192

Brain Research, 511 (1990) 192-196 Elsevier

BRES 15261

Cerebral glucose utilization during diazepam withdrawal in rats Cheryl A. Marietta 1, Michael J. Eckardt 2, Kerry L. Zbicz 1'* and Forrest F. Weight ~ Laboratories of 1Physiologic and Pharmacologic Studies, and of 2Clinical Studies, D1CBR, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20851 (U.S.A.)

(Accepted 8 August 1989) Key words: Diazepam withdrawal; 2-Deoxyglucose; Cerebral glucose utilization

The diazepam withdrawal syndrome in rats was characterized behaviorally by an increase in spontaneous motor activity, slight body tremors and a lack of convulsions. The 2-deoxyglucose (2-DG) technique was used to measure quantitatively cerebral glucose utilization during diazepam withdrawal and revealed changes in glucose utilization in 30% of the 54 structures evaluated. Areas of increased glucose utilization included medial geniculate, inferior colliculus, visual cortex, mamitlary body, dorsal hippocampus, cerebellar flocculus, and zona reticulata and zona compacta of the substantia nigra. Areas of decreased glucose utilization included columnar areas in frontal sensorimotor cortex, caudate, globus pallidus, olfactory cortex, nucleus accumbens and internal capsule. There was no single or consistent relationship between reported benzodiazepine receptor densities and glucose utilization.

INTRODUCTION The benzodiazepines are used as anxiolytics, muscle relaxants, and to treat insomnia, spasticity and convulsions. T h e y are one of the most widely prescribed group of drugs with d i a z e p a m (Valium, D Z ) accounting for up to 4% of all prescriptions 16. Initially, long-term D Z use was not thought to cause physical dependence; however, in the late 1970's withdrawal symptoms such as the ' r e b o u n d ' p h e n o m e n o n , in which anxiety is greater and m o r e intense after the withdrawal of D Z than before t r e a t m e n t was begun, were r e p o r t e d upon cessation of D Z t r e a t m e n t 19. Subsequently, W i n o k u r et al. 26 described the signs and symptoms of D Z withdrawal in man as including anxiety, dizziness, blurred vision, tinnitus, palpitations, constipation, tremulousness and p o o r coordination. In man, these signs and symptoms begin a p p r o x i m a t e l y 24 h after the last dose and can last for over a month. A n i m a l models have been d e v e l o p e d to study D Z withdrawal. F o r example, Rastogi and co-workers 18't9 have described a ' r e b o u n d ' p h e n o m e n o n of increased m o t o r activity in rats undergoing withdrawal after receiving 10 mg/kg of D Z daily (injected s.c.) for 20 days. M o r e o v e r , physical d e p e n d e n c e upon D Z has been achieved using a gastric fistula m e t h o d H as well as a per oral regime 2 with animals undergoing a withdrawal s y n d r o m e 10-60 h after their last dose of D Z . During

withdrawal from D Z , rats have been r e p o r t e d to exhibit an increase in spontaneous m o t o r activity but no convulsions2,11. The goal of the present study was to d e t e r m i n e if localized changes in glucose utilization a c c o m p a n y the D Z withdrawal s y n d r o m e using the quantitative 2deoxyglucose (2-DG) technique d e v e l o p e d by Sokoloff and co-workers 25. This technique has been used previously to evaluate changes in glucose utilization in animals undergoing withdrawal from ethanol 4'7, m o r p h i n e 8,27 and p h e n o b a r b i t a l 1°. We have previously r e p o r t e d preliminary, qualitative observations that there a p p e a r e d to be no generalized increase in cerebral glucose utilization in rats undergoing D Z withdrawal, but localized alterations in frontal sensorimotor cortex, cerebellar vermis, several thalamic nuclei and in the lateral geniculate were a p p a r e n t 9. MATERIALS AND METHODS Diazepam administration Female Sprague-Dawley rats (Charles River), weighing 200-250 g at the start of the experiment were maintained on a 12 h light:12 h dark cycle. A DZ suspension (95 ml tap water + 5 ml olive oil + 350 mg Carragheenan, an emulsifier, + DZ to make a 0.2% (20 mg/kg and 40 mg/kg doses) or 0.8% (80 mg/kg and 100 mg/kg) w/v suspension) was administered by means of oral gavage twice daily during the light phase with 10-11 h separating administrations of DZ, 7 days per week, on a rapidly increasing dosage schedule. For the first 4 days, rats were given a total daily dose of 20 mg/kg.

* Present address: GLAXO Inc., 5 Moore Drive, Research Triangle Park, NC 27709, U.S.A. Correspondence: Cheryl A. Marietta, LPPS/NIAAA, 12501 Washington Ave., Rockville, MD 20852, U.S.A.

193 During the following 4 days, the daily dose was increased to 40 mg/kg followed by a total daily dose of 80 mg/kg for the next 2 weeks. For the final 5 weeks, the animals were maintained on 100 mg/kg of DZ per day. Similar, rapidly increasing dosing schedules, achieving maximum daily doses of 90-133 mg/kg/day have been used by others TM. Control animals received an equivalent volume of suspension mixture without DZ throughout the treatment period. All rats were given 1 ml of a multi-vitamin supplement suspension formulated from the recommended daily allowance for rats 143 times per week. Food (Purina Rodent Chow) and water were available at all times.

Deoxyglucose method Approximately 52 h after the last dose of DZ, when withdrawal signs were subjectively judged to be the greatest, the rats were given light halothane/nitrous oxide anesthesia and cannulae were inserted into the femoral artery and vein. The rats were lightly restrained using a plaster cast applied to the lower torso and legs and allowed to recover for at least 2 h prior to the injection of 2-DG. The experimental protocol for animal preparation, injection of 2-DG, arterial blood sampling, sacrifice of the animals and preparation of the autoradiographs followed closely that described by Sokoloff et al.25. Fifty gCi of 2-[14C]deoxy-D-glucose (specific activity 51.1 mCi/mM; New England Nuclear) in saline were injected into the femoral vein, and the cannula was flushed with heparinized saline within 20 s. Arterial blood samples were taken for the determination of plasma 2-[14C]DG and glucose levels as prescribed by the protocol of Sokoloff et al.25. Arterial pO 2, pCO 2, pH, blood pressure, hematocrit, and temperature were also monitored. At 45 min after 2-DG administration, the rat was decapitated and the brain rapidly removed and frozen in Freon-12 chilled with dry ice (-70 °C). The brains were coated with Lipshaw Embedding Media and stored at -60 °C until sectioning. Twenty-gm sections were cut at -20 °C and every 200/~m, two sections were saved for analysis. One set of sections was mounted on cover slips and affixed to heavy cardboard along with a series of [14C]plastic standards (Amersham 196363) and placed in X-ray cassettes in contact with Kodak Min-R X-ray film for 20 days to produce autoradiographs. Adjacent sections were saved on glass slides and stained with Safranin O for histological comparison. Plasma [14C]DG concentration (determined by liquid scintillation counting) and plasma glucose concentration (Beckman Glucose Analyzer II) were used in the calculation of plasma integrals. Both tissue and plasma specific activities were used to calculate glucose utilization rates25. Densitometric measurements were made on 50 specific regions of gray matter, 4 white matter areas and radioactive standards using a 7.90-gm aperture on an image array processing system (Optronics photoscan P-1000, Gould-DeAnza image array processor). The regions analyzed were selected so as to facilitate comparisons with our previous studies7't°. Digital output of the image array processor was stored and analyzed on a PDP 11-34 computer. Local cerebral glucose utilization rates were calculated for each area examined using the equations of Sokoloff et al. 25. Student's two-tailed t-test was used to determine the significance of between-group differences. RESULTS

Behavioral and physiological observations during diazepam administration and withdrawal Following the administration of D Z , rats appeared somewhat sedated and slightly less active. Throughout the period of D Z administration, rats gained weight and appeared healthy and well groomed. Approximately 48 h after the last dose of D Z , rats showed increased activity and slight tremors which peaked approx. 54 h after the

last dose. No convulsions were seen in rats withdrawing from D Z . No significant differences ( P >

0.05) were found

between controls and DZ-withdrawing rats with regard to arterial p H , pOE, pCO2, hematocrit, blood pressure, plasma glucose level, body t e m p e r a t u r e , or body weight.

Quantitative autoradiography The optical density of autoradiographs from control and D Z withdrawing rats was d e t e r m i n e d using scanning microdensitometry, and glucose utilization rates were calculated using the equations of Sokoloff et al. 25. Because there were no consistent differences in values determined for left and right hemispheres of the brain, no distinction was made in computing average values. Table I depicts glucose utilization rates for 50 gray and 4 white areas in both control and DZ-withdrawing rats. The values are also expressed as percent of control. Glucose utilization rates in DZ-withdrawing rats approximated 100% of controls in most of the areas examined. Localized differences in glucose utilization in DZ-withdrawing rats were noted in some structures, however, with 16 showing statistically significant changes from control rates. A t the 0.001 level of significance, decreased glucose utilization in DZ-withdrawing rats was noted in olfactory cortex, c o l u m n a r areas of the frontal sensorimotor cortex and head of the caudate. A t the 0.01 level of significance, decreased glucose utilization was observed in nucleus accumbens and internal capsule, while an increase was found in cerebellar flocculus. At the 0.05 level of significance, decreased glucose utilization was noted in middle and tail of caudate nucleus and in globus pallidus. Increased glucose utilization was found in mammillary body, medial geniculate, dorsal hippocampus, both zona reticulata and zona compacta of substantia nigra, visual cortex and inferior colliculus. All other areas were not statistically different from control values. DISCUSSION No significant differences b e t w e e n DZ-withdrawing and control rats were noted in the physiological parameters of plasma glucose levels, hematocrit, pH, p C O 2, p O 2, blood pressure, weight or rectal temperature. In addition, there was no difference in peak serum 14C concentrations. These observations make it unlikely that alterations in these physiological parameters could account for the observed differences in cerebral glucose utilization. Although there were no quantitative indications of a generalized increase in cerebral glucose utilization in DZ-withdrawing rats, there were localized changes in

194 TABLE I

Comparison of glucose utilization (l~mol/lO0g/min) in selected brain regions of rats undergoing withdrawalfrom diazepam (D Z) and control Values are mean + S.E.M. (n = 6 for control and 7 for D Z withdrawing).

Region

Control

Gray matter Motor system Frontal cortex Columns Caudate head middle tail Globus pallidus Substantia nigra, reticulata compacta Red nucleus Cerebellar vermis ovoid areas Dentate nucleus Subthalamic nucleus Auditory system Cochlear nucleus Superior olive Lateral lemniscus Medial geniculate Auditory cortex Inferior colliculus Visual system Lateral geniculate dorsal ventral Visual cortex Superior colticulus Olfactory system Olfactory cortex Limbic system Hypothalamus ventral mammillary body Amygdala

176 + 9

DZ withdrawal % Control

125 + 3*** 170 + 7

71 97 71 81 87 84

178 + 178 + 143 + 94 +

10 13 6 5

127 + 6*** 144 + 10" 125 + 5* 79 _+4*

76 + 105 + 120 + 123 +

7 8 11 7

155 + 10 148 + 11

99 + 129 + 129 + 113 + 122 + 162 + 142 +

5* 5* 4 6 8 8 4

130 123 108 92 99 105 96

187 + 165 + 154 + 176 + 219 + 260 +

153 + 162 + 159 + 213 + 243 + 296 +

10 7 10 9* l0 ll*

82 98 103 131 111 114

166 + 134 + 142 + 150 +

18 13 12 13 15 13

20 14 7 11

210 _+ 12

71 + 6 159 + 13 65 + 5

141 + 110 + 172 + 154 +

6 5 9* 8

85 82 121 103

136 _+7***

74 + 7 209 + 11 61 _+4

65

'

104 131 94

Region

Control

Habenula 196 + 26 Interpeduncular 180 + 23 Hippocampus dorsal 128 + 7 ventral 121 + 8 dentate 95 + 7 Lateral septal 94 + 5 Cingulate cortex 200 + 5 Entorhinal cortex 106 + 10 Thalamus Anterior 144 + 7 Reticular 139 + 9 Paratenial 150 + 8 Dorsal medial 177 + 14 Lateral 143 + 11 Ventral 180 + 13 Reuniens 182 + 11 Posterior 176 + 18 Parafascicular 194 + 20 Miscellaneous gray structures Mesencephalic reticular formation 105 + 8 Pontine gray 76 + 6 Pontine reticular formation 79 + 7 Nucleus accumbens 163 + 11 Locus ceruleus 85 + 9 Raphe, median 159 + 17 Cerebellar flocculus 131 + 8 Cerebellar parafiocculus 93 + 8 Vestibular 176 + 12 White matter Corpus callosum 68 _+ 14 Genu of corpus callosum 50 + 6 Internal capsule 63 + 7 Cerebellar white 49 + 7

DZ withdrawal % Control 167 + 15 171 + 16

85 95

156 + 9* 131 + 9 119 + 9 93 + 8 190 + 15 107 + 4

122 108 125 99 95 101

139 + 9 142 + 8 139 + 11 155 + 6 157 _+ 12 169 + 7 195 + 15 160 + 16 170 + 16

97 102 93 88 110 94 107 91 88

119 + 4 85 + 5

113 112

81 + 124 + 86 + 144 + 164 + 90 + 193 +

4 7"* 3 3 7** 9 12

103 76 102 91 125 97 110

59 + 39 + 44 + 48 +

5 3 2** 2

87 78 70 98

*P ~

Cerebral glucose utilization during diazepam withdrawal in rats.

The diazepam withdrawal syndrome in rats was characterized behaviorally by an increase in spontaneous motor activity, slight body tremor and a lack of...
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