Evaluation of native GABA(A) receptors containing an alpha 5 subunit.

The type A receptor for gamma-aminobutyric acid (GABA), or GABA(A) receptor, is a pentamer of highly variable quaternary structure. It includes two alpha subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [(3)H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [(3)H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of alpha 5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [(3)H]flunitrazepam. The pattern mirrored alpha 5 mRNA distribution, and was similar to that previously reported for [(3)H]L-655,708 binding. Displacement of [(3)H]RY-80 bound to hippocampal homogenates, and of [(3)H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K(i)'s 5--7 nM). However, the K(i)'s for diazepam and for clobazam to displace [(3)H]RY-80 binding in hippocampus were about fourfold higher than for [(3)H]flunitrazepam, and the K(i) for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal alpha 5-containing receptors.

Down-regulation of benzodiazepine binding to alpha 5 subunit-containing gamma-aminobutyric Acid(A) receptors in tolerant rat brain indicates particular involvement of the hippocampal CA1 region.

Chronic benzodiazepine treatment can produce tolerance and changes in gamma-aminobutyric acid (GABA)(A) receptors. To study the effect of treatment on a selected population of receptors, assays were performed using [(3)H]RY-80, which is selective for GABA(A) receptors with an alpha 5 subunit. Rats were given a flurazepam treatment known to produce tolerance and down-regulation of benzodiazepine binding, or a diazepam treatment shown to produce tolerance but not receptor down-regulation. Quantitative receptor autoradiography using sagittal brain sections bound with [(3)H]RY-80 showed binding in areas known to express alpha 5 mRNA. Brains from flurazepam-treated rats showed significantly decreased 1 nM [(3)H]RY-80 binding in hippocampal formation (e.g., 32% decrease in CA1) and superior colliculus, but not other areas. Using 5 nM [(3)H]RY-80 showed similar decreases in hippocampus. A corresponding 29% decrease in B(max) but no change in K(d) was found with a filtration binding assay using hippocampal homogenates. Down-regulation of [(3)H]RY-80 binding had returned to control by 2 days after withdrawing flurazepam treatment. The magnitude of down-regulation of [(3)H]RY-80 binding suggested that GABA(A) receptors with an alpha 5 subunit may play a prominent role in the adaptive responses associated with benzodiazepine tolerance. Chronic diazepam treatment also resulted in decreased [(3)H]RY-80 binding. However, the regional selectivity was even more pronounced than in flurazepam-treated rats, and only the hippocampal CA1 region showed decreased binding (27%). This localized down-regulation persisted for several days after the end of diazepam treatment. These data indicate that synapses in the hippocampal CA1 region are particularly involved in the adaptive response to chronic benzodiazepine treatments.

Antagonist-induced reversal of functional and structural measures of hippocampal benzodiazepine tolerance.

One week oral flurazepam (FZP) administration in rats results in anticonvulsant tolerance in vivo, tolerance measured in vitro in hippocampal CA1 pyramidal cells, and regulation of hippocampal gamma-aminobutyric acid(A)-receptor subunit protein expression. A single injection (4 or 20 mg/kg i.p) of the benzodiazepine antagonist flumazenil (FLM) was given 1 day after FZP treatment, and tolerance and subunit protein expression were evaluated 1 day later. In vivo tolerance was measured by a reduced ability of the alpha(1)-subunit-selective agonist zolpidem to suppress pentylenetetrazole-induced seizures. This tolerance was reversed by 20 but not 4 mg/kg FLM. In in vitro hippocampal slices, there was tolerance to the effect of zolpidem to prolong the decay of pyramidal cell miniature inhibitory postsynaptic currents, which was reversed by FLM (4 mg/kg) pretreatment. A reduction in miniature inhibitory postsynaptic current amplitude ( approximately 50%) was also restored by FLM injection. [(3)H]Zolpidem binding measured 0, 2, and 7 days after FZP treatment was significantly decreased in the hippocampus and cortex at 0 days but not thereafter. Changes in alpha(1)- and beta(3)-subunit protein expression were examined via quantitative immunohistochemical techniques. alpha(1)-Subunit protein levels were down-regulated in the CA1 stratum oriens and beta subunit levels were up-regulated in the stratum oriens and stratum radiatum of the CA3 region. Chronic FZP effects on alpha(1)- and beta(3)-subunit protein levels were also reversed by prior FLM injection. FLM's effect on both functional and structural correlates of benzodiazepine tolerance suggests that each of these measures plays an interdependent role in mediating benzodiazepine tolerance.

Acute pentylenetetrazol injection reduces rat GABAA receptor mRNA levels and GABA stimulation of benzodiazepine binding with No effect on benzodiazepine binding site density.

The effects of a single convulsive dose of pentylenetetrazol (PTZ, 45 mg/kg i.p.) on rat brain gamma-aminobutyric acid type A (GABAA) receptors were studied. Selected GABAA receptor subunit mRNAs were measured by Northern blot analysis (with beta-actin mRNA as a standard). Four hours after PTZ, the GABAA receptor gamma2-mRNA was decreased in hippocampus, cerebral cortex, and cerebellum; alpha1-mRNA was decreased in cerebellum; and beta2 subunit mRNA was decreased in cortex and cerebellum. The alpha5 subunit mRNA level was not altered. Those mRNAs that had been reduced were increased in some brain regions at the 24-h time point, and these changes reverted to control levels by 48 h. PTZ effect on GABAA receptors was also studied by autoradiographic binding assay with the benzodiazepine agonist [3H]flunitrazepam (FNP), the GABAA agonist [3H]muscimol, and the benzodiazepine antagonist [3H]flumazenil. There was an overall decrease in [3H]FNP binding 12 but not 24 h after PTZ treatment. In contrast, [3H]muscimol binding was minimally affected, and [3H]flumazenil binding was unchanged after PTZ treatment. Additional binding studies were performed with well-washed cerebral cortical homogenates to minimize the amount of endogenous GABA. There was no PTZ effect on specific [3H]FNP binding. However, there was a significant reduction in the stimulation of [3H]FNP binding by GABA. The results showed that an acute injection of PTZ caused transient changes in GABAA receptor mRNA levels without altering receptor number but affected the coupling mechanism between the GABA and benzodiazepine sites of the GABAA receptor.

Decreased benzodiazepine binding with little effect on gamma-aminobutyric acid binding in rat brain after treatment with antisense oligodeoxynucleotide to the gamma-aminobutyric acidA receptor gamma-2 subunit.

Benzodiazepine potentiation of gamma-aminobutyric acid (GABA) neurotransmission is associated with the presence of a gamma-2 subunit in the GABAA receptor. A method was developed to modify the gamma-2 subunit expression in adult rat brain. Unilateral intracerebroventricular (i.c.v.) infusion of a 17-base phosphorothioate-modified antisense oligodeoxynucleotide (ASO) was performed every 12 hr for 3 days. Controls were treated with a sense oligodeoxynucleotide. Parasagittal brain sections were used for quantitative autoradiographic analysis of radioligand binding. ASO treatment caused a 15% to 25% decrease of specific [3H]flunitrazepam binding in most brain areas, with statistically significant decreases in frontal cortex, cerebellar molecular layer, zona reticulata of substantia nigra and CA3 of hippocampus. In contrast, [3H]muscimol binding was not changed. [3H]GABA binding was also unchanged, except for a 10% decrease in cerebellar granule cell layer. The effect on the chloride channel of the GABAA receptor complex was examined by 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate binding; most brain areas showed small decreases in 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate binding. However, hippocampal regions showed much larger decreases. Binding of the adenosine A1 receptor antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine was used to examine possible secondary effects of the ASO. There was a decrease in [3H]8-cyclopentyl-1,3-dipropylxanthine binding, but this was much smaller than the change in [3H]flunitrazepam binding, and no area showed a significant effect. Quantitative immunoblotting with a monoclonal antibody that recognizes GABAA receptor beta-2 and beta-3 subunits showed no change in immunoreactivity in cerebellar tissue after ASO treatment. The results indicate a selective effect on benzodiazepine binding to GABAA receptors and a possible change in receptor subunit composition.

Intravenous and oral propafenone for treatment of tachycardia in infants and children: pharmacokinetics and clinical response.

To elucidate contribution of an active metabolite to overall clinical responses to propafenone, steady-state disposition of propafenone and its active metabolite and the clinical responses to treatment were examined in pediatric patients receiving intravenous or oral propafenone. There were more than ten-fold interindividual differences in apparent clearance, resulting in a wide range of the steady-state trough plasma concentrations of propafenone. The active metabolite, 5-hydroxypropafenone, was detected in four of the six patients receiving oral propafenone; however, two neonates receiving oral propafenone and all eight receiving intravenous propafenone had no detectable levels of 5-hydroxypropafenone in plasma. In nine patients for whom electrocardiographic (ECG) data were available, the PQ interval was significantly increased, whereas the QRS duration and the QTc interval were not. There was no close relationship between plasma concentrations of propafenone or 5-hydroxypropafenone and ECG parameters. Lack of good correlation between serum concentrations and clinical response precludes using a serum-concentration targeting strategy with propafenone therapy.

The electrocardiogram and the secundum atrial septal defect: a reexamination in the era of echocardiography.

BACKGROUND: Ostium secundum atrial septal defects (ASDs) often present subtly and may be a diagnostic challenge to the community physician. Characteristic abnormalities of the electrocardiogram (ECG) have been described in adults. OBJECTIVE: To determine whether ECG abnormalities are consistently present in children with a hemodynamically significant ASD, and their potential for differentiating this group from patients with innocent murmurs. DESIGN: Retrospective evaluation of clinical characteristics, echocardiographic data, and ECGs was undertaken in 67 consecutive children with an ASD (mean age 2.9 +/- 2.8 years, 63% female) and 77 patients with innocent murmur (mean age 3.2 +/- 2.6 years, 61% male). Predetermined ECG criteria were derived from adult studies (rsR'-V1 with evidence of right ventricular hypertrophy, isolated rsR'-V1, and unequivocal right ventricular hypertrophy without rsR'-V1). ECGs were interpreted blindly by two observers. RESULTS: In the ASD group 58 (87%) patients had an ECG that met predetermined criteria compared with three (3.9%) controls (P < 0.001). Completely normal ECGs were found in only four (6.0%) ASD patients compared with 66 (86%) controls (P < 0.001). The ECG criteria had a sensitivity of 86% (95% CI 0.784 to 0.947) and a specificity of 96% (95% CI 0.918 to 1.000). When any ECG abnormality was considered the sensitivity increased to 94% (95% CI 0.884 to 0.997) with a decline in specificity to 86% (95% CI 0.779 to 0.935). CONCLUSIONS: The ECG is potentially a valuable adjunct to the physical examination in differentiating children with an ASD from those with an innocent murmur in the primary care setting.

Reversal of pulmonary arteriovenous malformation after diversion of anomalous hepatic drainage.

Pulmonary arteriovenous malformation can occur in up to 25% of patients after a classic Glenn shunt. Although unproven, exclusion of hepatic venous blood from the lungs has been proposed as a possible cause. We present a patient born with anomalous hepatic venous drainage into the left atrium with an intact atrial septum in whom pulmonary arteriovenous malformation developed in childhood. This was reversed after diversion of the hepatic venous drainage to the right atrium, supporting exclusion of hepatic venous flow as the cause of pulmonary arteriovenous malformation. The association with the hepatopulmonary syndrome is discussed.

Local pressure application of cannabinoid agonists increases spontaneous activity of rat substantia nigra pars reticulata neurons without affecting response to iontophoretically-applied GABA.

This study tested the hypothesis that cannabinoid agonists, applied locally into the pars reticulata of substantia nigra (SNpr), could modulate striatonigral transmission, without affecting the response of SNpr neurons to iontophoretically-applied GABA. Multibarreled glass capillary electrode assemblies were used for extracellular recording of the spontaneous electrical activity of single SNpr cells in anesthetized rats. Local pressure ejection of the cannabinoid agonists Win 55212-2 (WIN2) and CP 55940 increased SNpr spontaneous firing rate by 13-46%, similar to the effects of systemic injections. Neither WIN2 nor CP 55940 had an effect on the slowing of SNpr neuron activity in response to iontophoretic GABA. Local pressure application of Win 55212-3 (the much less active enantiomer of WIN2) produced an insignificant decrease in SNpr firing rate. Similarly, locally applied vehicle (45% 2-hydroxypropyl-beta-cyclodextrin) produced insignificant decreases in SNpr firing. A second application of cannabinoid agonist produced a much smaller effect, suggesting desensitization. Increasing the interval between CP 55940 applications to 45 min showed recovery of sensitivity to the agonist. Local application of the cannabinoid antagonist, SR 141716A, significantly decreased spontaneous cell firing by 34%. CP 55940, when given immediately following or concurrently with the antagonist application failed to produce the expected increase in discharge rate over baseline. A second application of CP 55940 45 min later produced a 26% increase in firing rate. Bicuculline methiodide (BMI) was applied locally causing a significant increase in SNpr cell firing. CP 55940, when locally administered concurrently with bicuculline methiodide, had no further effect on the firing rate of the cell. Based on the reported presynaptic localization of cannabinoid receptors in SNpr, these findings suggest that cannabinoids act within the SNpr to modulate striatonigral neurotransmission presynaptically. The effect of SR 141716A suggests that an endogenous cannabinoid may mediate striato-nigral transmission.

Treatment with an antisense oligodeoxynucleotide to the GABAA receptor gamma 2 subunit increases convulsive threshold for beta-CCM, a benzodiazepine "inverse agonist', in rats.

The gamma 2 subunit of the gamma-aminobutyric acid type-A (GABAA) receptor is associated with the actions of benzodiazepines and related drugs. A phosphorothioate-modified antisense oligodeoxynucleotide directed against the gamma 2 subunit was given by i.c.v. injection (18 micrograms in 2 microliters saline) to male Sprague-Dawley rats every 12 h for 3 days. Controls received the corresponding sense oligodeoxynucleotide. 4-6 h after the last i.c.v. treatment, rats were given methyl-beta-carboline-3-carboxylate (beta-CCM), a benzodiazepine "inverse agonist', by slow i.v. infusion. Compared to naive rats, the beta-CCM threshold dose was not affected by the sense oligodeoxynucleotide, but was increased 87% in antisense oligodeoxynucleotide-treated rats. The treatment had no effect on the seizure threshold for picrotoxin. Both antisense and sense oligodeoxynucleotide treatments slightly increased the threshold for strychnine seizures. The results suggest that antisense oligodeoxynucleotide treatment altered GABAA receptor composition and interfered with the actions of a benzodiazepine receptor ligand in vivo, and may provide a tool for studying regulation of receptor structure and function.

Referral of young adult patients with congenital heart disease to adult centres. The Canadian Adult Congenital Heart Network.

OBJECTIVE: To establish a process of referral for young adult patients with congenital heart disease from pediatric to adult centres. DESIGN: Directors of pediatric cardiology units across Canada were asked to complete a questionnaire detailing their process of referral of young adult patients with congenital heart disease to adult centres. They were also asked to respond to specific case scenarios. Adult cardiologist members of the Canadian Adult Congenital Heart Network were asked to respond to the same case scenarios. MAIN RESULTS: Most pediatric cardiology centres refer patients to adult cardiologists at 18 years of age. The process of referral generally involves a referral letter and relevant parts of the chart. Few centres arrange a booked appointment with the adult cardiologist, although this would be preferred by the majority of the responding adult cardiologists. Generally good agreement existed between pediatric and adult cardiologists with regard to the kind of patients who required specialized care at an adult congenital heart centre. CONCLUSION: Within Canada, a process is rapidly evolving to facilitate the transfer of care of young adults with congenital heart disease.

Diastolic flow across a ventricular septal defect: an invitation to a false diagnosis.

A case is reported of a child with a perimembranous ventricular septal defect with extensive aneurysm formation misdiagnosed as a ruptured aneurysm of the sinus of Valsalva on the basis of a distinct diastolic jet seen on colour Doppler echocardiography. The hemodynamic explanation of this rarely recognized finding is apparent from simultaneous pressure tracings taken at the time of cardiac catheterization. Diastolic flow across a ventricular septal defect can be distinguished from a ruptured aneurysm of the sinus of Valsalva by the low velocity of the jet and its tendency to peak in late diastole.

Differential expression of benzodiazepine anticonvulsant cross-tolerance according to time following flurazepam or diazepam treatment.

In previous studies in which the anti-pentylenetetrazol (PTZ) effect of benzodiazepines was used to measure tolerance, the results depended on the benzodiazepine used for chronic treatment as well as the benzodiazepine given acutely to test for tolerance. In this study, the time course of tolerance reversal was studied in rats given two treatments known to cause anticonvulsant tolerance, 1-week flurazepam (FZP), and 3-week diazepam (DZP). Neither treatment altered convulsive threshold for IV PTZ, but both treatments decreased the convulsive threshold for bicuculline. Withdrawing DZP, but not FZP, treatment resulted in a loss of body weight. Twelve hours after 1-week FZP treatment, all benzodiazepines were significantly less effective, showing tolerance. Forty-eight hours after the 1-week FZP treatment, tolerance was still observed with DZP, FZP, and zolpidem, but was no longer present with clonazepam or bretazenil. After the 3-week DZP treatment, rats were tolerant to all benzodiazepines tested at 12 h of withdrawal, but had lost tolerance to all the drugs except bretazenil by 48 h. The results suggest differences in the way these benzodiazepines interact with their receptors, allowing differential expression of tolerance, and that chronic DZP and FZP treatments affected interactions of the benzodiazepines with their receptors, but not in the same fashion.

Rapid down-regulation of [3H]zolpidem binding to rat brain benzodiazepine receptors during flurazepam treatment.

In a previous study, it was found that down-regulation of benzodiazepine (BZ) binding in rats treated 4 weeks with flurazepam was relatively greater and more widespread when measured with [3H]zolpidem, a selective 'BZ1 receptor' ligand, than that measured with the non-selective ligand, [3H]flunitrazepam. In the present study, the time course for down-regulation of [3H]zolpidem binding was studied in rats treated with flurazepam. [3H]Zolpidem binding was also studied in rats given a midazolam treatment shown to cause tolerance. Rats were chronically treated with flurazepam for 1 or 2 weeks, or with midazolam for 3 weeks, then killed immediately after the treatment. Another group of rats was acutely treated with desalkyl-flurazepam and killed 30 min later. After 2 weeks of flurazepam treatment, the Bmax of [3H]zolpidem binding was decreased by 22% in cerebral cortex, 26% in cerebellum and 33% in hippocampus, with no change in the Kd in any region. After 1 week of flurazepam treatment, the Bmax was decreased by 23% in cerebellum and 14% in hippocampus, but not changed in cerebral cortex. The Kd was increased in cerebral cortex, but not in cerebellum or hippocampus. Neither the Bmax nor the Kd of [3H]zolpidem binding was affected by acute desalkyl-flurazepam treatment, or by 3 weeks of midazolam treatment. These results, in combination with previous findings, which showed no change in [3H]flunitrazepam binding after 1 or 2 week flurazepam treatment, and no change in cerebellum even after the 4 week treatment, may indicate a shift in BZ receptor subtypes in flurazepam-tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc inhibition of GABA-stimulated Cl- influx in rat brain regions is unaffected by acute or chronic benzodiazepine.

Zinc modulation of GABAA receptor function was studied using GABA-stimulated 36Cl- influx into microsacs prepared from rat cerebral cortex, cerebellum and hippocampus. Zinc (10-100 microM) did not affect the basal influx, but significantly inhibited GABA-stimulated 36Cl- influx. The inhibition appeared to be noncompetitive. Zinc produced differing degrees of inhibition of GABA-stimulated 36Cl- influx in different brain regions. The order of sensitivity to zinc inhibition of GABA-stimulated 36Cl- influx was hippocampus > cerebral cortex > cerebellum. These regional differences may reflect the structural heterogeneity of GABAA receptors among brain areas. Zinc inhibition was not affected by the short-term addition of three benzodiazepines, diazepam, bretazenil and triazolam. The effect of diazepam and bretazenil to potentiate GABA-stimulated 36Cl- influx was not affected by zinc, but the effect of triazolam was decreased by zinc. In brain tissue prepared from flurazepam-treated rats, there was no difference compared with controls in zinc inhibition of GABA-stimulated 36Cl- influx. The results indicate that the effects of zinc on the GABAA receptor are largely independent of drugs acting on the benzodiazepine binding site.

Cardiac screening of children with Down's syndrome.

OBJECTIVE: To establish the role of clinical and laboratory investigation of the cardiovascular system in children with Down's syndrome. DESIGN: Prospective evaluation; examiners blinded to results of laboratory studies. SETTING: Tertiary pediatric referral centre. PATIENTS: Fifty consecutive children with Down's syndrome presenting to a regional genetic centre. Children less than six weeks of age or with known heart disease were excluded. MEASURES: Following independent examinations by a geneticist and a pediatric cardiologist, an electrocardiogram (ECG) and two-dimensional and Doppler echocardiograms were carried out. RESULTS: Assessment by the geneticist yielded two false positives and five false negatives (sensitivity 67%, specificity 88%). Addition of an ECG to clinical evaluation increased the sensitivity to 80% and specificity to 90%, a rate comparable with clinical assessment by a cardiologist. No lesion requiring surgical correction was missed by this combination. CONCLUSIONS: Where expertise in pediatric echocardiography is not readily available, careful clinical assessment coupled with the interpretation of an ECG is adequate and appropriate screening of the child with Down's syndrome.

Comparison of anticonvulsant tolerance, crosstolerance, and benzodiazepine receptor binding following chronic treatment with diazepam or midazolam.

In a previous study, rats treated chronically with flurazepam were tolerant to the anticonvulsant action of some benzodiazepines (BZs), but not others (34). To determine if this differential crosstolerance was unique to flurazepam, rats were treated chronically with diazepam or midazolam, and tested for tolerance to the anticonvulsant actions of diazepam, midazolam, clonazepam, and clobazam. Regional benzodiazepine receptor binding in brain was also studied. In contrast to previous findings with flurazepam, 1 week treatment with diazepam or with midazolam did not cause tolerance. Rats treated with diazepam for 3 weeks were tolerant to diazepam, clonazepam, clobazam, and midazolam. In contrast, rats treated 3 weeks with midazolam were tolerant to diazepam and midazolam, but not clobazam or clonazepam. Neither diazepam nor midazolam treatment for 3 weeks altered BZ binding in cerebral cortex, cerebellum, or hippocampus. The effects of chronic BZ treatment depended not only on the BZ given chronically, but also on the BZ used to evaluate these effects, suggesting drug-specific interactions of different BZs with their receptors.

Reduced expression of gamma-aminobutyric acid type A/benzodiazepine receptor gamma 2 and alpha 5 subunit mRNAs in brain regions of flurazepam-treated rats.

Previous studies showed that chronic benzodiazepine administration in rats affected the gamma-aminobutyric acid (GABA)A/benzodiazepine receptor. The present experiment investigated the effects of chronic flurazepam treatment on the mRNA levels for alpha 1, alpha 5, gamma 2, and gamma 2L (an alternatively spliced product of the gamma 2 gene) subunits of the GABAA/benzodiazepine receptor in rat cerebral cortex, cerebellum, and hippocampus. Rats were treated with flurazepam for 2 or 4 weeks, and the mRNA levels were measured while rats were still receiving drug or 48 hr after 4-week flurazepam treatment had been stopped. The level of alpha 5 mRNA was also measured in other rats 4 hr after a single injection of flurazepam or diazepam. The levels of mRNAs were analyzed by Northern blotting using digoxigenin-labeled oligonucleotide probes. Compared with the pair-handled controls, the levels of gamma 2 subunit mRNA in cortex and hippocampus were not changed after flurazepam treatment for 2 weeks. However, with rats treated with flurazepam for 4 weeks the levels of gamma 2 subunit mRNA were significantly reduced in cortex (31%) and hippocampus (39%) but not in cerebellum. The values returned to control levels by 48 hr after termination of the treatment. The regional distribution and time course of reduced gamma 2 levels matched the decrease in benzodiazepine binding produced by the same chronic flurazepam treatment. The amounts of alpha 5 mRNA were reduced in cortex (23%) and hippocampus (18%) 4 hr after a single dose of flurazepam but not diazepam. The levels of alpha 5 mRNA remained reduced in cerebral cortex and hippocampus (about 50%) after 2 weeks but returned to control after 4 weeks of chronic treatment with flurazepam. No change in alpha 1 or gamma 2L subunit mRNAs was observed in any of the three brain regions examined after 4 weeks of flurazepam treatment. These results suggest that benzodiazepine receptor down-regulation after chronic benzodiazepine treatment may be related to the reduced expression of gamma 2 subunit mRNA, and they also suggest differential temporal and regional regulation of alpha 5 and gamma 2 subunit mRNAs in rat brain.