Patent Grant
7816074
Alcohol is an addictive recreational drug that reduces anxiety at low doses and causes sedation at high doses (1). These effects are similar to those of drugs that enhance the function of gamma-amino butyric acid sub A. receptor, referred to herein as GABAA receptor, which gate the Cl− currents that mediate most inhibitory neurotransmission in the brain. Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABA (A) receptor. Acutely high doses of alcohol potentiate GABA-gated currents (1) at both native (1) and recombinant GABAA receptors (42), and chronically alter GABAA receptor expression (34). Low doses of alcohol have not been shown to directly modulate recombinant GABAA receptors (1), although there is indirect evidence for such effects at native receptors (6, 7, 29, 76).
It has been suggested that discrepancies between the alcohol sensitivity of native and recombinant receptors may be due to their subunit composition (64). Therefore there is a need to investigate the effects of low concentrations of alcohol on different subtypes of GABAA receptors.
Recently, a novel subunit combination of the GABAA receptor, α4β2δ, was identified (67), which is potentiated by very low concentrations of ethanol (1-3 mM), assessed using two electrode voltage clamp procedures and heterologous expression in oocytes. However, higher concentrations of ethanol (>10 mM) were ineffective in this regard, resulting in an “inverted U” concentration-response relationship. Because other subtypes were unresponsive to this low concentration of ethanol, including α4β2γ2 subunits of the GABAA receptor, this suggests that α4βδ subunits of the GABAA receptor possess a unique sensitivity to ethanol (67, 76).
Ethanol is known to exert behavioral (34) and electrophysiological (58) effects at relatively low concentrations. Low concentrations of ethanol can exert both postsynaptic and presynaptic effects in hippocampus and amygdala (8, 58, 76). This presynaptic effect of ethanol is enhanced when GABAB receptors are blocked (76), suggesting an effect of the drug on transmitter release, while effects on GABAergic transmission in amygdala are mediated via CRF1 receptors (54). Behavioral studies indicate that GABAA receptor antagonists/negative modulators can decrease ethanol consumption (32, 40), and ethanol can substitute for other GABA-modulatory drugs in drug discrimination tasks (18). Despite this evidence at native receptors, ethanol modulation of most recombinant GABAA receptor subtypes (50, 27), NMDA receptors (43), and glycine receptors (50) require comparatively high concentrations of the drug. In fact, the anesthetic effect of ethanol requires identified residues on the M2 region of the GABAA receptor (50). In contrast, the only recombinant receptor thus far identified as responsive to low concentrations of ethanol is the α4βδ subunit combination of the GABAA receptor.
The α4βδ subunits of the GABAA receptor have very low expression in most areas of the central nervous system (53, 81). The α4βδ subunits of the GABAA receptor also exhibit a distinctive response to GABA agonists, producing a greater maximum current in response to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP or gaboxadol), a GABA partial agonist, compared to the maximum current gated by GABA (6), suggesting that 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol is a full agonist at α4βδ subunits of the GABAA receptor. A similar increase in the ratio of maximum 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol response to maximum GABA response was noted in isolated hippocampal neurons after progesterone withdrawal (67, 25), consistent with increased expression of α4βδ subunits of the GABAA receptor at this time. Coexpression of α4 and δ subunits of the GABAA receptor was also determined directly in hippocampus using co-immunoprecipitation techniques (67).
Under these conditions of increased expression of α4βδ subunits of the GABAA receptor after progesterone withdrawal, GABA-gated current recorded from isolated hippocampal neurons was also potentiated by 1-3 mM, but not higher (>10 mM), concentrations of ethanol (67). Suppression of α4 expression prevented the response to 1-3 mM ethanol after P withdrawal, further suggesting that α4βδ subunits of the GABAA receptor possess a unique sensitivity to ethanol. Interestingly, ethanol withdrawal also increases expression of the α4 subunit of the GABAA receptor (44, 14) but decreases expression of the δ subunit of the GABAA receptor (7), an effect correlated with a decrease in response to ethanol.
Therefore, there is a need to understand the relationship between the α4βδ GABAA receptor and increased sensitivity to alcohol in order to provide a mechanism for identifying and treating members of the general population at increased risk for alcoholism and premenstrual anxiety, also known as premenstrual syndrome, herein designated PMS.
The present invention provides methods and compositions for determining whether a subject is at increased risk for alcoholism or premenstrual anxiety. In one aspect of the invention, there is provided a method comprising the steps of: (a) administering to a subject a therapeutically effective amount of a GABAA receptor modulator and determining whether the subject is sensitive or insensitive to such GABAA receptor modulator; (b) subsequently administering a therapeutically effective amount of a GABAA receptor agonist and determining whether the subject is sensitive or insensitive to such GABAA agonist; and (c) correlating a decreased sensitivity to a GABAA receptor modulator and an increased sensitivity to a GABAA agonist with an increased risk of alcoholism or premenstrual anxiety in the subject.
In another aspect of the present invention, there is provided a method of screening for a drug which decreases expression of the α4β2δ subunit of GABAA, comprising the steps of: (a) isolating and culturing neurons; (b) applying a drug to the cultured neurons; (c) measuring the level of δ subunit of GABAA from the treated neurons of step (b); (d) determining whether the drug applied in step (b) decreases expression of the δ subunit of GABAA receptors; and (e) correlating a decrease in expression of the δ subunit of GABAA receptors found in the treated neurons of step (b) when compared to a control neuron culture having no drug application, with the identification of a drug which decreases expression of α4β2δ GABAA receptors.
The present invention also provides an additional method of screening for a drug which decreases expression of the α4β2δ subunit of GABAA receptor. The method comprises the steps of: (a) expressing α4β2δ GABAA receptors ineukaryotic cells (b) applying a drug to the eukaryotic cells of (a); (c) measuring the level of δ subunit of GABAA from the treated eukaryotic cells of step (b); (d) determining whether the drug applied in step (b) decreases expression of the δ subunit of GABAA receptors; and (e) correlating a decrease in expression of the δ subunit of GABAA receptors found in the treated eukaryotic cells of step (b) when compared to a control eukaryotic cell population having no drug application, with the identification of a drug which decreases expression of α4β2δ GABAA receptors.
The present invention provides a drug, which blocks α4β2δ GABAA receptors and a method of treating a subject at risk for alcoholism, comprising administering a therapeutically effective amount of a drug which decreases or blocks the expression of α4β2δ GABAA receptors.
Also provided by the present invention is a method for identifying a drug, which blocks α4β2δ GABAA receptors. The method comprising the steps of: (a) isolating and culturing neurons; (b) applying a drug to the cultured neurons of (a); (c) measuring GABAA gated currents at α4β2δ GABAA receptors in the treated neurons of (b); and (d) correlating a decrease in GABAA-gated currents recorded at α4β2δ GABAA receptors when compared to a control culture with no drug application, with the identification of a drug which blocks α4β2δ GABAA receptors.
In yet another embodiment, the present invention provides an additional method for identifying a drug, which blocks α4β2δ GABAA receptors. The method comprises the steps of: (a) expressing α4β2δ GABAA receptors in eukaryotic cells; (b) applying a drug to the eukaryotic cells of (a); (c) measuring GABAA gated currents at α4β2δ GABAA receptors in the treated eukaryotic cells of (b); and (d) correlating a decrease in GABAA-gated currents recorded at α4β2δ GABAA receptors when compared to a eukaryotic cell population having no drug application, with the identification of a drug which blocks α4β2δ GABAA receptors.
In accordance with the present invention, it has been surprisingly found that very low concentrations of alcohol, e.g., as little as 1 mM ethanol, selectively increase GABA-gated currents at α4β2δ receptors by 45±11% (P<0.05). Also in accordance with the present invention, the α4β2δ GABAA receptor isoform is unique in its responsiveness (sensitivity) to such low concentrations of ethanol. Thus, members of the general population who have increased expression of the α4β2δ GABAA receptor isoform are likely to have an increased sensitivity to lower concentrations of alcohol coupled with a decreased sensitivity to higher concentrations of alcohol, a scenario frequently found in patients suffering from alcoholism and PMS.
Based on these findings, the present invention provides a screening mechanism for identifying members of the general population at increased risk for alcoholism.
The method comprises the steps of:
(a) administering to a subject a therapeutically effective amount of a GABAA receptor modulator and determining whether the subject is sensitive or insensitive to such GABAA receptor modulator; (b) subsequently administering a therapeutically effective amount of a GABAA receptor agonist and determining whether the subject is sensitive or insensitive to such GABAA agonist; and (c) correlating a decreased sensitivity to a GABAA receptor modulator and an increased sensitivity to a GABAA agonist with an increased risk of alcoholism and/or PMS in the subject.
An example of GABAA receptor modulator is a benzodiazepine. Examples of benzodiazepine include but are not limited to Valium (diazepam), Activan (lorazepam), Midazolam, Zolpidem or Flunitrazepam. Additional examples of GABAA receptor modulators include neurosteroids, such as allopregnanolone or 3α,5α-THP, benzodiazepine antagonists, such as flumazenil, benzodizepine inverse agonists, such as DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), barbiturates, such as pentobarbital, anesthetics, such as propofol, volatile anesthetics, such as halothane and ethanol. Dosages may vary and may include for example a dose range of about 1 to about 20 mg/kg.
An example of a GABAA receptor agonist includes but is not limited to gaboxadol, also known as THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and may be administered in the range of from about 1 to about 3 mg/kg. Additional examples of GABAA receptor agonists include muscimol, beta-alanine, taurine, THIP (gaboxadol), isoguvacine, trans-4-aminocrotonic acid, TACA, (z)-3-[(aminoiminomethyl)thio-2-propenoic acid, ZAPA, and piperidine-4-sulfonic acid (P-4S).
Also provided by the present invention is a method of screening for a drug, which decreases expression of the α4β2δ subunit of the GABAA receptor Drugs identified by this method are useful for treatment of alcoholism. The method comprises the steps of: (a) isolating and culturing neurons; (b) applying a drug to the cultured neurons; (c) measuring the level of δ subunit of GABAA from the treated neurons of step (b); (d) determining whether the drug applied in step (b) decreases expression of the δ subunit of GABAA receptors; and (e) correlating a decrease in expression of the δ subunit of GABAA receptors found in the treated neurons of step (b) when compared to a control neuron culture having no drug application, with the identification of a drug which decreases expression of α4β2δ GABAA receptors. Methods for culturing neurons are well known in the art as described e.g., in “Heterogeneity of GABA(A) receptor-mediated responses in the human IMR-32 neurobalstoma cell line.” J. Neuroscience Res. 60(4): 504-10, May 15, 2000, which article is incorporated by reference herein as if fully set forth. Examples of neurons suitable for use in the methods of the present invention include but are not limited to IMR-32 neurobalstoma, slice cultured neurons, neurobalstoma cell lines such as IMR-32 NTZ and other cultured rodent neurons.
Alternatively, a method of screening for a drug which decreases expression of the α4β2δ subunit of the GABAA receptor comprises the steps of: (a) expressing α4β2δ GABAA receptors in eucaryotic cells (b) applying a drug to the eukaryotic cells of (a); (c) measuring the level of δ subunit of GABAA from the treated eukaryotic cells of step (b); (d) determining whether the drug applied in step (b) decreases expression of the δ subunit of GABAA receptors; and (e) correlating a decrease in expression of α4β2δ GABAA receptors found in the treated eukaryotic cells of step (b) when compared to a control eukaryotic cell population having no drug application, with the identification of a drug which decreases expression of α4β2δ GABAA receptors.
Examples of eukaryotic cells suitable for use in the methods of the present invention include but are not limited to Xenopus laevis oocytes, Chinese hamster ovaries (CHO) cells, mouse fibroblast L929 cells, mouse L(−tk) fibroblast cell line, human embryonic kidney cells (HEK-293 cells) green monkey kidney cells, COS cells, or variants thereof.
Levels of the δ subunit of GABAA may be measured e.g., using an immunological detection method such as a Western blot procedure or the pharmacological tests described above. An antibody for the δ subunit of GABAA is readily available (56).
Expression of α4β2δ GABAA receptors in eukaryotic cells may be performed using methods well known in the art. For example, the nucleotide sequences for the α4β2δ GABAA receptor subunits may be subcloned into an appropriate expression vehicle such as a vector. There are various combinations which may be used to express such GABAA receptor subunits. For example, several expression vectors may be employed to express each subunit sequence separately. Alternatively, two or more subunit sequences may be expressed in the same expression vector.
Nucleotide sequences for the various GABAA receptor subunits, appropriate for use in an expression vector, are known and widely available. For example, mouse α4 (37), human α4 (52), rat β2 (83), mouse β2 (36), human δ (12) and rat δ (63) may be used.
Expression of the different GABAA receptor subunits sequence may be achieved by operably linking a promoter sequence to a receptor coding sequence using well known methods. Examples of promoters which function in eukaryotic cells and which may be used in the methods of the present invention include but are not limited to T7, CMV, and SV40. Methods of constructing expression vectors using such promoters are well known (59). Commercially available vectors may include, for example, PCDM8, pCDNA3.1 or pGHEMHE (Invitrogen, Carlsbad, Calif.).
Transfection of eukaryotic cells with nucleotide sequences for the α4β2δ GABAA receptor subunits may be performed using various well-known methodologies such as calcium phosphate, DEAE dextran, lipofection, electroporation, and injection. In order to express the α4β2δ GABAA receptor subunits in a eukaryotic cell, the expression vector may be introduced into the cell using the transfection methods described above. Alternatively, mRNA transcripts, or cRNA encoding the receptor subunits may be introduced into the eukaryotic cells. For example, oocytes may be injected with receptor subunit cRNAs. A range of from about 1 t about 100 ng of each receptor subunit sequence per 35 mm diameter dish of confluent cells may be used. Preferably, a range of from about 2 to about 20 ng of each receptor subunit sequence per 35 mm diameter dish of confluent cells is used
In still another embodiment of the present invention, there is provided a method of screening for a drug which blocks α4β2δ GABAA receptors. Drugs identified by this method are useful for treatment of alcoholism. The method comprises the steps of: (a) isolating and culturing neurons; (b) applying a drug to the cultured neurons of (a); (c) measuring GABAA gated currents at α4β2δ GABAA receptors in the treated neurons of (b); and (d) correlating a decrease in GABAA-gated currents recorded at α4β2δ GABAA receptors when compared to a control culture with no drug application, with the identification of a drug which blocks α4β2δ GABAA receptors. Alternatively, the method comprises the steps of: (a) expressing α4β2δ GABAA receptors in eukaryotic cells; (b) applying a drug to the eukaryotic cells of (a); (c) measuring GABAA gated currents at α4β2δ GABAA receptors in the treated eukaryotic cells of (b); and (d) correlating a decrease in GABAA-gated currents recorded at α4β2δ GABAA receptors when compared to a eukaryotic cell population having no drug application, with the identification of a drug which blocks α4β2δ GABAA receptors.
Also provided by the present invention are methods of treating a patient at risk for, or suffering from, alcoholism. The methods comprise administering to the patient a therapeutically effective amount of a drug which decreases expression of, and/or blocks α4β2δ GABAA receptors.
The following examples further describe the invention and are not meant in any way to limit the scope thereof.
Materials
Adult, female Long Evans rats (200-250 grams at the time of testing) were used in the following example. Animals were housed in pairs under controlled conditions (14:10 light:dark cycle, 24° C.) with free access to food and water. Progesterone (P, Sigma, crystalline)-filled capsules of silicone tubing (Nalgene Co. 1/16″×⅛″ o.d., 10 mm/100 g body weight) were implanted subcutaneously in the lower back after induction of halothane anesthesia (3-6% in O2, 1 liter/minutes) in the experimental animals. This paradigm has been shown to result in hippocampal and circulating levels of 3α,5α-THP and circulating levels of P in the physiological range (51), and halts estrous cyclicity until 8 hours after removal of the capsule. Capsules were removed under halothane anesthesia after a 21-day P administration period, and animals tested 24 hours after the last injection (“P withdrawal”). Control animals were tested on the day of diestrus, also referred to as diestrus controls, a low hormone state, as determined by vaginal lavage. This stage results in α4 levels similar to chronic P treatment (64). In addition, separate control animals, also referred to as sham controls, were implanted with empty capsules and tested with selected high or low doses of ethanol in the ASR paradigm or elevated plus maze 24 hours after implant removal. All animals were tested between 0730 and 1100 hour. Euthanasia was either by decapitation in the electrophysiology experiments or CO2 inhalation for the behavioral experiments following the indicated protocols. Both care and use of, as well as all procedures involving animals have been approved by the SUNY Downstate Medical Center Institutional Animal Care and Use Committee. In addition, all procedures were performed in accordance with the guidelines of the Institutional Care and Use Committee of the National Institute on Drug Abuse, National Institutes of Health, and the Guide for the Care and Use of Laboratory Animals (33).
Statistical Analysis
Average values for ethanol-induced changes in peak GABA-gated current and the ASR were evaluated across drug-treatment groups by one-way analysis of variance (ANOVA) with a post-hoc Tukey's test. Data from the plus maze were analyzed in an ANOVA followed by post hoc t-tests (Fisher's PLSD). A progesterone value <0.05 was used as an indication of statistical significance.
Patch Clamp Electrophysiology: Methods
Pyramidal neurons were acutely dissociated from CA1 hippocampus and GABA-gated Cl− currents analyzed using whole cell patch clamp procedures as previously described (67). Briefly, tissue was digested at 32° C. for 50-60 minutes under 100% O2 in PIPES-buffered saline containing (in mM): NaCl 120, KCl 5, CaCl2 1, MgCl2 1, D-glucose 25, PIPES 20 and trypsin (type XI, sigma) 0.8 mg/ml, pH 7.0. After 1 hour of enzyme-free incubation at room temperature, tissue was dissociated by trituration in 1 ml of 20 mM HEPES-buffered DMEM which was replaced by recording medium after transfer to the recording chamber. GABA-gated currents were recorded at room temperature (20-25° C.) at a holding potential of −50 mV in a bath containing (in mM): NaCl 120, CsCl 5, CaCl2 2, MgCl2 1, TEA Cl 15, 4-aminopyridine 5, HEPES 10 and glucose 25, pH 7.4, 320 mOsm/kg H2O. The pipette solution contained (in mM): N-methyl-D-glucamine chloride 120, Cs4BAPTA 5 and Mg-ATP 5. The ATP regeneration system Tris phosphocreatine (20 mM) and creatine kinase were added to minimize GABA rundown. All drugs were obtained from Sigma Chemical Co, (St. Louis, Mo.), except for BAPTA (Calbiochem, San, Diego, Calif.).
For the ethanol administration studies, drug delivery was accomplished with a superfusion system positioned within 50-100 μM of the cell, which yielded approximate 40-100 ms exposure times. Ethanol (3 or 20 mM) was applied concomitant with GABA (EC20=10 μM). Peak currents were calculated for all drug concentrations as relative to the current gated by 10 μM GABA alone using the equation: ([peak current: GABA+ETOH]−[peak current: GABA alone])/[peak current: GABA alone]. Currents were recorded using an Axopatch 1D amplifier (Axon Instruments) filtered at 2 kHz (four-pole Bessel filter) and detected at 500 Hz (pClamp 5.1).
Results
Ethanol Modulation of GABA-Gated Current
After progesterone withdrawal, GABA-gated responses of acutely isolated CA1 hippocampal pyramidal cells were potentiated by a mean 75±10% (P<0.01) with a 3 mM concentration of ethanol (
Acoustic Startle Response (ASR) Methods
Animals were tested in a stabilimeter device (SR-Lab Startle Response System, San Diego Instruments, Inc., San Diego, Calif.) that permits vertical movement of a Plexiglas platform contained within a lit, well-ventilated sound-attenuating chamber (26). The amplitude of movement is detected by a piezoelectric device with a voltage output proportional to platform displacement. The analog output of the accelerometer is then amplified and digitized before computer analysis. Five minutes after i.p. administration of 10% ethanol (0.05-1.6 g/kg) or saline vehicle (1.5 ml volume) or 25 minutes after 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol injection (3 mg/kg, i.p.), animals were placed in the chamber for a 5 minutes acclimation period. The drug latencies were previously established in this laboratory (25, 67) and others (34) as optimal for the anxiolytic effects of these two drugs, which have distinct pharmacokinetics. Four acoustic stimuli (116 dB, 40 ms duration) were then presented at random intervals once every 10-15 seconds via a white-noise generator through speakers positioned on either side of the cage. The 40 ms 116 dB acoustic stimulus has previously been demonstrated in a paradigm (26) as the minimum effective stimulus to produce a robust startle response in all animals. For each acoustic stimulus, the amplitude and integrated response (amplitude and duration) were determined and normalized to the weight of the animal. Values for the integrated response after administration of each dose of ethanol or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were averaged and expressed as a fraction of the saline control average for the indicated treatment (progesterone withdrawal versus control) group in order to minimize variability between groups. This paradigm has been used to demonstrate the anxiolytic effects of low doses of ethanol in ethanol-preferring rats (P rats, 34). It is also routinely used to assess behavioral excitability after withdrawal from BDZs and ethanol (73).
Results
Effects of Ethanol on the Acoustic Startle Response (ASR)
In order to compare the concentration dependence of ethanol effects in a behavioral paradigm, ethanol was tested across a concentration range (0.05-1.6 g/kg, i.p.) for its effect in modulating the ASR, a measure of behavioral excitability, which is increased after P withdrawal (26). To this end, animals were injected with ethanol or saline vehicle 5 minutes before placement in an acoustically isolated startle chamber. After an additional 5 minutes acclimation period, the startle responses to four 116 dB acoustic stimuli were averaged and adjusted for the weight of the animal. Both average peak and integrated startle responses were significantly (P<0.05) attenuated by about 70% with very low (0.2-0.4 g/kg) doses of ethanol after P withdrawal, an effect not observed in diestrous control animals or in sham control animals (
Capsule implantation was not responsible for the altered response to ethanol, as both diestrous (unimplanted) controls and sham controls (implanted with an empty capsule) reacted in a similar manner, with 0.8 g/kg effective, but 0.2 g/kg ineffective in decreasing the peak ASR (
Effects of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol on the Acoustic Startle Response
In the next step the effects of a GABA partial agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol was tested, which has greater potency at α4βδ subunit isoforms of the GABAA receptor than at the predominant native GABAA receptor isoforms expressed in CA1 hippocampus (6). In this case, either 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol or vehicle was administered 30 minutes before ASR testing. A 3 mg/kg i.p. dose of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol significantly (P<0.05) attenuated the ASR by 70% in P withdrawn animals, but exerted the opposite effect in control animals, increasing the ASR by about 40% (P<0.05,
Methods
Elevated Plus Maze
This device consists of four 10×50 cm arms at 90° angles, with two arms enclosed by 40 cm walls, and two open arms, all elevated 50 cm above the floor. The open arms are also partially bordered by small rails extending to the proximal half of the arm, and the floor of the maze is marked with grid lines every 25 cm. After a 30-45 minutes acclimation period, each animal was placed in the center of the maze 10 minutes after ethanol administration, and exploratory activity recorded for 10 minutes. In some cases, animals were tested 10 minutes following rapid insertion and removal of a needle coated with DMSO, i.p., to replicate the aversive qualities of an ethanol injection. Both number of entries and duration of time spent in the open and closed arms were tabulated, as were the total arm entries, a measure of general activity level. To be considered an open arm entry, the animal had to cross the line of the open platform with all four paws. An increase in open arm entries or time spent in the open arm versus the total time is considered to be a measure of decreased anxiety, (25).
Average values for ethanol-induced changes in peak GABA-gated current and the ASR were evaluated across drug-treatment groups by one-way analysis of variance (ANOVA) with a post-hoc Tukey's test. Data from the plus maze were analyzed in an ANOVA followed by post hoc t-tests (Fisher's PLSD). A P value <0.05 was used as an indication of statistical significance.
Results
Effects of Ethanol on the Elevated Plus Maze
It was observed that a low dose (0.5 g/kg) of ethanol significantly (P<0.05) increased the time in the open aim more than 20-fold and also increased the % of open arm entries by 7-fold compared to vehicle in P withdrawn animals (
An additional study was conducted to test for the possibility that the stress produced by an ethanol injection was different in control and P withdrawal animals. To this end, a separate group of animals was tested on the elevated plus maze following rapid insertion of a DMSO-coated needle to simulate the irritation produced by ethanol injection. There was no significant change in open arm entries in either group, where this stimulus decreased open arm entries by 10±4% for the control group, and by 8±3.6% for the progesterone withdrawal group. Closed arm entries were decreased by 31±11.5% for the controls, but increased 30±14% for the P withdrawal group, a change which was not significant. In addition, the stimulus produced no significant change in locomotor activity, increasing total arm entries by 18±9% (control group) and 28±12% (P withdrawal).
Materials
Adult, female Long Evans rats (140-180 g), housed in groups of three, were employed for all experiments. Animals were maintained under constant conditions of the light/dark cycle (14:10) and temperature (21° C.), with free access to food and water. In all cases, animals were sacrificed at 11:00-13:00 hour of the light cycle. Control animals were tested on the day of diestrus, a low hormone state. All procedures were carried out with the approval of the Institutional Animal Care and Use Committee.
Methods
Steroid and Ethanol Administration Procedures
Animals were administered steroids using one of two protocols: (i) For the 48 hour steroid administration protocol, animals were injected with 10 mg/kg 3α, 5β THP in oil, i.p., on a daily basis for three days and sacrificed 1-2 hour following the final injection. Controls were injected with oil vehicle. (ii) For the progesterone withdrawal paradigm, animals were implanted subcutaneously in the lower back with a silicone capsule (Nalgene Co.1=16 00 1=8 00 o.d., 10 mm/100 g body weight) filled with crystalline progesterone for 21 days, as previously described (51). At this time, the capsule was removed and the animals were injected with 17 β estradiol (2 μgs/animal) in order to reset their cycle. Animals were sacrificed 24 hours later. In the absence of progesterone withdrawal, this estradiol injection paradigm did not result in alterations in α4 expression. In all cases, implantation and removal of capsules were performed under halothane anesthesia. Both paradigms result in brain levels of steroids, which are in the physiological range (51). Although differences in potency and efficacy for the 5α and 5β isomers of THP have been reported (3), our results indicate that with in vivo administration across a 48 hour period, both isomers produce similar effects on GABAR physiology, kinetics and a subunit upregulation (23, 30). Therefore, this study employed the 5β isomer of the steroid.
In some groups of animals, either ethanol (0.5 g/kg in saline) or saline vehicle was administered i.p. (3×, every 40 minutes) for the final 2 hours of the respective steroid administration or withdrawal paradigm. The use of multiple injections of low doses of ethanol would approximate ethanol consumption in the human more closely than would a single injection. For the 48 hour steroid administration paradigm, animals received their first ethanol injection immediately after the steroid injection, and subsequent injections at 40 minute intervals. In all cases, animals were sacrificed 40 minutes after the final ethanol injection (2 hours after the first ethanol injection and final steroid injection). All animal protocols were carried out with the approval of the Institutional Animal Care and Use Committee.
Statistical Analysis
Average values for the optical density of the α band were standardized to the GAPDH control and compared between steroid and steroid/ethanol treatment groups versus control. Average values for LZM potentiation of peak GABA-gated current across the concentration range were compared between steroid/ethanol treatment groups versus control. In both cases, the statistical significance of the data was evaluated across drug-treatment groups using a one-way analysis of variance (ANOVA) with a post-hoc Tukey's test for unequal replications. A P value <0.05 was used as an indication of statistical significance.
Western Blot Protocol
Hippocampi removed from steroid or vehicle-treated animals were homogenized, and membranes prepared as previously described (65). Following electrophoresis on 8% SDS polyacrylamide gels, membranes were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, Calif.). α4 subunit levels were detected by evaluating the band density at 67 kDa after probing with a polyclonal antibody (64). Membranes were incubated for 1-2 hours at room temperature with a 1:5000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham, Arlington Heights, Ill.), and visualized with enhanced chemiluminescence with varying concentrations of protein in the linear range. Immunoreactive band densities were quantified using a CCD camera (UMAX Scanner) and one-Dscan software, which uses multiple Gaussian fits to achieve accurate band density profiles. Results were standardized to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH control protein (36 kDa band).
Electrophysiology Protocol
Whole cell patch clamp recordings were carried out on pyramidal neurons acutely dissociated from CA1 hippocampus, as described (65).
Initially, tissue was digested at 32° C. for 50-60 minutes under 100% O2 in PIPES-buffered saline containing (in mM): NaCl 120, KCl 5, CaCl2 1, MgCl2 1, d glucose 25, PIPES 20 and trypsin (type XI, sigma), 0.8 mg/ml, pH 7.0. Following a 1 hour enzyme-free incubation at room temperature, tissue was dissociated by trituration in 1 ml of 20 mM HEPES-buffered DMEM which was replaced by recording medium following transfer to the recording chamber. GABA-gated current was recorded at room temperature (20-25° C.) at a holding potential of −50 mV in a bath containing (in mM): NaCl 120, CsCl 5, CaCl2 2, MgCl2 1, TEA Cl 15, 4-aminopyridine 5, HEPES 10 and glucose 25, pH 7.4, 320 mOsm/kg H2O. The pipette solution contained (in mM): N-methyl-d glucamine chloride 120, Cs4 BAPTA 5 and Mg-ATP 5. The ATP regeneration system tris phosphocreatine (20 mM) and creatine kinase were added to minimize GABA rundown. All drugs were obtained from Sigma Chemical Co, (St. Louis, Mo.), or Calbiochem (BAPTA).
In order to determine functional expression of a containing GABAR, BDZ modulation of GABA (EC20)-gated current was determined across steroid and ethanol administration groups. GABA-gated current recorded from αχβγa2 GABAR is significantly potentiated BDZs, when x=1-3, 5 (75). However, GABARs containing α4 or α6 are relatively insensitive to BDZ modulation (75, 80). Therefore, potentiation of GABA-gated current by increasing concentrations of the BDZ lorazepam (LZM, 0.1-10 μM) was assessed in order to pharmacologically characterize the GABAR expressed following the treatment paradigms. Deliver of LZM to the isolated neuron was accomplished with a gravity-feed, solenoid controlled, superfusion system positioned within 50-100 μM of the cell, which yielded approximate 40-100 ms exposure times. Peak current was calculated for all drug concentrations as percentage GABA potentiation relative to the current gated b 10 l GABA. Currents were recorded using an Axopatch 1D amplifier (Axon Instruments) filtered at 2 kHz (four-pole Bessel filter) and detected at 500 Hz (pClamp 5.1). Decay time constants for macroscopic currents were approximated as monoexponential functions using non-linear curve fitting routines with Levenburg-Marquardt algorithms (Origin software, Microcal).
Results
Two-hour Ethanol Exposure and a Subunit Regulation
Because ethanol is known to regulate expression of the GABAR α4 subunit (14, 44) tests were run to determine whether acute ethanol administration would alter α expression during a 11-48 h 3α, 5β-THP administration paradigm known to increase α4 expression (23). Intraperitoneal administration of 0.5 g/kg ethanol across the final 2 hours of 3α, 5β-THP administration in fact reversed the increase in a expression normally seen at this time in hippocampus (FIGS. 4A,B). This ethanol-steroid paradigm resulted in levels of α4 expression not significantly different from control. In contrast, as previously shown, 48 hours of exposure to 3α, 5β-THP increased a expression threefold compared to control (
Similar effects were seen after withdrawal from 3α, 5β-THP following chronic treatment with the parent compound progesterone. The robust threefold increases in α4 expression produced by steroid withdrawal were also reversed by the 2 hour ethanol administration protocol, when levels of α4 expression were unchanged from control levels (
Alterations in α4 Expression and Benzodiazepine Sensitivity
Because GABARs containing the a subunit exhibit α4 unique pharmacological profile characterized by an insensitivity to modulation by classic BDZ agonists (75, 80), the responsiveness of acutely isolated CA1 hippocampal pyramidal cells to modulation by the BDZ LZM following 48 hour steroid exposure with or without 2 hour ethanol administration was tested. The ethanol-induced decrease in α4 expression restored the GABA-modulatory effects of LZM following 48 h 3α, 5β-THP treatment. Under these conditions, LZM potentiated GABA-gated current in a concentration-dependent manner, with a maximal 90% potentiation by 10 l LZM (FIGS. 5A,B).
In contrast, in saline-treated rats, the increased hippocampal expression of the α4 subunit produced by 48 hours of 3α, 5β-THP exposure was associated with a relative BDZ insensitivity. In this case, only the highest 10 μM concentration produced minimal (12-15%) potentiation of GABA-gated current, as expected for increased expression of BDZ-insensitive α4 containing GABARs.
In addition to restoring BDZ potentiation of GABA-gated current, sustained exposure to low dose ethanol during the final 2 hours of the 48 h 3α, 5β-THP administration also resulted in current with a decay time significantly slower (P<0:001) than observed following steroid administration (
Expression of Recombinant GABAA Receptors and Recording in Oocytes
Plasmids with inserts encoding GABAA receptor subunit sequence for rat α1 (62), rat β2 (83), rat α2 (62), rat α5 (62), mouse α4 (37), mouse β3 (82), rat β1 (83), rat γ2S (63), human δ (12), and human ε (11) were produced by subcloning each separately into an appropriate cloning vehicle such as PCDM8, pCDNA3.1 or pGHEMHE vectors (Invitrogen, Carlsbad Calif.) using a T7 promoter and standard subcloning techniques (59). Plasmids were linearized with an appropriate restriction enzyme at a site 3′ to the insert and cRNAs were synthesized using T3 or T7 polymerase (mMessage mMachine, Ambion, Tex.). The size of the cRNA was verified and RNA was quantified by ethidium bromide staining after electrophoresis on 1% agarose gels.
The preparation, injection, and maintenance of oocytes were carried out as described previously (78). Oocytes were injected with receptor subunit cRNAs (2 to 20 ng) in the following ratios: α1:β1:γ2S, α1:β2:γ2S and α1:β1:ε1, 1:1:1; α2:β1:γ2S, α5:β1:γ2S, α4:β1:γ2S, and α4:β2:γ2S, 10:1:1; α4:β1:δ, α4:β2:δ, α4:β3δ, 10:1:10; α1:β2:δ, 1:1:10 or 1:0.5:3; α1:β1:δ, 1:1:10 or 1:0.5:3; α2:β1:δ and α2:β2:δ, 1:0.5:3; α1:β1, α1:β2, α2:β1, α2:β2, β2:γ2S and α4δ, 1:1, α4:β1, α4:β2, α4:β3, 10:1, β2δ, 1:10.
Voltage-clamp recording was carried out 1 to 3 days after injection of cRNAs. Electrodes were filled with 3 M KCl and had resistances of 0.4-4 MΩ. Oocytes were positioned in a small Perspex chamber and continuously superfused with a saline solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.5). Agonists and modulators were both applied in the same saline solution. Oocytes were voltage-clamped at −70 mV and macroscopic currents were recorded with a two-electrode voltage-clamp using a Warner Instruments OC-725 amplifier as described previously (79). Currents were recorded on a chart recorder or by using Axograph on a Macintosh computer. Oocytes injected with β2, α4β2, β2γ2s or α4δ gave little (1-5 nA) or no response to even high concentrations of GABA. In oocytes injected with α4+β2+δ subunits, the ratio of currents induced by THIP: GABA was 1.24, consistent with an action of THIP at heteromeric α4β2δ receptors (1,16). In addition, lanthanum (La3+, 300 μM inhibited by 80% currents at α4β2δ receptors, an action specific for this receptor subtype (60).
Other subunits (α1, α2, β1, β2) were tested for co-expression with the δ subunit. Because functional receptors can assemble from some α+β subunits in the absence of a δ subunit, it is necessary to distinguish the properties of αβδ receptors from those of αβ receptors. Although α1β2δ receptors are inhibited by zinc, they are significantly less sensitive to zinc than are α1β2 receptors (61). Thus, the differential sensitivity to zinc was used (1 μM) to distinguish oocytes expressing α1β2δ receptors from those expressing α1β2δ receptors when injected with α1+β2+δ subunits. For 7 of 22 oocytes injected with α1+β2+δ, zinc inhibition (49.1±6.4%) was significantly less (P<0.0005) than for α1β2 receptors (72.4±6.9%; n=5), suggesting incorporation of the δ subunit within receptors in these oocytes. For the other 15 oocytes, zinc inhibited GABA currents by a mean 66.4±6.4%, a value not significantly different (P=0.338) from that for α1β2 receptors. Alcohol did not affect responses to GABA at these receptors, which were 88-95% of control in oocytes injected with α1+β2 or with α1+β2+δ. It was also determined that in the receptors containing the β1 rather than the β2 subunit, all oocytes injected with α1+β1+δ expressed receptors whose sensitivity to zinc was indistinguishable from that of α1β1 receptors. Again, GABA responses at these receptor subtypes were not altered by 1 mM alcohol. Oocytes injected with combinations of α2β1δ, α2β2δ, α4β1δ and α4β3δ did show any response to GABA, suggesting lack of expression of functional GABA receptors.
Statistical Analysis
Average values for peak current (THIP, GABA), alcohol-induced changes in peak GABA-gated current and the acoustic startle response were evaluated across drug-treatment groups by one-way analysis of variance (ANOVA) with a post-hoc Tukey's test. A P value <0.05 was used as an indication of statistical significance. (Supported by AA 13646 to N.L.H.)
Progesterone Withdrawal
Crystalline progesterone was administered via a silicone capsule ( 1/16″ i.d., ⅛″ o.d.) implanted subcutaneously (10 mm length/100 gm body weight) in female rats (110-120 grams, in vitro studies; 220-250 grams, behavioral studies) for 21 days. Control rats were implanted with an empty capsule. This paradigm has been shown to result in hippocampal levels of 3α, 5α-THP in the physiological range (65). Animals were tested 24 hours after removal of the implant.
Western Blot Analysis
Membranes prepared from hippocampal tissue were solubilized and electrophoresed on 9% SDS-polyacrylamide gels before transfer to a polyvinyldifluoride membrane, as described (65,69). Polyclonal antiserum against the δ subunit (Asr318-Leu400) was supplied by Dr. R. McKernan (Merck)(56) and is visualized as a 54 kDa band. Membranes were also probed with an antibody for the α4 GABAA receptor subunit (67 kDa) (64), developed according to a previously described protocol (38), and a commercial antibody for the control protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 36 kDa). Membranes were incubated for 1-2 hours at room temperature with 1:10,000 (α4), 1:7,000 (δ) or 1:20,000 (GAPDH) dilution of antibody followed by addition of 1:5,000 horseradish-peroxidase-conjugated donkey anti-rabbit IgG (Amersham). Band densities were quantified using densitometry with One-Dscan software after normalization to the GAPDH control. Reagents were obtained from Sigma/Genosys.
Co-Immunoprecipitation
Receptors were initially solubilized from hippocampal membranes using 0.5% Na deoxycholate and centrifugation at 120,000 rpm, as described (68,46). The solubilized receptor preparation (0.8 mg) was incubated overnight with 10 μl anti-δ (56) or a negative control antibody (anti-β-NAC, a cytosolic protein (77) bound to Protein A beads (68). This mixture was then washed three times with TBS/0.05% TWEEN-20/0.05% NP40 before eluting with 0.1 M glycine/HCl, pH 2.5, followed by low speed centrifugation. Proteins in the supernatant were precipitated with 10% trichloroacetic acid, washed with acetone and resuspended in sample buffer. Immunoprecipitated proteins in the final suspension were separated using gel electrophoresis (8% SDS-PAGE gel with an acrylamide:bis ratio of 30:1.2) and probed with a digoxygenin-labeled α4 antibody (39) (Roche Molecular Biochemicals) at a 1:400 dilution of the original serum. Initially, the α4 antibody (1 ml) was purified on a Sephadex G-25 column using Hepes-KOH, pH 8.3, and then labeled with digoxygenin (1.5 mg digoxygenin/1.9 mls α4 eluate) for 1.5 hours at room temperature, followed by re-purification on the Sephadex G-25 column using PBS/20 mM Tris. Anti-digoxygenin conjugated to horseradish peroxidase (poly, F(ab)2 fragments, Roche Molecular Biochemicals 1:45,000) was used as a secondary antibody, with 1.5% milk as the blocking agent. Following a 1 hour incubation, the resulting band for the digoxygenin-labeled α4 antibody was visualized with ECL chemiluminescence (ECL Plus, Amersham Pharmacia Biotech). Optical density could not be quantified because α4 levels were barely detectable under control conditions; therefore, it was virtually an all-or-none response.
Semi-Quantitative RT-PCR
Following isolation of total RNA from hippocampus, reverse transcription (SuperScript II, Rnase RT, GibcoBRL) yielded complementary DNA (cDNA) which was amplified with PCR methods (65) using oligonucleotide primers for the δ subunit (398 bp)(70) and GAPDH (657 bp, Operon). Twenty three to thirty amplification cycles were used for two different concentrations of cDNA (25 or 50 ng) to detect band densities within a linear range (15). Following gel electrophoresis (FMC Bioproducts), the density of DNA bands stained with ethidium bromide was quantified and normalized to the GAPDH control.
Whole Cell Patch Clamp Electrophysiology
Following progesterone withdrawal or control conditions, pyramidal cells were acutely isolated from CA1 hippocampus as described (65,64). GABA-gated Cl− currents were recorded using whole cell patch clamp techniques at a holding potential of −50 mV. An ATP-regeneration system was included in the pipet to prevent run-down of GABA-gated currents, and drugs were applied using a pipet array that yielded 40-100 msec exposure times.
Acoustic Startle Response
Animals were tested in a stabilimeter device (SR-Lab Startle Response System, San Diego Instruments, Inc.) that permitted vertical movement of a Plexiglas cage contained within a lit, well-ventilated, sound-attenuating chamber. The amplitude of movement was detected by a piezoelectric device with a voltage output proportional to cage displacement. The analog output of the accelerometer was amplified and digitized before computer analysis (15). Five minutes following intraperitoneal administration of alcohol (0.2 or 0.4 g/kg) or saline vehicle (1.5 ml volume), animals were placed in the chamber for 5 a minute acclimation period. Five acoustic stimuli (116 dB, 40 msec duration) were then presented once every 10-15 seconds via a white-noise generator through speakers positioned on either side of the cage. For each acoustic stimulus, the integrated response (amplitude and duration) was averaged across a 100 msec post-stimulus time period and the response latency determined. Both values were normalized to the weight of the animal. Values for the integrated response for the first noise stimulus following administration of each dose of alcohol were averaged and expressed as a fraction of the saline control average for the indicated treatment (P Wd versus control) group. This paradigm has been used to demonstrate the anxiolytic effects of low doses of alcohol in alcohol-preferring rats (34), a model of increased alcohol consumption. It is routinely used to assess behavioral excitability following withdrawal from benzodiazepiness (48) and alcohol (73), and is known to be modified by GABA modulators, such as diazepam (11), and by local infusion of excitatory compounds into dorsal hippocampus (85).
Results
The α4βδ GABAA receptors are expressed at very low levels in most regions of the brain (55). Predicting that physiological states that are associated with increased sensitivity to alcohol (such as PMS) may involve increased expression of α4βδ, the rodent model of PMS 1 was used to test this hypothesis. With chronic in vivo administration and withdrawal of progesterone, the hormonal and behavioral facets of PMS 2 were replicated. Expression of δ and α4 subunit proteins in the hippocampus was three-fold higher after progesterone withdrawal (
Concomitant with upregulation of α4βδ receptors in the progesterone-withdrawal model, 1 mM alcohol produced a 81±21% potentiation of GABA-gated currents recorded from hippocampal neurons in vitro (
To verify that native α4βδ receptors were sensitive to low doses of alcohol, the progesterone withdrawal experiments were repeated after suppressing α4 subunit expression. As previously shown that, following progesterone withdrawal, injection of a GABA modulator prevents expression of the α4 subunit (64). Thus, alcohol was used here to suppress expression of the α4 protein following progesterone withdrawal (P Wd+Alc;
It is thought that α4βδ receptors are expressed at extrasynaptic sites (55) where they may dampen neuronal excitability, primarily by acting as a resistive shunt (5). Enhanced function of these receptors by low concentrations of alcohol in women with PMS would further decrease neuronal excitability, leading to behavioral stress-reduction. Blood alcohol levels of 1-3 mM may result from consumption of a half glass of wine (35) or less (28). The reported increase in alcohol consumption and propensity for alcoholism in women with PMS (47) may thus be accounted for by these enhanced reinforcing properties of alcohol. More broadly, it is conceivable that alterations in GABAA receptors, perhaps including α4β2δ, are involved in the genetic predisposition for alcoholism in which there is an increased sensitivity to low doses of alcohol (9).
This application claims priority to U.S. Provisional Application Ser. No. 60/491,599 filed Jul. 31, 2003, which application is incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2004/024981 | 7/30/2004 | WO | 00 | 9/18/2006 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2005/011612 | 2/10/2005 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5189064 | Blum et al. | Feb 1993 | A |
| 20020115611 | Tallman et al. | Aug 2002 | A1 |
| 20030138776 | Goldman et al. | Jul 2003 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 9610637 | Apr 1996 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20070081943 A1 | Apr 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60491599 | Jul 2003 | US |
