Differential cerebrovascular and metabolic responses in specific neural systems elicited from the centromedian-parafascicular complex

0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd © 1992 IBRO

Neuroscience Vol. 49, No. 2, pp. 451466, 1992

Printed in Great Britain

DIFFERENTIAL CEREBROVASCULAR A N D METABOLIC RESPONSES IN SPECIFIC N E U R A L SYSTEMS ELICITED FROM THE C E N T R O M E D I A N - P A R A F A S C I C U L A R COMPLEX S. MRAOVITCH,*t Y. CALANDO,* E. PINARD,* W. J. PEARCE~ and J. SEYLAZ* *Laboratoire de Recherche Crrrbrovasculaire, C.N.R.S.U.A. 641, Universit6 Paris VII, 10, avenue de Verdun, 75010 Paris, France :~Division of Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California, CA 92350, U.S.A. Abstract--The effect of electrical stimulation of the centromedian-parafascicular complex on local cerebral blood flow and local cerebral glucose utilization was investigated in anesthetized, paralysed and ventilated rats. Local cerebral blood flow and local cerebral glucose utilization were measured in separate groups of animals using the autoradiographic (I4C)iodoantipyrine and (14C)2-deoxyglucose methods, respectively. Because of the well-established centromedian-parafascicular complex neuroanatomical connections, three functional neuronal systems were analysed and compared: the extrapyramidal motor system the limbic system and the reticular formation, also known as the ascending activating system. Cortical regions not included in the limbic system were considered separately. The validity of comparisons between changes in local cerebral blood flow and local cerebral glucose utilization across the brain was verified by assessing the reactivity and stability of the cortical blood flow during long-term centromedian parafascicular complex stimulation. Centromedian-parafascicular complex stimulation elicited a marked but heterogeneous increase in local cerebral blood flow in 50 of the 52 cerebral structures measured. The most pronounced increases were seen in the lateral habenular nucleus (331 ___30% of control), the zona incerta (400 + 55%), the mesencephalic reticular formation (415 __+122%) and the parietal cortex (211 +__35%). In contrast, local cerebral glucose utilization remained statistically unchanged (P > 0.05) in 28 of these 50 individual brain regions during centromedian-parafascicular complex stimulation. The most pronounced increases in local cerebral glucose utilization were seen in the zona incerta (123 + 28%) and the mesencephalic reticular formation (193 ± 26%). Local cerebral blood flow and local cerebral glucose utilization were linearly related in unstimulated controls, considering either all brain regions taken as a whole or the three systems separately. The significant increase in the slopes of the regression line between local cerebral blood flow and local cerebral glucose utilization for the reticular formation and the limbic system during centromedian-parafascicular complex stimulation indicates, however, that the coupling mechanisms for these systems, but not for the extrapyramidal motor system, were reset. The local cerebral blood flow to local cerebral glucose utilization ratio was heterogeneous in controls and differentially increased during centromedian-parafascicular complex stimulation, being markedly pronounced in the parietal cortex and in the reticular formation. We conclude that these results, for the first time, provide evidence that, the functionally well-defined neural networks may have different mechanisms whereby changes in vascular and metabolic demands are regulated.

A number of recent studies have suggested that specific neural systems in the brain participate in the regulation of the cerebral blood flow (CBF) by different mechanisms (for recent reviews see Refs 25, 29). An overall cerebral vasodilation secondary to an increase in cerebral glucose utilization can

tTo whom correspondence should be addressed. Abbreviations: AP, systemic arterial pressure; CBF, cerebral

blood flow; CM-Pf, centromedian-parafascicular complex; CSD, cortical spreading depression; EEG, electroencephalogram; EMS, extrapyramidal motor system; IAP, 4-[N-methyl-C 14] iodoantipyrine; LCBF, local cerebral blood flow; LCGU, local cerebral glucose utilization; LS, limbic system; MABP, mean arterial blood pressure; RF, ascending activating system or reticular formation; 2-DG, 2-[14]-deoxy-D-glucoSe. 451

be elicited by electrical stimulation of the dorsal medullary reticular formation, 6 while stimulation of the medial parabrachial nucleus of the pons elicits widespread cerebral vasoconstriction and a reduction in glucose metabolism. 2° In contrast, electrical stimulation of two neural systems results in altered C B F which is independent of metabolism: cortical C B F is increased by stimulating the cerebellar fastigial nucleus, 27 and decreased by stimulating the lateral parabrachial nucleus, 19 without significant changes in cerebral glucose utilization. We have recently obtained evidence that electrical stimulation of the centromedian-parafascicular complex ( C M - P f ) in rats elicits a marked increase in regional cortical and subcortical C B F ) l The increase in C B F was modulated by the sympathetic nervous

452

S, MRAOVITCH el al.

system a n d circulating adrenal h o r m o n e s , a n d was not c o n c o m i t a n t with electroencephalogram ( E E G ) activation. W e also observed t h a t the cerebral vasodilation elicited by the C M - P f stimulation m a y be i n d e p e n d e n t of changes in glucose metabolism. 23 In the present study we sought further evidence to indicate w h e t h e r the changes in local cerebral b l o o d flow ( L C B F ) a n d local cerebral glucose utilization ( L C G U ) elicited by electrical stimulation of the C M - P f are widespread or specific to neural networks h a v i n g well-established a n a t o m i c a l relation with the C M - P f . W e c o m p a r e d three systems: the extrap y r a m i d a l m o t o r system (EMS), the limbic system (LS), a n d the ascending activating system or reticular f o r m a t i o n (RF). The validity o f c o m p a r i n g changes in L C B F a n d L C G U was evaluated by verifying the stability of the CM-Pf-elicited cerebrovascular responses using the mass spectrometry m e t h o d 35 during c o n t i n u o u s long-term C M - P f stimulation. W e shall d e m o n s t r a t e t h a t during p r o l o n g e d electrical stimulation o f the CM=Pf, elevations in L C B F were maintained, t h a t the increases in L C B F in individual regions were heterogeneous t h r o u g h o u t the b r a i n a n d were largely dissociated from glucose metabolism, a n d finally t h a t the linear regression analysis a n d / o r L C B F to L C G U ratio comparison, suggest t h a t the m e c h a n i s m s c o n t r i b u t i n g to the vascular a n d metabolic changes in individual b r a i n regions a n d specific neural systems are different. Some of the d a t a from this study have been included in preliminary reports. 24 EXPERIMENTAL PROCEDURES

Animal preparation Wistar rats (IFFA CREDO, Lyon, France) weighing 300-370 g were anesthetized with ~-chloralose (40 mg/kg, s.c.) after induction with halothane (0.5-2% in 25% O: and 75% N2). Thin-wall vinyl catheters were inserted into both right and left femoral arteries and veins for arterial blood sampling, systemic pressure monitoring, intravenous infusion of tracer, and KC1 injection at the end of the experiment. The trachea of anesthetized rats was cannulated, and animals were placed in a stereotaxic frame (bite bar position: - 5 . 0 ram). Halothane was discontinued and the rats were paralysed with d-tubocurarine (initial dose of 0.5 mg/kg i.v. supplemented with 0.2 mg/kg every hour), and artificially ventilated (Rodent Respirator, Harvard Apparatus Co.) with 25% 02 and 75% N 2. One of the arterial catheters was connected to a Statham P 23 Db transducer for continuous monitoring of systemic arterial pressure (AP) and heart-rate using a polygraph recorder (Gould). Body temperature was maintained at 37 _+ 0.5°C throughout the experiment. The parietal bone of the animal was perforated unilaterally with a dental drill at a point 4.0 mm posterior to the bregma and 1 mm lateral to the midline. The drill-hole was then enlarged laterally to approximately 2.5ram from the midline and 5.5 mm posterior to the bregrna. Small samples (0.2 ml) of arterial blood were periodically taken for measurement of 02 and CO 2 partial pressures (POz and pCO2) and pH (ABL 2, blood gas analyser Radiometer), Measurement of local cerebral blood flow LCBF was measured using 14C-iodoantipyrine (IAP) as diffusible inert indicator. 31 Tissue concentrations of IAP

were assessed by autoradiography) t The effect of longterm CM-Pf stimulation on LCBF in the parietal cortex (chosen for its high cerebrovascular reactivity to CM-Pf stimulation) was assessed using the method of mass spectrometry. 35 Determination of the arterial concentration-time curve of 4-[N-methyl-CJ4]iodoantipyrine. IAP in ethanol (Amersham International; specific activity: 40-60 mCi/mmol) was dried under a stream of N 2 and dissolved in 1.2 ml normal saline. The IAP was infused (10 pCi/100 g of body weight) at a constant rate over approximately 30-35 s with an infusion pump. During the infusion, about 50 #1 of blood was sampled every 3-4 s through a femoral arterial catheter to determine the arterial concentration-time curve of IAP. The blood samples (40 #1) were placed in scintillation vials containing 0.6 ml tissue solubilizer (Solulyte, Baker), incubated for 20rain and decolorized with 30% hydrogen peroxide. Ten milliliters of Dynagel was added and the sample radioactivity was determined in a liquid scintillation spectrometer. Measurement of local brain concentration of 4-[N-methylCt4]iodoantipyrine. Rats were killed approximately 30-35 s after the start of lAP infusion by an i.v, bolus injection of saturated KC1. The brain was rapidly removed from the skull, frozen in isopentane ( - 4 5 ° C to -50°C), coated with an embedding medium (Lipshaw), and sectioned (20-/~m thick) in a cryostat (Bright). Four consecutive sections of the brain were removed from the knife every 140 # m using coverslips. The sections were dried immediately on a.hot plate, and placed sequentially in an X-ray cassette, together with a set of autoradiographic 14C-microscale standards (Amersham). Sections together with calibrated standards were exposed to X-ray film (Kodak SB-5) for six days. The films were developed according to the manufacturer's instructions. Alternate sections were stained with Cresyl Violet. Autoradiograms were analysed using a computerized image analysis system (Histopericolor, MS2I Matra Sep, France). The mean optical density of each brain region was determined bilaterally over four consecutive autoradiograms. The calibrated standards (nCi/g) were used to quantify the concentration of the tracer within specific brain regions. Calculation of local cerebral blood flow. LCBF (ml/100 g per min) was calculated from the concentration-time curve of IAP and the local brain concentration of the tracer using a computerized approximation of the equation developed by Kety. N The blood-brain partition coefficient for IAP was set at 0.8. 31 Measurement of local blood flow within the parietal cortex by mass spectrometry. In the seven experiments in which LCBF was measured by mass spectrometry, 35 the animal preparation was as previously described. 2a Briefly, the surgical procedures were performed in two phases. Animals were first anesthetized with sodium pentobarbital (35 mg/kg i.p.) and a gas sampling cannula (o.d. = 0.7 mm) for measuring blood flow was stereotaxically and chronically implanted in the parietal cortex of the right hemisphere (A, 8.0 + 0.5 mm) rostrally from the interaural line, laterally (L, - 5 . 5 ram) from the midline, and horizontally (H, - 5 . 0 m m ) from the surface of the cortex. The gas sampling cannula was fixed to the skull with dental cement and protected by a fiberglass cover. Fifteen days later, after any possible inflammation or edema had resorbed, the animals were reanesthetized with ct-chloralose (40mg/kg s.c.) after induction with halothane (0.5-2% in 25% 02-75% N2). The general procedures for surgical preparation and electrical stimulation are summarized above. The LCBF was determined repeatedly by helium clearance according to the Fick principle: CBF = lambda in 2/T½, where lambda is the tissue-blood partition coefficient for helium. CM-Pf stimulation was initiated after the blood gases reached a steady state. Stimulus intensity was adjusted during the initial first

C M - P f stimulation and LCBF/LCGU regulation in specific neural systems

453

minute of C M - P f stimulation, so that arterial pressure remained stable. The LCBF was measured during the period of stable arterial pressure. Determination of local cerebral glucose utilization L C G U was measured using the 2-deoxyglucose (2-DG) method and quantitative autoradiography. 36 Determination of the arterial concentration-time curve of 2-[14C]-deoxy-D-glucoseand glucose. 2-DG (NEN, specific activity 50-60mCi/mmol) was diluted in about 1.2ml normal saline. 2-DG was then infused (12.5/~Ci/100g of body weight) at a constant rate over about 35 s through a femoral venous catheter using an infusion pump. Approximately 70/~1 of arterial blood were sampled every 10s during the first minute following the start of the infusion, at 90 s, and then at two, three, five, 10, 15, 25, 35 and 45 min. The blood samples were immediately centrifuged and stored on ice. Aliquots of plasma (20 #1) were transferred to scintillation vials containing 0.6ml Solulyte. Ten milliliters Dynagel was added to each vial, and the radioactivity was measured by liquid scintillation. Plasma glucose concentration was measured using a spectrophotometer (Turner). Calculation of local cerebral glucose utilization. L C G U ~ m o l / 1 0 0 g per min) was calculated from the arterial time-course of 2-DG and glucose concentration, and local brain radioactivity was calculated using the operational equation developed by Sokoloff et al. 36 The values adopted for the lumped constant and rate constant were those determined by Sokoloff et al. 36 Calculations were made using a form of the operational equation that allows plasma glucose to vary during the experimental period. 37 Preparation and analysis of the autoradiograms, and measurements of local brain radioactivity were the same as for LCBF method.

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Physiological procedure In order to establish appropriate controls for C M - P f stimulated rats, we first determined the changes in LCBF and/or L C G U associated with mechanical injury to the brain produced by introducing a stimulating electrode into the cerebral cortex. Such mechanical injury is known t o result in a cortical spreading depression. ]5 Two separate groups were considered. First, no-sham rats, in which the stimulating electrode was not introduced into the brain prior to LCBF and/or L C G U measurement and second, shamoperated rats in which an electrode was introduced into the brain (to reach the CM-Pf) 30 min after the completion of surgery, but without stimulation throughout the experiment. In 12 rats a stimulating electrode was lowered into the brain to a site in the C M - P f complex from which electrical stimulation elicited an increase in AP. This site was then used for stimulation during the measurement of LCBF or LCGU. Stereotaxic zero was established at the level of the bregma for the rostro-caudal coordinate, at the midline for the lateral coordinate and at the pial surface for the horizontal coordinate. The electrode was then inserted vertically into the brain 4.3 m m caudal from the bregma, 1,1 mm lateral (right side) from the midline and lowered to horizontal - 5 . 0 mm from the surface of the cortex. From this point, the stimulating electrode was lowered in 0.25-ram steps~ The brain was stimulated at each step with a 9-s train of pulses delivered at 200 Hz, (previously found to be the optimal frequency for a rise in AP) with a pulse duration of 0.5 ms and a stimulus current intensity of 50 #A. Electrodes were made from 150-#m diameter stainless steel, and were insulated with epoxylite except at the tip, which was left bare for 100 #m. The anode (ground) was a clip attached to the scalp muscle. The stimulus current was measured by passing the stimulus output across a 10-f~ resistor and displayed on a Cathode ray oscilloscope. A positive site (a site used

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for stimulation during LCBF or L C G U measurement) was defined as a site along an electrode track from which a just-detectable increase in mean arterial blood . . . . ~ure (MABP; < 10mmHg) was elicited with the lowest (threshold) stimulus current. Care was taken during C M P f exploration for positive sites and during LCBF or L C G U measurement that the MABP during the electrical stimulation never exceeded 150 mmHg, a value considered to be w~ll within the autoregulatory range of A P for the rat. 4 The electrode was left in place at the positive site until the end of the experiment. Blood gases were carefully adjusted. The arterial pCO 2 was maintained within the normocapnic range (34-38 mmHg) by adjusting the respirator stroke volume. Ninety minutes after introduction of the stimulating electrode, the blood gases had reached a steady state, C M - P f stimulation for LCBF measurement was initiated. This time, however, the C M - P f was stimulated with an intermittent stimulus train (1 s on/1 s off, 200 Hz). The intensity of the stimulus was gradually increased to reach three-times threshold current (threshold current = 20-30#A). After an initial period of stimulus intensity adjustment (2-3 min), C M - P f stimulation was continued ( l ~ 1 5 m i n for LCBF measurement and approximately 50-55rain for L C G U measurement), during which the MABP remained stable. The general procedures used in the experiments in which LCBF was measured continuously by mass spectrometry during electrical stimulation of the brain have been described. 2z

Histological location of electrode sites The stimulation site was marked by passing 50/~A d.c. for 40 s through the stimulating electrode at the end of each experiment. The location of the stimulated site was then precisely reconstructed from Cresyl Violet-stained serial sections using a Baush and Lumb vertical projector.

Statistical analysis Two groups were compared and statistically evaluated with the Mann Whitney U-test; side-to-side comparisons

were analysed by paired t-test and multiple comparisons were evaluated by analysis of variance and Scheffe's test. To evaluate the correlation between LCBF and LCGU, a simple linear regression analysis was performed. The significance o f the difference between the slopes was analysed using a t-test. The significance of the difference between the ratios was evaluated using analysis of variance and Scheffe's test.

RESULTS

C o n s i d e r i n g the a n a t o m i c a l c o n n e c t i o n s b e t w e e n the C M - P f a n d b r a i n areas i m p l i c a t e d in m o t o r control, b e h a v i o r a l a n d e m o t i o n a l e x p r e s s i o n a n d arousal, the values for L C B F a n d L C G U in 48 structures were g r o u p e d into t h r e e f u n c t i o n a l systems: E M S (nine structures), L S (22 structures) a n d R F (17 structures). T h e cortical regions (frontal, parietal, occipital) a n d c o r p u s c a l l o s u m were a n a l y s e d separately. T h e d a t a are s u m m a r i z e d in Tables 3, 4, 5 a n d 6.

Physiological variables during the experiments T h e p h y s i o l o g i c a l variables are s u m m a r i z e d in T a b l e 1. A l t h o u g h M A B P w a s elevated d u r i n g m e a s u r e m e n t o f L C B F a n d L C G U in the C M - P f s t i m u l a t e d rats, it r e m a i n e d well w i t h i n the a u t o r e g u l a t o r y range. 4 T h e values o f arterial pCO2, POE a n d p H for t h e u n s t i m u l a t e d c o n t r o l s a n d stimulated a n i m a l s were n o t significantly different. T h e p l a s m a glucose c o n c e n t r a t i o n o f u n s t i m u l a t e d rats varied f r o m 180-218 rag/100 ml. Electrical stimulation o f t h e C M - P f elicited a significant ( P < 0.05) increase in the p l a s m a glucose c o n c e n t r a t i o n (up to 276 ± 23 m g / 1 0 0 m l d u r i n g first 5 m i n ) , w h i c h

Table 2. Local cerebral blood flow and glucose utilization within the extrapyramidal motor system in anesthetized, paralysed rats, with or without stimulation of centromedian-parafascicular complex LCBF (ml/100 g per min)

Region Caudate nucleus Globus pallidus Accumbens nucleus Entopeduncular nucleus Anteroventral thalamic nucleus

Ventrolateral thalamic nucleus Subthalamic nucleus Substantia nigra compacta Substantia nigra reticulata

R L R L R L R L R L R L R L R L R L

Unstimulated no sham sham n=5 n=6

CM-Pf stimulated n=6

119 + 6 113_+5 81 -+ 3 77_+1 115 + 11 126_+ 10 74 -+ 2 72 -+2 123 -+ 10 124__.11 116 +_.4 112-+4 150 + 16 150 _+ 8 98 + 7 103_+6 86 _+ 3 91 -+ 4

224 __+34¢ 159+15 c 150 +__17c 127±11 ¢ 228 _+ 28 ¢ 224+ 18c 165 _ 6 c 122 _+ 8c 304 -+ 28 ¢ 282_+_19c 223 __+23 ¢ 162-+15 ¢ 343 __+56 "c 209 + 5¢ 307 + 44 a¢ 187_+ l P 187 -+_17ac 147 +__12¢

101 -I- l 0 b 1 0 5 + 7 n.s. 68 ___7 n.s. 69_+6 n.s. 115 _+ 13n.s. 131 _+ 22n.s. 77 __+7 n.s. 77 + 7 n.s. 118 + 13n.s. 133 + 12n.s. 111 + 8 n.s. 114_+10n.s. 127 _ 10n.s. 128 + 12n.s. 98 + 9 n.s. 98_+8 n.s. 87 + 7 n.s. 94 _+ 6 n.s.

LCGU (/~mol/100 g per min) Unstimulated no sham sham n=5 n =5 53 + 3 53+3 40 __+2 41_+2 51 _+ 4 51 ___4 39 __+1 38 _+ 1 72 _+ 2 72-+2 52 + 1 51___2 64 _+ 2 65 _ 2 50 + 4 49_+4 44 _ 3 43 +_ 2

48 _ 2n.s. 51 _+ 3n.s. 36 -+ 2n.s. 37 _ 2n.s. 52 +__3n.s. 53 _+ 3n.s. 36 _+ ln.s. 36 _+ ln.s. 57 + 2 b 58__+3b 49 __+2n.s. 5 0 _ 2n.s. 57 _ 3b 58 -+ 1b 44 + 3n.s. 44 -+ 3n.s. 39 + 2n.s. 41 _+ 3n.s.

CM-Pf stimulated n=6 64 _ 6c 60 -+ 5n.s. 53 __+6¢ 47+4 c 65 __+6n.s. 63 + 7n.s. 43 __+6n.s. 39 + 5n.s. 79 _+ 7° 81+7 ¢ 64 + 7n.s. 57 -+ 4n.s. 80 _ 10n.s. 71 _+ 7n.s. 75 + 8"c 62_+5 c 56 -+ 6¢ 55 _+ 6n.s.

R, right; L, left; values are means + S.E.M. ap < 0.05 (paired t-test), significance refers to side-to-side difference; bp < 0.05 (Mann-Whitney test), significance refers to difference from unstimulated no sham group; n.s., not significant; cP < 0.05 (Mann-Whitney test), significance refers to difference from unstimulated sham group.

CM-Pf stimulation and LCBF/LCGU regulation in specific neural systems

455

Table 3. Local cerebral blood flow and glucose utilization within the limbic system in anesthetized, paralysed rats, with or without stimulation of centromedian-parafascicular complex LCBF (ml/100 g per min) Unstimulated no sham sham n = 5 n = 6

CMPfstimulated n = 6

R L R L R L R L R L

123 ___ 11 124+ 11 123 ___9 119 + 8 108 _+ 6 111 + 6 125 ___ 16 107+ 11 132+ 12 120 + 9

113 ___14an.s. 122+ 14n.s. 121 + 13an.s. 125 + 14n.s. 87 _ 8an.s. 9 8 + 10n.s. 61 + 20 ab 9 3 + 10n.s. 7 7 + 11ab 96 ___9n.s.

335 + 64~ 244 + 21 c 355 __+49 ~ 247 + 25 c 201 + 33 c 184_+20 ¢ 103 __+11ac 142+ 15~ 114+ 11~ 141 -+ 8~

59 __+4 59+4 69 + 3 70 _ 3 57 ___4 57+4 47 __+3 45_+3 62+ 1 61 ___ 1

58 + 3n.s. 6 0 _ 3n.s. 56 ___3n.s. 58 + 3b 60 + 3n.s. 58__+ 3n.s. 42 + 3n.s. 4 6 _ 3n.s. 53 4 n . s . 57 -+ 3n.s.

70 __+4n.s. 69 +4n.s. 70 _+ 7~n.s. 67 __+6n.s. 71 ___5n.s. 70_+ 5n.s. 52 ___6n.s. 54 + 6n.s. 52__+6n.s. 59 _+ 8n.s.

R L R L R L

90 __+10 86 _ 7 103 + 10 99_+6 91 _+ 5 92+7

50 __+6~b 68 + 7n.s. 66 _ 7 ab 9 6 + 1ln.s. 63 _+ 6ab 87 + 9n.s.

71 ___8~ 87 ___4n.s. 86 _+ 9~n.s. 108 __+5n.s. 84 + 8an.s. 111 -+ 6n.s.

46 -+ 2 45 ___ 1 54 + 2 52_+2 52 __+2 51__+2

49 +__2n.s. 47 -+ 2 61 + 2an.s. 52 + 2n.s. 68 + 4"b 55 + 3n.s.

43 -+ 5n.s. 42 __+4n.s. 57 _+ 5~n.s. 50 + 5n.s. 60 ___5"n.s. 55 __+5n.s.

R L R L R L R L R L

143 + 18 126___12 143 ___ 16 125+8 126 __+16 116 __+12 152 + 25 150 ___25 99 ___5 95 ___4 122 __+10 115 -+ 11 126 _+ 10 166 __+9 168+11 139 + 6 133__+6 241 __+10 144 __+14 97 + 4 93+3 123 + 10 124 ___ 11 116 ___ 11 112+ 11

73 + 7"b 115 + 16n.s. 80 __+8ab 111 ± lln.s. 79 + 6b 96 + 10n.s. 103 __+10an.s. 132 + 17n.s. 87 ___9~n.s. 92 + 10n.s. 113 + 14n.s. 115 + 13n.s. 131 ___22n.s. 148 + 15n.s. 164 + 22n.s. 136 + 39n.s. 128 + lln.s. 196 + 17n.s. 186 ___29n.s. 92 + 10n.s. 98 + lln.s. 118 ___13n.s. 133 + 12n.s. 73 + 5ab 8 7 _ 9n.s.

158 __+24 ¢ 46 + 3 178 + 22 c 45+2 112 + !2an.s. 52 + 3 146 ___ 10c 51+4 149 + 12c 41 ___2 138 + 14c 40 ___2 205 __+27 c 53 + 4 205 _+ 29 c 51 ___3 165 _+ 5c 40 + 2 136 ___6~ 40 + 2 226 + 18c 47 __+3 228 + 28 ~ 51 + 4 224 ___ 18c 51 + 4 639 ___44 a~ 75 + 4 165+15 c 77+4 306 ___39 ¢ 59 + 1 276 ___21 ~ 59___1 415 + 61 ~ 100 +__2 300 + 29 ~ 55 __+3 240 __+31 a~ 42 + 1 165+15 ~ 42+1 304 +__28 ~ 72 + 2 282 +__19c 72 + 2 168 + 21 c 42 ___ 1 149_+9 c 42+ 1

50 __+3n.s. 49 + 3n.s. 57 + 4an.s. 50 + 2n.s. 39 + 2n.s. 38 + ln.s. 51 __+2n.s. 50 _+ 2n.s. 41 + ln.s. 40 + ln.s. 47 + ln.s. 52 + 3n.s. 53 ___3n.s. 68 ___3n.s. 70 + 3n.s. 55 ___ 1b 54+1 b 90 + 5n.s. 56 + 4n.s. 38 + 1b 37+1 b 57 __+2b 58 __+3b 40 _+ 3n.s. 40 + 2n.s.

61 + 6n.s. 58 _ 7n.s. 69 ___5n.s. 62 __+6n.s. 52 ___5c 54 -+ 5~ 65 + 6n.s. 63 +__6n.s. 45 + 3n.s. 45 ___3n.s. 55 +__2° 65 + 6n.s. 63 + 7n.s. 115 ___7~ 84__5 c 67 + 5n.s. 61 _ 5n.s. 116 +__8c 82 + 6~ 51 + 5"n.s. 45 +__5n.s. 79 d- 7ac 81 __+7~ 50 __+5an.s. 45 __+4n.s.

Region Cingular cortex (area 24) Cingular cortex (area 29) Cingular cortex (area 32) Entorhinal cortex (area 28) Piriform cortex (area 51) Hippocampus anterior dorsal Field CA1 Field CA3 Field CA4 Posterior ventral Field CA1 Field CA3: Dentate gyrus Subiculum Lateral septal nucleus Medial septal nucleus Accumbens nucleus Lateral habenular nucleus Medial habenular nucleus Interpeduncular nucleus Mammillary complex Lateral hypothalamus Anteroventral thalamic nucleus Central amygdaloid nucleus

L C G U (,umol/100 g per min)

R L R L R L

R L R L R L

Unstimulated no sham sham n = 5 n = 5

CM-Pfstimulated n = 6

For designations see Table 2.

t h e r e a f t e r slowly declined to 254 + 9 m g / 1 0 0 ml, r e m a i n i n g stable t h r o u g h o u t the s t i m u l a t i n g p e r i o d . T h e s e values were well w i t h i n the limits o f the o p e r a t i o n a l e q u a t i o n f o r calculating L C G U . 36

Local cerebral blood flow and glucose utilization in unstimulated rats I n n o - s h a m r a t s (n = 5), L C B F (see T a b l e s 2, 3, 4 a n d 5) r a n g e d f r o m 54 ___ 5 m l / 1 0 0 g p e r m i n in the c o r p u s c a l l o s u m ( T a b l e 5) to 241 + 10 ml/100 g p e r m i n in the i n t e r p e d u n c u l a r n u c l e u s (Table 3). I n s h a m - o p e r a t e d rats (n = 6), L C B F , m e a s u r e d 90 m i n after p o s i t i o n i n g the electrode, w a s r e d u c e d in several b r a i n areas, p a r t i c u l a r l y in the limbic system. T h e r e were significant bilateral decreases

in L C B F ( P < 0 . 0 5 ) in the e n t o r h i n a l a n d pirif o r m cortices, h i p p o c a m p a l c o m p l e x a n d a m y g d a l o i d nucleus. T h e r e were also significant differences ( P < 0.05, p a i r e d t-test) b e t w e e n right a n d left sides in the cingular, the e n t o r h i n a l a n d the p i r i f o r m cortices, the h i p p o c a m p a l c o m p l e x , the lateral septal n u c l e u s a n d the central a m y g d a l o i d n u c l e u s ( T a b l e 3). L C B F did n o t differ b e t w e e n sides ( P > 0.05, p a i r e d t-test) w i t h i n the R F a n d the E M S (except in the c a u d a t e nucleus, T a b l e 2). T h e L C G U m e a s u r e d in a n o - s h a m g r o u p o f r a t s (n = 5) r a n g e d f r o m 25 + 1 p m o l / 1 0 0 g p e r m i n in the c o r p u s c a l l o s u m to 100 + 2 / ~ m o l / 1 0 0 g p e r m i n in the i n t e r p e d u n c u l a r nucleus. L C G U r a n g e d from 27_2#mol/100g p e r m i n in the c o r p u s

S. MRAOVITCH et al.

456

Table 4. Local cerebral blood flow and glucose utilization within the reticular formation in anesthetized, paralysed rats, with or without stimulation of centromedian-parafascicular complex LCBF (ml/100 g per min)

Region Zona incerta Reticular thalamic nucleus Ventral tegmental area Anterior pretectal area Mesencephalic reticular formation Reticular tegmental nucleus Ventral tegmental nucleus Dorsal tegrnental nucleus Median raphe Dorsal raphe Raphe pontis Raphe magnus Pontine reticular nucleus oral Pontine reticular nucleus caudal Dorsal gigantocellular reticular nucleus Ventral gigantocellular reticular nucleus Nucleus reticular ventral

R L R L R L R L R L R L R L R L

R L R L R L R L R L

Unstimulated no sham sham n =5 n-=6

CM-Pfstimulated n =6

140+10 140__+11 103+5 I00 + 4 113+6 115___9 156-t-14 155_+15 117_+10 118_+13 141_+13 137_+12 144+9 142 4- 8 164 4- 17 161+17 151 -+ 13 111+6 93 + 1 98 4- 2 110+6 108_+6 93 -+ 3 94 _+ 3 104 _ 3 102_+3 117 _+ 4 116 -+ 4 111_+5 110-t-5

6 5 0 + 7 2 ac 201+16 ¢ 165+9 c 147 + 9¢ 3594-56 ac 226+25 ¢ 4454-123 c 2244-21 ~ 5 6 4 + 1 3 4 ac 202 + 34c 416___ 114ac 287+51 c 477+__57~ 302 4- 25 c 329 _ 42 ac 274+31 ¢ 437 4- 78c 370+67 c 269 4- 52c 254 + 37~ 562+111 a¢ 240+32 c 319 + 64 ~¢ 210 + 30~ .210 + 29 ¢ 183_ 22 c 308 _+ 62¢ 214 _+ 33¢ 2304-42 c 202 + 28 c

130 + 8n.s. 128 _+ 10n.s. 97 __+5n.s. 105 ___5n.s. 130 _ 7n.s. 134 + 9n.s. 132± lln.s. 131 _+ lln.s. 110 + 10n.s. 113 + 10n.s. 132 -+ 9n.s. 133 + 8n.s. 130 -+ lln.s. 127 _ 9n.s. 138 + 15n.s. 137 + 14n.s. 137 _ 14n.s. 123 -+ 10n.s. 107 4- 9n.s. 100 + 7n.s. 1 1 2 + 9 n.s. 112 -+ 10n.s. 124 -+ 6n.s. 120 ___7n.s. 106 + 6n.s. 105 -+ 6n.s. 118 -+ 8n.s. 121 -+ 10n.s. 115_+5n.s. 114-+ 6n.s.

L C G U ~ m o l / 1 0 0 g per min) Unstimulated no sham sham n =5 n=5 53+3 52+3 51+2 50 + 2 43+3 43+2 54+2 54_+2 51_+3 51+3 52_+2 52_+I 59+2 59 4- 2 64 ___2 66+1 63 -+ 1 61+1 43 + 1 41 + 1 51-+2 49_+2 43 _+ 1 43 4- 1 46 ___2 46_+1 46 -+ 1 46 -+ 1 48_+I 48_+1

51 _ ln.s. 50 + ln.s. 47 -+_2n.s. 46 ___2n.s. 4 4 _ 2n.s. 43 + 2n.s. 48 _+_ln.s. 48 _+ 2n.s. 44 + 2n.s. 43 -+ 2n.s. 50 _ 3n.s. 52-+ 2n.s. 53 + 2n.s. 53 + 2n.s. 61 + 3n.s. 60 + 2n.s. 59 ___3n.s. 54+2 b 42 -+ 2n.s. 42 -+ 2n.s. 424-1 b 43_1 b 42 + 2n.s. 42 + 2n.s. 41 + 2n.s. 40+2 b 42 _ ln.s. 42 _+ 1b 434-1 b 42+1 b

CM-Pfstimulated n =6 1 1 4 _ 1 4 a¢ 64 + 6n.s. 48 -+_ln.s. 41 + ln.s. 59__+5¢ 55+4 ¢ 1 0 6 + 6 "~ 64+4 c 130+11 ac 61+4 c 7 3 + 3 ac 59 -+ 6n.s. 80_+7 ac 68 + 4~ 72 -+ 4an.s. 67 -+ 4n.s. 74 4- 8n.s. 6 5 _ 5n.s. 61 -+ 6~ 54 + 5n.s. 9 8 + 9 ac 61+4 c 68 _+ 6~c 60 + 6¢ 56 + 6an.s. 54-+ 5n.s. 66 _+ 7ac 54 _+ 6n.s. 564-5 c 53±4 ~

Designations as in Table 2.

c a l l o s u m to 90 _ 5 / ~ m o l / 1 0 0 g p e r m i n in the interp e d u n c u l a r n u c l e u s in s h a m - o p e r a t e d rats (n = 5). T h e r e were significant ( P < 0.05) decreases in L C G U in the a n t e r o v e n t r a l t h a ! a m i c n u c l e u s a n d the s u b t h a lamic n u c l e u s ( T a b l e 2), the c i n g u l a r cortex, the m e d i a l h a b e n u l a r n u c l e u s a n d the lateral h y p o t h a l a m u s (Table 3), the d o r s a l r a p h e a n d the p o n t i n e reticular nuclei (Table 4) o f these rats. L C G U w a s significantly increased in the C A 3 a n d C A 4 fields o f the h i p p o c a m p u s .

All b r a i n r e g i o n s d e m o n s t r a t i n g significant c h a n g e s in L C B F a n d / o r L C G U d u e to s p r e a d i n g d e p r e s s i o n were e x c l u d e d f r o m f u r t h e r analysis,

Effect of long term centromedian-parafascieular complex stimulation on local cerebral blood flow T a b l e 6 s h o w s that, after an initial rise ( u p to 86_+ 15%, m e a s u r e d 5 m i n f o l l o w i n g the o n s e t o f s t i m u l a t i o n ) , L C B F r e m a i n e d stable a n d significantly elevated ( P < 0 . 0 5 ) d u r i n g 5 0 m i n o f c o n t i n u o u s

Table 5. Local cerebral blood flow and glucose utilization within the cortex and the corpus callosurn in anesthetized, paralysed rats, with or without stimulation of centromedian-parafascicular complex LCBF (ml/100 g per min) Unstimulated no sham sham n =5 n=6

Region Frontal cortex (area 10) Parietal cortex (area 2; 40) Occipital cortex (area 17; 18) Corpus callosum Designations as in Table 2.

R L R L R L R L

119 + I1 117_+11 135 ___ 14 130 4- 16 112 + 6 102 ___2 54 + 5 54+4

115 _+ 17n.s. 115 _ 17n.s. 136 + 20n.s. 130 _+ 15n.s. 86 4- 12n.s. 92_+ 8n.s. 43 4- 7n.s. 43 _ 7n.s.

CM-Pfstimulated n =6 280 + 24 b 187_ 31 b 425 + 48 ab 216 -+_24 b 168 _ 24 b 135+9 b 85 4- 6b 74 ± 4 b

L C G U (#mol/100 g per min) Unstimulated no sham sham n =5 n =5 53+3 53-t-3 56 -+_4 56 +_ 3 57 -t- 4 57+4 25 4- 1 25 _+ 1

50 _+ 2n.s. 5 4 _ 3n,s. 54 _+ 2n.s. 58 _+ 3n.s. 53 ± 3n.s. 56 _+ 4n.s. 27 _+ 2n.s. 28 4- ln.s.

CM-Pfstimulated n =6 604-3 b 57 + 4n.s. 61 + 4n.s. 59 _+ 3n.s. 58 4- 5n.s. 60 ± 6n.s. 29 ___2n.s. 28 _ 2n.s.

CM-Pf stimulation and LCBF/LCGU regulation in specific neural systems

457

stimulation. LCBF returned to its pre-stimulus level when stimulation was stopped.

Effect of centromedian-parafascicular complex stimulation on local cerebral blood flow and glucose utilization

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Electrical stimulation of the C M - P f resulted in an overall but differentiated increase in LCBF and LCGU (Figs 1, 2, 3). LCBF in the extrapyramidal motor system (n = 6) increased significantly (P < 0.05) and bilaterally in all structures, including the substantia nigra pars compacta (212___44%), the substantia nigra pars reticulata ( 1 1 5 _ 2 0 % ) , and the globus pallidus (118 + 2 4 % ) , as shown in Fig. 3. LCBF increased in the LS, particularly in the lateral habenular nucleus (331 + 30%) and the cingular cortex area 24 (194+56%). The most pronounced increase in LCBF occurred in the brain regions comprising the mesencephalic R F (415-+ 122%), the zona incerta including Forel's field (400 ± 55%) and the ventral tegmental nucleus (267 _+ 43%). In addition to its effect on subcortical regions, electrical stimulation of the C M - P f significantly increased (P < 0.05) cortical LCBF (Fig. 4, Table 5). The most pronounced increase was seen in the parietal cortex on the stimulated side (211 _ 35%). In contrast to the marked increase in LCBF, electrical stimulation of the C M - P f had a rather small effect on subcortical LCGU (Fig. 3). LCGU increased significantly (P < 0.05) in two of the six regions comprising the EMS: the globus pallidus (45 ___15%) and the substantia nigra pars compacta (72 -+ 19%). LCGU increased in only four of the nine limbic structures analysed. The greatest increases were seen in the lateral habenular nucleus (68 +__10%) and the mammillary body (45-+ 11%). In regions comprising the RF the most marked increases in LCGU during the C M - P f stimulation were in the mesencephalic R F (193-+ 26%), the zona incerta (123 + 2 8 % ) and the anterior pretectal area (121 _+ 13%). Stimulation of the C M - P f elicited a significant increase in LCGU in the frontal cortex, but not in the parietal and occipital cortices (Fig. 4, Table 5).

Relationship between local cerebral blood flow and glucose utilization in unstimulated sham-operated ra ts

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The relationship between LCBF and LCGU in unstimulated sham-operated rats was examined by linear regression analysis across the brain as a whole and for each system separately. There were significant positive correlations, similar to those reported previously in anesthetized rats, 6'19'25 as well as correlations computed for the three systems separately. The overall regression line (Fig. 4) was expressed by the equation LCBF = 2.0 L C G U + 20 (r = 0.75, P < 0.05). The regression equations for the three systems are indicated in Fig. 5. The correlation

458

S. MRAOVITCHet al.

coefficients for the three systems were: r = 0.99, P < 0.05 (EMS); r = 0.63, P < 0.05 (LS); r = 0.72, P < 0.05 (RF). The LCBF to LCGU ratio ranged from 1.45 ml/ /lmol in the cingular cortex (area 32) to 3.32 ml/#mol in the mammillary body. The mean LCBF to L C G U ratios (Table 7) for the three systems (EMS: 2.18 + 0.05 ml//lmol; LS: 2.22 _+ 0.11 ml/#mol; and RF: 2.56 _+ 0.06 ml//~mol) were significantly different (RF being higher than LS and EMS), indicating heterogeneity between the systems in unstimulated rats. Effect o f centromedian-parafascicular complex stimulation on the relationship between local cerebral blood flow and glucose utilization Electrical stimulation of the C M - P f preserved and improved the correlation of the overall relationship (r = 0.85, P < 0.05) between LCGU and LCBF (Fig. 5) and those for the three systems analysed separately (EMS: r = 0.89, P < 0.05; LS: r =0.82, P