• Abstract
  

• Introduction
  

• Materials and Methods
  

• Results
  

• Discussion
  

• Acknowledgments
  

• References
  

ABSTRACT

Benzodiazepines allosterically modulate the inhibitory effects mediated by the GABA_A receptor, and the g₂ subunit of the receptor is necessary for this action. Since no antagonists selective for the g₂ subunit are available, our goal was to determine whether expression of this subunit could be decreased in vivo using antisense phosphorothioate oligonucleotides (S-ODNs) to prevent its expression. S-ODNs were administered into the lateral ventricle of Long Sleep mice for 3.5 days via miniosmotic pump. Saturation binding parameters were determined using [³H]-flunitrazepam ([³H]-FNZ) and washed membrane preparations. In initial experiments, Bmax values were decreased significantly in cortex, striatum, and cerebellum by antisense S-ODN infusion compared to phosphate buffered saline controls. However, these effects were not consistent in subsequent experiments and Bmax values in cortex were also decreased by random-base S-ODN treatment. Approximately 30% of the animals infused with S-ODNs with 100% S-ODN linkages displayed overt signs of toxicity. Modified S-ODNs produced no apparent toxicity, but had no significant effect on [³H]FNZ binding parameters. These results demonstrate that antisense S-ODN treatment can decrease [³H]FNZ binding in vivo, but also suggest that toxic effects of the S-ODN derivatives may interfere with the interpretation of the data.

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INTRODUCTION

GABA_A receptors are hetero-oligomeric complexes which are presumed to be pentameric in structure. These receptors are assembled and inserted into the membrane to form Cl^- channels which have distinct binding sites for GABA, benzodiazepines (BZDs), barbiturates, and convulsants such as picrotoxin. Brain GABA_A receptor subunits have been classified into four families based on sequence homology, and multiple subtypes are known within each family. To date, 6 a, 3 b, 3 g, 1 d, and 2 r subunit cDNAs have been cloned (see Rabow et al., 1995), making the potential for GABA_A receptor heterogeneity quite large. Immunoprecipitation studies with subunit specific antibodies have shown that the majority of brain GABA_A receptors contain a, b, and g₂ receptor subunits (Stephenson et al., 1990; Weiland et al., 1992). The subunit composition of GABA_A receptors determines their pharmacology. For example, the g subunit (co-expressed with a and b subunits) is required for BZD binding and function (Pritchett et al., 1989) and plays a role in mediating the effects of ethanol (Mihic and Harris, 1995). BZDs, such as flunitrazepam (FNZ) and diazepam, allosterically potentiate GABA action by increasing the frequency of Cl^- channel opening (Rogers et al., 1994). Establishing the pharmacological profile of the GABA_A g₂ subunit is complicated by the lack of highly selective ligands and the multiplicity of possible subunit combinations.

Advances in molecular biology have provided potentially powerful methods to examine receptor protein expression and function. One such method, the use of antisense oligonucleotides (ODNs), could be of considerable value when selective ligands are unavailable or when the receptor protein of interest is composed of multiple subunits which allosterically modulate receptor function. Antisense ODNs, complimentary to a select region of DNA or messenger RNA (mRNA) encoding the protein of interest, can potentially interfere with transcription and translation thereby decreasing gene expression. Although the precise mechanisms involved in the action of antisense ODNs are unknown, they are thought to inhibit gene expression by hybridizing to a specific region of mRNA thereby blocking translation (Crook, 1992). ODN/mRNA hybrids also provide a substrate for RNase H degradation (Walder and Walder, 1988), and ODNs are known to hybridize to double-stranded DNA forming a DNA triplex (Moser and Dervan, 1987). Antisense ODNs have been used successfully to inhibit the expression of several G-protein-linked receptors including dopamine D₂ (Zhang and Creese, 1993), muscarinic m₁ (Zang et al., 1994) and m₂ (Holopainen and Wojcik, 1993), neuropeptide Y-Y₁ (Wahlestedt et al., 1993b), and d opioid receptors (Standifer et al., 1994). In addition, decreased expression of select subunits of ligand-gated ion channels including the g₂ subunit of the GABA_A (Karle and Nielsen, 1995; Karle et al., 1995), the GluR-1 subunit of the AMPA receptor (Vanderklish et al., 1992), and the NMDA-R₁ subunit of the NMDA receptor (Wahlestedt et al., 1993a) has been reported.

The antisense strategy of manipulating gene expression in vivo typically does not result in a complete loss of a given protein and may represent an important alternative to techniques such as targeted disruption select genes (mutant animals). Targeted disruption of the mouse g₂ subunit gene of the GABA_A receptor results in a 94% decrease in BZD binding sites; however, the life-span of the animal is drastically reduced (Günther et al., 1995). The purpose of our present study was to determine whether antisense ODNs could be used to selectively decrease the g₂ subunit of the GABA_A receptor in mouse brain. The g₂ subunit is thought to play a role in the ethanol sensitivity of Long Sleep (LS) mice (Deitrich et al., 1989), therefore, these animals were used in all experiments. Phosphorothioate ODNs (S-ODNs) with 100% phosphorothioate linkages or modified S-ODNs (capped or alternating phosphorothioate linkages) were continuously infused into the lateral ventricle of mice via miniosmotic pumps for 3.5 days. We found that S-ODNs with 100% phosphorothioate linkages inconsistently reduced the density of [³H]FNZ binding sites in particular brain regions. The effects, however, were not entirely specific as random-base S-ODN control treatment also decreased [³H]FNZ Bmax values. In addition, approximately 30% of mice treated with S-ODNs (100% phosphorothioate linkages) displayed signs of toxicity. Modified S-ODN treatment did not show evidence of toxicity but had no effect on [³H]FNZ binding.

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MATERIALS AND METHODS

Male Long Sleep (LS)/Ibg mice, selectively bred for sensitivity to ethanol, were obtained from the Institute for Behavioral Genetics, University of Colorado, Boulder, CO. The mice (60-80 days of age) were housed five mice/cage at 25°C on a 12 hr. light/dark cycle.

A 23-mer phosphorothioate antisense oligodeoxynucleotide (S-ODN) complementary to the translational start site of the mouse g₂ subunit (5'-CATGTATTTGGCGAACTCATCGC-3') and a control S-ODN (consisting of randomized nucleotides with identical base content as the antisense sequence; 5'-CGTAGAGTTGTATCTCACTCACG-3') were synthesized on an Applied Biosystems PCR mate synthesizer using tetraethylthiuram disulfide provided by the manufacturer, or obtained from either DNA Express, Colorado State University (Ft. Collins, CO) or The Midland Certified Reagent Company (Midland, TX). In different experiments, the S-ODN's were either partially purified (precipitation with n-butanol) or HPLC-purified, and differed in the placement and number of phosphorothioate linkages (Figure 1).

Fig.1: Location of phosphorothioate linkages present in antisense and random base phosphorothioate oligonucleotides (S-ODNs).

Alzet miniosmotic pumps (Alza Corp., Palo Alto, CA) were assembled according to manufacturer's instructions. S-ODN's were suspended in phosphate buffered saline (PBS) and administered intracerebro-ventricularly (ICV; 35 and 70 µg S-ODN/day; 4-6 animals/group) via osmotic pumps designed to deliver 1 µl/hr for 3 days. ICV administration of PBS alone was used as an additional control. Animals were anesthetized to a deep surgical level with sodium pentobarbital (120 mg/kg; i.p.) and chloral hydrate (80 mg/kg; i.p.) and a subcutaneous pocket was made in the midscapular region by blunt dissection. A 1 mm diameter hole was drilled through the skull 1 mm lateral to bregma, above the left lateral ventricle, and the cannula was lowered to a depth of 3 mm from the skull surface. The osmotic pump was positioned into the subcutaneous pocket, the pocket was sutured shut, and the cannula was glued to the surface of the skull. The exposed area surrounding the cannula was covered with dental cement. Animals were placed in an incubator (32°C) for 2 h following surgery, and allowed to recover with food and water available ad libitum.

[³H]FNZ binding was used as a marker for the g₂ subunit since this subunit is required for binding of BZDs to the GABA_A receptor. The method of Pritchett et al. (1993a) was used with minor modifications. Crude washed membrane preparations were prepared by homogenization in 10 mM potassium phosphate buffer (pH 7.4) and pelleted by centrifugation (20,000 x g). After 4 subsequent washes, the membranes were suspended in 10 mM potassium phosphate buffer (pH 7.4) containing 100 mM KCl. GABA_A receptors were measured in cortex, striatum, hippocampus and cerebellum by indirect saturation binding. Binding parameters were determined using [³H]FNZ (1 nM) in the absence or presence of 10 concentrations of unlabeled flunitrazepam (FNZ; 0.316 pM - 1 µM) in 10 mM KH₂PO₄/K₂HPO₄ buffer containing 100 mM KCl incubated at 0°C for 1 hr. Nonspecific binding was defined with clonazepam (1 µM). For A₁ adenosine receptor measurements (Mayfield and Zahniser, 1996), the tissue was preincubated with adenosine deaminase (2 IU/ml) for 30 min at 37°C . The tissue was then incubated with a saturating concentration of [³H]N6-cyclohexyladenosine ([³H]CHA; 12 nM) in 50 mM Tris buffer containing 10 mM MgCl₂ at 25°C for 2 hr. Nonspecific binding was defined with 2-chloroadenosine (20 mM).

Receptor binding parameters (Bmax and Ki) were estimated by iterative nonlinear curve fitting (GraphPad Software, San Diego, CA). Parameter estimates were analyzed by one-way ANOVA followed by Newman-Keuls post-hoc comparisons.

Four separate experiments were conducted with phosphorothioate linkages in different positions in the ODNs. In the first two experiments animals were treated with partially purified S-ODN's containing 100% phosphorothioate linkages. In the third experiment animals were treated with HPLC-purified S-ODNs in which the first 5 and last 5 linkages were sulfurized (capped). In the fourth experiment partially purified and HPLC-purified S-ODNs (100% phosphorothioate linkages) were compared to S-ODNs containing alternating phosphorothioate linkages.

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RESULTS

Indirect [³H]FNZ saturation curves were constructed to determine the effects of the S-ODN treatment on GABA_A receptor g₂ subunit expression in the LS mouse brain (Figure 2). Density (Bmax) and affinity (Ki) of [³H]FNZ binding sites were determined from these curves. Infusion via miniosmotic pump of partially purified antisense S-ODN (100% phosphorothioate linkages; 35 or 70 µg/day) in the left lateral ventricle of LS mice for 3.5 days resulted in brain region-specific decreases in maximal [³H]-FNZ binding (Figure 3). Compared with PBS controls, Bmax values were decreased significantly in membrane preparations of left and right cerebral cortex (28% decrease), striatum (20% decrease), and cerebellum (36% decrease) by antisense S-ODN (70 µg/day). Similar decrements were produced by the 35 µg/day dose although the decrease was not significant in striatum. These changes in Bmax values were not associated with changes in non-specific binding (7% of total binding). Bmax values were unchanged by either dose in hippocampal membrane preparations. Ki values (striatum 8-10 nM; all other regions 1-2 nM) were unchanged (data not shown). The yield of partially purified randomized S-ODN (100% phosphorothioate linkages) was not sufficient to treat a complete group of control animals in this experiment.

Fig.2 : Flunitrazepam (FNZ) competition of [3H]FNZ (1 nM) binding to membranes of right cortex from Long Sleep (LS) mice treated intracerebro-ventricularly (ICV) into the left ventricle for 3.5 days with phosphate buffered saline (PBS) or partially purified S-ODNs. Curves were generated by nonlinear least-squares regression. Density (Bmax) and affinity (Ki) values were determined from this type of indirect saturation experiment for the entire study. Symbols represent the mean±sem for N=5-6.

 

Fig.3 : Density of [3H]FNZ binding sites in washed membrane preparations of cerebral cortex (CTX; left and right values), hippocampus (HIP), striatum (STR), and cerebellum (CBL) after ICV administration of PBS or partially purified antisense S-ODN (100% sulfurized linkages) for 3.5 days. S-ODN treatment resulted in a similar decrease in Bmax values in the left and right hemispheres of the cort ex, thus, the data were pooled. Data are expressed as mean±sem for N=5-6. Data were analyzed by analysis of variance followed by Newman-Keuls post-hoc tests; *p<0.05, **p<0.01 compared to PBS control.

Experiment 1 was repeated using the 70 µg/day dose to determine the between-experiment variance associated with the treatment protocol and included a randomized S-ODN control group (Figure 4). Partially purified antisense S-ODN (100% phosphorothioate linkages) treatment significantly decreased Bmax values in cortex (40% decrease) and cerebellum (22% decrease) compared to PBS controls. No significant effects were observed in striatum or hippocampus. Bmax values in cortex were also decreased significantly after randomized S-ODN administration (22% decrease). However, in cortex, the antisense-induced decrease in Bmax values was significantly greater than that resulting from random-base control treatment (25% decrease). As was observed in Experiment 1, changes in Bmax values were not associated with changes in non-specific binding. No significant changes in Ki values were observed (data not shown). As an additional control for non-specific effects, adenosine A₁ receptor binding was performed in the same membrane preparations (cortex and hippocampus). Neither the density nor the affinity (data not shown) of A₁ receptors as measured with [³H]CHA were altered by any of the treatments.

Fig.4 : Density of [3H]FNZ binding sites in washed membrane preparations of cerebral cortex (CTX), hippocampus (HIP), striatum (STR), and cerebellum (CBL) after ICV administration of PBS, partially purified randomized S-ODN (100% sulfurized linkages; 70 µg/day), or partially purified antisense S-ODN's (100% sulfurized linkages; 70 µg/day) for 3.5 days. Data are expressed as mean±sem for N=5-6 and were analyzed by analysis of variance followed by Newman-Keuls post-hoc tests; **p<0.01 compared to PBS control, **†p<0.01 compared to random-base control.

In the third experiment, animals were administered HPLC-purified S-ODNs in which the first 5 and last 5 internucleoside linkages contained the nonbonding sulfur (capped; Figure 1) to test whether the nonspecific effects of randomized S-ODN treatment observed in Experiment 2 could be attributed to S-ODN purity and/or the degree of ODN sulfurization. In cortex and cerebellum (the only regions examined), small, statistically nonsignificant decreases (13 and 6%, respectively) in maximal [³H]FNZ binding were observed from antisense S-ODN treated animals (35 and 70 µg/day) compared to PBS controls. Bmax values were also unaffected by randomized S-ODN treatment. Ki values were not significantly changed by any treatment (data not shown).

Lastly, partially purified and HPLC-purified S-ODNs which were either 100% sulfurized or had the nonbonding sulfur at alternating internucleoside linkages (100% and alternating, respectively; Figure 1) were compared. [³H]FNZ binding parameters were determined only in membrane preparations of left cortex. Bmax and Ki values (data not shown) were not changed by any of the treatments.

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DISCUSSION

ICV administration of S-ODNs (100% phosphorothioate linkages; Figure 1) both purified and partially purified (antisense and randomized) resulted in significant ataxia, tachypnea, and loss of appetite in approximately 30% of the animals per group. These side-effects were not observed in animals treated with PBS or S-ODNs which were not 100% sulfurized (capped and alternating phosphorothioate linkages; Figure 1). However, the occurrence of toxic side-effects was not correlated with the changes in [³H]FNZ binding observed in these experiments (data not shown). The antisense strategy for inhibiting gene expression has been used to specifically inhibit the expression of a variety of proteins including neurotransmitter receptors. The present studies were undertaken to determine whether the g₂ subunit of the GABA_A receptor could be selectively and substantially reduced after ICV infusion of an antisense S-ODN. [³H]FNZ binding was used as a marker for the g₂ subunit since this subunit is required for BZD binding to the GABA_A receptor (Pritchett et al., 1993b). S-ODN's were continuously infused into the lateral ventricle of mice for 3.5 days via miniosmotic pumps. This delivery system avoids high transient concentrations of ODN and has been shown to provide uniform distribution in cerebrospinal fluid (Whitesell et al., 1993). The concentrations of S-ODN administered to mice in these experiments were based on a concentration of continuously infused S-ODN which has been shown to effectively decrease the density of dopamine D₂ receptor binding sites in rats (Zhang and Creese, 1993), and on the difference in intraventricular volume and CSF production for rats and mice (Pardridge, 1991). The concentrations of S-ODNs used in the present study (1.5 or 2.9 µg/µl) were below those reported to be lethal when infused continuously in rats (Whitesell et al., 1993).

In our initial experiments, ICV infusion of a partially purified antisense S-ODN with 100% phosphorothioate linkages decreased [³H]FNZ binding in a brain region-dependent manner, but the magnitude and brain regional specificity of this effect varied from experiment to experiment. In general, when significant effects of antisense treatment were observed, [³H]FNZ binding decreased 20-40% in cortex and cerebellum compared to PBS controls (figures 3 and 4). However, in a subsequent experiment, an identical treatment protocol had no effect on BZD receptor binding. Karle and Neilsen (1995) reported small but statistically significant decreases of 9-11% in [³H]FNZ binding in rat striatum and cerebral cortex, respectively, after 2 days of ICV bolus injections of a similar antisense S-ODN directed at the initiation start sequence of the g₂ subunit. Differences in experimental procedures are likely to explain the differences in response magnitude found in these studies. First, even though the concentrations of S-ODNs administered were similar between the two studies [2.0 or 4.0 µg/ul (Karle and Nielsen, 1995); 1.5 or 2.9 µg/µl present study], the total amount of S-ODN delivered was considerably greater in our experiments (40 versus 245 µg for the high doses). Secondly, S-ODNs were infused continuously for a greater period of time (2 days versus 3.5 days) in our experiments; and thus, steady-state levels of S-ODN should have been higher. S-ODNs have been shown to be rapidly cleared from the ventricular space after bolus ICV injections (Whitesell et al., 1993). Receptor turnover rate, subunit mRNA levels, and duration of S-ODN treatment are all undoubtedly important factors which determine the magnitude of protein loss in response to antisense treatment. A shorter treatment duration might be adequate for receptors with low basal expression and rapid turnover whereas receptors with high basal expression and slower turnover may require longer treatment periods. Currently, the in vivo turnover rate for the GABA_A receptor or its individual subunits is unknown; thus, the optimal treatment duration awaits evaluation of time-course experiments. In a recent report, an antisense ODN complementary to the g₂ subunit was infused continuously directly into the rat hippocampus for 3 or 5 days (Karle et al., 1995). After 5 days of antisense treatment BZD binding was not decreased to a greater extent than that observed after 3 days of treatment suggesting that at least one population of g₂ subunit may be sensitive to short duration antisense treatment.

In experiments in which antisense treatment did result in a decreased density of [³H]FNZ binding sites, it is unclear whether the total number of heteromeric GABA_A receptors decreased or whether receptors were assembled which either lacked g₂ subunits or had alternate subunits included in the receptor complex. However, the decreases in [³H]FNZ binding site density were not accompanied by changes in affinity (Ki) suggesting that the total number of GABA_A receptors decreased as a result of S-ODN treatment. However, it is also possible that S-ODN treatment resulted in a shift from a high- to a low-affinity state of a population of g₂ subunit containing receptors which might not be detectable under the receptor binding conditions used in these experiments. Such an occurrence could result in an apparent decrease in Bmax values. A loss of total GABA_A receptors is supported by the recent report that the binding of [³⁵S]tert-butyl-bicyclo-phosphorothionate and [³H]muscimol, which bind to the ion channel domain and GABA recognition site of the GABA_A receptor, respectively, decrease in parallel with [³H]FNZ binding as a result of continuous g₂ antisense S-ODN administration directly into the hippocampus (Karle et al., 1995).

Infusion of a control S-ODN (random-base; 100% phosphorothioate linkages) also decreased maximum [³H]FNZ binding in cortex but not in the other brain regions examined. Nonspecific effects of S-ODN have been reported in other experimental paradigms (Gao et al., 1992); however, it is unclear whether the decreases in [³H]FNZ binding observed in the present experiments were due to non-sequence specific effects of the S-ODN or whether generalized toxicity resulted from S-ODN infusion. An unexpected finding in our experiments was that approximately 30% of the mice infused with S-ODNs containing 100% phosphorothioate linkages (antisense and random-base sequences; 35 or 70 µg/day) exhibited some degree of ataxia, tachypnea, and loss of appetite. In addition, tissue in close proximity to the lateral ventricle (particularly striatum) appeared pale and swollen compared to tissue from PBS control animals. Adenosine A₁ receptor binding, which was assayed in the same tissue preparations and served as an additional control, was not affected by S-ODN infusion. The A₁ receptor system was chosen because of its widespread distribution in brain (Goodman and Snyder, 1982) and because it belongs to the G-protein coupled family of receptors. The lack of antisense or randomized S-ODN effects on A₁ binding argues against a generalized neuronal toxicity, however, this possibility can not be ruled out. Potential S-ODN toxicity was reported by Karle et al. (1995) in that tissue protein content as well as a marked change in tissue histology (neuronal cell death) occurred as a result of continuous infusion of a S-ODN (1.7 µg/µl). It was hypothesized that the observed neuronal cell death was the result of GABA_A receptor loss, but S-ODN toxicity could not be ruled out. In unrelated experiments we have observed very similar toxic effects of ICV infused S-ODNs (antisense and random-base sequences) directed to the dopamine D₂ receptor in rats (unpublished observations). We conclude that the toxic and nonselective effects of S-ODNs observed in these studies are a direct effect of S-ODN administration and not sequence-dependent effects related to the loss of a targeted protein.

We examined the possibility that these nonselective and toxic effects of S-ODN infusion were dependent on S-ODN purity and/or degree of sulfurization. The toxic side-effects of S-ODNs with 100% phosphorothiate linkages was not dependent on S-ODN purity. Approximately the same number of animals were affected adversely by S-ODNs purified by precipitation with n-butanol as were affected adversely by HPLC-purified S-ODNs. Furthermore, a lack of consistency in treatment effects was observed in these sets of experiments in that neither antisense nor randomized S-ODNs with 100% phosphorothioate linkages changed [³H]FNZ binding parameters. Modified S-ODNs, which differ in the number and position of phosphorothioate linkages, could be a potential alternative to S-ODNs with 100% phosphorothioate linkages. Modified ODNs ranging in length from 15 to 28 bases have been shown to retain a high degree of hybridization specificity and resistance to nuclease activity (Gao et al., 1992). In contrast to the toxic side-effects we observed after infusion of S-ODNs with 100% phosphorothioate linkages, S-ODNs with capped or alternating linkages produced no apparent changes in the behavior of the animals or in the macroscopic appearance of the brain tissue. However, no consistent changes in [³H]FNZ binding were observed after infusion of these S-ODNs. Small, statistically nonsignificant, decreases in [³H]FNZ binding were observed in one set of experiments while no change whatsoever was observed in another.

The results of these experiments indicate that ICV infusion of S-ODNs may produce a number of effects that are independent of effects on gene expression. Clearly, a number of questions remain to be answered if the antisense approach is to be of value when examining a complex ion channel such as the GABA_A receptor or to be of value as a general pharmacological tool. For example, the mechanisms involved in gene regulation are largely unknown thus targeting a single antisense ODN to the translational start site of a given mRNA sequence may not provide the most viable means of blocking transcriptional/translational events. The use of multiple ODNs complimentary to different sites along the target sequence(s) might improve the overall effectiveness of antisense treatment. This lack of mechanistic information regarding gene expression is complicated by the fact that the extent of uptake and distribution of ODNs are difficult to assess directly.

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ACKNOWLEDGMENTS

This work was supported by grants: AA 03527, NRSA GM07635, and NIH Neurobiology Development Training Grant. The authors wish to thank Dr. R.A. Harris for careful review of this manuscript and Rene Meyers for surgical assistance.

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REFERENCES

Crook, S.T. (1992) Therapeutic applications of oligonucleotides. Ann. Rev. Pharmacol. Toxicol., 32: 329-376.

Deitrich, R.A., Dunwiddie, T.V., Harris, R.A., and Erwin, V.G. (1989) Mechanism of action of ethanol: Initial central nervous system actions. Pharmacol. Rev., 41: 489-537.

Gao, W.-Y., Han, F.-S., Storm, C., Egan, W., and Cheng, Y.-C. (1992) Phosporothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: Implications for antisense technology. Mol. Pharm., 41: 223-229.

Goodman, R.R., and Snyder, S.H. (1982) Autoradiographic localization of adenosine receptors in rat brain using [³H]cyclohexyladenosine. J. Neurosci., 2: 1230-1241.

Günther, U., Benson, J., Benke, D., Fritschy, J.-M., Reyes, G., Knoflach, F., Crestani, F., Aguzzi, A., Arigoni, M., Lang, Y., Bluethmann, H., Mohler, H., and Lüscher, B. (1995) Benzodiazepine-insensitive mice generated by targeted disruption of the g₂ subunit gene of g-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci., 92: 7749-7753.

Holopainen, I., and Wojcik, W.J. (1993) A specific antisense oligodeoxynucleotide to mRNAs encoding receptors with seven transmembrane spanning regions decreases muscarinic m2 and gamma-aminobutyric acid_B receptors in rat cerebellar granule cells. J. Pharmacol. Exp. Ther., 264: 423-30.

Karle, J., and Nielsen, M. (1995) Modest reduction of benzodiazepine binding in rat brain in vivo induced by antisense oligonucleotide to GABA_A receptor g₂ subunit subtype. Eur. J. Pharmacol., 291: 439-441.

Karle, J., Witt, M.-R., and Nielsen, M. (1995) Antisense oligonucleotide to GABA_A receptor g₂ subunit induces loss of neurones in rat hippocampus. Neurosci. Lett., 202: 97-100.

Mayfield, R.D., and Zahniser, N.R. (1996) Opposing actions of adenosine A_2a receptors and dopamine D₂ receptor activation on GABA release in the basal ganglia: Evidence for an A_2a/D₂ receptor interaction in globus pallidus. Synapse, 22: 132-138.

Mihic, S.J., and Harris, R.A. (1995) Alcohol actions at the GABA_A receptor/chloride channel complex. In: Pharmacological Effects of Ethanol on the Nervous System, R.A. Deitrich, and V.G. Erwin, eds., CRC Press, Boca Raton, Fla., pp. 55-72.

Moser, H.E., and Dervan, P.B. (1987) Sequence-specific cleavage of double helical DNA by triple helix formation. Science, 238: 645-650.

Pardridge, W.M. (1991) In: Peptide drug delivery to the brain, Raven Press, New York, 226pp.

Pritchett, D.B., Lüddens, H., and Seeburg, P.H. (1989a) Type I and Type II GABA_A-benzodiazepine receptors produced in transfected cells. Science, 245: 1389-1392.

Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R., and Seeburg, P.H. (1989b) Importance of a novel GABA_A receptor subunit for benzodiazepine pharmacology. Nature, 338: 582-585.

Rabow, L.E., Russek, S.J., and Farb, D.H. (1995) From ion currents to genomic analysis: Recent advances in GABA_A receptor research. Synapse, 21: 189-274.

Rogers, C.J., Twyman, R.E., and MacDonald, R.L. (1994) Benzodiazepine and beta-carboline regulation of single GABA_A-receptor channels of mouse spinal neurons in culture. J. Physiol., 475: 69-82.

Standifer, K.M., Chien, C.-C., Wahlestedt, C., Brown, G.P., and Pasternak, G.W. (1994) Selective loss of d opioid analgesia and binding by antisense oligodeoxynucleotides to a d opioid receptor. Neuron, 12: 805-810.

Stephenson, F.A., Duggan, M.J., and Pollard, S. (1990) The g₂ subunit is an integral component of the g-aminobutyric acidA receptor but the a₁ polypeptide is the principal site of the agonist benzodiazepine photoaffinity labelling reactions. J. Biol. Chem., 265: 21160-21165.

Vanderklish, P., Neve, R., Bahr, B.A., Arai, A., Hennegriff, M., Larson, J., and Lynch G. (1992) Translational suppression of a glutamate receptor subunit impairs long-term potentiation. Synapse, 12: 333-337.

Wahlestedt, C., Golanov, E., Yamamoto, S., Yee, F., Ericson, H., Yoo, H., Inturrisi, C.E., and Reis, D.J. (1993a) Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions. Nature, 363: 260-263.

Wahlestedt, C., Pich, E.M., Koob, G.F., Yee, F., and Heilig, M. (1993b) Modulation of anxiety and neuropeptide Y-Y₁ receptors by antisense oligodeoxynucleotides. Science, 259: 528-531.

Walder, R.Y., and Walder, J.A. (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl. Acad. Sci., 85: 5011-5015.

Whitesell, L., Geselowitz, D., Chavany, C., Fahmy, B., Walbridge, S., Alger, J.R., and Neckers, L.M. (1993) Stability, clearance, and disposition of intraventricularly administered oligodeoxynucleotides: Implications for therapeutic application within the central nervous system, Proc. Natl. Acad. Sci., 90: 4665-4669.

Weiland, H.A., Lüddens, H., and Seeburg, P.H. (1992) A single histidine in GABA_A receptors is essential for benzodiazepine agonist binding. J. Biol. Chem., 267: 1426-1429.

Zang, Z., Florijn, W., and Creese, I. (1994) Reduction in muscarinic receptors by antisense oligodeoxynucleotide. Biochem. Pharmacol., 48: 225-228.

Zhang, M., and Creese, I. (1993) Antisense oligodeoxynucleotide reduces brain dopamine D₂ receptors: Behavioral correlates. Neurosci. Lett., 161: 223-226.