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cages for 7 days before the first measurement of loco- motor activity was ..... surement of local cerebral glucose utilization: theory, pro- cedure, and normal values ...
Brain Research, 456 (1988) 350-356 Elsevier

350 BRE 13801

Local cerebral glucose utilization of the awake rat during chronic administration of nicotine F r a n k G r i i n w a l d 2, H e l m u t S c h r 6 c k l, H e r m a n n T h e i l e n 1, A n t o n B i b e r 3 a n d

Wolfgang Kuschinsky 1 1Department of Physiology, 21nstitutefor Clinical and Experimental Nuclear Medicine, University of Bonn, Bonn (F.R.G.) and 3Forschungslaboratorium Prof. Schievelbein, Munkh (F.R. G. ) (Accepted 16 February 1988)

Key words: Nicotine; 2-Deoxyglucose; Rat brain; Local cerebral glucose utilization; Nicotinic receptor; Chronic nicotine administration; Locomotor activity

Local cerebral glucose utilization (LCGU) was measured in 45 regions of the rat brain during chronic nicotine infusion using the quantitative autoradiographic 2-deoxy-D-[1-14C]glucosemethod described by Sokoloff et al. [J. Neurochem., 28 (1977) 897-916]. Osmotic minipumps, filled with L-nicotine, were implanted 14 days before the measurement of LCGU. The infused nicotine dose of 12.5 ~tg/kg/minresulted in a plasma nicotine concentration of 77 + 17 and a plasma cotinine concentration of 504 + 137 (mean + S.E.M.) ng/ml plasma. One day before the LCGU experiment was performed, spontaneous locomotor activity was measured and found to be reduced significantly. Measurement of LCGU showed a significant increase in 6 of the 45 brain structures examined, i.e. globus pallidus, septal nucleus, lateral geniculate body, superior colliculus (superficial grey layer), interpeduncular nucleus and optic chiasm. These results are partly congruent with previous data of our group obtained during acute nicotine infusion, insofar as LCGU was increased in the optic chiasm, the lateral geniculate body, the superior colliculus, and the interpeduncular nucleus. On the other hand, the increased LCGU in the globus pallidus and septal nucleus occurred during chronic infusion only; other structures were not affected by chronic infusion although their LCGU had been raised during acute infusion. It is concluded that chronic nicotine infusion has distinct effects on the functional activity of several brain structures which are partly congruent with those affected during acute nicotine infusion and partly divergent from them.

INTRODUCTION

structures by an acute administration of nicotine. Chronic effects may be different. This may be indi-

The central nervous effects of nicotine are of interest with respect to the mechanism of nicotine addiction. Our previous approach to detect the mechanism of nicotine addiction has been to measure local cerebral glucose utilization ( L C G U ) in several structures of the rat brain during acute infusion of L-nicotinetl. In this study, L C G U was increased in several structures of the limbic system (anteroventral and anteromedial nucleus of thalamus, interpeduncular nucleus, mammillary body, cingulate cortex), the visual system (optic chiasm, superior colliculus, lateral geniculate body), and the substantia nigra, compact part, thus indicating a functional activation of these

cated by receptor binding studies of nicotine in vitro in the rat brain. After chronic nicotine infusion, the density of cholinergic nicotinic receptors is increased 2s when compared to untreated controls. It was the aim of the present study to detect potential long-term changes of brain function by the measurement of the L C G U in several structures of the rat brain during chronic administration of nicotine. The 2-deoxy-D-[1-14C]glucose method described by Sokoloft et al. 29 was used as a tool to trace alterations of functional activity in the brain since local rates of energy metabolism are normally correlated with local functional activity 3°.

Correspondence: F. Griinwald, Institute for Clinical and Experimental Nuclear Medicine, University of Bonn, Sigmund-Freud-Str. 25, D-5300 Bonn 1, F.R.G. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)

351 MATERIALS AND METHODS The experiments were performed on male Sprague-Dawley rats (Versuchstieranstalt Hannover, F.R.G.) weighing 300-380 g. LCGU was measured by the method described by Sokoloff et al. 29. The animals were anaesthetized with halothane and N 2 0 , and osmotic minipumps (Alzet model 2002, Alza Corp., Palo Alto, CA, U.S.A.), filled with L-nicotine (free base, Sigma, Deisenhofen, F.R.G., diluted with 0.9% NaCl), were implanted subcutaneously. The pumps were filled with L-nicotine diluted with saline solution such as to result in a dosage of 12.5/tg/ kg/min. Control animals received minipumps filled with saline. Rats were weighed daily. The spontaneous locomotor activity was measured on the 7th and 13th day after implantation. Locomotion was recorded in 5-min periods for 30 min as brightness intensity change in one or several of 16 areas of a black cage by photo cells placed over the cage (Motilimat, H. Albrecht Instruments, Munich, F.R.G.). Since it was found that the locomotor activity of a single rat is positively correlated with the amount of rats kept in the cage during the days before the measurements, all rats were housed in single cages for 7 days before the first measurement of locomotor activity was performed. All measurements of locomotor activity took place in the afternoon between 14.00 and 17.00 h. Each measurement of a control rat (with implanted minipump containing saline) was either preceded or immediately followed by a measurement of locomotor activity in a rat with chronic infusion of nicotine. On the 14th day after implantation (one day after the last measurement of spontaneous locomotor activity), the anaesthesia described above was performed again and polyethylene catheters were inserted into a femoral artery and vein. The rats were then placed in loose-fitting plaster casts and immobilized from the abdomen to the hind legs; the head, forelegs, and thorax were unrestrained. After 210 minutes following the end of anaesthesia, 125 /zCi/kg of 2-deoxy-D-[1-14C]glucose (spec. act. 45-55 mCi/mmol; New England Nuclear Corp., Dreieich, F.R.G.) was injected as a pulse via the left femoral vein within 40 s, and timed arterial blood samples of about 80/A each were collected through the femoral arterial catheter at 15, 30 and 45 s, and 1,

2, 3, 5, 7.5, 10, 15, 25, 35 and 45 min. The blood samples were immediately centrifuged and stored on ice until assayed for [lac]deoxyglucose and glucose concentration as previously described 29. At 45 min an additional sample of 2 ml was collected for the measurement of nicotine and cotinine concentration in plasma, and the animal was decapitated. The brain was rapidly removed and frozen in isopentane chilled to -40 to -50 °C with dry ice. The frozen brain was coated with chilled embedding medium (Lipshaw Manufacturing Co., Detroit, MI, U.S.A.), stored at -80 °C in a plastic bag, sectioned into 20-/~m sections at -20 °C in a cryostat, and autoradiographed along with calibrated [14C]methylmethacrylate standards as described previously29. Local tissue concentrations of ~4C were determined from the autoradiographs by densitometric analysis with a densitometer (DT 1105 R, Parry, Newbury, U.K.) equipped with a 0.2 mm aperture. Local rates of cerebral glucose utilization were calculated from the local concentration of 14C and the time courses of the plasma [14C]deoxyglucose and glucose concentrations 29. Body temperature was measured before the experiment. Blood pressure, heart rate, plasma glucose concentration, hematocrit, pH, pCO2, and pO2 were measured before and 35 min after administration of deoxyglucose. LCGU was measured in two groups of 5 animals each (saline control and 12.5 pg/kg/min nicotine). The values of LCGU obtained for each structure, the locomotor activity rates and body weights on each day for both groups were compared using a t-test (saline versus nicotine group). The plasma concentrations of nicotine and cotinine were determined by the radioimmunoassay described by Langone and Van Vunakis 21. RESULTS LCGU was measured in 45 brain structures during control conditions and during chronic nicotine infusion. In 39 structures LCGU was unchanged, whereas in 6 structures LCGU was increased significantly. No significant decrease was observed in any structure. The LCGU in structures of the sensorimotor and extrapyramidal system is shown in Table I for control conditions and chronic infusion of nicotine. A moder-

352 TABLE I

TABLE III

Local cerebral glucose utilization in sensorimotor and extrapyramidal system (~mol/lO0 g/min) during chronic infusion o f nicotine (means + S.E.M.)

Local cerebral glucose utilization in the visual and auditory system (#mol/lO0 g/min) during chronic infusion o f nicotine (means + S.E.M.)

Sensorimotor cortex Inferior olivary nucleus Cerebellar cortex Vestibular nucleus Caudate nucleus Globus pallidus Substantia nigra reticular part compact part Thalamus posterior lateral nucleus ventral nucleus anteromedial nucleus anteroventral nucleus Pontine gray Dentate nucleus

Control group (n = 5)

Nicotine group (n = 5)

96 66 44 101 84 40

100 + 2 71 + 1 49 + 4 110 _+ 2 92 _+ 3 44 + 1"*

+ 4 + 3 +_ 1 _+ 3 + 4 + 1

46 _+ 1 53 _+ 3 84 _+ 3 82 + 3 111 + 8 114 _+ 6 50 +_ 1 77 + 2

50 _+ 2 70 _+ 11 93 + 3 90 _+ 3 123 _+ 2 133 _+ 5 53 ___2 81 _+ 2

**P