280

Brain Research, 558 (1991) 280-288 © 1991 Elsevier Science Publishers B.V. All rights reserved. (I006-8993191/$03.50 ADONIS 000689939116950R

BRES 16950

Muscimol-associated changes in local cerebral glucose use following chronic diazepam administration Ros R. Brett and Judith A. Pratt Department of Physiology and Pharmacology, University of Strathclyde, Glasgow (U.K.) (Accepted 23 April 1991) Key words: Benzodiazepine; Diazepam; Muscimoi; [14C]Deoxyglucose technique; Glucose utilization; High-affinity GABA A receptor

Local cerebral glucose use (LCGU) was determined in parallel groups of conscious rats receiving museimol (1.5 mg/kg i.v.) after either saline pretreatment (28 days i.p.), saline pretreatment (27 days i.p.) followed by a single dose of diazepam (5 mg/kg i.p,) 24 h prior to muscimol administration, or chronic diazepam pretreatment (5 mg/kg i.p. daily for 28 days). Acute administration of muscimol produced a significant reduction in LCGU in 25 out of 66 structures examined compared with vehicle-treated controls. The pattern of reductions was heterogeneous. Thalamic and most cortical areas showed reductions of the order of 30--45%, whereas more modest depressions of 15-20% were observed in some limbic structures (e.g. basolateral amygdala, anterior thalamic nuclei, nucleus aceumbens, subicuhm). This contrasts with the more extensive and homogeneous pattern of LGCU reductions (around 20%) produced by diazepam. Neither acute diazepam treatment the previous day nor chronic diazepam pretreatment altered the LGCU response to mnscimol. These data suggest that high-affinity GABA receptor-mediated responses are unchanged by both acute and chronic benzodiazepine pretreatment. It would appear unlikely that alterations in these responses contribute to the mechanism of benzodiazepine tolerance. INTRODUCTION There is now considerable evidence for the ability of the benzodiazepines to produce tolerance and dependence when administered chronically in therapeutic doses :5. The mechanisms underlying these phenomena are still unclear, but they do not appear to be attributable to altered pharmacokinetic properties TM. Benzodiazepines are generally agreed to exert their effects by enhancing the inhibitory effects of y-aminobutyric acid ( G A B A ) via a benzodiazepine receptor/GABA A receptor/chloride ionophore complex 4a. Tolerance might therefore involve alterations at the benzodiazepine receptor itself, or may be due to changes in other components of the complex. There is no clear consensus regarding benzodiazepine receptor alterations. Radioligand binding studies have shown downregulation 9'32'3s, no change 4A9" 33, or even upregulation 1°'a9 subsequent to chronic treatment; the lack of agreement between these results is probably due to widely differing methodologies, and, specifically, differences in dosage, length of treatment, benzodiazepine used, time between last dose and sacririce, and preparation of tissue for radioligand binding. The inevitable use of grossly dissected brain regions may also mean that very specific regional changes are missed. A receptor autoradiographic study, which overcomes this

limitation, has shown regional differences in receptor downregulation, ranging from 12% to 25% after 1 week of high-dose flurazepam treatment 45. Other studies have concentrated on the effect of chronic benzodiazepine treatment on binding to the G A B A receptor, or on measures of coupling between the benzodiazepine and G A B A receptors, again without a clear consensus, Both reduction 14"31 and increase 42 in the ability of G A B A to enhance benzodiazepine binding have been found. An increase in [3H]bicuculline, but not in [3H]museimol, binding sites has been observed by one group 14'16, but another group has demonstrated an increase in [31-1]muscimol binding sites 29. Electrophysiological studies have shown subsensitivity to exogenously applied G A B A in the dorsal raphe nucleus 14A5, but not in the substantia nigra pars reticulata ~, following chronic diazepam treatment. Reduction in GABA-stimulated 3° and muscimol-stimulated 32 36C1- uptake has been found, suggesting subsensitivity to G A B A , while another study has found GABA-stimulated chloride uptake itself unchanged but its modulation by benzodiazepines and pentobarbital reduced 47. There is also evidence from behavioural experiments that long-term treatment with benzodiazepines modulates the GABAergic system. Bagetta et al.2 found reductions in muscimol-induced changes in behaviour and

Correspondence: J.A. Pratt, Department of Physiology and Pharmacology, University of Strathclyde, George Street, Glasgow G1 1XW, U.K.

281 electrocortical activity 1 w e e k after the cessation o f 30 days of d i a z e p a m t r e a t m e n t . Tietz and R o s e n b e r g 44 have also d e m o n s t r a t e d b o t h tolerance to the effect of intranigral flurazepam and subsensitivity to the effect of intranigral muscimol on rotational b e h a v i o u r after chronic flurazepam t r e a t m e n t . H o w e v e r , two groups have failed to show significant alterations in the anticonvulsant effect of p r o g a b i d e after chronic b e n z o d i a z e p i n e treatment, although such t r e a t m e n t abolished or r e d u c e d prog a b i d e ' s h y p o t h e r m i c effect 27"35. E x p e r i m e n t s in o u r l a b o r a t o r y using the quantitative 2-deoxy-[14C]glucose ([14C]2-DG) autoradiographic m e t h o d for the d e t e r m i n a t i o n of local c e r e b r a l glucose use ( L C G U ) 41 have suggested brain structures which m a y be i m p o r t a n t in the d e v e l o p m e n t o f tolerance to the sedative and to the anxiolytic and anticonvulsant p r o p erties of d i a z e p a m , which occur at different rates 26. Since rates of L C G U are believed primarily to reflect ion p u m p activity in the nerve terminal 28, this technique gives information related principally to alterations in the activity of neuronal circuits consequent on r e c e p t o r occupation, rather than to the characteristics of the receptors themselves. A s such it provides a useful c o m p l e m e n tary a p p r o a c h to the study of b e n z o d i a z e p i n e tolerance. The high resolution o f this technique permits analysis of metabolic changes in small but discrete brain structures, which m o r e o v e r n e e d not be p r e s e l e c t e d for study. If chronic d i a z e p a m t r e a t m e n t results in a reduction in G A B A function, then it should be possible to identify circuits affected as a consequence of altered G A B A function by examining the changes in p a t t e r n s of L C G U in response to G A B A o r a G A B A A agonist which such t r e a t m e n t m a y produce. A d d i t i o n a l l y , comparison with d a t a from our previous study 26 would allow evaluation of w h e t h e r subsensitivity to G A B A is expressed in identical n e u r o a n a t o m i c a l circuits to those affected during tolerance to benzodiazepine. Since G A B A itself does not a p p e a r to p e n e t r a t e the b l o o d - b r a i n barrier, the G A B A agonist muscimol has b e e n used in the present study. Muscimol itself a p p e a r s to be rapidly m e t a b o l i s e d in the b l o o d , and only a small p e r c e n t a g e o f the dose given intravenously m a y e n t e r the brain; however, this small a m o u n t m a y be sufficient to explain muscimol's pharmacological actions; m o r e o v e r , metabolites a p p e a r to cross the b l o o d - b r a i n b a r r i e r and m a y contribute to the pharmacological effects 3. Muscimol has a l r e a d y b e e n shown to have clear effects on L C G U 22'37. A p r e l i m i n a r y account of this w o r k has a p p e a r e d in abstract form 6. MATERIALS AND METHODS Experiments were performed on 23 male Long-Evans hooded rats (weight on initiation of treatment 389 -+ 13 g, on the experimental day 437 - 12 g (mean -+ S.E.M.), bred at Glasgow College

of Technology, maintained on a natural day/night light cycle and allowed food and water ad libitum. Animals were randomly assigned to 4 groups. Groups 1 and 2 received daily injections of vehicle (1% Tween 20 in 0.9% saline) for 28 days. Group 3 received 27 daily injections of vehicle and on day 28 one injection of diazepam 5 mg/kg mierosuspended in vehicle by ultrasound. Group 4 received 28 daily injections of diazepam 5 mg/kg. All injections were given i.p. in a volume of 1 mi/kg body weight. The treatment schedule for group 4 was one which would be expected to produce tolerance to the anxiolytic action of benzodiazepines 13, permits direct comparison with our previous work on tolerance to diazepam effects on LCGU 26 and is similar to a protocol which, in an electrophysiological study, has produced subsensitivity to exogenously applied GABA TM. Local cerebral glucose use was measured in the conscious rat using the quantitative [14C]2-DG autoradiographic technique of Sokoloff et al.41. Details of the procedure were essentially as previously described26. Briefly, on the day of the experiment (day 29), rats were lightly anaesthetised, cannulae inserted into both femoral veins and both femoral arteries; the rats were then partially restrained in a loosely fitting plaster cast, supported on blocks, and allowed to recover from the anaesthesia for a minimum of 2 h. Muscimol (Sigma) was dissolved in distilled water and injected i.v. in animals in groups 2 (saline-pretreated), 3 (acute diazepam) and 4 (chronic diazepam) at a dose of 1.5 mg/kg in a volume of 1 ml/kg 20 min prior to administration of [14C]2-DG. This timeschedule was based on that of other workers 22"37 to ensure the continued action of muscimol during the 45-min experimental period. Animals assigned to group 1 (controls) received i.v. injections of distilled water. Measurement of LCGU was initiated by an i.v. injection of [14C]2-DG (125 #Ci/kg). Over the next 45 min, timed arterial blood samples were withdrawn, from which plasma was separated for the determination of 14C by scintillation counting and of glucose by a glucose oxidase assay. The rats were killed by decapitation 45 min after the injection of the radioisotope. Brains were dissected out, frozen at -42 °C, and 3 consecutive 20-#m coronal sections cut every 200 gm. Autoradiograms were prepared from the sections, together with standards of known 14C concentration (83-1087 nCi/g tissue equivalent for 20-/~m sections). Optical density readings were made using a computer-based densitometer, and local tissue concentrations of 14C were calculated by comparison of the optical density measurements with those of the standards. Local rates of LCGU were calculated, using the operational equation derived by Sokoloff et al. 41, from final local tissue concentrations of 14C, the histories of glucose and of 14C in the arterial plasma and the appropriate rate constants for the rat. Comparisons of LCGU values for individual brain areas and of physiological parameters were made by one-way analysis of variance, followed by Newman-Keuls multiple range tests to assess differences between individual groups. In any individual brain structure all possible inter-group comparisons were made.

RESULTS

Behaviour Rats which had received 1.5 mg/kg muscimol a p p e a r e d slightly sedated, c o m p a r e d with the control group. T h e y r e m a i n e d responsive to environmental stimuli. T h e r e were no overt behavioural differences b e t w e e n the muscimol-treated groups which had received different pretreatments.

Physiological parameters T h e r e were no significant differences in b l o o d pres-

282 TABLE I

Physiological parameters Data are presented as m e a n values -+ S.E.M. with the n u m b e r of animals in parentheses. The injection referred to is the i.v, muscimol or distilled water injection. M A B P , m e a n arterial blood pressure.

Structure

Control

Muscimol 1.5 mg/kg i.v. Pretreatment Saline

Acute

Chronic

D Z P (5 mg/kg)

(6) Weight on test day (% of initial weight) pO 2 (mmHg) pCO 2 (mmHg) M A B P ( m m H g ) (5 m i n after injection) Plasma glucose (mmoi/I) (5 rain after injection)

113 94 36 135 7.8

± 2 ± 5 -+ 2 -+ 3 ± 0.1

sure, blood gas tensions, or plasma glucose between any of the groups, indicating that acute muscimol did not alter any of these parameters, either alone, or following pretreatment with a single or repeated doses of diazepam (Table I). There were also no differences in rate of weight gain between groups, indicating no differences in food consumption (Table I). Local cerebral glucose use Acute muscimol. The i.v. administration of muscimol

1.5 mg/kg to vehicle-pretreated rats caused a generalised depression of LCGU compared with control animals, which reached significance in 25 out of 66 brain areas measured (Tables II-VI). The pattern of reduction was

(6)

(5)

(6)

114 ± 3 98-+2 39--- 2 124-+ 3 8.2 -+ 0.3

115 -+ 3 92-+4 38- 2 127 ± 3 8.1 -+ 0.3

110 +_ 2 99.+_5 38 ± 2 126 -+ 3 8.1 ± 0,2

not homogeneous. It was of the order of 30-40% in most cortical areas (Table II) and some primary auditory and visual structures (Table III). The anterior thalamic nuclei, mediodorsal thalamus and the ventrolateral and ventromedial thalamic nuclei were similarly changed by 30-45% (Tables IV and V), while reductions in other limbic areas were more typically 15-20% (Tables IV and VI). In the septohippocampal formation, only the subiculum showed a significant reduction in LCGU in response to muscimol in the vehicle-pretreated group (Table VI). Single and repeated pretreatments with diazepam. The pattern of muscimol-induced LCGU changes after 28 days treatment with diazepam 5 mg/kg, and after a sin-

T A B L E II

Local cerebral glucose use following acute administration o f muscimol (cerebral cortex) D a t a are presented as m e a n glucose use ~ m o l / 1 0 0 g/rain) ± S.E.M. with the n u m b e r of animals indicated in parentheses.

Structure

Control

Muscimol 1.5 mg/kg i.v. Pretreatment Saline

Acute

Chronic

D Z P 5 mg/kg

Senaorymotor cortex (layer IV) Visual cortex (layer IV) Auditory cortex (layer IV) Parietal cortex (layer IV) Frontal cortex (layer IV) Entorhinai cortex Anterior cingnlate cortex Primary olfactory cortex Infralimbic cortex

(6)

(6)

93 --- 6 92 ± 6 125 -+ 9 93 ± 6 87 ± 7 63 ± 3 91 ± 5 92+--5 90 -+ 7

55 56 ± 69-+ 58 ± 53 -+ 51 ± 57 ± 86+5 62 -+

(5) 5* 5* 9* 6* 4* 3* 4* 5*

*P < 0.05, significantly different from control; N e w m a n - K e u l s multiple range test.

49 + 52 -+ 63 -+ 51 ± 49 ± 49-+ 52 -+ 77±7 54 ±

(6) 1" 3* 3* 3* 2* 4 3* 5*

52 +- 4* 53 ± 4* 63 ± 6* 53 - 4* 51 -+ 5* 49 -+ 4* 54 -+ 5* 81-+9 58 +- 5*

283 T A B L E III

Local cerebral glucose use following acute administration of musciraol (primary auditory and visual areas) Data are presented as mean glucose use ~mol/100 g/rain) ± S.E.M. with the n u m b e r of animals indicated in parentheses.

Structure

Control

Muscimol 1.5 mg/kg i.v. Pretreatment Saline

Acute

Chronic

D Z P 5 mg/kg

(6) Lateral geniculate body Superior colliculus superficial layer deep layer Anterior pretectal area Medial geniculate body Inferior colliculus Superior olive Cochlear nucleus Paraflocculus

(6)

100 +-- 6 93 79 83 98 162 109 106 71

--± ± ± ± ± ± ±

5 4 4 4 9 9 7 8

(5)

55 ± 5* 53 64 65 66 153 106 93 49

± ± ± --± ± ± ±

7* 6* 4* 6* 13 5 5 5*

(6)

48 ± 4* 44 58 58 59 150 98 97 46

± ± -+ ± ± -+ ± +-

3* 4* 3* 3* 13 9 14 3*

50 ± 3* 47±3* 59 ± 5* 63 ± 3* 61 ± 5* 158 ± 14 107±7 91±7 45±5*

*P < 0.05, significantly different from control; N e w m a n - K e u l s multiple range test.

gle dose of diazepam 5 mg/kg the previous day, were essentially the same as those described above for acute muscimol treatment after vehicle pretreatment (Tables I I - V I I ) . There was a tendency for the L C G U values in

all structures in the acute and chronic diazepam groups to be slightly lower than in the animals pretreated with vehicle; this may be an effect of a small amount of residual diazepam from the treatment 24 h previously.

TABLE IV

Local cerebral glucose use following acute administration of muscimol (limbic and functionally associated areas) Data are presented as mean glucose use (,umol/100 g/rain) ± S.E.M. with the n u m b e r of animals indicated in parentheses.

Structure

Control

Muscimol 1.5 mglkg i.v. Pretreatment Saline

Acute

Chronic

D Z P 5 mg/kg

(6) Dorsal tegmental nucleus Locus coeruleus Dorsal raphe Median raphe Mammillary body Amygdala (basolateral) Amygdala (medial) Amygdala (cortical) Anteroventral thalamic nucleus Anteromedial thalamic nucleus Ventral tegmental area Interpeduncular nucleus Nucleus accumbens Nucleus reuniens Lateral habenula Mediodorsal thalamic nucleus Lateral hypothalamus Hypothalamus (lateral preoptic area)

91 49 85 91 104 74 48 65 111 114 53 100 62 83 92 98 54 60

(6) -+ 6 --- 3 ± 4 --+ 5 ± 7 -+ 3 --- 2 ± 6 ± 7 ± 5 ± 3 ± 5 ± 3 ± 4 - 4 ± 7 ± 3 ± 4

75 47 73 76 86 58 43 62 74 71 47 91 50 62 92 65 49 52

(5) ± ± ± ± ± --± --± --± ± --± --±

4 2 5 6 6 6* 3 5 7* 8* 3 6 3* 5* 6 5* 5 3

73 42 68 68 78 53 40 57 64 54 41 81 47 53 80 57 44 46

(6) -+ 6 ± 1 ± 4* -+ 4* -+ 6 -+ 5* -+ 3 -+ 4 ± 7* -+ 5* --- 2* ± 4 ± 3* ± 5* - 5 ± 4* ± 3 _+ 4

78 45 69 74 86 56 41 56 69 68 44 87 50 56 90 62 49 49

± ± ± ± ± ± ± -+ ± ± ± ± ± ------±

5 3 4* 5 8 5* 3 7 4* 6* 2 6 5* 5* 6 4* 3 5

284 TABLE V

Local cerebral glucose use following acute administration of muscimol (extrapyramidal and sensorymotor areas) Data are presented as mean glucose use ~mol/100 g/min) ± S.E.M. with the number of animals indicated in parentheses.

Structure

Control

Muscimol 1.5 mg/kg i.v. Pretreatment Saline

Acute

Chronic

DZP 5 rng/kg

(6) Caudate nucleus (lateral) Substantia nigra (retieulata) Substantia nigra (compacta) Globus pallidus Subthalamic nucleus Ventromedial thalamic nucleus Ventrolateral thalamic nucleus Inferior olive Cerebellax hemisphere Pontine nucleus

92 49 61 48 73 100 87 62 50 59

(5)

(6) ± ± ± ± ± ± ± ± -+ ±

4 3 3 1 5 6 5 2 2 5

53 46 57 39 65 69 49 59 45 46

-+ 6* ± 3 ±3 -+ 3 ± 4 ± 6* ± 5* ± 6 ± 2 -+ 3*

49 42 53 39 58 58 44 54 41 42

~6) ± 2* -+ 3 -+ 3 -+ 2 -+ 4 -+ 4* ± 4* ± 3 -+ 3 -+ 3*

50 43 54 41 62 63 48 58 41 43

-+ ± ± ± ± ± ± ± ± +-

4* 2 3 3 4 4* 4* 4 3 3*

*P < 0.05, significantly different from control; N e w m a n - K e u l s multiple range test.

However, no significant differences were found between the 3 different pretreatment groups which received muscimol on the experimental day.

DISCUSSION

A c u t e muscimol: comparison with acute d i a z e p a m

Administration of museimol produced a generalised

TABLE VI

Local cerebral glucose use following acute administration of muscimoi (septohippocampal formation) Data are presented as mean glucose use ~mol/100 g/rain) ± S.E.M. with the n u m b e r of animals indicated in parentheses.

Structure

Control

Muscimol 1.5 mg/kg i.v. Pretreatment Saline

Acute

Chronic

DZP 5 mg/kg (6) CA1 oriens CA1 molecular layer CA1 radiatum CA2 oriens CA2 molecular layer CA2 radiatum CA3 oriens CA3 molecular layer CA3 radiatum Subiculum Dentate gyrus (tool. layer, rostral) Dentate gyrus (mol. layer, caudal) Dentate gyrus (hilus) Medial septal nucleus Lateral septal nucleus Septohypothalamic nuclei Diagonal band

58 78 63 58 77 62 59 75 60 80 57 81 63 80 66 57 74

± 4 +- 4 ± 3 + 4 ± 4 ± 4 ± 2 ± 3 ± 2 ± 3 ± 2 ± 7 ± 3 -+ 3 ± 3 ± 2 ± 3

(6)

(5)

49 + 4 64 ± 5 53 - 4 50--- 4 65 + 5 53 ± 4 57 ± 3 66 -+ 5 57 ± 4 63 ± 4* 53 ± 3 73 ± 6 56 ± 5 65 ± 7 57 ± 4 50 --- 4 63 ± 6

49 62 52 49 61 52 51 58 52 63 50 67 54 61 52 40 53

*P < 0.05, significantly different from control, N e w m a n - K e u l s multiple range test.

(6) - 4 ± 6 ± 5 - 4 ± 6 ± 4 ± 3 ± 4 ± 3 ± 5* ± 2 -+ 5 ± 4 ± 6 - 4 ± 2* ± 5*

48 61 51 48 64 52 53 62 56 63 52 69 54 61 58 46 57

___ 5 -+ 6 -+ 5 ± 4 ± 5 ± 4 -+ 3 ± 5 ± 4 ± 4* --- 4 ± 4 -+ 3 ± 5 ± 6 - 5 ± 5

285 TABLE VII Local cerebral glucose use following acute administration of muscimol (myelinated fiber tracts)

Data are presented as mean glucose use (.umol/100g/min) +-- S.E.M. with the number of animals indicated in parentheses. Structure

Control

Muscimol 1.5 mg/kg i.v. Pretreatment Saline

Acute

Chronic

DZP 5 mg/kg

Cerebellar white Genu Internal capsule

(6)

(6)

(5)

(6)

41 --- 2 30 --- 2 34 +- 1

36 -+ 2 27 +-- 1 29 --- 2

34 --- 2 25 +-- 1 27 --- 1

34 --- 2 28 -+ 2 28 -+ 2

depression of LCGU, an effect which is consistent with the action of G A B A as an inhibitory neurotransmitter believed to be involved in up to 40% of neurons in the mammalian nervous system. The pattern of reduction was not homogeneous, ranging from as much as 40-45% in some cortical and thalamic areas, through 15-20% in many limbic areas, to areas which were little affected, such as the primary olfactory cortex, inferior colliculus, superior olives, locus coeruleus, substantia nigra and inferior olives. This pattern is broadly consistent with previous studies by Kelly and McCulloch 22 and Palacios et al.37. It contrasts markedly with the pattern of reduction in glucose use in response to diazepam, which is much more homogeneous 21'26. The overall mean reduction of LCGU in the 64 areas measured in both our previous diazepam study 26 and the present study is only slightly more for acute muscimol 1.5 mg/kg than for diazepam 0.3 mg/kg (muscimol: 19.0 +-- 1.6%; diazepam: 16.7 +0.7%), suggesting a comparable depressant effect of the two drugs at these doses. However, with the exception of the entorhinal cortex and primary olfactory cortex, cortical areas revealed a more marked depression (3045%) with muscimol than with diazepam (13-20%). All regions of the thalamus showed greater depression (3045%) with muscimol than with diazepam (15-25%). This contrasts with the limbic system and the hippocampus where both drugs reduced L C G U to a broadly similar extent in most structures. Muscimol, however, produced much more marked depressions of the anteroventral, anteromedial and mediodorsal thalamic nuclei (33%, 38% and 33% respectively) than did diazepam (24%, 23%, 15%). Interestingly, in the visual and auditory circuits, there was considerable difference in response patterns between the two drugs. In several components of the auditory pathway (superior colliculus, lateral geniculate and visual cortex) muscimol produced marked reductions in

LCGU, whereas reductions were smaller after diazepam (15-27%). Diazepam produced similar homogeneous reductions in LCGU in components of the auditory pathway (cochlear nucleus 29%, superior olives 9%, inferior colliculus 15%, medial geniculate 20%, auditory cortex 20%), whereas muscimol produced differential reductions in this pathway. Marked reductions were evoked in the medial geniculate and auditory cortex, whereas non-significant reductions occurred in the components of this pathway which lie between the ear and the medial geniculate (cochlear nucleus 13%, superior olives 3%, inferior colliculus 6%). These comparisons between acute administration of muscimol and diazepam in the previous and the present study, conducted under similar experimental conditions, beg the question why the effect of a G A B A agonist and a positive modulator of GABAergic function believed to act at the same complex should produce such different patterns in cerebral glucose use. Muscimol and its metabolites are heterogeneously distributed in the brain after intravenous injection3; however, the heterogeneous reductions in LCGU do not appear to parallel the distribution of muscimol, nor do they reflect the known distributions of the G A B A receptor. Kelly and McCulloch, while advocating caution in drawing conclusions from fine details of the two patterns, conclude that the binding of these two agents is expressed quite differently in terms of their functional activity21. It is possible that fine regional differences in binding characteristics, or in the coupling between benzodiazepine and G A B A receptors and between G A B A receptors and the ion channels they gate may underlie this. The recent demonstration of considerable complexity in the possible subunit combinations of the GABAA receptor complex 4° may provide a basis for such regional differences. It has been known for some time that both high- and low-affinity GABAA receptor sites exist in mammalian

286 brain. Both a high- and a low-affinity site can be labelled with [3H]muscimol; however, the low-affinity site has usually been defined by antagonists such as bicuculline and the pyrazinyl-GABA derivative SR95531. The distribution of high- and low-affinity sites is known to be different. A recent study by Olsen et al.36 has compared the autoradiographic localisation of sites bound by various ligands of the G A B A receptor/benzodiazepine receptor/C1- ionophore complex. This study found considerable mismatches between ligands; pertinent to our study is the finding of little correlation between muscimol and flunitrazepam binding sites. Good correlation ws found between the binding of SR-95531 and the BZ2 subtype of the benzodiazepine receptor. Sites labelled by bicuculline methochloride seemed to correlate with the sum of benzodiazepine-preferring and muscimol sites. If these autoradiographic correlations represent true interactions in the brain, then diazepam and muscimol may be acting on different subsets of the G A B A A receptor complex. A number of investigations have suggested a role for G A B A agonists in the regulation of cerebral blood flow. Any such action could of course complicate the interpretation of cerebral metabolism studies on muscimol. Muscimol has been shown to produce a transient increase in cerebral blood flow in the goat 1 and the rat 12. Specific G A B A A receptors have been demonstrated in bovine and rat cerebral vasculature 24"34 and in vitro vasodilation of cerebral arteries in response to G A B A has been shown in cat, dog and human vessels n. However, Kelly and McCulloch 23 have shown a decrease in cerebral blood flow in conscious rats 30 rain after muscimol administration, and more recently Kelly et al. 20 have demonstrated that this decrease may be detected as early as 30 s after the beginning of a muscimol infusion into conscious rats. This, together with the present and previous 2-DG studies 22'37, suggests that if G A B A agonists do have a direct vasodilatory role, this is minor in comparison to the reduction in blood flow consequent upon reduction in metabolic demand.

Chronic diazepam pretreatment There was no effect of chronic diazepam pretreatment on the L C G U response to acute muscimol. Chronic diazepam treatment produces tolerance to the acute effects of diazepam administration on L C G U in some brain areas 26. This may be due to subsensitivity to G A B A induced by the repeated benzodiazepine receptor occupation. We were using a dose of muscimol which in the study of Kelly and McCulloch 22 showed clear reductions in glucose use in most areas, but was not maximal, so the lack of tolerance is unlikely to be attributable either to the reductions being too slight to permit detection of

tolerance, or being too great to be affected by the changes in the receptor complex consequent upon a mild diazepam dosing regime. As the present data show no subsensitivity to muscimol, this may suggest that a highaffinity G A B A response is involved in the LCGU changes induced by this dose of muscimol, and that highaffinity G A B A responses are unchanged by chronic diazepam treatment. This would be consistent with the previously discussed suggestion that benzodiazepine receptors are coupled to low-affinity sites. However, some studies have shown subsensitivity to muscimol after longterm benzodiazepine treatment. Miller et al. 32 found decreased muscimol-stimulated 36C1- uptake in mice after 7 days of lorazepam treatment. Reductions in muscimolinduced behavioural changes have also been observed by Bagetta et al. after chronic diazepam treatment 2 and by Tietz and Rosenberg after chronic flurazepam treatment 44. Alternatively, the development of functional tolerance to benzodiazepines may proceed at a different rate from the development of functional subsensitivity to G A B A A agonists. There is some evidence for this in the work of Tietz and Rosenberg 44 who have shown differences in the time course of the development of tolerance to flurazepam's and subsensitivity to G A B A ' s effect upon rotational behaviour in rats. Similarly, differences exist for the time courses of return to control values on withdrawal. Whilst Gonsalves and Gallager note that the time course of tolerance to the anticonvulsant effects of diazepam paralleled to some extent subsensitivity to G A B A in the dorsal raphe, this subsensitivity continued to develop despite the anticonvulsant tolerance being complete. Yu et al. 47 could show no reduction in G A B A stimulated C1- influx into rat brain microsacs after 4 weeks of flurazepam treatment, at a time when tolerance to the ability of benzodiazepines to enhance CI flux was evident. A further possibility is that chronic diazepam treatment may not induce subsensitivity to muscimol itself, but reduce the coupling between the benzodiazepine and the G A B A receptors. This effect might not be detectable in our study, since the experiment was initiated 24 h after the last diazepam treatment, at a time at which we would expect there to be no diazepam present. Some studies have addressed the question of a reduction in the interaction between the benzodiazepine and G A B A receptors14"31'42; however, the usual measure of coupling in such studies is the ability of GABA to enhance benzodiazepine binding, and it is not always clear whether an alteration in the G A B A receptors themselves might be responsible for a reduction in G A B A enhancement of benzodiazepine binding. Behavioural factors could also influence these results.

287 F o r e x a m p l e , the chronic handling p r o c e d u r e could have o p p o s e d any changes at the G A B A A r e c e p t o r induced by r e p e a t e d drug administration. Biggio has p r o p o s e d that h a b i t u a t i o n to a mild stressor (handling) upregulates the G A B A e r g i c system, which cannot then b e further m o d u l a t e d by b e n z o d i a z e p i n e s s. We have also found that r e p e a t e d handling m a y modify the G A B A A receptor complex and a t t e n u a t e the anxiolytic effects of diaze p a m in b e h a v i o u r a l tests 5'7. It is possible, in the present study, that r e p e a t e d handling has c o u n t e r a c t e d G A B A e r gic subsensitivity accompanying b e n z o d i a z e p i n e tolerance. This would require further investigation. In s u m m a r y , chronic d i a z e p a m (5 mg/kg i.p. daily) does not a p p e a r to reduce the m a r k e d L C G U response to the G A B A agonist muscimol in the rat, suggesting

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