vector+Ligase+lnsert plate.
5. Small-scale preparation of pBluescript SK+ plasmid (Miniprep) Miniprep procedure was used to select a colony with purer insert for doing maxiprep (large scale plasmid preparation). A sterile platinum wire was used to pick up 8-12 individual colonies, and each was transferred to a 15ml sterile polypropylene tube containing 5ml LB medium with 75pg/ml ampicillin, and incubated overnight in a 37°C waterbath with agitation. If the bacteria were grown successfully, the LB medium became turbid. The samples were stored at 4°C. A quantity of 1.5ml bacterial culture from each 50 tube prepared from the ligation steps was transferred to an Eppendorf tube for the small-scale preparation of the plasmid (Miniprep). Each tube was centrifuged for 5 minutes at 12,000 x g at 4°C, and the supernatant was discarded. Care was taken to remove all the supernatant since any medium remaining on the pellet could dramatically reduce the yield of plasmid. The pellet was then suspended in 100pl of Miniprep lysis buffer (25mM Tris-CI, 10mM EDTA, 50mM glucose), and incubated for 5 minutes at room temperature. Two hundred microliter of freshly prepared solution containing 0.2M NaOH, and 1% SDS was then added. Tubes were mixed by inversion, and incubated for 5 minutes on ice. After incubation, the suspension became viscous but transparent, and 150pl of ice- cold potassium acetate (3M KOH, 5M acetic acid, pH 4.8) was added and mixed by inversion, without vortexing. After 5 minutes of incubation on ice, the suspension formed a white precipitate. The precipitate was then sedimented by centrifugation for 10 minutes at 12,000 x g at 4°C. The pellets were reconstituted with 20pl of
0.5mg/ml DNase free RNAase A (Boehringer), and incubated at 37°C for 20 minutes (final volume was 300jil). The plasmid was extracted by the addition of the same volume of phenol:chloroform:isoamyl alcohol (25:24:1). The mixture was vortexed for 1 minute and centrifuged for 5 minutes at 12,000 x g at 40C. The upper phase was carefully transferred to a new set of test tubes, and re-extracted by chlorofornrisoamyl alcohol mixture (24:1) by vortexing for 1 minute. The upper aqueous phase was transferred to a new set of test tubes (about 0.25ml per tube), 51 followed by addition of 0.63ml of ice-cold absolute ethanol to precipitate the plasmid. The solution was mixed by inversion, incubated for 10 minutes on dry ice, and centrifuged for 15 minutes at 12,000 x g at 4°C. The pellets were washed with 1ml of 70% ethanol, and vacuum dried. Dried pellets were dissolved in 16jil of deionized water, and 3.2pl of 5M NaCI and 20pl of 13% w/v PEG 8000 (Boehringer) was added. The mixture was vortexed, incubated for 20 minutes on ice, and centrifuged for 15 minutes at 12000 x g at 4°C. After discarding the supernatant, the pellet was washed with 1ml of pre chilled 70% EtOH, vacuum dried, and solubilized in 40pl of water by heating 5 minutes at 65°C. The Bam HI and Hind III endonucleases were added to each tube. These restriction enzymes were used to check the presence of insert and purity of plasmid. The enzymes (0.5pl of each endonuclease) were applied to 17pl of plasmid solution, and supplemented with 2pl of restriction buffer B (supplied with restriction enzymes kit; 10mM Tris-HCI, 300mM KCI, 1mM EDTA, 1mM dithiothreitol, 0.015% Triton X-100, 50% glycerol, pH 7.4; Boehringer). The mixture was incubated for 120 minutes at 37°C, and 10 minutes at 70°C, then soaked at 4°C in the Perkin-Elmer Cetus DNA thermal cycler. The restricted plasmids were separated on a 2% agarose gel, and the same volume of non-treated plasmid was taken as a control. The suspension which produced the plasmid with the highest purity was taken for the large scale plasmid preparation. 52
6. Large-scale plasmid isolation (Maxiprep) One ml of the suspension with the purest plasmid found in the Miniprep was inoculated into 250ml of the LB medium containing 75(ig/ml ampicillin, and cultivated overnight at 37°C in a 4 liter conical flask within a shaking waterbath in order to ensure the proper aeration for the suspension. The cultivated suspension was dispensed into 6 sterile 50ml Corning centrifugation tubes and centrifuged at 5000 x g (6500 rpm in JA-20 Beckmann rotor) at 4°C for 10 minutes. The supernatant was discarded, and the cell pellets were resuspended in 10ml of freshly prepared lysis buffer (25mM Tris/CI, pH 8.0, 10mM EDTA, 15% (w/v) sucrose, 2mg/ml muramidase lysozyme (Boehringer) using a sterile 10 ml pipette. The suspension was then incubated in ice/water mixture for 20 minutes. Thereafter, 12 ml of 0.2M NaOH and 1% SDS was added, with carefully and thoroughly mixing by inversion, and incubated in ice/water mixture for 10 minutes. After the initial incubation, 7.5 ml of 3 M Na acetate, pH 4.6, was added to the suspension, followed by incubation for 20 minutes on ice. The suspension was then centrifuged at 12,000 x g (10,500 rpm in Beckman JA-20 rotor) at 4°C for 15 minutes. The supernatant with plasmids was transferred to other test tube without disturbing the white pellet; 5-7jil of 10mg/ml RNAase solution was added to the supernatant, and incubated for 20 minutes at 37°C. 53 The plasmids were extracted with one volume of phenol:chloroform:isoamyl alcohol mixture (25:24:1) by vortexing for 5 minutes, centrifuged at 12,000 x g for 10 minutes, and the upper aqueous phase was collected and extracted again as follows. The upper aqueous phase was collected, mixed with one volume of chloroform:isoamylalcohol mixture (24:1), and centrifuged as above. The upper phase was then transferred to a fresh tube, and two volumes of absolute ethanol were added; the solution was mixed, incubated on dry ice for 30 minutes, then centrifuged for 20 minutes at 12,000 x g at 4°C in 10 ml polyethylene tubes with rubber adapters. The pellet was dissolved in 1.6ml water, and mixed with 0.32ml of 5M NaCI and 0.2ml of 13% w/v PEG 8000 solution (Boehringer) to separate small nucleotides from plasmid DNA. The plasmid DNA was then incubated in ice/water for 60 minutes, and centrifuged for 10 minutes at 12,000 x g at 4°C. The pellet was then washed with 70% EtOH, vacuum dried in a desiccator overnight, and dissolved in 100pl DEPC water at 65°C for 5 minutes. The concentration of the plasmid (1jil of plasmid diluted with
1ml H2 O) was checked spectrophotometrically at A260 and A280.
One optical density unit was equivalent to 50 pg/ml of plasmid. The ratio of A260 to A280 was usually 1.5, an indication of plasmid purity. The amount of plasmid obtained from one large-scale preparation was between 250 and 750|jg. The plasmid was stored in aliquots at -20°C. 54 7. Linearization of the insert-containing plasmids for transcription Bluescript plasmid containing al, a6, (52, p3 or y2 cDNA inserts were linearized with Hind III and Bam HI restriction enzymes (Boehringer, Indianapolis) for the subsequent production of antisense and sense RNA probes. Each plasmid (10pg) was linearized with SOU of either Bam HI, or Hind III, in a final volume of 100|il, using a PCR program, BamHind (120 minutes at 37°C, 10 minutes at 70°C, and infinitely at 4°C). The linearized templates, together with 400pl of reagent A (100mM Tris-CI, pH 7.7, 10mM triethylamine, 1mM disodium or dipotassium EDTA), were purified through Nensorb 20 columns (see section 4, page 45). A volume equivalent to 500 ng of plasmid was taken for DNA electrophoresis in 2% agarose gel to check whether the plasmids were cut successfully by both enzymes.
C, Sequencing double-stranded plasmid template 1. Alkali denaturation of supercoiled plasmid DNA The sequence of the insert was checked to see if it was the desired segment. The double stranded plasmids were converted to a single stranded form prior to sequencing. This was accomplished by alkali denaturation of supercoiled plasmid DNA by the following procedures: A volume containing 4pg (about 2pmol) of supercoiled plasmid DNA and 18pl of deionized H 2 O was pipetted to a microcentrifuge tube, followed by addition of 2pl of a 2M NaOH and 2mM EDTA solution. The plasmid was then incubated for 5 minutes 55 at room temperature. The mixture was neutralized with 2pl of ammonium acetate, pH 4.6, mixed with 75pl of absolute ethanol, and incubated on dry ice (or at -70°C) for 10 minutes. After incubation, the tube was centrifuged for 10 minutes in a microcentrifuge at 4°C; the supernatant was decanted and plasmid pellet was washed with 200pl cold 70% ethanol.
2. Probe labeling The purified single strand plasmid DNA was mixed with 4|il of
M13 primer (1.3 pmol/pl stock), 11|il of H2 O, 1pl of a-35S-labeled dATP (Amersham, SJ.1304, 1000 Ci/mmol, 10 mCi/ml), and 4pl of labeling mix (0.57 U/pl AmpliTaq DNA polymerase, 0.86pM each dGTP, dATP, dCTP, and dTTP, 143mM Tris-CI, pH 8.8, 20mM MgCI2, stabilizers; AmpliTaq sequencing kit, Part No. N808-0035; Perkin Elmer Cetus). This solution was mixed, centrifuged briefly, incubated for 5 minutes at 45°C, and then put on ice for the following procedures. The end-labeling reaction was terminated by the addition of G, A, T, C termination mix (as shown below). Four microliter of termination mix was dispensed into 4 eppendorf tubes, each containing 4pl aliquots of the labeling solution prepared from the previous step, and incubated for 5 minutes at 72°C. After incubation,, tubes were kept on ice, and 4|il of the stop solution (95% formamide, 20mM EDTA, 0.05% Bromphenol Blue, 0.02% Xylene Cyanol FF) was dispensed into each tube. The mixture was stored at -20°C up to one week before sequencing. 56 The composition of each termination mix
G termination mix: 20pM dGTP, 20pM dATP, 20pM dCTP, 20pM dCTP, 60pM ddGTP A termination mix: 20pM dGTP, 20pM dATP, 20pM dTTP, 20pM dCTP, 800(iM ddATP, T termination mix: 20pM dGTP, 20(iM dATP, 20pM dTTP, 20pM dCTP, 1200pM ddTTP C termination mix: 20pM dGTP, 20(iM dATP, 20pM dTTP, 20pM dCTP, 40(iM ddCTP
3. Gel preparation and electrophoresis The 6% acrylamide gel used for sequencing the plasmid DNA was prepared as follows: Three components, 28.5g acrylamide (DNA- sequencing grade), 1.5g N.N'-methylenebisacrylamide, and 210g ultrapure urea, were dissolved in 50ml 10 x TBE buffer (0.89M Tris base, 0.89M boric acid, 0.01 M EDTA, pH 8.2), and adding water up to 500ml in a waterbath at 55°C, and filtered through 0.45pm nitrocellulose fitter; 75ml of this solution was mixed gently with 50pl TEMED (N.N.N'.N'-tetramethylethylenediamine, Bio Rad), and 500pl of 10% (w/v) ammonium persulfate. The mixture was then poured into the sequencing mold. The bonded glass (upper plate) of the sequencing mold was siliconized with Sigmacote (10% dichlorodimethylsilane in 1,1,1-trichloroethane, Sigma) to prevent the gel sticking to the glass plate. The gel was poured continuously with extreme care, so as not to create any bubbles in the sequencing mold. While pouring the gel, the top of the mold was elevated a few 57 inches to allow the gel to flow easily. Immediately after the gel solution flowed out of the bottom of the mold, the mold was laid horizontally for even distribution of gel on the glass plate. After the gel was mounted in the frame, the flat side of the sequencing comb was inserted into the top of the gel to create wells for loading the sample solution. After one hour of initial polymerization, the bottom of the mold was then covered with 1 x TBE buffer-soaked kimwipes, and both sides of the mold were wrapped with Saran wrap to prevent the gel from drying. The gel was polymerized overnight at room temperature. The gel mold was inserted into the base of the sequencing apparatus and both upper and bottom reservoirs were filled with the 1 x TBE buffer (up to 500 ml each). The comb was removed, and a 10 cc syringe equipped with a thin hypodermic needle was inserted between the plates to remove excessive gel and urea. The comb was then re-inserted with teeth downwards and pressed gently into the gel so that the teeth entered the gel about 1mm deep, and the space between each comb tooth served as wells for sample loading. The sequencing apparatus was connected to the power supply, and the gel was run for about 40-60 minutes at 1700 V until the temperature of the gel was about 45°C. Thereafter, the power supply was turned off, and the wells were cleaned with 1 x TBE buffer by using the syringe and needle as described above. The [35S]dATP labeled G, A, T, C termination mixes were denatured immediately before loading by heating 5 minutes at 80°C, and 2pl of each mix was loaded in each well. The temperature was checked 58 regularly, since a temperature over 55°C increases the risk of breaking the glass plate. After xylenol cyanine tracking dye migrated to the middle of the mold (about 60 minutes after sample loading), another 2pl of each mix was loaded to the wells next to the first set of samples, and the procedure was repeated.
After running the gel, the power supply was turned off, and the gel mold was unlocked. It was transferred to a bowl, washed with water, and laid on a metal tray covered with water-resistant Benchcote in a horizontal position. After unmounting clamps from the gel mold, a razor blade was inserted to separate the plates, and the gel fell down to the lower plate. The glass plate with the gel was then transferred to a bowl filled with 2 liters of fixing solution (10% methanol, 10% acetic acid in water). After 15 minutes of washing in the fixing solution, the gel was covered by a piece of 3MM Whatman chromatographic paper and transferred to a porous support. The upper part of the gel and paper was cut with a razor blade to fit the porous support. The gel was covered by the Saran wrap and vacuum dried on a gel dryer (Bio Rad) for 7 hours at 80°C; a concentrated solution of NaOH or KOH was used in a trap for acetic acid remaining from the fixing solution. When gel was completely dry, the Saran wrap was removed carefully and the gel was ready for autoradiography. 59 4. Autoradiography
The gel was exposed to Kodak XOMAT film for 2 days at room temperature in a X-ray cassette with amplifying plates. After exposure, the film was developed in Kodak X-ray developer (1:20 dilution with water) for 4 minutes, briefly washed in water and fixed for 5 minutes in Kodak X-ray fixer. After drying at room temperature, the film was then placed on a light box and the sequence of the inserted nucleotides was assessed.
D. Northern blot analysis Northern blot analysis was utilized to determine the molecular size of each GABAa/BZ receptor subunit mRNA (ai,a6,
P2. P3. and y2 )■ The procedures are shown as follows: 1. Labeling riboprobes with [32pjuTP The labeling mixture was prepared at room temperature by pipetting components in the following order: 1. 5 x transcription buffer (200mM Tris, pH 8.0, 40mM MgCI2, 10mM spermidine, 250mM NaCI) 2. 0.3pl of 0.75M DTT 3. 0.5|il of RNAase block II (Stratagene) 4. 1.2pl of each NTP (excluding UTP) 5. 0.5(il of template (Hind III restricted a1, a6, p2, p3, or y2
tem plates) 6. 0.5pl of T3 DNA-dependent RNA polymerase (Stratagene) 7. 5.0pl of a 32P- labeled UTP (Amersham PB 40383, 800 Ci/mmol,
40 mCi/ml 60 The labeling mixture was mixed, centrifuged, and incubated at 3 7 °C for 60 minutes. It was then treated with DNAse for 30 minutes at 37°C by adding the following reagents: 3jil of 5 x transcription buffer, 10.4pl of water, 0.6pl of 0.75M DTT, 0.5pl of RNAase-free DNAase (stock 10 U/|il), and 0.5pl RNAase block II (1 U/pl). Transcripts were then purified through a Nuctrap column.
2. RNA electrophoresis Messenger RNAs were extracted from whole mouse brains, and run through a gel to be separated based on their sizes. The mRNA was electrophoresed through a formaldehyde gel under RNAase-free conditions (Fourney et al., 1988). The gel was prepared by dissolving 0.3g agarose in 25.5ml boiling water. When the gel was cool, 3ml of 10 x MOPS buffer (0.2M MOPS, 50mM Na acetate, 10mM EDTA, pH 7.0) was added, followed by 1.5ml of 37% formaldehyde. The gel was covered with 1 x MOPS and pre-run for 5 minutes at 70 V just before loading the RNA sample. About 10-15jig of mRNA dissolved in 2jil, and O.Sjil of 125mM EDTA and 0.5% SDS solution was added, so that the final concentration of each reagent was 25 mM and 0.1%, respectively. The samples were then diluted with 12.5pl of sample buffer (150pl of 10 x MOPS, 240pl of formaldehyde, 100pl of water, 100|il of glycerol, 80pl of 10% bromophenol blue). The samples were heated at 65°C for 15 minutes, and 1pl of 1 mg/ml EtBr was added into each sample. The samples were then mixed and loaded into the pre-run gel, and run for 120 minutes at 70 V. RNA molecular weight marker II ( Boehringer) was used in parallel to 61 check the size of each subunit mRNA. The gel, together with a ruler underneath, was photographed to examine the bands of mRNA. Two main bands of mRNA with molecular sizes of 5 kb (28S RNA) and 1.9 kb (19S RNA) were seen under UV light. After electrophoresis, the gel was washed for 5 minutes in 200ml of DEPC H 2 O, twice; 30 minutes in 200ml of a solution containing 50mM NaOH and 150mM NaCI; and 30 minutes in 200ml of a solution containing 100mM Tris- Cl (pH 8.0) and 150mM NaCI.
3. Northern blot Messenger RNAs on the gel were transferred to a Immobilon- S™ membrane (Millipore) by using the following blotting technique. The membrane with the same dimensions as the gel was soaked in 10 x SSC for 10 minutes. A long strip of Whatman 3MM paper was placed on a solid support in a container containing about 500ml 10 x SSC; both ends of the paper strip were immersed in the solution to supply hydrophilic force for blotting. The gel was laid on the Whatman 3MM strip surface upside down. The presoaked Immobilon- S membrane was then placed on the gel. Two pieces of Whatman 3MM paper pre-soaked with 10 x SSC with the same dimension as the gel was put on top of the membrane. Two additional dry strips were then laid on top of the wet 3MM paper. The paper was covered with Saran wrap membrane in which the middle portion was cut with the size slightly smaller than the dimension of the gel. This was to prevent solution coming up from the sides of membrane since such a side flow of 10 x SSC could diminish the efficacy of the blotting. A 62 tower of paper towels about 15 cm high was placed on the Saran wrap, and a glass bowl, about 0.5 kg, was loaded on top of the paper tower to produce weight for increasing soaking ability. After blotting overnight, the gel, together with the Immobilon-S membrane, was removed carefully and put on chromatographic paper with the membrane on the bottom. The position of wells were marked on the membrane and the gel was discarded. The membrane was then placed face up in the UV source (Stratagene linker) to crosslink RNA to the membrane. Thereafter, the membrane was rinsed briefly in 2 x SSC and kept in between 2 sheets of chromatographic paper in RNAase - free conditions.
4. Hybridization Membranes were prehybridized in 25 ml of prehybridization washing solution (5 x SSC, 50% formamide, 0.1% N-laurylsarcosine
Na salt, 0.02% SDS, 5% blocking reagent, DEPC-H 2 O) in a plastic container, and incubated for 2 hours at 62°C with agitation. Membranes were hybridized as follows: The membrane blots were transferred to another container with 25 ml of prehybridization washing solution containing 1 x 10® cpm/ml of [32P] labeled probe. The membrane was incubated overnight at 62°C with shaking. The membranes were then washed twice in 50 ml of posthybridization washing solution I (2 x SSC and 0.1% SDS) for 10 minutes at room temperature, and twice in 50ml of posthybridization washing solution II (0.1 x SSC and 0.1% SDS) for 15 minutes at 62°C. After washing, the blots were placed between 63 two sheets of Saran wrap and taped to prevent the blots from drying.
The well positions of the blots were marked with phosphorescent marker (Stratagene) which was illuminated for about 3 minutes with white light prior to exposure. The blots were then exposed to the Kodak XOMAT film. After 2-7 days of exposure, the film was developed in Kodak X-ray developer diluted 1:20 for 4 minutes, washed with water, and fixed for 10 minutes in Kodak X- ray fixer.
E. In vitro transcription of linearized template The restriction enzyme-treated templates were used to synthesize sense and antisense probes. The reagent used in this step was the Stratagene RNA transcription kit (Stratagene, La Jolla). The reaction mixture for the synthesis of sense probe was composed of 1 x transcription buffer (40mM Tris, pH 8.0; 8mM MgCI2; 2mM spermidine; 50mM NaCI), 20mM DTT, 0.5U RNAase Block II, 400pM each of GTP, ATP, and CTP, 10nM of Bam Hl-treated template, 20pM of 35S-labeled UTP (800 Ci/mmol), and 25U of T7 RNA polymerase. The reaction mixtures were prepared at room temperature because the DNA might be precipitated by spermidine when prepared on ice. The components for the antisense probe reaction mixture were the same as those of sense probe mixture; however, Bam Hl-treated template and T7 polymerase were replaced by Hind Ill-treated template and T3 polymerase. Samples were incubated for 60 minutes at 37°C. 64 After incubation, 15pl of the digestion mixture (6pl of 5 x transcription buffer, 20.8pl water, 1.2pl 0.75M DTT, 1.0pl of RNAase-free DNAase, and 1.0pl RNAase Block II) was added to both antisense and sense transcription mixtures, and mixed by flicking the tip of the tubes. The mixtures were then briefly centrifuged and incubated for 30 minutes at 37°C. Thereafter, the samples were purified through a Nuctrap push column (Stratagene, La Jolla) by the following procedures: The transcription mixture was diluted with 1x STE buffer (100mM NaCI, 20mM Tris-CI, pH 7.5, 10mM EDTA) to 70jil. The push Nuctrap column was moistened with 70pl 1x STE buffer, and the sample was pushed through the column by a syringe. Another 70pl of 1 x STE buffer was applied to wash away the residue. The fluid was collected in the Eppendorf tube, and 1pl of the eluate was taken for measuring radioactivity. One microliter of 100mM dithiothreitol was added to prevent disulphide bond formation and the final volume of eluate was about 130pl. The probes were stored at -20°C.
F. Jn situ hybridization 1. Tissue preparation
Mice were decapitated after inhalation anesthesia (CO 2 or Metofane, Pitman-Moore Inc., NJ). The brains from the ethanol treated and control animals were mounted onto cryostat chucks and frozen in dry ice. Coronal sections of 20|im were cut in a cryostat at between -10 to-15°C, and thaw-mounted onto 3 x subbed slides (1g gelatin and 0.1 g chromium potassium sulfate added to 200ml 65 DEPC-H 2 O; the mixture was stirred and heated to boiling, then filtered and allowed to cool before use). Adjacent sections were divided into set A, B, C and D, and stored at -70°C. Different sets of sections were used for riboprobes or radioligand binding to determine the effect of ethanol on GABAa/BZ receptor subunit mRNA or protein levels, respectively.
2. Prehybridization wash Prior to the hybridization procedures, slides stored at -70°C were brought to room temperature to dry for 5 minutes. The prehybridization washing procedures were performed at room temperature in Coplin jars (Fisher). All solutions and laboratory supplies were RNAase-free. All water was DEPC-treated (1ml of DEPC in 1000ml of deionized water, autoclaved for one hour). Sections were fixed in 4% paraformaldehyde (freshly prepared by mixing 8% formaldehyde with 2 x PBS, PH 7.4) for 30 minutes, followed by 2 x 5 minutes washes in 1 x PBS. PBS was rinsed off with 0.1 M TEA for 1 minutes. Sections were then acetylated (0.25% acetic anhydride diluted in 0.1M triethanolamin/0.9% NaCI, pH 8.0) for 10 minutes at room temperature. Thereafter, the sections were washed twice in 2 x SSC for 2 minutes and dehydrated through 50%, 70%, 90%, 95% and 100% EtOH for 1 minutes. The sections were then dried at room temperature in a dust-free environment. 66 3. Hybridization
The compositions of solution A, B, and C needed for hybridization mix were as follows: Solution A: 5% formamide was deionized by mixed anion/cation AG 501-X8 (Bio Rad), and filtered through a 0.45pm nitrocellulose f ilt e r . Solution B: 40ml of solution containing 4g dextran sulfate (M.W. 500,000), 8ml 20 x SSC, 0.8ml Denhard's solution (10mg/ml Ficoll, 10mg/ml bovine serum albumin, 10mg/ml polyvinylpyroldione, Sigma), 0.8ml tRNA (yeast) of 12.5mg/ml stock, and 4 ml 1M DTT. Solution C: 10mg/ml salmon testes DNA (Sigma). The hybridization mix was prepared by mixing 0.5ml of solution A with 0.38 ml of solution B, 50pl of solution C which was boiled 10 minutes at 90°C just before mixing, and 70pl of DEPC-
H 2 O and probe mixture. The radioactivity of the probe is approximately 1.0- 2.5 x 106 cpm/100pl. For the hybridization step, the slides were heated on a slide warmer (50°C) covered by a sheet of paper to prevent possible radioactive contamination. Hybridization mix (100pl) prepared as above (preincubated to 50°C) was distributed in several drops on each slide. The slides were covered with parafilm cut to the same size as slides, distributing the hybridization mix evenly across the slides; slides were then put on a paper-covered tray and incubated at 50°C for 20-24 hours in an incubator with 100% humidity. 67 4. Posthybridization washing
Three sets of autoclaved Coplin jars were filled with different washing solutions: the first set of jars contained 1 x SSC for rinsing the slides to remove excessive radioactivity; the second set of jars contained 2 x SSC for incubation before RNase treatment, and the third set of jars were used for incubating slides with RNase and different concentrations of SSC solution. These jars were kept separately from each other and from the other glassware and labware in order to avoid contamination with the RNase. Pipettes and forceps were also stored separately for RNAase-free and RNAase-treatments. After hybridization, the jars were filled with 1 x SSC, 2 x SSC and RNAase mix. Individual slides were picked up with a forceps and immersed into jars containing 1 x SSC. The parafilm paper was removed carefully while slides were still inside 1 x SSC. The slides were then incubated in 2 x SSC for 10 minutes at room temperature, followed by incubation with RNase (20pg/ml RNase A in 10mM Tris-HCI, 500mM NaCI, 5mM EDTA, pH 8.0). Jars were put into beakers containing pre-warmed water and incubated in a 37°C water bath for 30 minutes, and then washed at increasing
stringency, as follows: 2 x SSC (10 minutes), 1 x SSC (10 minutes), 0.5 x SSC (10 minutes), 0.25 x SSC (60 minutes) at 70° C, and 0.25 x SSC (10 minutes) at room temperature. After the last wash, the slides were dehydrated in increasing concentrations of EtOH (50%, 70%, 90%,95% and 100%) for 1 minutes each. Slides were then stored with desiccant at room temperature until used for 68 autoradiography. Ten mM 2-mercaptoethanol was added to all washing solutions, with the exception of RNase A solution, to prevent disulphide bond formation. The non-specific hybridization signal was obtained by adding sense RNA probe to adjacent control sections and processing under the same conditions as antisense probes.
& ImmunQcylQchemiatry Adult mice were deeply anesthetized with pentobarbitol (Nembutal sodium, Abbott Laboratories; 75 mg/kg) and perfused intracardially with 10 ml 1 x phosphate buffer saline (PBS), followed by 30-50 ml 4% paraformaldehyde in 1 x PBS, using a syringe and 27-30 gauge needles (with short bevels) under a dissection microscope. After perfusion, the brains were dissected and further fixed by immersion in 4% paraformaldehyde in 1 x PBS for 4 hours. The brains were then immersed in 10% sucrose in 1 x PBS at 4°C and allowed to equilibrate for cryoprotection for approximately 12-18 hours. The fixed brains were mounted onto cryostat chucks with Tissue-Tek (Lab-Tek Products, Naperville) and quickly frozen in powdered dry ice. Coronal sections (60pm) were cut on a cryostat at -20°C, removed from the knife with a 1xPBS- moistened fire-polished Pasteur pipette, and collected in a test- tube.
Immunocytochemical localization of GABAa receptor (32/3 subunits and Calbindin protein were obtained by use of an avidin- biotin complex labeling method. The (32/3 monoclonal antibody, mAb 69 62-3G1 {Dr. Angel de Bias, University of Missouri, Kansas City; de Bias et al., 1988) and Calbindin monoclonal antibody (Sigma) were used in the ethanol treatment studies. All incubations were performed with gentle agitation in 1.5ml polypropylene microcentrifuge tubes. Transferals of sections from tube to tube was made with a Pasteur pipette, fire-polished and curved at the tip . Sections were pre-incubated with 0.2% Triton X-100 in 1 x PBS (pH 7.4) for 30 minutes. Following incubation with 0.3% hydrogen peroxide {H 2 O 2 ) in methanol for 30 minutes to destroy endogenous peroxidase activity, sections were placed in 10% normal horse serum (NHS) for 1 hour at room temperature prior to immunoreaction. After 2 x 5 minutes washes in cold 1 x PBS, sections were incubated for 16 hours at 4°C with the mAb 62-3G1 (1:100 dilution in 1 x PBS) or Calbindin antibody (1:1,000 dilution in 1 x PBS) containing 0.2% Triton X-100, 1% NHS, and 1% bovine serum albumin. After incubation with mAb, sections were then washed 3 x 10 minutes in cold 1 x PBS. Antibody binding to corresponding sites were revealed by reaction with a biotin-labeled antibody (horse anti-mouse IgG), followed by an avidin-biotin horseradish peroxidase complex (ABC "Vectastain" procedure, Vector Labs).
Following 3 x 10 minutes washes, sections were transferred to a solution of 0.5% diaminobenzidine (Sigma) in 0.05 M Tris/ 0.9%
NaCI/ 0.01% H2 O 2 for approximately 30 seconds to one minutes at room temperature to express the brown color reaction. Finally, the sections were rinsed in 1 x PBS, mounted onto glass slides and 70 allowed to dry on a warm plate for about 2 hours. Sections were then washed and dehydrated in ascending concentrations of ethanol (50%-100%) and then immersed in xylenes, coverslipped and examined by light microscopy. Controls were processed as above, in the absence of the primary antibodies.
H. Radioligand binding Alternate sets of sections were processed for the measurement of GABAa /BZ receptor binding sites:
[3H]Flunitrazepam binding: Sections were incubated in 170mM Tris- HCI buffer, pH 7.4, containing 2nM [^Hjflunitrazepam (80Ci/mmole, Amersham) for 40 minutes at 4°C. Thereafter, sections were washed twice for 5 minutes in ice-cold buffer alone to remove unbound radioactivity, and then dipped for 10 seconds into ice-cold deionized water to remove residual salts. Control slides for nonspecific binding were labeled in the presence of 1 pM diazepam, and processed as above. [3H] Muscimol binding: Sections were preincubated in 1 x phosphate buffered saline (PBS), pH 7.4, for 30 minutes at 4°C, followed by a 40 minutes incubation in 1 x PBS containing 5 nM [3H]muscimol (12 Ci/mmole, Amersham) at 4°C. Sections were then washed (2 x 30 sec) in ice cold buffer to remove unbound radioactivity, followed by dipping into ice cold deionized water to remove residual salts. Control slides for nonspecific binding were labeled in the presence of 200 pM GABA, and processed as above. The labeled sections were then dried in a stream of cold air and stored at 4°C until required 71 for autoradiography.
L Autoradiography The slides of both in situ hybridization and radioligand binding studies were processed for autoradiography. Number 0 coverslips (25 x 75mm, Corning, New York), previously cleaned with 20% sulfuric acid/80% nitric acid and rinsed with double-distilled H 2 O , were coated with a uniform layer of Kodak NTB-2 photographic emulsion (diluted 1:1 with double distilled H 2 O) at 42°C , briefly cooled on a cold plate, solidified at room temperature overnight, and then stored at 4°C with desiccant. Before use, emulsion coated coverslips were brought out to room temperature for 4-5 hours to assure that they were completely dry; they were then apposed to the slide-mounted sections. A backing slide was then placed on top of the emulsion coated coverslip forming a sandwich-like assembly. These slides were clamped together with binder clips under minimum sodium safelight illumination, and put into lightproof boxes containing desiccant. The exposure time for each ligand or cRNA probe varied, and was determined experimentally. In general, cRNA probes were exposed for 4-6 days, while radiolabeled ligands were exposed for 2-3 weeks. After specific period of exposure, the emulsion-coated coverslips were developed in Kodak Dektol developer (diluted 1:1) for 2.5 minutes at 17° C, washed with deionized water for 10 seconds, and fixed for 3.5 minutes in Kodak Rapid Fix. Sections were then dehydrated in different concentrations of alcohol (50%, 75%, 90%, 95% and 100%) for 1 72 minutes each. The coverslips were finally mounted onto clean microscope slides with DePeX (Biomedical Specialities, Santa Monica, CA), and used for quantification analysis. After exposure to emulsion coated coverslips, sections used for in situ hybridization experiments were dipped in liquid emulsion (42°C, diluted 1:1) in order to localize the autoradiographic grains to a particular cell type. Sections were dipped, dried overnight at room temperature and stored in light proof boxes with desiccant at 4°C. Autoradiographic grains were developed as above, and sections were counterstained with 0.5% cresyl fast violet (0.5g Cresyl Violet in 250 ml of 0.1 M Na acetate buffer, pH 3.6). Because reversible ligands dissociate from their receptors at the temperature that liquefies emulsion, this method of dipping sections with emulsion was not appropriate for radioligand autoradiography. Autoradiograms were observed and photographed (Ilford Pan-F film) under dark or bright-field illumination using either a Nikon SMZ-10 binocular dissecting microscope (low power/whole sections), or a Nikon Optiphot compound microscope (high power/grain counts).
J. Quantification The Image-1 optical density measurement program (Universal Imaging Corporation) was used to measure grain density in autoradiograms which was a measure of the level of subunit mRNA expression. Through such measurements, the effect of ethanol on 73 the expression of various GABAa/BZ subunit mRNAs was then determined. Optical density within known areas were obtained from 10 randomly selected regions within the cerebellar Purkinje cell layer, granule cell layer, molecular layer and deep cerebellar nuclei of the same section. Ten sections from each animal for each probe were selected and measured. The average of these optical density measurements from each ethanol-treated and control animal was expressed as mean ± standard deviation (S.D.), and the sample number (n) was 100. The results for each probe were reproduced in triplicate (3 animals), and the average data was expressed as mean ±_ standard error (S.E.), where the n was equal to 3. The measurements for control and ethanol-treated sections were performed under the same conditions, such as light intensity, magnification, gray and black level, and image contrast, to reduce the experimental variation to the minimum. Since the relationship between optical density and radioactivity was not linear, a standard curve was required to convert the internal video pixel gray level value, which has a linear relationship with the optical density value, to a calibrated number. Because of its very long half life (5730 years), and similar decay energy to that of 35S, 14C-labeled brain paste standards were used to estimate optical density by quantitative autoradiography (Miller, 1991). The 14C-labeled standard was exposed along with other tissue samples to calibrate the relationship between the optical density of the autoradiogram and concentration of the 35S-labeled probes used in in situ hybridization experiments. 74 Additional measurements utilizing an object counting program was used to count the number of grains within a known area. An additional program was used to mathematically separate overlapping grains which would normally be defined as a single object and, thus, lead to an under-estimate of grain density. The data obtained from optical density measurement and object counting program was analyzed by using Systat 5.2.1 statistical program {SYSTAT Inc.) to test for significant changes in subunit mRNA levels induced by ethanol in various cerebellar cell types. Data were analyzed using Student's t test. CHAPTER III RESULTS
A. Probe design and specificity
Of the presently known GABAa /BZ receptor subunits, only the a t, p2, P3 and Y2 subunit variants are present in the cerebellar Purkinje, granule, and deep cerebellar neurons (Malherbe et al., 1990; Laurie et al., 1992; Zdilar et al., 1992; Luntz-Leybman et al., 1993); the a6 subunit, which contains binding sites for the ethanol antagonist Ro 15-4513 (Palmer and Hoffer, 1989; Lister and Nutt, 1987; Britton et al., 1988; Suzdak et al., 1988a, b), is uniquely expressed in granule cells (Kato, 1990 ; Laurie et al., 1992; Varecka et al., 1993). In the present studies, ribonucleotide probes against these subunits were synthesized to study the effects of ethanol on
GABAa /BZ receptor. Figure 3 shows the laminar organization of the cerebellar cortex, and the autoradiographic distribution of these five mRNAs is shown in figure 4. Mouse-specific sense and antisense ribonucleotide probes were designed to recognize a specific segment of mRNA which codes for the most variable portion of the intracellular loop between the M3 and M4 transmembrane spanning domains of the five subunits, in order to maximize specificity of hybridization. This intracellular loop exhibits the least sequence identity among the
75 76 various GABAa/BZ receptor subunit cDNAs, and is, thus, unique to each subunit mRNA. The sequence of the upstream and downstream primers, based on the published rat or mouse sequence (a1: Wang et al., 1992; a6: Kato, 1990; (52/3: Ymer et al., 1989a; y2: Kofuji et al.,
1991), with Hind III or Bam HI restriction sites and three additional nucleotides, are shown in table 4. Northern blot hybridization, using mouse cRNA riboprobes conducted under high stringency conditions, was used to determine the number and size of the mRNA species recognized by theai, a6, 02, 03 and y2 probes. Each of the riboprobes was specifically designed to include regions complementary to the most variable portion of the corresponding mRNA. These experiments showed a different molecular size for each subunit mRNA: 4.0 and 4.3 kilobases for a i, 1.4 and 2.2 kilobases for a6. 8.2 kilobases for (52. 6.1 kilobases for 03, and 4.3 kilobases for y2 (Table 5). No cross hybridization between these subunit mRNAs was observed.
B. Blood ethanol concentration and body weight Blood ethanol concentration and individual body weight, were measured during the period of ethanol administration. Blood ethanol concentration was used as an index to monitor whether the animal was under the effect of ethanol, and to determine the point at which the peak concentration was reached. The period of acute ethanol administration was determined by the time at which the mouse had the highest blood ethanol concentration after a single injected dose of ethanol. As shown in figure 5, the maximal blood ethanol 77 concentration (332mg/100ml) was reached at 30 minutes after a single intraperitoneal injection of 4g/Kg of ethanol. One hour after ethanol injection, the blood ethanol level (319mg/100ml) remained similar to the maximal level. Thereafter, the blood ethanol was gradually metabolized. Two hours after ethanol injection, less than two thirds of maximal blood ethanol concentration {189mg/100ml) remained in the blood stream. The majority of ethanol was metabolized after 6 hours of injection (24mg/100ml). Twenty four hours after injection, the blood ethanol concentration returned to baseline. Therefore, the period of 30 minutes after injection was selected to determine the acute effect of ethanol on GABAA/BZ receptor subunit expression. Blood ethanol concentration of control and ethanol-treated mice in acute and chronic treatment groups was measured at the end of each ethanol administration to ensure animals were under the effect of ethanol. In acute ethanol treatment, the average blood ethanol concentration of six ethanol-treated mice was 332.1 mg/IOOml, and not detectable in saline-treated mice. After chronic ethanol treatment, six mice receiving ethanol-containing liquid diet had a blood ethanol concentration of 106.1 mg/100ml; the blood ethanol concentration in animals treated with equicaloric dextrose-containing liquid diet was not detectable. The blood ethanol concentration of chronic ethanol-treated mice was less than that of acute ethanol-injected mice, since the ethanol concentration in the liquid diet mixture was much lower than that used for injections. The blood ethanol concentrations of acute 78 ethanol- and chronic ethanol-treated mice are shown in table 6. Animals were fed a normal laboratory diet of pelleted chow before ethanol treatment, and were therefore unused to the liquid diet. The body weight of each control and chronically ethanol- treated mouse was monitored throughout the experiment to ascertain the general health of each animal. Figure 6 shows the mean body weight of both control and ethanol-treated mice with and without a nutritionally complete liquid diet administration. The body weight of both groups was measured for 3 consecutive days prior to liquid diet administration as a baseline. Mice of both groups gradually gained weight from an average of 20 grams to 22 grams. However, when the mice were first given plain liquid diet, their body weight (19 grams) decreased to slightly below the baseline levels. The decreased body weight may be due to the change of chow pellet food to liquid diet. After 5 days of acclimation, their body weight gradually returned to the normal (21 grams). From then on, both control and ethanol-treated mice expressed a steady increase in body weight showing no significant differences between groups. This suggests that chronic ethanol treatment does not alter the normal murine growth pattern.
C. The effect of chronic ethanol treatment on Purkinie
££U number In previous studies, cerebellar Purkinje cells have been shown to be affected by exposure to ethanol (Phillips and Cragg, 1982; Gruol, 1991; Hamre and West, 1993). In order to determine whether 79 ethanol administration affects Purkinje cell number, the Purkinje cells were counted in a defined cerebellar lobule following chronic ethanol treatment. A monoclonal antibody against the Calbindin and cersyl fast violet was used to identify Purkinje cells. In Calbindin studies, 53 Purkinje cells/mm for control groups and 50 Purkinje cells/mm for ethanol-treated groups were observed in cerebellar vermis; in the hemisphere, about 49 Purkinje cells/mm for control groups and 52 Purkinje cells/mm for ethanol-treated groups were observed (Fig. 7). Purkinje cells stained with cersyl fast violet also showed a similar results: 47 Purkinje cells/mm for controls and 45 Purkinje cells/mm for ethanol-treated groups were observed in the vermis; and 45 Purkinje cells/mm for controls and 44 Purkinje cells/mm for ethanol-treated groups in the hemisphere (Fig. 8). The differences between the results shown in the present studies and in the work of other laboratories may be due to differences in dose, exposure periods, age of animals tested, and routes of ethanol administration. The present results suggest that Purkinje cell number is not changed by the chronic ethanol treatment used in this study. Therefore, any downregulation of the in situ hybridization signal in Purkinje cells reflect a change in GABAa/BZ receptor subunit mRNA expression, rather than a generalized loss of Purkinje ce lls. The direct effect of ethanol on the expression of GABAa/BZ receptor subunit mRNAs was also determined. Prior to in situ hybridization, tissue sections were incubated with 100mM ethanol for 30 minutes; the ai subunit riboprobe was then applied to assess 80 the direct effect of ethanol on GABAa /BZ receptor ai subunit mRNA expression. Table 7 shows that direct application of ethanol did not change the ai subunit mRNA expression.
IL Acute ethanol administration
1. In situ hybridization with GABA a /BZ receptor ai, <*6* P2» P3, and y2 subunit cRNA probes Granule cells, Purkinje cells, and deep cerebellar neurons are major cellular components of cerebellar circuitry. Granule cells activate Purkinje cells, which in turn inhibit deep cerebellar neurons, thereby influencing a variety of neuronal systems in the
CNS. The effects of ethanol on the expression of GABAa /BZ receptor a l. a6. P2. P3. and y2 subunit mRNAs were measured in each of the three neuronal types. The mRNA hybridization signal was measured in optical density units. In each brain section hybridized with a specific subunit cRNA probe, Purkinje, granule, and deep cerebellar neurons in ten different regions were randomly selected. Six to ten sections for each animal were measured. Three mice were used for each subunit probe. Therefore, total measurements for each subunit cRNA probe in each of the three cerebellar cell types range from 180 to 300. The data shown in the following figures represent the mean with standard error, and were analyzed using Student's t test. Since the absolute value of optical densities derived from the same subunit probe may vary from one experiment to another, in addition to the raw data, the differences between control and ethanol- 81 treated animals were converted to a percentage of control values. The effect of acute ethanol administration on the expression
of the GABAa/BZ receptor a i, a 6 . P2. P3 , and 72 subunit mRNAs within the cerebellum is shown in figure 9. Both at and p3 subunit
mRNAs were increased in the adult C57/BL6J mouse cerebellum by acute ethanol treatment, while the hybridization signal generated
by a 6 , P2. and 72 probes w®r© not changed significantly. In animals acutely treated with ethanol, there was 40% increase of at mRNA in
Purkinje cells, 33% increase in the granule cells, and 55% increase in the deep cerebellar neurons (Fig. 10). An increased expression of p3 subunit mRNA was also observed in Purkinje cells (50%), granule cells (40%), and deep cerebellar neurons (55%) (Fig. 1 1 ).
In contrast to the ai and P 3 subunits, a 6 , P2 and 72 subunit mRNAs did not show significant changes in response to acute ethanol administration. Although the a6 subunit has been shown to carry an ethanol antagonist binding site (Palmer and Hotter, 1989; Lister and Nutt, 1987; Britton et al., 1988; Suzdak et al., 1988a, b), this property did not affect the a6 subunit mRNA level in granule cells 30 minutes after ethanol injection (Fig. 12). The expression of p2 and 72 subunit mRNAs in Purkinje cells, granule cell, and deep cerebellar neurons were only minimally affected by acute ethanol treatment (Figs. 13 and 14). 82 2. The effect of acute ethanol treatment on [3H]muscimol and [3H]flunitrazepam binding sites The binding sites for [3H]flunitrazepam and [3H]muscimol are located on a 1 and p2/3 subunits, respectively. Previous results have shown that the direct application of ethanol to sections did not change the levels of GABAa/BZ receptor subunit mRNA, suggesting that the direct effect of ethanol on GABAa/BZ receptor is minimal.
Therefore, the observed changes in subunit mRNA levels and the associated changes in the number of ligand binding sites are likely to be due to the effect of ethanol on mRNA translation, rather than to the direct effect of ethanol on physical structure of the receptor. The effect of acute ethanol administration on the expression of the two binding sites was examined autoradiographically. Figure 15 shows the autoradiographs of 3H]Flunitrazepam and [3H]Muscimol binding sites following acute ethanol administration. Optical density measurements were taken over the cerebellar molecular layer, granule cell layer, and deep cerebellar nuclei. Since the majority of the GABAa/BZ receptor proteins are expressed on the
Purkinje cells dendrites which arborize in the molecular layer, whereas the Purkinje cell bodies contain most of the subunit mRNAs, the radioligand binding signals were measured in the molecular layer. Both [3H]flunitrazepam and [3H]muscimol binding were increased by acute ethanol exposure: [3H]Flunitrazepam binding was increased in the molecular layer (26%), granule cell layer (37%), and deep cerebellar nuclei (35%) (Fig. 16). [3H]Muscimol binding was increased in the molecular layer (23%) and granule cell 83 layer (25%) (Fig. 17). These results are consistent with the increased expression of a1 and (33 subunit mRNAs by acute ethanol tre a tm e n t.
3. The effect of acute ethanol administration on the expression of (32/3 subunit protein
The increases in [3H]flunitrazepam and [3H]muscimol binding suggest that the expression of a and (3 subunit proteins were changed by acute ethanol administration. Immunocytochemical studies using 62-3G1 monoclonal antibody against GABAa/BZ receptor (32/3 subunits revealed an increase in antibody labeling in the cerebellar granule cells, 30 minutes after a single dose of4g/Kg ethanol injection. Figure 18 shows increased immunostaining over the granule cell layer of an ethanol treated mouse when compared to a control animal. Uncalibrated optical density measurements in the granule cell layer shows a 56% increase after acute ethanol administration (Fig. 19). This suggests that the GABAa/BZ receptor (32/3 subunit protein is upregulated by acute ethanol treatment. This result is in agreement with the increased (33 subunit mRNA and
[3H]muscimol binding sites within the same region.
Chronic ethanol administration
1. The expression of GABA a /BZ receptor subunit mRNAs after chronic ethanol treatment The effect of chronic ethanol treatment (fourteen day liquid diet containing 5-7.5% (V/V) of ethanol) on the expression of
GABAa /BZ receptor subunit mRNAs was examined. The 84
autoradiography of a i, a6, p2. P3 and 72 subunit mRNAs is shown in figure 20. In contrast to acute effect of ethanol, chronic ethanol administration reduced ai subunit mRNA levels by approximately
40% in Purkinje cells, 23% in granule cells, and 39% in the deep cerebellar neurons (Fig. 21). Conversely, ae subunit mRNA expression was substantially increased in cerebellar granule cells (approximately 40%) (Fig. 12). Even larger increases were observed in 72 subunit mRNA expression in all three cerebellar cell types
(85% in Purkinje cells; 45% in granule cells; 56% in the deep cerebellar neurons) (Fig. 22). As with acute administration of ethanol, chronic ethanol ingestion did not greatly influence the expression of p2 subunit
mRNA levels in Purkinje cells, granule cells, or deep cerebellar neurons (Fig. 23). In addition, the increased expression of p3 subunit
mRNA levels in granule cells observed after acute administration of ethanol was not observed after chronic ethanol ingestion. Instead, after chronic ethanol treatment, 03 subunit mRNA levels were slightly, but not significantly, decreased in Purkinje cells (7.3%), granule cells (6.2%), and deep cerebellar neurons (10.7%) (Fig. 24). The effects of acute and chronic ethanol administration on the expression of the GABAa/BZ receptor a i, a6, P2. P3. and 72 subunit mRNAs in various cerebellar regions are summarized in table 6. 85 2. The effect of chronic ethanol treatment on [3H]flunitrazepam and [3H]muscimol binding sites in mouse cerebellum The effect of chronic ethanol exposure on the expression of a and [3 subunit binding sites was measured. Figure 25 shows the autoradiographs of 3H]Flunitrazepam and [3H]Muscimol binding sites following chronic ethanol administration. Rather than an increase in binding as observed in acute treatment, chronic ethanol treatment decreased [3H]flunitrazepam binding in the cerebellar molecular layer, the granule cell layer, and the deep cerebellar nuclei by 25%, 22%, and 24%, respectively (Fig. 26). The decreased [3H]flunitrazepam binding is consistent with the decreased a1 subunit mRNA levels following the chronic ethanol treatment. Unlike the binding increased after acute ethanol treatment, [3H]muscimol binding over the cerebellar granule cell layer and molecular layer was unchanged by chronic ethanol administration (Figure 27).
3. The effect of chronic ethanol administration on the expression of (32/3 subunit protein
The lack of any change in [3H]muscimol binding sites in the cerebellar granule cell layer and molecular layer after chronic ethanol administration suggested that the expression of (3 subunit proteins, which contain the muscimol binding sites (Mamalaki et a I., 1987; Vitorica et al., 1987), was not affected by long term ethanol exposure. The monoclonal antibody 62-3G1 was used to examine the 86 expression of the 32/3 subunit protein after chronic ethanol treatment. Figure 28 shows that immunostaining over the granule cell layer and molecular layer was unchanged by chronic ethanol exposure. These results are consistent with the unchanged expression of P2/3 subunit mRNAs following chronic ethanol administration.
F. The effect of chronic maternal ethanol treatment on the expression of GABA^/BZ receptor and y2_ subunit mRNAs in the developing mouse cerebellum The effect of ethanol on the expression of GABAa/BZ receptor a i , a6, 32. 33. and y2 subunit mRNAs was studied in mice chronically exposed to ethanol from embryonic day 12 to postnatalday 7. Figure 29 shows the effect of chronic maternal ethanol administration on the expression of these five suubnit mRNAs in the cerebellar vermis (lobules 8 and 9). The cerebellar pattern of GABAa/BZ receptor subunit expression was, in general, similar to that of adult mice. At postnatal day 7, ai subunit mRNA levels were decreased in Purkinje cells (23%), granule cells (27%), and deep cerebellar neurons (22%) (Fig. 30. The a6 subunit mRNA level in granule cells was increased by 58% (Fig. 31). The levels of
32 and 3 3 subunit mRNAs in all three cell types were not significantly altered when compared to controls (Figs. 32 and 33), thus following an expression similar to that observed in chronically ethanol-treated adults. However, unlike the effect of chronic ethanol exposure on levels of y2 subunit mRNA in adult mice, 87 embryonic and early postnatal ethanol exposure did not substantially alter 72 subunit mRNA expression in either Purkinje cells, granule cells, or deep cerebellar neurons (Fig. 34). The effect of embryonic and early postnatal ethanol exposure on the expression
GABAa/BZ receptor a-|, a 6 , 02. P3. and 72 subunit mRNAs in various cerebellar regions is summarized in table 9. 88 Table 4. Sequence of upstream and downstream primers of GABAA/benzodiazepine receptor a-| t a6, P2, P3 and Y2 subunits.
Primer Sequence ai 5'-ATA AAG CTT AAG AGA GGG TAT GCG TGG GAT-3' upstream a i 5’-ATA GGA TCC AGG CTT GAC TTC I I I CGG TTC-3’ dow nstream
a6 5*-ATA AAG CTT GAA GGC TGA AAG GCA GGC ACA-3’ upstream a 6 5’-ATA GGA TCC AGT GGC TGG TAA GAG CAA TGG-3' dow nstream
P2 5'-ATA AAG CTT GAG AAG ATG CGC CTG GAT GTC-3' upstream
P2 5'-ATA GGA TCC GCA CGT CTC CTC AGG CGA CTT-3' dow nstream
P3 5’-ATA AAG CTT CTA GCA CCG ATG GAT GTT CAC-3* upstream
P3 5’-ATA GGA TCC TGC TTC TGT CTC CCA TGT ACC-3' dow nstream
Y2 5'-ATA AAG CTT TCA GCA ACC GGA AGC CAA GCA-3' upstream 5'-ATA GGA TCC ACT GGC ACA GTC CTT GCC ATC-3' Y2 dow nstream 89
Table 5. Northern blot analysis of size of the mRNA species recognized by the mouse-specific riboprobes for GABAa/BZ receptor a t, a6, P2. P3 and 72 subunits
mRNA size a i «6 P2 P3 Y2
Upper band 4.0Kb 1.4Kb 8.2Kb 6.1Kb 4.3Kb
Lower band 4.3Kb 2.2Kb - - - 90
Table 6. Blood ethanol concentration of acute and chronic ethanol treated mice. Acutely treated mice received either a single I.P. dose of 4g/Kg ethanol or same volume of saline. Chronically treated mice were fed with liquid diet containing 5-7.5% ethanol or equicaloric dextrose for 14 days. Data represent mean ± S.D. (n = 6 for both group).
T reatment Ethanol group Control group
Acute 332.1 ±5.7 mg/dl ND
Chron ic 106.1 ±7.2 mg/dl ND
ND: not detectable 91
Table 7. The direct effect of ethanol on the expression of GABAa /BZ receptor ai subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). Data are expressed as the number of optical density units within a defined area due to specific 35S-labeled riboprobe labeling (mean ±. S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 2 animals; n-2).
Treatment Purkinje cell Granule cell Deep layer layer cerebellar neurons
Control 10.78 ± 1.98 0.74 ± 0.13 2.55 ± 0.39
Ethanol 11.50 ± 1.92 0.83 ± 0.15 2.68 ± 0.47 92 Table 8. The summary of acute (A) and chronic (B) effects of ethanol treatment on the expression of GABAa /BZ receptor a i , a6, p2. P3. and y2 subunit mRNAs in adult mouse cerebellum. Data are expressed as percentage change of control (mean ± S.E.M.; n«3)
A.
Acute ethanol treatment (% change of control)
Purkinje cell Granule cell Deep layer layer cerebellar nuclei al +40.4 ± 4.4 +33.0 ± 4.4 +54.2 ± 5.0
a6 - +2.60 ± 3.7 - P2 -5.80 ± 3.3 -11.0 ± 3.6 -4.90 ± 2.2 P3 +49.7 ± 5.2 +55.0 ± 4.5 +39.6 ± 5.4 V2 +3.10 ± 1.6 +8.10 ± 1.1 -0.50 ± 2.1
b .
Chronic ethanol treatment (% change of control)
Purkinje cell Granule cell Deep layer layer cerebellar nuclei al -41.5 ± 3.9 -22.6 ± 2.5 -38.6 ± 2.8
a6 - +41.1 ± 5.7 - 02 +7.50 ± 2.1 +5.80 ± 2.5 +8.10 ± 3.2 (33 -7.30 ± 3.3 -6.20 ± 5.8 -10.7 ± 3.2 Y2 +84.1 ± 6.5 +44.9 ± 8.6 +55.8 ±11.2 93 Table 9. The summary of the effects of chronic maternal ethanol exposure on the expression of GABAa /BZ receptor a i , a6, P2. P3, and y2 subunit mRNAs in the mouse cerebellum. Data are expressed as percentage change of control (mean ± S.E.M.; n-3)
Fetal ethanol treatment (% change» of control)
Purkinje C. Granule C. DCN
a l -22.8 ± 2.5 -27.4 ± 0.3 -22.1 ±. 1.7
a6 - +58.3 ± 0.4 -
p2 +5.90 ± 1.5 +4.90 ± 0.1 +3.30 ±2.0
P3 +3.90 ± 1.9 +2.40 ± 0.2 +8.90 ±2.8
Y2 -6.70 ± 2.9 -5.10 ± 4.6 -4.90 ± 2.0 Fig. 3. The laminar organization of the cerebellar cortex. A: A cerebellar sagittal section stained with cresyl fast violet. B: A cerebellar coronal section photographed under darkfield illumination showing the in situ hybridization signal of the a i cRNA probe. C: Cresyl fast violet stained section photographed at high power (50x) under brightfield illumination. D: Autoradiogram of in situ hybridization signal of ai cRNA probe photographed at high power (50x) under brightfield illumination. E: A cresyl fast violet stained section photographed at high power (100x) under brightfield illumination. F. Autoradiogram of in situ hybridization signal of ai cRNA probe photographed at high power (100x) under brightfield illumination, p, Purkinje cells; g, granule cell layer; m, molecular layer; e, external germinal layer; den, deep cerebellar nuclei; b, basket cells; s, stellate cells
94 95
Fig. 3 Fig. 4. The autoradiographic distribution of the GABAa /BZ receptor <*1, <*6, P2, P3 and y2 subunit mRNAs in mouse cerebellum. A: distribution of ai subunit mRNA. B: distribution of a6 subunit mRNA. C. distribution of (32 subunit mRNA. D. distribution of (33 subunit mRNA. E. distribution of y2 subunit mRNA. F. Sense probe, p, Purkinje cells; g, granule cell layer; m, molecular layer; den, deep cerebellar neuron; w, white matter.
96 97 Fig. 5. The blood ethanol concentration of mice at different time intervals after receiving a 4g/Kg ethanol IP injection. Values shown here represent the mean ±_ S.D. (n - 3 at each time point)
98 i. 5 Fig.
Blood ethanol concentraion (mg/dl) 0 -i 400 i atrehnl treatment ethanol after e Tim 99 Fig. 6. Body weights of mice maintained on liquid diet supplemented with either ethanol or dextrose. Values shown represent the mean ± S.D. (n » 6 for both group).
100 Fig. 6
Average body weight (g) 20 - 5 2 -, 0 3 0 iud diet Liquid loo o dextrose or alcohol iud it with diet Liquid 5 10
Days 5 1 20 5 2 Control Alcohol 101 Fig. 7. The number of Purkinje cells labeled by the Calbindin monoclonal antibody (Sigma) in the cerebellar vermis (v) and hemisphere (h) of chronic ethanol treated mice and dextrose controls. Data are expressed as the number of Purkinje cells within a defined region (mean ±. S.E.M. determined from 5 paired sections, 10 measurements from each section; 2 animals; n-2).
102 i. 7 Fig.
No of Purkinje cells/mm COtsTT ■ 2 EtOH E2
103 Fig. 8. The number of Purkinje cells stained by cersyl fast violet in the cerebellar vermis (v) and hemisphere (h) of chronic ethanol treated mice and dextrose controls. Data are expressed as the number of Purkinje cells within a defined region (mean ±. S.E.M. determined from 5 paired sections, 10 measurements from each section; 2 animals; n-2).
104 31 to' cd No of Purkinje cells / mm
o cn Fig. 9. The effect of acute ethanol administration on the expression of the GABAa/BZ receptor a i, <*6, p2. P3. and 72 subunit mRNAs within the cerebellum. Left column: Saline controls. A: a i. C: a6- E: P2- G- P3- I: Y2- Right colmun: Acute ethanol treatment. B: a i. D: a6- F: P2- H: P3 . J: 7 2 . p, Purkinje cells; g, granule cell layer; m, molecular layer; w, white matter.
106 107
a> m P - 9
G
Fig. 9 Fig. 10. The effect of acute ethanol treatment on the expression of GABAa/BZ receptor ai subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n»3) due to specific 35S-labeled riboprobe labeling. * shows difference from saline- injected control at P < 0.01, paired Mest. (B) Conversion of the data listed in panel (A) to percentage change of control.
108 1 09 A.
T reatment Purkinje cell G ranule cell Deep layer layer cerebellar neurons
Control 12.43 ± 1.73 1.20 ± 0.32 3.60 ± 0.49
Ethanol 17.79 ± 1.79* 1.64 ± 0.21* 5.58 ± 0.83*
B.
o c o o O)© c © .c CJ
DC N
Fig. 10 Fig. 1 1 . The effect of acute ethanol treatment on the expression of GABAa/BZ receptor P 3 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 3 5 S-labeled riboprobe labeling. * shows difference from saline- injected control at P < 0.01, paired f-test. (B) Conversion of the data listed in panel (A) to percentage change of control.
110 111 A.
Treatment Purkinje cell G ra n u le cell Deep layer layer cerebellar neurons
Control 1.05 ± 0.17 1.15 ± 0.15 0.30 ± 0.05
Ethanol 1.55 ± 0.24* 1.77 ± 0.24* 0.40± 0.05*
100 -
o 80 - c o
P C G C D C N
Fig. 11 Fig. 12. The effects of acute and chronic ethanol treatment on the expression of GABAa/BZ receptor <*6 subunit mRNA in mouse cerebellar granule cells (GC). (A) Data are expressed as the number of optical density units within a defined area (mean ±_ S.E.M. determined from 10 paired sections, 10 measurements from each section; 3 animals; n«3) due to specific 35S-labeled riboprobe labeling. * shows difference from saline-injected control at P < 0.01, paired Mest. (B) Conversion of the data listed in panel (A) to percentage change of control. 113 A.
Treatment Acute Chronic
Control 8.79 ± 1.24 3.85 ± 0.51
Ethanol 8.86 ± 1-17 5.23 ± 0.77*
B.
100 -i O c 80 - o O 60 - 20 - GC Fig. 12 Fig. 13. The effect of acute ethanol treatment on the expression of GABAa/BZ receptor p2 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DON). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 35S-labeled riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 11 4 1 15 A. T reatment Purkinje cell Granule cell Deep layer layer cerebellar neurons Control 1.87 ± 0.35 1.69 ± 0.34 0.79 ± 0.11 Ethanol 1.78 ± 0.57 1.51 ± 0.35 0.75 ± 0.12 2 0 "l o> c ” - 6 0 - o 5s - 8 0 - . 100-1 ------P C G C D C N Fig. 13 Fig. 14. The effect of acute ethanol treatment on the expression of GABAa/BZ receptor 72 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ±. S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 3 5 S-labeled riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 11 6 1 1 7 A. Treatment Purkinje cell Granule cell Deep layer layer cerebellar neurons Control 15.15 ± 1.80 1.00 ± 0.18 2.37 ± 0.50 Ethanol 15.69 ± 1.98 1.08 ± 0.19 2.33 ±. 0.46 B. 100 i -20------P C G C DC N Fig. 14 Fig. 15. Autoradiographs of [ 3 H]flunitrazepam binding and [3 H]muscimol binding sites following acute ethanol administration. A: [3 H]Flunitrazepam binding in a control animal. B: [ 3 H]Flunitrazepam binding in an ethanol-treated animal. C. [3 H]Muscimol binding in a control animal. D. [ 3 H]Muscimol binding in an ethanol-treated animal, g, granule cells; m, molecular layer. 118 Fig. 16. The effect of acute ethanol treatment on [ 3 H]flu nitrazepam binding sites in the mouse cerebellar molecular layer (ML), granule cells (GC), and deep cerebellar nuclei (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ±. S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 2 animals; n-2). * shows difference from control at P < 0.01, paired f-test. (B) Conversion of the data listed in panel (A) to percentage change of control. 120 121 A. T reatment Molecular Granule cell Deep layer layer cerebellar nuclei Control 1.90 ± 0.34 0.36 ± 0.07 1.09 ± 0.21 Ethanol 2.40 ± 0.43* 0.49 ± 0.10* 1.44 ± 0.26* M L GC DC N Fig. 16 Fig. 17. The effect of acute ethanol treatment on [^HJmuscimol binding in the mouse cerebellar molecular layer (ML) and granule cells (GC). (A) Data are expressed as the number of optical density units within a defined area (mean ±. S.E.M. determined from 10 paired sections, 10 measurements from each section; 2 animals; n-2). * shows difference from control at P < 0.01, paired f-test. (B) Conversion of the data listed in panel (A) to percentage change of co n tro l. 122 1 23 A. Treatment Molecular G ra n u le cell layer layer Control 0.62 ± 0.11 2.66 ± 0.50 Ethanol 0.76 ± 0.14* 3.55 ± 0.45* B. o c o o O) Fig. 17 Fig. 18. The effect of acute ethanol administration on monoclonal antibody 62-3G1 immunostaining in the mouse cerebellar granule cell layer and molecular layer. Panel A: Dextrose-treated control. Panel B: Ethanol-treated group, p: Purkinje cell; g: granule cell layer; m: molecular layer; w: white matter. 124 125 , •« t ■» t -v . , ^ , 3f v • *» * m > Fig. 18 Fig. 19. The effect of acute ethanol administration on the expression of GABAa/BZ receptor (32/3 subunits in the mouse cerebellar molecular layer (ML) and granule cells (GC). Data are expressed as the number of optical density units converted from immunostaining of monoclonal antibody 62-3G1 (mean ± S.E.M. determined from 5 paired sections, 10 measurements from each section; 2 animals; n-2). * shows difference from saline-injected control at P < 0.01, paired Mest. Optical Density Units 200 1 27 Fig. 20. The effect of chronic ethanol administration on the expression of the GABAa/BZ receptor a i, a6, 32. 33. and 72 subunit mRNAs within the cerebellum. Left column: Dextrose controls. A: a i. C: a6- E: 32- G: 33- Y2- Right column: Acute ethanol treatment. B: a-|. D: a6- F: 32- H: 33- J: Y2- P. Purkinje cells; g, granule cell layer; m, molecular layer; w, white matter. 128 129 Fig. 20 Fig. 21. The effect of chronic ethanol treatment on the expression of GABAa/BZ receptor a i subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n»3) due to specific 35S-labeled riboprobe labeling. * shows difference from saline- injected control at P < 0.01, paired f-test. (B) Conversion of the data listed in panel (A) to percentage change of control. 130 131 A. Treatment Purkinje cell G ran u le cell Deep layer layer cerebellar neurons Control 29.01 ± 5.57 1.06 ± 0.16 21.06 ± 4.92 Ethanol 17.33 ± 3.48* 0.82 ±. 0.14* 12.73 ± 4.32* B. o c o u o PC GC D C N Fig. 21 Fig. 22. The effect of chronic ethanol treatment on the expression of GABAa/BZ receptor 72 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 35S-labeled riboprobe labeling. 4 shows difference from saline- injected control at P < 0.01, paired f-test. (B) Conversion of the data listed in panel (A) to percentage change of control. 132 1 33 A. T reatment Purkinje cell Granule cell Deep layer layer cerebellar neurons Control 11.95 ± 1.98 0.87 ± 0.12 2.44 ± 0.50 Ethanol 21.73 + 2.81* 1.27 0.22* 3.96 ± 0.65* B. 100 1 PC GC DC N Fig. 22 Fig. 23. The effect of chronic ethanol treatment on the expression of GABAa/BZ receptor P 2 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n«3) due to specific 35S-labeled riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 134 135 A. Treatment P u rk in je cell Granule cell Deep layer layer cerebellar neurons Control 1.76 ± 0.31 1.28 ± 0.24 1.60 ± 0.23 Ethanol 1.89 ± 0.35 1.34 ± 0.25 1.75 ± 0.28 B 100 80 c o o 60 o>® c 40 nCO o 20 Fig. 24. The effect of chronic ethanol treatment on the expression of GABAa/BZ receptor (33 subunit mRNA in mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 35S-labeled riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 136 1 37 A. T reatment Purkinje cell Granule cell Deep layer layer cerebellar neurons Control 3.07 ± 0.50 3.21 ± 0.44 0.33 ± 0.06 Ethanol 2.78 ± 0.38 2.83 ± 0.43 0.29 ± 0.05 B. ko . ——c o o O)® c CO JZo o' -100 -* ' ------PC GC DC N Fig. 24 Fig. 25. Autoradiographs of [3H]flunitrazepam binding and [3H]muscimol binding sites following chronic ethanol administration. A: [3H]Flunitrazepam binding in a control animal. B: [3H]Flunitrazepam binding in an ethanol-treated animal. C. [3H]Muscimol binding in a control animal. D. [3H]Muscimol binding in an ethanol-treated animal, g, granule cell layer; m, molecular layer. 138 Fig. 2 5 Fig. 26. The effect of chronic ethanol treatment on the [3H]flunitrazepam binding in the mouse cerebellar molecular layer (ML), granule cells (GC), and deep cerebellar nuclei (DON). (A) Data are expressed as the number of optical density units within a defined area (mean ± S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 2 animals; n-2). * shows difference from control at P < 0.05, paired Mest. (B) Conversion of the data listed in panel (A) to percentage change of control. 140 141 A. T reatment Molecular G ranule cell Deep layer layer cerebellar nuclei Control 2.71 ± 0.45 0.63 ± 0.08 1.65 ± 0.34 Ethanol 2.13 ± 0.37* 0.53 ± 0.09* 1.31 ± 0.26* 20 i O) | -60- o S2 -80 - .100 -I------■------ML GC DCN Fig. 26 Fig. 27. The effect of chronic ethanol treatment on the [3H]muscimol binding in the mouse cerebellar molecular layer (ML) and granule cells (GC). Data are expressed as the number of optical density units within a defined area (mean ±. S.E.M. determined from 10 paired sections, 10 measurements from each section; 2 animals; n-2). 142 1 43 A. T reatment Molecular Granule cell layer layer Control 0.77 ± 0.14 4.61 ± 0.61 Ethanol 0.73 ± 0.16 4.46 ± 0.63 20 1 o> | -60“ o a* -80- .100 -* ------GC ML Fig. 27 Fig. 28. The effect of chronic ethanol administration on monoclonal antibody 62-3G1 immunostaining in the mouse cerebellar granule cell layer and molecular layer. Panel A: Dextrose-treated control. Panel B: Ethanol-treated group, p: Purkinje cell; g: granule cell; m: molecular layer; w: white matter. 144 145 irr** Fig. 28 Fig. 29. The effect of chronic maternal ethanol administration on the expression of the GABAa/BZ receptor a i, <*6, P2. P3. and 72 subunit mRNAs in the P7 mouse cerebellar vermis (lobules 8 and 9). Left column: Dextrose controls. A: at. C: a6- E: (32- G' P3- I: Y2- Right colmun: Acute ethanol treatment. B: a i . D: a6- F: P2 P3- 72- Arrow, Purkinje cells: g, granule cell layer; e, external germinal layer. 146 147 Fig. 29 Fig. 30. The effect of chronic maternal ethanol exposure on the expression of GABAa /BZ receptor ai subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (OCN). (A) Data are expressed as the number of optical density units within a defined area (mean ±_ S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 35S-iabeled riboprobe labeling. * shows difference from saline-injected control at P < 0.01, paired Mest. (B) Conversion of the data listed in panel (A) to percentage change of control. 148 149 T reatm en t Purkinje cell Granule cell Deep layer layer c e re b e lla r neurons C o n tro l 21.06 ± 3.61 1.71 ± 0.41 11.73 ± 2.64 Ethanol 16.22 ±. 3.76* 1.24 ±. 0.44* 9.09 ± , 2.18* B. 0 c o o E -20 o a -40 <0 -60 O9 m*<0 c 9 -80 wO Q.® -100 PC GC DCN Fig. 30 Fig. 31. The effect of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor a6 subunit mRNA in P7 mouse cerebellar granule cells. (A) Data are expressed as the number of optical density units within a defined area (mean ±_ S.E.M. determined from 10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 35S-labeled riboprobe labeling. * shows difference from saline-injected control at P < 0.01, paired f-test. (B) Conversion of the data listed in panel (A) to percentage change of control. 150 151 A. T reatm en t Acute Chronic C o ntro l 8.79 ± 1.24 3.85 ± 0.51 Ethanol 8.86 ±1.17 5.23 ± 0.77* B. 100-1 8 0 - 6 0 - o> 4 0 - 2 0 - GC Fig. 31 Fig. 32. The effect of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor 3 2 subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ±_ S.E.M. determined from 6*10 paired sections, 10 measurements from each section; 3 animals; n«3) due to specific 35S-labefed riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 152 1 53 A. T reatm en t Purkinje cell Granule cell Deep la ye r layer c e re b e lla r neurons C ontrol 2.37 ± 0.44 1.73 ± 0.32 2.57 ± 0.46 Ethanol 2.49 ± 0.50 1.81 ± 0.32 2.69 ± 0.56 B. 100 80 c o 60 - © cO) CO 40 - -Co 20 - m z z m m 'm ssssosessm 'S/S/S/SSSSSSSSS. PC cc DC N Fig. 32 Fig. 33. The effect of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor P 3 subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (OCN). (A) Data are expressed as the number of optical density units within a defined area (mean ±_ S.E.M. determined from 6 -1 0 paired sections, 10 measurements from each section; 3 animals; n»3) due to specific 3 5 S-labeled riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 1 55 A. T reatm ent Purkinje cell Granule cell Deep layer layer c ereb ellar neurons C ontrol 3.54 ± 0.62 3.76 ± 0.56 0.42 ± 0.09 Ethanol 3.74 ± 0.54 3.86 ± 0.64 0.45 ± 0.09 B. 100 ' 80 - L.o c o O 60 - o ® PC GC OCN Fig. 33 Fig. 34. The effect of chronic maternal ethanol exposure on the expression of GABAa/BZ receptor 72 subunit mRNA in P7 mouse cerebellar Purkinje cells (PC), granule cells (GC), and deep cerebellar neurons (DCN). (A) Data are expressed as the number of optical density units within a defined area (mean ±_ S.E.M. determined from 6-10 paired sections, 10 measurements from each section; 3 animals; n-3) due to specific 3SS-labeled riboprobe labeling. (B) Conversion of the data listed in panel (A) to percentage change of control. 156 157 A. T reatm en t Purkinje cell Granule cell Deep layer layer c e re b e lla r neurons C o n tro l 5.77 ± 1.23 1.94 ± 0.47 6.25 ± 1.64 Ethanol 5.33 ± 0.99 1.83 ± 0.36 5.97 ± 1.20 B X C o o - 2 0 - e o ® o> -40 tflc -Cu a> -60 Fc O -100 PC GC DCN Fig. 34 CHAPTER IV DISCUSSION Ethanol addiction has been a serious social problem for a extensive period of time. Physical dependence on ethanol is associated with a number of organ pathologies, including neurological defects, liver disease, degenerative changes in heart and muscle, and dietary deficiencies. While much is known about the clinical outcomes of ethanol consumption, relatively little is understood about the direct effects of ethanol on the individual cell types comprising the neuronal circuits most likely to be involved in ethanol intoxication and dependence. Although ethanol research has been conducted on rats and human subjects, the mouse serves as an excellent model for studying the effect of ethanol on physiological systems, for a number of reasons. Firstly, there is a large body of literature, including behavioral (Bowers and Wehner, 1989; Stichcomb et a I., 1989), physiological (Buck and Harris, 1990; Buck et al., 1991), pharmacological (Unwin and Taberner, 1980; Ticku and Burch, 1980), biochemical (Allan and Harris, 1987), and genetic (Harris and Allan, 1989; Wafford et al., 1991), based on the mouse model, and the results are generally applicable to humans (Tran et al., 1981). Secondly, there are mouse strains with different sensitivities to 1 58 1 59 ethanol, for example, short-sleep (SS) and long-sleep (LS) mice which have been bred selectively for different ethanol sensitivities as measured by the loss of righting responses and a differential sleep-time in response to ethanol (Bowers and Wehner, 1989; Stinchcomb et al., 1989). In addition, a selective breeding program has been carried out to produce two Swiss-Webster mouse lines that are withdrawal seizure prone (WSP) or withdrawal seizure resistant (WSR) to the development of seizures caused by ethanol withdrawal (Crabbe et al., 1983). It has been shown that about 26% of the total phenotypic variance in ethanol withdrawal seizure severity in mice is genetically controlled (Crabbe et al., 1985). Therefore, the diversity of mouse strains may provide good models for genetic studies of ethanol susceptibility. Thirdly, on the practical aspects, the size of mouse is small and easy to handle, and the cost is usually lower than that of other animals. On the basis of the above advantages, mice have been the animals of choice for studies of the effects of ethanol on various physiological system s. The cerebellum is an excellent region in CNS to study the effects of ethanol since it is involved in the control of locomotor activity which is severely affected in response to ethanol. The cerebellum has been proposed as coordinator, rather than an initiator or actuator, of movements (Ito, 1984). Damage to the cerebellum results in difficulty in coordinating contractions of numerous muscles which results in abnormal movements of various body components (Ito, 1984). Abnormal behaviors, such as gross 1 60 tremulousness in conjunction with disorientation, resemble ethanol withdrawal syndromes (Little, 1991). Therefore, it is believed that the cerebellum is, at least in part, involved in ethanol-induced behavioral impairment. The laminated structure of the cerebellum is highly anatomically regular with very few neuronal types; these include stellate, basket, Purkinje, Golgi, and granule cells, distributed within the cerebellar cortex (see fig. 2). The deep cerebellar nuclei are located ventral to the cerebellar cortex on either side of the midline. This anatomical regularity provides relatively easy access to the study of the effects of ethanol on neuronal activity and synaptic transmission in CNS. Moreover, the physiology and neurochemistry of cerebellum has been described in great detail (Ross et al., 1989, 1990; Mignery et al., 1989), making it a favored site for analyzing the effects of ethanol. The various neurotransmitters released by different cerebellar neurons have been welt defined. Granule cells innervate dendrites of Purkinje cells via excitatory parallel fibers, which release glutamate (Ross et al., 1990). Glutamate activates Purkinje cells by initiating a phosphatidylinositol (PI) cycle via a G-protein-linked second messenger system, which activates the release of Ca2+ from non- mitochondrial stores (Berridge and Irvine, 1989). The increased intracellular Ca2+ concentration in turn triggers numerous Ca2 + - dependent reactions, which are responsible for Purkinje cell activation (Ross et al., 1990). Three inhibitory interneurons, stellate, basket, and Golgi cells, as well as Purkinje cells, all 161 release GABA as neurotransmitter which induces hyperpolarization of the cell membrane and reduces cellular activity (Wuenschell et al., 1986). Stellate and basket cells provide inhibitory contact to Purkinje cells, whereas Golgi cells make inhibitory synapses with the granule cells. Purkinje cells project inhibitory GABAergic terminals to deep cerebellar neurons. There is a significant number of GABAa receptors localized on granule cells, Purkinje cells, and deep cerebellar neurons, and the in situ hybridization technique has been used to identify which cerebellar neurons synthesize the various GABAa receptor subunit mRNAs (Laurie et al., 1992; Zdilar et al., 1992; Luntz-Leybman et al., 1993). A number of behavioral and biochemical studies have indicated that GABAa /BZ receptor function is affected by ethanol (Hunt, 1982; Allan and Harris, 1987; Frye and Fincher, 1988; Buck et al., 1991; Mhatre et al., 1993). Results from behavioral studies show that ethanol-induced sedation is enhanced by systemic application of GABA agonists (Martz et al., 1983; Mehta and Ticku, 1989), and audiogenic seizures induced by ethanol withdrawal can be antagonized by treatment with GABA-mimetic agents (Cooper et al., 1979). Ethanol-enhanced GABA-stimulated chloride flux in microsacs, synaptoneurosomes and cultured spinal cord neurons has also been reported in many biochemical studies (Ticku et al., 1986; Suzdak et al., 1986 a, b; Allan and Harris, 1987; Morrow et al., 1988). 162 The effects of both ethanol and benzodiazepines share many similarities, including CNS depression, muscle relaxation, decreased locomotor activity, anticonvulsant activity, respiratory depression, and hypothermia (Yu and Ho, 1990). The above ethanol- induced motor impairments have been reported to be antagonized by the imidazodiazepine Ro 15-4513, a benzodiazepine inverse agonist (Suzdak et al., 1986a; Lister and Nutt, 1987). This "anti-ethanol" effect is not shared by other benzodiazepine partial inverse agonists, such as FG-7142 and p-CCE (Suzdak et al., 1986b). Taking the above observations together, it is believed that GABAa /BZ receptors play an important role in mediating the physiological effects of ethanol, and this receptor may be important for understanding the molecular and cellular mechanisms underlying ethanol intoxication. More recently, the effects of ethanol on GABAa/BZ receptors have been studied by molecular approaches using brain homogenates (Suzdak et al., 1986; Bowers and Wehner, 1989; Buck et al., 1991; Mhatre et al., 1993). However, these homogenate studies, including Cl' flux, ligand binding and Northern blot experiments, are unable to localize the ethanol-induced changes to specific identified cell types. For example, if one cell type shows a reduction in the level of mRNA, while another cell type in the same brain region shows an increase, the net result shown by the Northern blot technique will be an average of the two results; this may, and has, caused misleading conclusions. Therefore, in the present studies, anatomical approaches, including in situ hybridization, 163 immunocytochemistry, and autoradiography, have been used to supplement the information obtained from the biochemical studies on the influence of ethanol on GABAa /BZ receptors. The in situ hybridization approach, using 35S-labeled probes to identify a particular type of mRNA in different anatomical loci throughout the brain, along with the autoradiographic localization of [3H] labeled ligand binding sites, creates high resolution images which are quantifiable and cell type specific. From these images, it is possible to identify the cell type containing a particular subunit mRNA, and to ascertain, by the presence of a specific binding site, if the mRNA is translated into a protein within the dendritic membrane. The probes used to label GABAa/BZ receptor subunit mRNAs are highly specific, since they were designed to recognize a specific segment of mRNA which codes for the least conserved nucleotide sequence of the intracellular loop between transmembrane spanning domains M3 and M4 of the GABAa/BZ receptor subunits. In addition, after probe hybridization, tissue was washed under high stringency conditions at the melting temperature (Tm) of the individual probe, which was calculated taking into account the Na+ concentration in the washing solution, the G and C nucleotide content of the probe, and the probe length. Therefore, the signals produced by 35S-labeled riboprobes were very specific to the GABAa/BZ receptor subunit mRNAs concentrated in cell bodies. The effect of acute and chronic ethanol exposure on the localization of two receptor subunit binding sites ([3H]flunitrazepam and [3H]muscimol) was also examined in order to 1 64 determine the distribution on the cell bodies and dendrites. In addition, changes in immunostaining produced by the subunit- specific antibody (62-3G1 mAb) revealed how P 2 /3 subunit protein expression affected by ethanol treatment. A. The effect of acute ethanol treatment on GAB A ^/B Z re c e p to r When mice receive a single hypnotic dose of ethanol they lose their motor coordination: Their behavioral performance on an elevated plus-maze and rotarod was suppressed dramatically when compared to controls (Stinchcomb et al., 1989). Since the pharmacological effects of ethanol, such as muscle relaxant, antianxiety, and sedative-hypnotic properties, are similar to those of the benzodiazepines (Burch and Ticku, 1980; Skolnick and Paul, 1981; Deitrich et al., 1989), it has been suggested that ethanol acts as a GABAa/BZ receptor agonist (Suzdak et al., 1986a; Allan and Harris, 1987). Previous in situ hybridization studies (Malherbe et al., 1990; Laurie et al., 1992; Zdilar et al., 1992; Luntz-Leybman et al., 1993) have shown that Purkinje cells and deep cerebellar neurons contain the cti,fJ2,P3, and 72 GABAa/BZ subunit variants. Granule cells contain a significant amount of ot6 subunit mRNA, in addition to these subunits (Kato, 1990; Laurie et al., 1992; Varecka et al., 1993). These five subunit variants are, therefore, obvious choices for studying the effect of ethanol on the GABAa/BZ receptor in a major cerebellar circuit. In the present studies, acute ethanol treatment resulted in increased levels of GABAa/BZ receptor ai and P 3 subunit mRNA levels in Purkinje cells, granule cells, and deep cerebellar neurons, whereas a 6 , p2 and y2 subunit mRNAs were unchanged in these cell types. The lack of uniform changes in the subunit mRNAs indicates that the compositions of GABAa/BZ isoreceptors are differentially regulated by acute ethanol treatment. Previous studies have shown that different receptor subunit variant combinations change receptor function. For example, various GABAa/BZ receptor subunit subtypes, ax (x= 1 , 2, 3, 5), Pi and y 2 , expressed in human kidney 293 cells, showed functional diversity in electrophysiological and pharmacological characteristics (Puia et al., 1991). Cultures of subunit-transfected kidney cells were studied with the single electrode voltage-clamp technique in the whole cell configuration. Upon the application of GABA, a Cl- current was generated by these cells. Several drugs were used to test the effects of GABA on various receptor subunit combinations. Zolpidem, a benzodiazepine agonist (Hugh et al., 1993), was shown to potentiate GABA-induced Cl" current in a iP iY 2 recombinant receptors, but failed to enhance the GABA response in a5piY2 receptors (Puia et al., 1991); this suggests that the ai subunit is more related to the effects of benzodiazepines than the 0 5 subunit. Since the ai subunit carries the majority of high affinity benzodiazepine binding sites (Casalotti et al., 1986; Vitorica et al., 1987), the increased levels of ai subunit mRNA in response to acute ethanol treatment may imply that more mRNA is available for 166 translation into the receptor subunit protein. The increase in [ 3 H]flunitrazepam binding in the cerebellar molecular layer following acute ethanol administration observed in the present studies strongly supports this assumption. In addition to the increased levels of ai subunit mRNA and [3 H]flunitrazepam binding sites, [ 3 H]muscimol labeling and mAb 62- 3G1 immunostaining also were increased by acute ethanol treatment. This suggests that the GABAa/BZ receptor p subunits are also susceptible to the effect of acute ethanol administration. However, only the P 3 subunit mRNA level was increased after acute ethanol injection, while the p 2 subunit mRNA remained unchanged. Therefore, the increased [ 3 H]muscimol ligand labeling and immunostaining of 62-3G1 monoclonal antibody are likely to be generated by the increased expression of P 3 subunit mRNA. The increased level of P 3 subunit mRNA is of particular interest as ethanol-induced phosphorylation of the GABAa/BZ receptor may be one of the mechanisms of acute ethanol intoxication. The P 3 subunit carries a unique cAMP-dependent serine phosphorylation consensus sequence as the substrate for cAMP- dependent protein kinase (PKA) in the region between transmembrane domain 3 and 4 (Feramisco et al., 1980; Sigel et al., 1991). It has been reported that GABAa/BZ receptor phosphorylation by PKA may increase GABA-induced suppression of neuronal activity (Walaas and Greengard, 1991): Application of isoproterenol, a p- adrenergic agonist, was found to enhance the inhibitory effects of locally-applied GABA on cerebellar Purkinje neuron firing rate by 167 increasing cAMP levels and activating PKA phosphorylation; however, isoproterenol together with timolol, a (5-adrenergic antagonist, showed no enhancement of GABA-induced inhibition (Lin et al., 1991). It is possible, therefore, that acute ethanol administration increases (53 subunit expression in the cerebellum, which, in turn, increases the possibility of receptor phosphorylation by PKA. Ethanol, as a benzodiazepine agonist, may then act on the phosphorylated GABAa/BZ receptor and enhance GABA-mediated neuronal inhibition. Therefore, acute ethanol-induced sedative and anticonvulsant actions may be associated with the phosphorylation process of GABAa /BZ receptor (53 subunit. The increased levels of GABAa/BZ a-j and (53 subunit mRNAs in response to acute ethanol treatment may suggest that the composition of this receptor is undergoing a form of "subunit switching". This process has been described in a variety of receptors, including the nicotinic and glycine receptors, both of which belong to the superfamily of ligand-gated ion channels. During early postnatal development, a transient appearance of receptor subunits is observed in the nicotinic receptor: the embryonic y subunit is replaced by an e subunit (Mishina et al., 1986). A similar process also occurs in glycine receptors on developing spinal neurons, where the embryonic a.2 subunit is changed to the adult a i isoform (Takahashi et al., 1992). Since the GABAa/BZ receptor also belongs to the ligand-gated ion channel superfamily, similar changes in subunit composition may occur in this receptor. Indeed, in the inferior olive, GABAa/BZ receptor (53 168 subunit mRNA has been found to be transiently expressed: high levels of P 3 subunit mRNA were observed at birth and increased during the first postnatal week, after which they decreased to low adult levels {Frostholm et al., 1992); during this time the 71 subunit is rapidly upregulated. Therefore, gene regulatory mechanisms may exist for the control of the subunit switching process. Like other members of ligand-gated ion channel superfamily, the GABAa/BZ receptors exist as pentamers of several distinct subunits (Schofield et al., 1987). Increased levels of a i and p 3 subunit mRNAs by acute ethanol administration could be due to activating the gene regulation of subunit switching in the adult animal. For example, ot‘|aiP2P372 is a possible combination in Purkinje cells, and aiot6P2P3Y2 is another possibility in the granule cells. If the subunit switching mechanism is turned on by acute ethanol administration, the increased levels of the GABAa/BZ receptor ai and P 3 subunit mRNAs may suggest more copies of ai and P3 subunit in the pentameric structures, for example, multiple a i and P3 subunits with no p 2 or 72 subunits, to form GABAa/BZ receptor complexes. Compared to a putative receptor combination containing only one oh and one P 3 subunit, the new receptor would have more benzodiazepine binding sites and PKA phosphorylation sites, and thus be more sensitive to the pharmacological effects of benzodiazepines and GABA mimetics which open the chloride ion channel. This may be one of the explanations why ethanol increases GABA-induced 3 6 CI‘ uptake into the cerebral cortex synaptoneurosomes (Suzdak et al., 1986a). 169 Unlike a i and (33 subunits, a6, P2 and y2 subunit mRNA levels in the cerebellum were not changed by acute ethanol treatment when compared to saline injected controls, suggesting the effect of acute ethanol administration does not cause generalized alterations in GABAa /BZ receptor mRNA transcription. B. The effect of chronic ethanol treatment on the G A B A & /BZ receptor When animals were chronically treated with ethanol, the GABAa/BZ receptor ai subunit mRNA level in Purkinje cells, granule cells, and deep cerebellar neurons was decreased, whereas the levels of 72 subunit mRNA in these three cell types, and a6 subunit mRNA in the granule cells were increased. In addition to the decreased expression of ai subunit mRNA, [3H]flunitrazepam binding sites were also decreased in the cerebellar molecular layer, granule cell layer, and deep cerebellar neurons. These results suggest that chronic ethanol exposure is accompanied by a downregulation in the transcription of GABAa/BZ receptor subunit genes, and a concomitant decrease in subunit protein expression. The reduction in ai mRNA expression after long term ethanol exposure may be due to an adaptive response to the enhancement of GABAergic transmission by ethanol. It is well known that the extended interaction of many neuronal transmitters with their receptor leads to decreased physiological function (Taylor and Insel, 1990). Since ethanol appears to act as an benzodiazepine agonist which augments GABAa /BZ receptor function (Suzdak et al., 1 70 1986a; Allan and Harris, 1987), the prolonged presence of ethanol could decrease the function of the receptor by downregulating the subunit mRNAs, and thus the density of GABAa /BZ receptor binding sites (Ticku and Burch, 1980). In contrast to acute ethanol administration, the level of GABAa /BZ receptor a6 subunit mRNA in cerebellar granule cells was increased following chronic ethanol treatment. The oc6 subunit is uniquely expressed in these cells, and has been reported to carry the diazepam-insensitive binding site for Ro 15-4513 (Suzdak et al., 1986a,b; Mhatre et al., 1988; Mehta and Ticku, 1989; Luddens et al., 1990; Kato 1990). Ro 15-4513, is a partial benzodiazepine- inverse agonist which acts on the GABAa /BZ receptor, and antagonizes the electrophysiological (Palmer and Hoffer, 1989), behavioral (anticonvulsant action) and biochemical effects (Lister and Nutt, 1987; Britton et al., 1988; Suzdak et al., 1988a, b) of ethanol. Ro 15-4513 not only antagonized ethanol-induced 36CL uptake into isolated brain synaptoneurosomes (Suzdak et al., 1986b), but also reversed anxiolytic effect of ethanol in behavioral studies (Stinchcomb et al., 1989). It appears that these effects were mediated via the GABAa /BZ receptor since they were abolished by the benzodiazepine receptor antagonist Ro 15-1788 (Lister and Nutt, 1987). The effects of Ro 15-4513 on the CNS depressant effects of ethanol through the GABAa /BZ receptor appears specific and selective, since other benzodiazepine inverse agonists, such as FG7142, p-CCM and (5-CCE, were not capable of reversing the effects of ethanol (Suzdak et al., 1986a; Lister and 171 Nutt, 1987). The increased level of a6 subunit mRNA found in the present study may suggest a concomitant increase of subunit protein by chronic ethanol treatment. Indeed, a 77% increase in Ro15-4513 binding sites has been shown in the rat cerebellum following chronic ethanol administration (Mhatre et al., 1988). Since Ro 15- 4513 is an ethanol antagonist, an increase in number of binding sites for this drug might be accompanied by an increase in the number of endogenous Ro 15-4513 analogs bound. It is not clear whether endogenous Ro 15-4513 analogs exist; however, several endogenous benzodiazepine inverse agonists with pharmacological effects similar to that of Ro 15-4513 have been isolated, such as octadecaneuropeptide and ethyl-p-carboline-3-carboxy late (Braestrup and Nielsen, 1981). If endogenous Ro 15-4513 compounds do exist, the increased binding sites for these compounds might tend to reduce the potentiation effect of ethanol on GABA-induced chloride influx. Following withdrawal, in order to restore normal GABAa /BZ receptor function, more ethanol would have to be consumed. This may be one of the reasons why long term ingestion of ethanol produces tolerance. After long term exposure to ethanol, y2 subunit mRNA increased dramatically in cerebellar Purkinje cells, granule cells, and deep cerebellar neurons. Using the whole cell patch-clamp technique, it has been shown that the y2 subunit is essential for the benzodiazepine potentiation of GABA-induced inward currents (Pritchett et a., 1989; Knoflach et al., 1991). Expression of a 1 72 GABAa /BZ receptor in Xenopus oocytes, which was composed only of a and p subunit mRNAs, showed only a weak modification of GABA responses by benzodiazepines (Schofield et al., 1987; Malherbe et al., 1990); benzodiazepine sensitivity was conferred on recombinant GABAa /BZ receptors only when the y2 subunit was included in combination with a and p subunits (Pritchett et al., 1989b; Knoflach et al., 1991). Alternative RNA splicing produces two forms of the 72 su bu nit variant (Whiting et al., 1990); The long form ( 7 2 L. examined in the present studies) contains an extra 8 amino acid, an insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. In the short form (y2S). this sequence is absent (Whiting et al., 1990; Wafford et al., 1991). The mRNAs of these two forms are differentially expressed in various brain regions (Mirales et al., personal communication). It has been reported that ethanol potentiation of GABA-stimulated 36CI" uptake requires the presence of the long form of 72 subunit (Wafford and Whiting, 1992). Electrophysiological studies have shown that expression of aipiy2L receptor and aiPiy2S in oocytes displays different sensitivities to ethanol; Ethanol potentiates only receptors containing the 72L subunit, whereas those containing the 72S subunit are not affected (Wafford and Whiting, 1992). Protein phosphorylation plays an extremely important role in receptor modulation, cell excitability, and neuronal function (Walaas and Greengard, 1991). The additional splice insert in the 72 subunit contains a consensus sequence which can be phosphorylated 173 by protein kinase C, and is thought to be essential for ethanol modulation of the GABAa /BZ receptor (Whiting et al., 1990; Macdonald and Angelotti, 1993). Disruption of the phosphorylation site by in vitro mutagenesis abolishes the effects of ethanol (Wafford et al., 1991; Wafford and Whiting, 1992). In these studies, the GABAa /BZ receptor subunit mRNAs were expressed in Xenopus oocytes, with the combinations of a-|PiY2L and aiPiY2S. both of which elicited Cl" current in responses to application of GABA. The GABA-induced current was potentiated by ethanol only at receptors containing y2L subunit but not at those containing the y2S subunit. Mutating a serine to an alanine within the eight amino acid insertion of the y2L subunit abolished the ethanol potentiation of the GABA-induced current (Wafford and Whiting, 1991). The increased level of GABAa /BZ receptor y2L subunit mRNA may result in an increased amount of subunit protein, thus generating an increased number of phosphorylation sites for PKC. Single channel analysis has shown that an increase in phosphorylation decreases the function of the GABA-mediated Cl" channels (Suzdak et al., 1986b; Sigel et al., 1991; Browning et al., 1993). This decreased current is due to a reduction of opening frequency of GABAa /BZ receptors, with no alteration in the conductance or mean open time (Macdonald and Angelotti, 1993). If the available functional GABAa /BZ receptors are decreased due to receptor phosphorylation, more ethanol then may be needed to obtain its original cellular effect; thus, the phosphorylation process may also be associated with ethanol tolerance and dependence. 1 74 Autoradiographic studies of ethanol effects in different brain regions also support the involvement of 72 L subunit in ethanol intoxication. The GABAa/BZ receptors of hippocampus and lateral septum, where have more abundant y2S. are more resistant to ethanol potentiation effects, whereas GABA response in cortex, cerebellum, spinal cord, and medial septum, where have more 7 2 L. are enhanced by ethanol (Suzdak et al., 1986a; Mehta and Ticku, 1988). The GABAa/BZ receptor (32 and (33 subunits are less affected by chronic ethanol administration. In Purkinje cells, granule cells, and deep cerebellar neurons, the levels of these subunit mRNAs were only changed to a minimal degree. To determine if the unaltered subunit transcripts result in unaltered protein expression, the levels of the (32 and (33 subunit polypeptides were investigated using a subunit specific monoclonal antibody. The immunostaining expressed in the granule cells and molecular layer was similar in both ethanol-treated and control groups, suggesting the (32 and (33 subunit proteins were not affected by long term exposure of ethanol. Radioligand binding studies also showed similar results in that [3H]muscimol binding, associated with the (3 subunits in granule cells and molecular layer, remained unchanged when compared to controls. These results that the (3 subunits are not changed by chronic ethanol treatment, suggesting that chronic ethanol intoxication may be mediated by parts of the GABAa /BZ receptor other than GABAergic binding components, such as the benzodiazepine or picrotoxin binding sites. 1 75 The unchanged mRNA level of (53 subunit following chronic ethanol treatment suggests that the cAMP-dependent phosphorylation sites on this subunit are less affected by long term than acute ethanol treatment. The differential expression of (33 and Y2L subunit mRNAs in the two treatment paradigms indicates that ethanol exerts its effect on the GABAa/BZ receptors at least via two different phosphorylation sites, one associated with protein kinase A and the other with protein kinase C (Macdonald and Angelotti, 1993). C, The effects of maternal ethanol treatment during gestation on the expression of GABA ^ /BZ receptor subunit mRNAs There is significant evidence showing that ethanol exerts neurotoxic effects in the developing as well as the adult central nervous system ( West, 1987; Gruol, 1991; Ledig et al., 1993; Hamre and West, 1993). Since ethanol can cross the placenta (Lee and Becker, 1989), consumption of ethanol during pregnancy may result in teratogenic effects in the offspring, the various symptoms of which have been defined as fetal alcohol syndrome. These symptoms include delays in motor development, problems with fine motor tasks, retarded body growth, microencephaly, congenital heart defects, and mental retardation (Jones and Smith, 1973). In the developing rodent cerebellum, ethanol treatment has been shown to cause delays of neuronal differentiation, reduced synapse formation, change of membrane properties, and decreased cerebellar 1 76 size (Phillips, 1985; Nathaniel et al., 1986; Smith et al., 1986). Results from the studies presented here show that, as in adult rodents, the expression of cerebellar GABAa /BZ receptor subunit mRNAs in neonatal mice is changed differentially by maternal exposure to ethanol between embryonic day 12 and postnatal day 7 of gestation and prior to weaning. These changes may be due partially to the above morphological alterations of cerebellum following ethanol exposure. The signals which turn on the transcription process of subunit mRNA may be disrupted by disconnection of synaptic contacts, or reduced cellular differentiation. The decreased level of ai subunit mRNA, increased level of a6 subunit mRNA, and unchanged levels of P2. P3 and Y2 subunit mRNAs in the cerebellum indicate that, at early ages, a subunits are more sensitive to ethanol effects than the other subunits. The downregulation of ai subunit mRNA may result in decreased function of GABAa/BZ receptor. If inhibitory GABAa/BZ receptor function is decreased, the projections from cerebellum via brain stem to motoneurons, such as pontine reticulospinal tract, rubrospinal tract, and corticospinal tract, to control muscle activity may then be affected; this may contribute to the problems with fine motor control seen in fetal alcohol syndrome (Jones and Smith, 1973). Since the a6 subunit has been reported to carry an ethanol antagonist binding site (Mhatre et al., 1988; Mehta and Ticku, 1989; Palmer and Hoffer, 1989), it is plausible that the ethanol-induced increase of a6 subunit mRNA may, in turn, increase 1 77 ethanol antagonist binding sites; which, this might counteract the ethanol-enhanced GABA-induced hyperpolarization. Unlike in the adult, the level of y2L subunit mRNA in P7 mouse remains unchanged by chronic maternal ethanol treatment. As mentioned earlier, the y2L subunit carries a protein kinase C phosphorylation site which is required for the effect of ethanol on GABAa /BZ receptors (Wafford et al., 1991). However, at the early age, the mechanism leading to phosphorylation of this subunit may not be functional, and, therefore, y2L may not be involved in chronic ethanol intoxication. Although the y2 subunit mRNA is clearly expressed in developing Purkinje cells and deep cerebellar neurons at P7 (Luntz-Leybman et al., 1993), the granule cells at this stage are only starting their migration into the internal germinal layer. Receptors may not be functionally mature at this age, and, therefore, the y2 subunit components of these receptors are not as sensitive to ethanol as in the adult. D. Summary The effect of ethanol on the GABAa/BZ receptor is subunit specific; some but not all subunit types are affected. Only a i and p 3 subunit mRNAs, following acute treatment, and ai,a6 and y2 subunit mRNAs, following chronic treatment, are affected by ethanol. These results support the hypothesis that the GABAa/BZ receptor subunit mRNA levels are differentially affected by ethanol treatment. However, the effect of ethanol is not cell-type specific, since some of the subunit mRNAs in granule cells, Purkinje cells 178 and deep cerebellar neurons are all affected by ethanol. This general effect of ethanol on GABAa /BZ receptor in these cell types indicates that the effect of ethanol on motor control is mediated by all GABAa /BZ receptor-containing synapses in the cerebellum. The effect of ethanol on GABAa /BZ receptor subunit mRNAs is dependent on mode of administration. Acute or chronic ethanol treatment produces different effects on the expression of GABAa /BZ receptor subunit mRNAs. For example, the level of «i subunit mRNA is increased by acute ethanol treatment, whereas it is decreased by chronic ethanol treatment. This supports the hypothesis that different time periods of ethanol exposure produces different effects on the GABAa /BZ subunit mRNA expression. The effect of ethanol is also dependent on the stage of neuronal development during administration of ethanol. The results indicate that some of the GABAa /BZ receptor subunits in the immature nervous system are less sensitive to ethanol than in the adult. REFERENCES 1. Abel E. L. "Prenatal effects of alcohol" Drug Alcohol Depend. 14 (1984) 1-10. 2. Abraham W. C. and Hunter B. E. "An electrophysiological analysis of chronic ethanol neurotoxicity in the dentate gyrus: distribution of entorhinal afferents" Exp. Brain Res. 47 (1982) 61- 68 . 3. Algar E. M. and Holmes R. S. "Kinetic properties of murine liver aldehyde dehydrogenases" Pro. Clin. Biol. Res. 290 (1989) 93-103. 4. Allan A. M. and Harris R. A. "Acute and chronic ethanol treatment alter GABA receptor-operated chloride channels" Pharmacol. Biochem. Behav. 27 (1987) 665-670. 5. Altman J. "Morphological development of the rat cerebellum and some of its mechanisms" Exp. Brain Res. Suppl. 6 (1982) 8-46. 6. Badley J. E., Bishop G. A. and Frelinger J. A. "A simple, rapid method for the purification of poly A+ RNA" BioTechniques 6 (1988) 1 14-116. 7. Baile A., Hoffer B. and Dunwiddie T. "Differential sensitivity of cerebellar Purkinje neurons to ethanol in selectively outbred lines of mice: maintenance in vivo independent of synaptic transmission" Brain Res. 264 (1983) 69-78. 8. Bateson A. N., Lasham A. and Darlison M. G. "y-am inobutyric acidA receptor heterogeneity is increased by alternative splicing of a novel p-subunit gene transcript" J Neurochem. 56 (1991) 1437- 1440. 179 1 80 9. Baude A.t Sequier J. M., McKernan R. M., Olivier K. R. and Somogyi P. "Differential subcellular distribution of the aQ subunit versus the a 1 and P2/3 subunits of the GABAA/benzodiazepine receptor complex in granule cells of the cerebellar cortex" Neurosci 51(4) (1992) 739-748. 10. Benke D., Mertens S., Trzeciak A., Gillessen D. and Mohler H. "GABAa receptors display association of y 2 -subunit with a i- and p2/ 3 -subunits" J. Biol. Chem. 266(7) (1991) 4478-4483. 1 1 . Berne R. M. and Levy M. N.: The cerebellum, In: Physiology 2nd ed. Mosby 1988. 12. Berridge M. J. and Irvine R. F. "Inositol phosphate and cell signalling" Nature 431 (1989) 197-205. 13. Biggio G., Brodie B. B., Costa E. and Guidotti A. "Mechanism by which diazepam, muscimol, and other drugs change the content of cGMP in cerebellar cortex" Proc. Natl. Acad. Sci. USA 74 (1977) 3 5 9 2-3 59 6. 14. Biosse N. R. and Okamoto M.: Ethanol as a sedative-hypnotic: comparison with barbiturate and nonbarbiturate sedative-hypnotic, In: Alcohol Tolerance and Dependence. Elsevier 1980. 15. Bloom R. E. and Iversen L. L. "Localizing (3 h ]GABA in nerve terminals of cerebral cortex by electron microscopic autoradiography" Nature 229 (1971) 628-630. 16. Bomann J. "Electrophysiology of GABAa and GABAb receptor sybtypes" TINS 11 (1985) 112. 17. Bowers R. J. and Wehner J. M. "Interaction of ethanol and stress with the GABA/BZ receptor in LS and SS mice" Brain Res. Bull. 23 (1989) 53-59. 18. Bowery N. G., Hill D. R. and Hudson A. L. "Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes" Br. J. Pharmacol. 78 (1983) 191. 181 19. Braestrup C. and Nielsen M. "[3H]Propyl p-carboli ne-3- carboxylate as a selective radioligand for the BZ1 benzodiazepine receptor subclass" J. Neurochem. 37 (1981) 333-341. 20. Breese G. R., Frye G. D., McCown T. J. and Muller R. A. "Comparison of the CNS effects induced by TRH and bicuculline after microinjection into medial septum, substantia nigra and inferior colliculus: absence of support for a GABA antagonist action for TRH" Pharmacol. Biochem. Behav. 21 (1984) 145-149. 21. Breese G. R., Lundberg D., Mailman R. B., Frye G. D. and Mueller R. A. "Effect of ethanol on cyclic nucleotides in vivo: consequences of controlling motor and respiratory changes" Durg Alcohol Depned. 4 (1979) 321- 326. 22. Brioni J. D., Nagahara A. H. and McGaugh J. L. "Involvement of the amygdala GABAergic system in the modulation of memory storage" Brain Res. 487 (1989) 105-112. 23. Britton K. T., Ehlers C. L. and Koob G. F. "Is ethanol antagonist Ro 15-4513 selective for ethanol? " Science 239 (1988) 648-649. 24. Browning M. D., Endo S., Smith G., Dudek E. M. and Olsen R. W. "Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: analysis of the substrate domain" Neurochem. Res. 18 (1993) 95-100. 25. Buck K. J. and Harris R. A. "Benzodiazepine agonist and inverse agonist actions on GABA^ receptor-operated chloride channels. I. Acute effects of ethanol” J. Pharmacol. Exp. Ther. 253(2) (1990a) 7 0 6 -7 1 2 . 26. Buck K. J. and Harris R. A. "Benzodiazepine agonist and inverse agonist actions on GABA^ receptor-operated chloride channels. II. Chronic effects of ethanol" J. Pharmacol. Exp. Ther. 253(2) (1990b) 7 1 3 -7 1 9 . 27. Buck K. J. and Harris R. A. "Neuroadaptive responses to chronic ethanol" Alcoholism 15 (1991) 460-470. 28. Buck K. J., Hahner L., Sikela J. M. and Harris R. A. "Chronic 1 82 ethanol treatment alters brain levels of y-aminobutyric acidA receptor subunit mRNAs: relationship to genetic difference in ethanol withdrawal seizure severity" J. Neurochem. 57 (1991) 1452-1455. 29. Buck K. J., Sikela J. M. and Harris R. A. "Expression of GABAa receptor subunit mRNAs in long-(LS) and short-sleep (SS) mouse brain regions: analysis by polymerase chain reaction (PCR)" Ale. Clin. Exp. Res. 15 (1991) 320-331. 30. Buckle V. J., Fujita N., Ryder-Cook A. S., Derry J. M., Barnard P. J., Lebo R. B., Schofield P. R., Seeburg P. H., Bateson A. N., Darlison M. G. and Barnard E. A. "Chromosomal localization of GABAa receptor subunit gene: relationship to human genetic disease" Neuron 3 (1989) 647-654. 31. Burch T. P. and Ticku M. K. "Ethanol enhances [^H]diazepam binding at the benzodiazepine-GABA-receptor-ionophore complex” Eur. J. pharmacol. 67 (1980) 325-326. 32. Burt D. R. and Kamatchi G. L. "GABAa receptor subtypes: from pharmacology to molecular biology" FASEB J. 5 (1991) 2916-2923. 33. Carlen P. L., Gurevich N. and Durand D. "Ethanol in low doses augments calcium-mediated mechanisms measured intracellularly in hippocampal neurons" Science 215 (1982) 306-309. 34. Casalotti S. O., Stephenson F. A. and Barnard E. A. "Separate subunits for agonist and benzodiazepine binding in the y - aminobutyric acidA receptor oligomer" J. Biol. Chem. 261 (1986) 15013-15016. 35. Celentano J. J., Gibbs T. T. and Garb D. H. "Ethanol potentiates GABA- and glycine-induced chloride currents in chick spinal cord neurons" Brain Res. 455 (1988) 377-380. 36. Chin J. H. and Goldstein D. B. "Drug tolerance in biomembrane: a sin label study of the effects of ethanol" Science 196 (1977a) 684- 686 . 183 37. Chin J. H. and Goldstein D. B. "Effects of low concentrations of ethanol on the fluidity of spin labeled erythrocytes and brain membranes" Mol. Pharmacol. 13 (1977b) 435-441. 38. Cooper B. R., Viik K.( Ferris R. M. and White H. L. "Antagonism of the enhanced susceptibility to audiogenic seizures dureing alcohol withdrawal by y-amonobutyric acid (GABA) and GABA-mimetic agents" J. Pharmacol. Exp. Ther. 209 (1979) 396-403. 39. Cowen P. J., Green A. R., Nutt D. J. and Martin I. L. "Ethyl- betacarboline carboxylate lowers seizure threshold and antagonizes flurazepam-induced sedation in rats" Nature 290 (1981)54-55. 40. Crabbe J. C., Kosobud A. and Young E. R. "Genetic selection for ethanol withdrawal severity: differences in replicate mouse lines" Life Sci. 33 (1983) 955-962. 41. Crabbe J. C., Kosobud A., Young E. R., Tam B. R. and McSwigan J. D. "Bidirectional selection for susceptibility to ethanol withdrawal seizures in Mus musculus" Behav. Genet. 15 (1985) 521-536. 42. Crepel F. J. "Regression of functional synapses in the immature mammalian cerebellum" TINS 5 (1982) 266-270. 43. Cutting G. R. , Lu L., O’Hara B. F., Kasch L. M., Montrose- Rafizader C., Donovan D. M., Shimada S., Antonarakis S. E., Guggino W. B., Uhl G. R. and Kazazian H. H. Jr. "Cloning of the g-aminobutyric acid (GABA) r 1 cDNA: a GABA receptor subunit highly expressed in the retina" Proc. Natl. Acad. Sci. USA 88 (1991) 2673-2677. 44. Cutting G. R., Curristin S., Zoghbi H., O’Hara B., Seldin M. F and Uhl G. R. "Identification of a putative y-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalizaiton of the genes encoding rho2 (GABAR2) and rhol (GABAR1) to human chromosome 6q14-21 and mouse chromosome 4" Genomics 12 (1992) 801-806. 45. Danciger M., Farber D. B. and Kozak C. A. "Genetic mapping of three GABAa receptor-subunit gene in the mouse" Genomics 16 (1993) 361-365. 46. Daniell L. C. and Harris R. A. "Ethanol and inositol 1,4,5- 1 84 trisphosphate release calcium from separate stores of brain microsomes" J. Pharmacol exp. Ther. 250 (1989) 875*881. 47. Davis W. C. and Ticku M. K. "Ethanol enhances [3H]-diazepam binding at the benzodiazepine-gamma-aminobutyric acid receptor ionophore complex" Mol. Pharmacol. 20 (1981) 287-294. 48. Davis W. C. and Ticku M. K. "Ethanol enhances [3H]diazepam binding at the benzodiazepine-gamma-aminobutyric-acid receptor- ionophore complex" Mol. pharmacol. 20 (1981) 287-294. 49. De Bias, A. L., Vitorica, J., and Friedrich, P. "Localization of GABAa receptor in the rat brain with a monoclonal antibody to the 57,000Mr peptide of GABAa receptor/benzodiazepine receptor/CI‘ channel complex" J. Neurosci. 8 (1988) 602-614. 50. Deitrich R. A., Dunwiddie T. V., Harris R. A. and Erwin V. G. "Mechanism of action of ethanol: initial central nervous system actions" Pharmacol. Rev. 41(4) (1989) 489-537. 51. Dixon L., Weiden P. J., Frances A. J. and Sweeney J. "Alprazolam intolerance in stable schizophrenic outpatients" Psychopharmacol. Bull. 25 (1989) 213-214. 52. Dobbing J. and Sands J. "Comparative aspects of the brain growth spurt" Early Hum. Dev. 3 (1979) 79-83. 53. Dobbing J. "Maternal nutrition in pregnancy and later achievement of offspring: A personal interpretation” Early Hum. Dev. 12 (1985) 1-8. 54. Doble A. and Martin I. L. "Multiple benzodiazepine receptors-no reason for anxiety" TIPS 13 (1992) 76-81. 55. Feramisco J. R., Glass D. B. and Krebs E. G. "Optimal spatial requirements for the location of basic residues in peptide substrates for the cyclic AMP-dependent protein kinase" J. Biol. Chem. 255(1980)4240-4245. 56. Fourney R. M., Miyakoshi J., Day III R. S., Paterson M. C. "Northern blotting: efficient RNA staining and transfer" FOCUS 10(1) 185 (1988) 5-7. 57. Franklin C. L. and Gruol D. L. "Acute ethanol alters the firing pattern and glutamate response of cerebellar Purkinje neurons in culture" Brain Res. 416 (1987) 205-218. 58. Freund G. "Benzodiazepine receptor loss in brains of mice after chronic alcohol consumption" Life Sci. 27 (1980) 987-992. 59. Freund R. K., van Horne C. G., Harian T. and Palmer M. R. "Electrophysiological interactions of ethanol with GABAergic mechanisms in the rat cerebellum in vivo" Alcohol Clin. Exp. Res. 17(2) (1993) 321-328. 60. Frostholm, A., Zdilar, D., Luntz-Leybman, V., Janapati, V. and Rotter, A. "Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: Transient appearance of p 3 subunit mRNA and [^H] muscimol binding sites" Molecular Brain Res. 16 (1992) 246-254. 61. Frye G. D. and Fincher A. S. "Effect of ethanol on gamma-vinyl GABA-induced GABA accumulation in the substantia nigra and on synaptosomal GABA content in six rat brain regions" Brain Res. 449(1-2) (1988) 71-79. 62. Geokas M. C. "Ethyl alcohol and disease" Med. Clin. North Am. 68 (1984) 1-23. 63. George F. and Chin H. S. "Effects of ethanol on Purkinje cells recorded from cerebellar slices" Alcohol 1 (1984) 353-358. 64. Ghez C. and Gahn S.: The cerebellum, In Kandel E. R. and Schwartz J. H.: Principles of neuronal science. Elsevier Sci. New York 1981. 65. Givens B. S. and Breese G. R. "Ethanol reduces single unit activity and disrupts the rhythmically bursting pattern of neurons in the rat medial septum/diagonal band" Soc. Neurosci Abstr. 13 (1987) 502. 6 6 . Goldstein D. B. "The alcohol withdrawal syndrome. A view from 1 86 the laboratory" Rec. Dev. Ale. 4 (1986) 231-240. 67. Goodchild C. S. "GABA receptors and benzodiazepines" Br. J. Anaesth. 71 (1993) 127-133. 6 8 . Greenberg D. A., Cooper E. C., Gordon A. and Diamond I. "Ethanol and the Y-aminobutyric acid-benzodiazepine receptor complex" J. Neurochem. 42 (1984) 1062-1068. 69. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundelfinger D. E. and Betz H. "The strychnine-binding subunit of the glycine receptor shows homology with nicotine acetylcholine receptors" Nature 328 (1987) 215-220. 70. Gruol D. L. "Chronic exposure to alcohol during development alters the membrane properties of cerebellar Purkinje neurons in culture" Brain Res. 558 (1991) 1-12. 71. Gruol D. L. "Ethanol alters synaptic activity in cultured spinal neurons" Brain Res. 243 (1982) 25-33. 72. Haefely W. : Allosteric Modulation of Amino Acid Receptors. In Garmard E. A. and Costa E. (Ed): Therapeutic implications, Raven Press, New York. 1989. 73. Hakkinen H. M. and Kulonen E. "Ethanol intoxication and GABA" J. Neurochem. 27 (1976) 631-633. 74. Hamdi A. and Prasad C. "Bidirectional changes in striatal D 2- dopamine receptor density during chronic ethanol intake" Alcohol 9 (1992) 133-137. 75. Hamre K. M. and West J. R. "The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles" Alcohol. Clin. Exp. Res. 17(3) (1993) 610-622. 76. Harris R. A., Burnett R., McQuilkin S., McC.ard A. and Simon F. R. "Effects of ethanol on membrane order: fluorescence studies" Ann. NY Acad. Sci. 492 (1987) 125-135. 187 77. Harris R. A., Fenner D., Feller D., Sieckman G., Lloyd S., Mitchell M., Dexter J. D., Tumbleson M. E. and Bylund D. B. "Neurochemical effects of long-term ingestion of ethanol by Sinclair (S-1) swine" Pharmacol. Biochem. Behav. 18 (1983) 363-367. 78. Howerton T. C. and Collins A. C. "Ethanol-induced inhibition of GABA release from LS and SS mouse brain slices” Alcohol 1 (1984) 4 7 1 -4 7 7 . 79. Huidobro-Toro J. P., Blec V., Allan A. M. and Harris R. A. "Neurochemical actions of anesthetic drugs on the gamma- aminobutyric acid receptor-chloride channel complex" J. Pharmacol. Exp. Ther. 242 (1987) 963-969. 80. Hunt W. A.: Alcohol and biological membranes, In: The Guilford Alcohol Studies Series. The Guilford Press. 1985. 8 *. Ito M. : Climbing fibers. In: The cerebellum and neuronal control. New York, Raven Press 1984. 82. Jackson H. C. and Nutt D. J. "Effects of benzodiazepine receptor inverse agonists on locomotor activity and exploration in mice" Eur. J. Pharmacol. 221 (2-3) (1992) 199-203. 83. Jones D. G. "Influence of ethanol on neuronal and synaptic maturation in the central nervous system-morphological investigations" Prog. Neurobiol. 31 (1988) 171-197. 84. Jones K. L., Smith D. W. "Recognition of the fetal alcohol syndrome in early infancy" Lancet 2 (1973) 999-1001. 85. Kalant H.: Comparative aspects of tolerance to, and dependence on, alcohol, barbiturates and opiates. In: Alcohol Intoxication and Withdrawal. Plenum Press, New York. 1977. 8 6 . Kalvakolanu D. V. R. and Livingston W. H. Ill "Rapid and inexpensive protocol for generating greater than 95% recombinants in subcloning experiments" Biotechniques 10(2) (1991) 176-17. 87. Kato K. "Novel GABAa receptor a subunit is expressed only in cerebellar granular cells" J. Mol. Biol. 214 (1990) 619-624. 188 8 8 . Keir W. J., Kozak C. A., Chakraborti A., Deitrich R. A. and Sikela J. M. "The cDNA sequence and chromosomal location of the murine GABAa al receptor gene" Genomics 9 (1991) 390-395. 89. Kerr D. I. B., Ong J., Prager R. H., Gynther B. D. and Curtis D. R. "Phaclofen: a peripheral and central baclofen antagonist" Brain Res. 405 (1987) 150. 90. Khrestchatisky M., MacLennan A. J., Chiang M. Y., Xu W., Jackson M. B., Brecha N., Sternini C., Olsen R. W. and Tobin A. J. "A novela subunit in rat brain GABAa receptors" Neuron 3 (1989) 745-753. 91. Khrestchatisky M., MacLennan A. J., Tillakaratne N. J. K., Chiang M. Y., and Tobin A. J "Sequence and regional distribution of the mRNA encoding the a2 polypeptide of rat y-aminobutyric acidA receptors" J. Neurochem. 56 (1991) 1717-1722. 92. Klatsky A. R., Friedman J. D. and Siegelaub A. B. "Alcohol and mortality: A ten year Kaiser Permanent experience" Ann. Intern Med. 95 (1981) 139-146. 93. Knoflach F., Rhyner Th., Villa M., Kellenberger S., Drescher U., Malherbe P., Sigel E. and Mohler H. "The y 3 -subunit of the GABAa- receptor confers sensitivity to benzodiazepine receptor ligands" FEBS 293(1,2) (1991) 191-194. 94. Knoll J. H. M., Sinnett D., Wagstaff J., Glatt K., Wilcox A. S., Whiting P. M., Wingrove P., Sikela J. M. and Lalande M. "FISH ordering of reference markers and of the gene for the a5 subunit of the y- aminobutyric acid receptor (GABRA5) within the Angelman and Prader-Willi syndrome chromosomal regions" Hum. Mol. Gene. 2(2) (1993) 183-189. 95. Kofuji P., Wang J. B.f Moss S. J., Huganir R. L. and Burt D. R. "Generation of two forms of the y-aminobutyric acidA receptor y2- subunit in mice by alternative splicing" J. Neurochem. 56 (1991) 7 1 3 -7 1 5 . 96. Kulkarni S. K. and Ticku M. K. "Interaction between GABAergic 1 89 anticonvulsants and the NMDA receptor antagonist MK801 against MES and picrotoxin induced convulsions in rats" Life Sci. 44 (1989) 1317-1323. 97. Lai H., Mann P. A., Shearman G. T. and Lippa A. S. "Effect of acute and chronic pentylenetetrazol treatment on benzodiazepine and cholinergic receptor binding in the rat brain" Eur. J. Pharmacol. 75 (1981) 75-81. 98. Laurie D. J., Seeburg P. H. and Wisden W. "The distribution of 13 GABA-A receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum" J. Neurosci. 12(11) (1992) 1063-1076. 99. Ledig M., Simler S., Ciesielski L. and Mandel P "Alcohol exposure before pregnancy: effect on GABA levels and turnover in rat offspring" Alcohol 28(2) (1993) 175-179. 100. Lee N. M. and Becker C. E.: The alcohols. In Katzung B. G. (ed.): Basic and clinical pharmacology. Lange 1989. 101. Leguicher A., Beague F. and Mordmann R. "Concomitant changes of ethanol partitioning and disordering capacities in rat synaptic membranes" Biochem. Pharmacol. 36 (1987) 2045-2048. 102. Leidenheimer N. J., Machu T. K., Endo S., Olsen R. W., Harris R. A. and Browning M. D. "Cyclic AMP-dependent protein kinase decreases gamma-aminobutyric acidA receptor-mediated 3®CI- uptake by brain microsacs" J. Neurochem. 57(2) (1991) 722-725. 103. Leonard B. E. "Ethanol as a neurotoxin" Biochem. Pharmacol. 36 (1987) 2055-2059. 104. Levitan E. S., Blair L. A. C., Dionne V. E. and Barnard E. A. "Biophysical and pharmacological properties of cloned GABAa receptor subunits expressed in Xenopus oocytes" Neuron 1 (1988a) 7 73 -7 8 1 . 105. Levitan E. S., Schofield P. R., Burt D. R., Rhee L. M.t Wisden W., Kohler M., Fujita N., Rodriguez H., Stephenson F. A., Darlison M. G., Barnard E. A. and Seeburg P. H. "Structural and functional basis for GABAa receptor heterogeneity" Nature 335 (1988b) 76-79. 1 90 106. Liljequist S. and Engel J. "Effects of GABAergic agonists and antagonists on various ethanol-induced behavioral changes" Psychopharmacol. 78 (1982) 71-75. 107. Lin A. M. Y., Freund R. K. and Palmer M. R. "Ethanol potentiation of GABA-induced electrophysiological responses in cerebellum; requirement for catecholamine modulation" Neurosci. Lett. 122 (1991) 154-158. 108. Linnoila M., Stowell L., Marangos P. J. and Thurman R. G. "Effect of ethanol and ethanol withdrawal on ^H-muscimol binding and behavior in the rat; a pilot study" Acta Pharmacol. Tox. 49 (1981) 4 0 7 -4 1 1 . 109. Lister R. G. and Nutt D. J. "Is Ro 15-4513 a specific alcohol antagonist?" TINS 10(6) (1987) 223-225. 110. Little H. J. "Mechanism that may underlie the behavioral effects of ethanol" Prog. Neurobiol. 36 (1991) 171-194. 111. Little H. J. "The effects of benzodiazepine agonists, inverse agonists and Ro 15-1788 on the responses of the superior cervical ganglion to GABA in vitro" Br. J. Pharmacol. 83 (1984) 57-68. 112. Ludden H. and Wisden W. "Function and pharmacology of multiple GABAa receptor subunits" TIPS 12 (1991) 49-51. 113. Luddens H., Pritchett D. B., Kohler M., Killisch I., Keinanen K., Monyer H., Sprengel R. and Seeburg P. H. "Cerebellar GABAa receptor selective for a behavioral alcohol antagonist" Nature 346 (1990) 648-651. 114. Luntz-Leybman V., Frostholm A., Fernando L., De Bias A. and Rotter A. "GABAA/Benzodiazepine receptor y2 subunit gene expression in developing normal and in mutant mouse cerebellum" Mol. Brain Res. 19 (1993) 9-21. 115. Luttinger D., Hemeroff C. B., Mason G. A., Frye G. D., Greese G. R. and Prange A. J. Jr. "Enhancement of ethanol-induced sedation and 191 hypothermia by centrally administered neurotensin, p-endorphin, and bombesin" Neuropharmacol. 20 (1981) 305-309. 116. Macdonald R. L. and Angelotti T. P. "Native and recombinant GABAa receptor channels" Cell Physiol. Biochem. 3 (1993) 352-373. 117. Majewska M. D., Harrison N. L., Schwartz R. D.t Barker J. L. and Paul S. M. "Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor" Science 232 (1986) 1004-1007. 118. Majewska M. D., Menville J. and Viccini S. "Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons. Neurosci. Lett. 90 (1988) 279-284. 119. Malherbe P., Draguhn A., Multhaup G., Beyreuther K. and Mohler H. "GABAA-receptor expressed from rat brain a- and p-subunit cDNAs displays potentiation by benzodiazepine receptor ligands" Mol. Brain Res. 8 (1990a) 199-208. 120. Malherbe P., Sigel E., Baur R., Persohn E., Richards J. G. and Mohler H. "Functional characteristics and sites of gene expression of the a i, pi, Y2-isoform of the rat GABAa receptor" J. Neurosci. 10(7) (1990b) 2330-2337. 121. Malherbe P., Sigel E., Baur R., Persohn E., Richards J. G. and Mohler H. "Functional expression and sites of gene transcription of a novel a subunit of the GABAa receptor in rat brain" FEBS Lett. 260 (1990c) 261-265. 1 2 2 . Mamalaki C., Stephenson F. A. and Barnard E. A. "The GAB A A/benzodiazepine receptor is a heterotetramer of homologous a and p subunits" EMBO J. 6 (1987) 561-565. 123. Mariani J. and Changeux J.-P. " Ontogenesis of olivocerebellar relationships: II. Spontaneous activity of inferior olivary neurons and climbing fiber mediated activity of cerebellar Purkinje cells in developing rats" J. Neurosci. 1(1981b) 703-709. 124. Mariani J. and Changeux J.-P. "Ontogenesis of olivocerebellar relationships: I. Studies by intracellular recordings of the multiple 1 92 innervation of Purkinje cells by climbing fibers in the developing rat cerebellum" J. Neurosci. 1(1981 a) 696-702. 125. Martin D. L. and Rimvall K. "Regulation of y-aminobutyric acid synthesis in the brain" J. Neurochem. 60 (1993) 395-407. 126. Martz A. Deitrich R. A. and Harris R. A. "Behavioral evidence for the involvement of y-aminobutyric acid in the actions of ethanol" Eur. J. Pharmacol. 89 (1983) 53-62. 127. Mehta A. K. and Ticku M. K. "Chronic ethanol treatment alters the behavioral effects of Ro 15-4513, a partially negative ligand for benzodiazepine binding sites" Brain Res. 489 (1989) 93-100. 128. Mehta A. K. and Ticku M. K. "Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma- aminobutyric acid A-gated chloride channels" J. Pharmacol. Exp. Ther. 246 (1988) 558-564.& 129. Mhatre M. C. and Ticku M. K. "Chronic ethanol administration alters y-aminobutyric acidA receptor gene expression" Mol. Pharmacol. 42 (1992) 415-422. 130. Mhatre M. C., Pena G., Sieghart W. and Ticku M. K. "Antibodies specific for GABAa receptor a subunits reveal that chronic alcohol treatment down-regulates a-subunit expression in rat brain regions" J. Neurochem. 61 (1993) 1620-1625. & 131. Mhatre M., Mehta A. K. and Ticku M. K. "Chronic ethanol administration increases the binding of the benzodiazepine inverse agonist and alcohol antagonist J3H]-Ro15-4513 in rat brain" Eur. J. Pharmacol. 153 (1988) 141-145. 132. Miale, I. L. and Sidman, R. L. "An autoradiographic analysis of histogenesis in the mouse cerebellum" Exp. Neurol. 4 (1961) 277- 296. 133. Mignery G. A., Sudhof T. C., Takei K. and De Camilli P. "Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor" Nature 342 (1989) 192-195. 193 134. Mihic S. J., Kalant H., Liu J. F. and Wu P. H. "Role of the y- aminobutyric acid receptor/chloride channel complex in tolerance to ethanol and cross tolerance to diazepam and pentobarbital" J. Pharmacol. Exp. Ther. 261{1) (1992) 108-113. 135. Miller J. A. "The calibration of 35S or 32P with 14C-labeled brain paste or 14C-plastic standards for quantitative autoradiography using LKB Ultrofilm or Amersham Hyperfilm" Neurosci. Lett. 121 (1991) 211-214. 136. Mishina, M., Takai, T., Imoto, K., Noda, M. and Takahashi, T., Numa, S., Methfessel, C. and Sakman, B. "Molecular distinction between fetal and adult forms of muscle acetylcholine receptor" Nature. 321 (1986) 406-411. 137. Mohamed S. A., Nathaniel E. J., Nathaniel D. R. and Snell L. "Altered Purkinje cell maturation in rats exposed prenatally to ethanol" Exp. Neurol. 97 (1987) 35-52. 138. Mohler H., Battersby M. K., Richards J. G. "Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label" Proc. Natl. Acad. Sci. USA 77 (1980)1666-1670. 139. Montpied P., Ginns E. I., Martin B. M., Stetler D., O’Carroll A. M., Lolait S. J., Mahan L. C. and Paul S. M. "Multiple GABAa receptor a subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain" FEBS Lett. 258 (1989) 94-98. 140. Montpied P., Martin B. M., Cottingham S. L., Stubblefield B. K., Ginns E. I. and Paul S. M. "Regional distribution of the GABAA/benzodiazepine receptor (a subunit) mRNA in rat brain" FEBS Lett. 258 (1989) 94-98. 141. Montpied P., Morrow A. L., Karanian J. W..Ginns E. I., Martin B. M. and Paul S. M. "Prolonged ethanol inhalation decreases y- aminobutyric acidA receptor a subunit mRNA in the rat cerebral cortex" Mol. Pharmacol. 39 (1991) 157-163. 142. Morrow A. L., Herbert J. S. and Montpied P. "Differential 1 94 effects of chronic ethanol administration on GABAa receptor <*1 subunit and a 6 subunit mRNA levels in rat cerebellum" Mol. Cell. Neurosci. 3 (1992) 251-258. 143. Morrow A. L., Montpied P. , Lingford-Hughes A. and Paul S. M. "Chronic ethanol and pentobarbital administration in the rat: effects on GABAa receptor function and expression in brain* Alcohol 7 (1990) 237-244. 144. Morrow A. L., Suzdak P. D., Karanian J. W. and Paul S. M. "Chronic ethanol administration alters GABA, pentobarbital and ethanol mediated 36 q |- uptake in cerebral cortical synaptoneurosomes" J. Pharmacol. Exp. Ther. 246 (1988) 158-164. 145. Nakatsu Y., Tyndale R. R., DeLorey T. M., Durham-Pierre D., Gardner J. M., McDanel H. J., Nguyen Q., Wag staff J., Lalande M., Sikela J. M., Olsen R. W., Tobin A. J., Brilliant M. H. "A cluster of three GABAa receptor subunit genes is deleted in a neurological mutant of the mouse p locus" Nature 364 (1993) 448-450. 146. Nathaniel E. J., Nathaniel D. R., Mohamed S. A., Nathaniel L., Kowalzik C. and Nahnybida L. "Prenatal ethanol exposure and cerebellar development in rats" Exp. Neurol. 93 (1986) 601-609. 147. Nordberg A. and Wahlstrom G. "Cholinergic mechanisms in physical dependence on barbiturates, ethanol and benzodiazepines" J. Neural Transm. 88 (1992) 199-221. 148. Olsen R. W. and Tobin A. J. "Molecular biology of GABAa receptors" FASEB J. 4 (1990) 1469-1480. 149. Olsen R. W. "Drug interactions at the GABA receptor-ionophore complex" Ann. Rev. Pharmacol. Toxicol. 22 (1982) 531-539. 150. Olsen R. W. "The GABA postsynaptic membrane receptor- ionophore complex. Site of action of convulsant and anticonvulsant drugs" Mol. Cell. Biochem. 39 (1981) 261-279. 151. Olsen, R. W. and Venter, C. J. (Eds), Benzodiazepine/GABA receptors and chloride channels; structural and functional properties. Alan R. Liss, New York (1986). 152. Palmer M. R. and Hoffer B. J. "GABAergic mechanisms in the electrophysiological actions of ethanol on cerebellar neurons" Neurochem. Res. 15 (1989) 145-151. 153. Palmer T. and Paul A. I.: Molecular basis of drug action, In: William B. P. and Palmer T.(eds), Principles of drug action 3rd Edn., Churchill livingstone, New York. 1990. 154. Perez J., Zucchi I. and Maggi A. "Estrogen modulation of the y- aminobutyric acid receptor complex in the central nervous system of rat" J. Pharmacol. Exp. Ther. 244 (1988) 1005-1010. 155. Peters J. A., Kirkness E. F., Callachan H., Lambert J. J. and Turner A. J. "Modulation of the GABA-A receptor by depressant barbiturates and pregnane steroids" Br. J. Pharmacol. 94 (1988) 1257-1269. 156. Phillips S. C. "Age-dependent susceptibility of rat cerebellar Purkinje cells to ethanol exposure" Drug Alcohol Depend. 16 (1985) 273-277. 157. Phillips S. C. and Cragg B. G. "Change in susceptibility of rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal and adult life" Neuropathol. Appl. Neurobiol. 8 (1982) 441- 454. 158. Pierce D. R., Goodlett C. R., West J. R. "Differential neuronal loss following early postnatal alcohol exposure" Teratology 40 (1989) 113-126. 159. Porter N. M., Twyman R. E., Uhler M. D. and Macdonald R. L. "Cyclic AMP-dependent protein kinase decreases GABAa receptor current in mouse spinal neurons" Neuron 5(6) (1990) 789-796. 160. Pregenzer J. F., Im W. B., Carter D. B. and Thomsen D. R. "Comparison of interactions of [3 Hjmuscimol, t- butylbicyclophosphoro [35s]thionate, and [^HJflunitrazepam with cloned y-aminobutyric acidA receptors of the aiP2 and aiP2Y2 subtypes" Mol. Pharmacol. 43 (1993) 801-806. 1 96 161. Pritchett D. B. and Seeburg P. H. "y-Aminobutyric acidA receptor oc5-subunit creates novel type II benzodiazepine receptor pharmacology" J. Neurochem. 54 (1990)1802-1804. 162. Pritchett D. B., Luddens H. and Seeburg P. H. "Type I and type II GABAa benzodiazepine receptors produced in transfected cells" Science 245 (1989a) 1389-1392. 163. Pritchett D. B., Sontheimer H., Shivers B. D., Ymer S., Kettenmann H., Schofield P. R. and Seeburg P. H. "Importance of a novel GABAa receptor subunit for benzodiazepine pharmacology" Nature 338 (1989b) 582-585. 164. Puia G., Vicini S. Seeburg P. H. and Costa E. "Influence of recombinant y-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of y-aminobutyric acid-gated Cl'currents" Mol. Pharmacol. 39 (1991) 691-696. 165. Rassnick S., D'Amico E., Riley E. and Koob G. F. "GABA antagonist and benzodiazepine partial inverse agonist reduce motivated responding for ethanol" Alcohol Clin. Exp. Res. 17(1) (1993) 124-130. 166. Ritchie J. M.: The aliphatic alcohols. In Goodman L. S. and Gilman A. (Eds): The pharmacological basis of therapeutics. (4th ed.) 1970. 167. Ross C. A., Bredt D. and Snyder S. H. "Messenger molecules in the cerebellum" TINS 13(6) (1990) 216-222. 168. Ross C. A., Meldolesi J., Milner T. A., Satoh T., Supattapone S. and Snyder S. H. "Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons" Nature 339 (1989) 468-470. 169. Schofield P. R., Darlison M. G., Fujita N., Burt D. R.t Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A., Seeburg P. H. and Barnard E. A. "Sequence and functional expression of the GABA^ receptor shows a ligand-gated receptor 197 super-family" Nature 328 (1987) 221-227. 170. Schofield P. R., Pritchett D. B., Sontheimer H.t Kettenmann H. and Seeburg P. H. "Sequence and expression of human GABAa receptor a1 and p1 subunits" FEBS Lett. 244 (1989) 361-364. 171. Schwartz R. D. "The GABA-A receptor-gated ion channel: Biochemical and pharmacological studies of structure and function" Biochem. Pharmacol. 37 (1988) 3369-3375. 172. Seeburg P. H., Wisden W., Verdoorn T. A., Pritchett D. B., Werner P., Herb A., Luddens H., Sprengel R. and Sakmann "The GABAa receptor family: molecular and functional diversity" Cold Spring Harbor Symposia on Quantitative Biology LV (1990) 29-40. 173. Shivers B. D., Killisch I., Sprengel R., Sontheimer H., Kohler M., Schofield P. R. and Seeburg P. H. "Two novel GABAa receptor subunits exist in distinct neuronal subpopulations" Neuron 3 (1989) 3 27 -3 3 7 . 174. Sigel E., Baur R. and Malherbe P. "Activation of protein kinase C results in down-modulation of different recombinant GABAa - channels " FEBS Lett. 291 (1991) 150-152. 175. Sigel E., Baur R. and Malherbe P. "Recombinant GABAa receptor function and ethanol" FEBS 324(2) (1993) 140-142. 176. Sigel E., Baur R., Trube G., Mohler H. and Malherbe P. "The effect of subunit composition of rat brain GABAa receptors on channel function" Neuron 5 (1990) 703-711. 177. Sigel E., Stephenson F. A., Mamalaki C. and Barnard E. A. "A y- aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex: purification and partial characterization" J. Biol. Chem. 258 (1983) 6865-6971. 178. Siggins G. R., Pittman Q. J. and French E. D. "Effects of ethanol on C A 1 and CA3 pyramidal cells in the hippocampal slice preparation: an intracellular study" Brain Res. 414 (1987) 22-34. 198 179. Sinclair J. G. and Lo G. F. "The effects of ethanol on cerebellar Purkinje cell discharge pattern and inhibition evoked by local surface stimulation" Brain Res. 204 (1980) 465-471. 180. Skolnick P. and Paul S. M. "Benzodiazepine receptors" Annual Reports in Medicinal Chemistry 16 (1981) 21-29. 181. Smith D. E. and Davies D. L. "Effect of perinatal administration of ethanol on the CA1 pyramidal cell of the hippocampus and Purkinje cell of the cerebellum: an ultrastructural survey" J. Neurocytol. 19 (1990) 708-717. 182. Smith D. E., Foundas A. and Canale J. "Effect of perinatally administered ethanol on the development of the cerebellar granule cell" Exp. Neurol. 92 (1986) 491-501. 183. Smith S. S. "Progesterone enhances inhibitory responses of cerebellar Purkinje cells mediated by the GABA^ receptor subtype" Brain Res. Bull. 23 (1989) 317-322. 184. Sommer B., Poustka A.t Spurr N. K. and Seeburg P. H. "The murine GABAa receptor 5-subunit gene: structure and assignment to human chromosome 1" DNA and Cell Biol. 9(8) (1990) 561-568. 185. Sorensen S., Carter D., Marwaha J., Baker R. and Freedman "Disinhibition of rat cerebellar Purkinje neurons from noradrenergic inhibition during rising biood ethanol" J. Stud. Alcohol 42 (1981) 9 0 8 -9 1 7 . 186. Squires R. F. and Saederup E. "A review of evidence for GABAergic predominance/glutamatergic deficit as a common etiological factor in both schizophrenia and affective psychoses: more support for a continuum hypothesis of 'Functional' psychosis" Neurochem. Res. 16(10) (1991) 1099-1111. 187. Squires R. F., Benson D. I., Braestrup C., Coupet J., Klepner C. A., Muers V. and Beer B. "Some properties of brain specific benzodiazepine receptors: new evidence for multiple receptors" Pharmacol. Biochem. Behav. 10 (1979) 825-830. 188. Squires R. F., Casida J. E., Richardson M. and Saederup E. 1 99 ”[35s]t-Butylbicyclonphosphorothionate binds with high affinity to brain-specific sites coupled to y-aminobutyric acid-A and ion recognition" Mol. Pharmacol. 23 (1983) 326-336. 189. Stinchcomb A., Bowers B. J. and Wehner J. M. "The effects of ethanol and Ro 15-4513 on elevated plus-maze and rotarod performance in long-sleep and short-sleep mice" Alcohol. 6(5) (1989) 369-376. 190. Suzdak P. D., Glowa J. R., Crawley J. N., Schwartz R. D., Skolnick P. and Paul S. M. "A selective imidazobenzodiazepine antagonist of ethanol in the rat" Science 234 (1986a) 1243-1247. 191. Suzdak P. D., Glowa J. R., Crawley J. N., Skolnick P. and Paul S. M. "Is ethanol antagonist Ro 15-4513 selective for ethanol? Response" Science 239 (1988a) 649-650. 192. Suzdak P. D., Schwartz R. D., Skolnick P. and Paul S. M. "Alcohols stimulate gamma-aminobutyric acid receptor-mediated chloride uptake in brain vesicles: correlation with intoxication potency" Brain Res. 444 (1988b) 340-345. 193. Suzdak P. D., Schwartz R. D., Skolnick P. and Paul S. M. "Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes" Proc. Natl. Acad. Sci. USA 83 (1986b) 4071-4075. 194. Takada R., Saito K, Matsura H. and Inoki R. "Effect of ethanol on hippocampal receptors in the rat brain" Alcohol 6 (1989) 115-119. 195. Tallman J. R., Paul S. M., Skolnick P. and G allager D. W. "Receptors for the age of anxiety: Pharmacology of the benzodiazepines" Science 202 (1980) 274-281. 196. Taylor P. and Insel P. A.: Molecular basis of drug action, In Pratt W. B. and Taylor P. (Ed): Principles of drug action, Churchill Livingstone, New York. 1990. 197. Teichberg V. L., Tal N., Goldberg O. and Luini A. "Barbiturates, alcohols and the CNS excitatory neurotransmission: specific effects on the kainnate and quisqualate receptors" Brain Res. 291 (1984) 2 0 0 285-292. 198. Ticku M. K. (1980) "The effect of acute and chronic ethanol administration and its withdrawal on gama-aminobutyric acid receptor binding in rat brain" Br. J. Pharmacol. 70 (1980) 403-410. 199. Ticku M. K. and Burch T. "Alterations in g-aminobutyric acid receptor sensitivity following acute and chronic ethanol treatment" J. Neurochem. 34 (1980) 417-423. 200. Ticku M. K. and Olsen R. W. "Interaction of barbiturates with dihydropicrotoxinin binding sites in mammalian brain" Life Set. 22 (1978) 1643-1652. 201. Ticku M. K., Ban M. and Olsen R. W. "Binding of [3|H)a- dihydropicrotoxinin, a -y-aminobutyric acid-A synaptic antagonist, to rat brain membranes" Mol. Pharmacol. 14 (1978) 391-402. 202. Ticku M. K., Burch T. P. and Davis W. C. "The interactions of ethanol with the benzodiazepine-GABA-receptor ionophore complex" Pharmacol. Biochem. Behav. 18 (1983) 15-18. 203. Ticku M. K., Lowrimore P. and Lehoullier P. "Ethanol enhances GABA-induced 36ci-influx in primary spinal cord cultured neurons" Brain Res. Bull. 17 (1986) 123-126. 204. Tomita Y., Haseba T., Kurosu M. and Watanabe T. "Effects of chronic ethanol intoxication on aldehyde dehydrogenase in mouse liver" Alcohol and Alcoholism 27(2) (1992) 171-180. 205. Tran V. T., Snyder S. H., Major L. F. and Hawley R. J. "GABA receptors are increased in brains of alcoholics" Ann. Neurol. (1981) 2 8 9 -2 9 2 . 206. Treistman S. N. and Wilson A. "Alkanol effects on early potassium currents in Aplysia neurons depend on chain length" Proc. Natl. Acad. Sci. USA 84 (1987) 9299-9303. 207. Treistman S. N. and Wilson A. "Effect of ethanol on early potassium currents in Aplysia: cell specificity and influence of channel state" J. Neurosci. 7 (1987) 3207-3214. 201 208. Twyman R. E., Green R. M. and Macdonald R. L. "Kinetics of open channel block by penicillin of single GABAa receptor channels from mouse spinal cord neurons in culture" J. Physiol. 445 (1992) 97- 127. 209. Ueha T. and Kuriyama K. "Direct action of ethanol on cerebral GABAa receptor complex: analysis using purified and reconstituted GABAa receptor complex" Neurochem. Int. 19(3) (1991) 319-325. 210. Unwin J. W. and Taberner P. V. "Sex and stain differences in GABA receptor binding after chronic ethanol drinking in mice" Neuropharmacol. 19 (1980) 1257-1259. 211. Varecka L., Wu C. H., Rotter A. and Frostholm A. "GABAA/Benzodiazepine receptor a 6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice" J. Comp. Neurol. 383 (1993) 1- 12. 212. Verdoorn T., Draguhn A., Ymer S., Seeburg P. H. and Sakmann B. "Functional properties of recombinant rat GABAa receptors depend upon subunit composition" Neuron 4 (1990) 919-928. 213. Vitorica J.T Park D. and De Bias A. L. "Immunocytochemical localization of the GABAa receptor in the rat brain" Eur. J. Pharmacol. 136 (1987) 451-453. 214. Volicer L. and Biagione T. M. "Effect of ethanol administration and withdrawal on benzodiazepine receptor binding in the rat brain" Neuropharmacol. 21 (1982) 283-286. 215. Wafford K. A. and Whiting P. J. "Ethanol potentiation of GABAa receptors requires phosphorylation of the alternatively spliced variant of the y 2 subunit" FEBS Lett. 313(2) (1992) 113-117. 216. Wafford K. A., Burnett D. M.( Leidenheimer N. J., Burt D. R., Wang J. B., Kofuji P., Dunwiddie T. V., Harris R. A. and Sikela J. M. "Ethanol sensitivity of the GABAa receptor expressed in Xenopus Oocytes requires 8 amino acids contained in the y2L subunit" Neuron 7 (1991) 27-33. 2 0 2 217. Wagstaff J., Chaillet J. R. and Lalande M. "The GABAa receptor {33 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7" Genomics 11 (1991) 1071-1078. 218. Walaas S. I. and Greengard P. "Protein phosphorylation and neuronal function" Pharmacol. Rev. 43(3) (1991) 299-349. 219. Wang J. B., Kofuji P., Fernando J. C. R., Moss S. J., Huganir R. L. and Burt D. R. "The oc-|, a 2 , and a 3 subunits of GABAa receptors: comparison in seizure-prone and -resistant mice and during development" J. Mol. Neurosci. 3 (1992) 177-184. 220. West J. R. "Fetal alcohol-induced brain damage and the problem of determining temporal vulnerability: a review" Alcohol and Drug Res. 7 (1987) 423-441. 221. West J. R.: Alcohol and brain development, In: Alcohol and Brain Development. Oxford Press, New York. 1986. 222. Whiting P., McKernan R. M. and Iversen L. L. "Another mechanism for creating diversity in y-aminobutyrate type A receptors: RNA splicing directs expression of two forms of y2 subunit, one of which contains a protein kinase C phosphorylation site" Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 9966-9970. 223. Wilcox A. S., Warrington J. A., Gardiner K., Berger R., Whiting P., Altherr M. R., Wasmuth J., Patterson D. and Sikela J. M "Human chromosomal localization of genes encoding the yi and y 2 subunits of the y-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome" Proc. NatL Acad. Sci. USA 89 (1992) 5857-5861. 224. Wilson-Shaw D., Robinson M., Gambarana C., Siegel R. E. and Sikela J. M. "A novel y subunit of the GABAa receptor identified using the polymerase chain reaction" FEBS Lett., 284 ( 2 ) (1991) 2 1 1 -2 1 5 . 225. Wisden W., Herb A., Wieland H. A., Keinanen K., Luddens H. and 203 Seeburg P. H. "Cloning, pharmacological characteristics and expression pattern of the rat GABAa receptor a 4 subunit" FEBS Lett. 289 (1991) 227-230. 226. Wisden W., Laurie D. J., Monyer H. and Seeburg P. H. "The distribution of 13 GABAa receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon" J. Neurosci. 12(3) (1992) 1040-1062. 227. Wisden W., Morris B. J., Darlison M. G., Hunt S. P. and Barnard E. A. "Localization of GABAa receptor a-subunit mRNAs in relation to receptor subtypes" Mol. Brain Res. 5 (1989) 305-310. 228. Wuenschell C. W., Fisher R. S., Kaufman D. L. and Tobin A. J. "In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum" Proc. Natl. Acad. Sci. USA 83 (1986) 6193-6197. 229. Xu D., Heng J. K. M. and Palmer T. N. "The mechanisms(s) of the alcohol-induced impairment in glycogen synthesis in oxidative skeletal muscles" Biochem. Mol. Biol. Int. 30(1) (1993) 169-176. 230. Yakushiji T., Shirasai T., Munakata M., Hirata A. and Akaike N. "Differential properties of type I and type II benzodiazepine receptors in mammalian CNS neurons" Br. J. Pharmacol. 109 (1993) 8 19-825. 231. Ymer S., Draguhn A., Kohler M., Schofield P. R. and Seeburg P. H. "Sequence and expression of a novel GABAa receptor a subunit" FEBS Lett., 258 (1989a) 119-122. 232. Ymer S., Draguhn A., Wisden W., Werner P., Keinanen K., Schofield P. R., Sprengel R., Pritchett D. B. and SeeburgP. H. "Structure and functional characterization of the yi subunit of GABAA/benzodiazepine receptors" EMBO J. 9 (1990) 3261-3267. 233. Ymer S., Schofield P. R., Draguhn A., Werner P., Kohler M. and Seeburg P. H. "GABAa receptor (} subunit heterogeneity: functional expression of cloned cDNAs" EMBO J. 8 (1989b) 1665-1670. 204 234. Yoshida M. "The neuronal mechanism underlying Parkinsonism and dyskinesia, and differential roles of the putamen and caudate nucleus" Adv. Neurol. 60 (1993) 71-77. 235. Zdilar D., Luntz-Leybman V., Frostholm A. and Rotter A. "Differential expression of GABAA/benzodiazepine receptor p1, 02, and P3 subunit mRNAs in the developing mouse cerebellum" J. Comp. Neurol. 326 (1992) 580-594. 236. Zdilar D., Rotter A. and Frostholm A "Expression of GABAA/benzodiazepine receptor ai -subunit mRNA and [3H]flunitrazepam binding sites during postnatal development of the mouse cerebellum" Develop. Brain Res. 61 (1991) 63-71. 237. Zhang J-H, Sato M. and Tohyama M. "Region-specific expression of the mRNAs encoding p-subunits (pi, 02, and 0 3 ) of GABAa receptor in the rat brain" J. Comp. Neurol. 303 (1991) 637-657. c0 5 CO c . o