- Email: [email protected]
Vasodilatation and enhanced oxidative metabolism of the cerebral cortex provoked by the periaqueductal gray matter in anaesthetized rats
Neuroscience Vol. 72, No. 4, pp. 1133-1140, 1996
~
Pergamon
S0306-4522(96)00013-9
Elsevier Science Ltd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00
VASODILATATION A N D E N H A N C E D OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX PROVOKED BY THE P E R I A Q U E D U C T A L GRAY MATTER IN ANAESTHETIZED RATS M. N A K A I * f and M. MAEDA:~ tNational Cardiovascular Center Research Institute, Suita, Osaka 565, Japan :~Department of Systems Physiology, University of Occupational and Environmental Health, Kita-Kyushu, Fukuoka 807, Japan Abstract--The aim of the present study was to evaluate the possible contribution of the cerebral cortical oxidative metabolism to the cortical vasodilator action of the periaqueductal gray. In 70 rats with cervical cordotomy, we found that unilateral stimulation of the caudal third of the lateral longitudinal column of the periaqueductal gray with N-methyl-D-aspartate bilaterally provoked the greatest increase in cortical blood flow (laser-Doppler flowmetry and/or microsphere flowmetry). The response was widespread over the entire neocortical regions, elicited in a dose-dependent manner, with little change in arterial blood pressure. The flow was increased effectively by a submaximal dose of the amino acid (1 mM, 100 nl), attaining a peak increase by 99 + 41% of the baseline level (mean +__S.D., n = 30), and was associated with an enhancement of the cortical metabolic rate for oxygen by 51 + 26%. We then compared the flow increase with that induced by cold exposure (by 52_ 29%, n = 27), the latter response being tightly coupled to an enhanced metabolic rate for oxygen (by 41 _ 23%). It was thus found that the increase in cortical blood flow provoked by the subdivision was dependent on the cerebrovasodilator mechanism that may be coupled to the cortical oxidative metabolism to the extent of one-half, and on certain other mechanisms for the remaining half. In view of the fact that this particular region serves to generate vigorous defence reactions that involve flight behaviour, the region should also help to meet the urgent demand for an increased cortical blood flow, so as to prepare for the possible generation of cortical hyperactivity in coping vigorously with a threatening emergency. Key words: central gray matter, cerebral blood flow, cerebral metabolic rate, cold exposure, defence
reaction, thermal stimulus.
A distinctive neuron pool within the midbrain periaqueductal gray matter (PAG) has recently been identified as a reflex centre located at the top of the central processing and organization of the natural behavioural and haemodynamic defence reactions to noxious-sensory and threatening stimuli) ,2,6,12 We recently reported for the first time that, when chemically stimulated, the neural circuitry emanating from the intermediate and caudal thirds of the lateral longitudinal column extending along the rostrocaudal axis of the P A G 2 also serves to increase the cerebral blood flow, as revealed by microsphere flowmetry in rats. 23 It has long been considered that an enhancement of the cerebral oxidative metabolism provoked by brain activation is capable of achieving a powerful and opportune cerebrovasodilatation to ensure an adequate supply of blood and substrates for energy
*Towhom correspondence should be addressed. CoBF, cortical blood flow; CoMRO2,
Abbreviations:
cortical metabolic rate for oxygen; NMDA, N-methylD-aspartate; PAG, periaqueductal gray matter.
generation to the brain region within which neurons are activated (see Ref. 11 for review). The magnitude of the cerebral blood flow could therefore be tightly coupled to the oxidative metabolic state for achieving adequate execution of brain functions. Indeed, during brain activation provoked by sudden exposure of rats to a thermal stimulus, we have demonstrated such tight coupling37 It is conceivable, therefore, that the P A G subdivision could have exerted its cerebrovasodilator action by enhancing the cortical oxidative metabolism, since the subdivision is one of the preferential target sites of nociceptive and thermoreceptive afferents. 3'16'17 We found in our subsequent study, 24 however, that the increase in cerebral cortical blood flow (CoBF) was accompanied by a minor change in oxidative metabolic state in the cortex, as estimated from the observation of the cortical metabolic rate for oxygen (CoMRO2). In this context, by devising an improved experimental approach in the present study, we attempted to reassess whether or not the cerebrovasodilator action of the P A G subdivision is really minimally
1133
1134
M. Nakai and M. Maeda
a t t r i b u t a b l e to the m e c h a n i s m t h a t is c o u p l e d to the c e r e b r a l o x i d a t i v e m e t a b o l i s m . A s a result, a clear-cut m o d e r a t e e n h a n c e m e n t o f the oxidative m e t a b o l i s m , a l t h o u g h still d i s p r o p o r t i o n a t e l y s m a l l e r t h a n t h a t o f the flow, was actually d e t e c t e d . W e t h e r e f o r e subj e c t e d a f u r t h e r s e p a r a t e g r o u p o f a n i m a l s to s u d d e n e x p o s u r e to a t h e r m a l stimulus so as to o b t a i n d a t a for t h e w e l l - e s t a b l i s h e d r e l a t i o n s h i p involving tight c o u p l i n g b e t w e e n b l o o d flow a n d o x i d a t i v e m e t a b olism. 27 A c o m p a r i s o n o f t h e results o b t a i n e d was t h e n u n d e r t a k e n to assess the e x t e n t to w h i c h the c e r e b r o v a s o d i l a t o r a c t i o n o f t h e P A G is a t t r i b u t a b l e to t h e oxidative m e t a b o l i c m e c h a n i s m .
EXPERIMENTAL PROCEDURES
General surgical procedures Experiments were conducted on 70 male Wistar rats (Oriental Bioservice, Kyoto, Japan) weighing from 240 to 350 g. The animals were divided into three groups. These groups were subjected to either a basic characterization study (the extra-PAG stimulation group, 18 rats) with chemical stimulation of the P A G with N-methyl-D-aspartate (NMDA), a flow-metabolism study with chemical stimulation of the P A G with N M D A (the P A G stimulation group, 27 rats) or a flow metabolism study with sudden exposure to cold (the cold exposure group, 25 rats). At one to three days before the experiment, the animals of each group were anaesthetized with a mixture of 2 2.5% halothane in oxygen gas blown over the nose. We cut notches along the bilateral surfaces of the rostrolateral quarter of the parietal bones with a dental drill, outlining the cranial windows which were to be opened later on the day of the experiment. This preparatory procedure served to minimize untoward cerebrovascular effects of the surgical manipulations. The exposed skull surface was covered with a sheet of bacteriocidal wound dressing (Sofratulle, Laboratories Roussel, Uxbridge, U.K.) and the skin wounds were sutured. The animals were then kept in individual cages. On the day of the experiment, the animals were anaesthetized initially with 2-2.5% halothane only during surgery, and subsequently with urethane (1.25 g/kg body weight, i.p.) during the experiments. Supplemental urethane was given at intervals of 2 h at a dose of 250 mg/kg. The adequate depth of halothane anaesthesia was maintained, as confirmed by inspection of the immobility of the animal during an earlier part of surgery. Once anaesthesia with urethane had been established, no further surgical manipulations were performed. In the animals of the cold exposure group, the chest and belly were shorn with clippers. Subsequent experimental procedures and techniques were as detailed by us in preceding reports, and will be described only briefly here. Each o f our previous reports will be cited below as appropriate. In the animals of the PAG stimulation and cold exposure groups, a left cardiac ventricular catheter for microsphere injection was threaded into place via the right brachial artery. The femoral arteries and veins were catheterized bilaterally. In the extra-PAG stimulation group, only a unilateral femoral artery and the bilateral femoral veins were catheterized. The trachea was then intubated. The animals were paralysed (D-tubocurarine, 0.3mg/kg, i.v., given at hourly intervals), artificially ventilated and maintained in a homoeothermic state at 37°C. The head was held in a stereotaxic frame with the incisor bar lowered by 17 mm from the horizontal plane passing through the interaural line. Following laminectomy, cervical cordotomy was undertaken at the C4 or C5 level in order to provide
generalized sympathectomy. For achieving cordotomy, we preferred that level to the higher cervical level, otherwise the procedure would have easily caused injury or oedema of the lower medulla. The normal level of arterial blood pressure was then maintained by continuous i.v. infusion of phenylephrine (Sigma, St Louis, MO, U.S.A.). The cranial windows were opened in the bilateral parietal bones by peeling off the pieces of bone whose outline had been notched previously. The dural surfaces so exposed were then covered with silicone oil (Medical Fluid, Dow-Corning, Asia, Tokyo, Japan). In the animals of the extra-PAG stimulation and PAG stimulation groups, the dorsal surfaces of the medulla and cerebellum were visualized by occipital craniectomy and covered with warm saline. Basic characterization of periaqueductal gray stimulation with N-methyl-D-aspartate in the extra-periaqueductal gray stimulation group The tips of a laser-Doppler flowmeter (Biomedical Science, Kanazawa, Japan) were fitted bilaterally over each of the dural surfaces. Moment-to-moment monitoring of the CoBF was commenced. The halothane anaesthesia was then replaced with urethane anaesthesia. After a subsequent resting period of 30 min, the tip of a multi-barrelled glass micropipette (Clark Electromedical Instruments, Reading, U.K.), 28 mounted on a micro-manipulator and inclined caudally at an angle of 30 ° from the vertical, was lowered into the PAG. The cerebrovasodilator sites within the PAG were explored in a series of 0.5-mm steps by microinjecting N M D A solution into the relevant brain region. N M D A had previously been dissolved (30-100 pmol as 30-100 nl of a I mM solution) in a vehicle (Dulbecco's phosphate-buffered saline, pH 7.3-7.65; Nissui, Tokyo, Japan), and delivered over 5-16 s through one lumen of the pipette using a 10-/~1 Hamilton syringe installed on a Narishige infusion pump. The tip of the micropipette was then fixed in place unilaterally within the PAG at the most potent cerebrovasodilator site so identified. The lateral (side-to-side) difference in CoBF response to unilateral PAG stimulation was evaluated. Furthermore, the relationship between N M D A concentration and the resultant CoBF response was investigated, by injecting vehicle or an N M D A solution (100nl at 0.316, 1 or 3.16mM, over 16s) into the PAG from the individual lumina of a quadruple-barrelled pipette, with a separation of 10 20 min between each injection. We also undertook functional mapping of the cerebrovasodilator sites and tests for reproducibility of the flow response. For histological confirmation, the location of the pipette tip was marked with 2% India ink solution (100 nl), delivered via another lumen of the multi-barrelled pipette into the tissue. Microsphere flowmetry was also employed to evaluate the side-to-side difference in responsiveness of the CoBF and to assess the regional difference in responsiveness of the blood flow within the neocortex. Details of these procedures are given below. Preparation for measurement of cortical blood flow and cortical metabolic rate for oxygen in the periaqueductal gray stimulation group and cold exposure group For later sampling of blood from the dorsal sagittal sinus to estimate the CoMRO2, 27 a burr hole (3 mm in diameter) was made acutely so that the dorsal surface of the stem of the sinus was exposed at its junction with the confluence of the sinuses. A device (NTM-1R, Inter Medical, Nagoya, Japan), designed by us for the sampling of sinus blood, was fitted over the burr hole and secured to the skull with dental cement. The exposed dorsal wall of the sinus was later incised, shortly before the start of the experimental runs. This procedure allowed the sinus blood (150/~1) to drain (150~tl/min) through a Minipulse-3 tube pump (Gilson, Villiers-le-Bel, France) out through the device into a glass capillary.
Cerebrovasodilator action of the central gray The halothane anaesthesia was then switched to urethane anaesthesia. During the subsequent resting period of 30 min, we injected heparin (600 U/kg, i.v.) and set up a crosscirculation circuit with a chronically catheterized donor rat, in preparation for microsphere flowmetry. 25'3° A laserDoppler flowmeter was additionally applied to the animal as described earlier, and moment-to-moment monitoring of the CoBF with the flowmeter was commenced.
The flow-metabolism study undertaken in the periaqueductal gray stimulation group The cerebrovasodilator sites within the P A G were explored by microinjecting N M D A through one lumen of a double-barrelled micropipette as described above. The tip of the pipette was fixed in place unilaterally at the most potent site. We then carried out the first experimental run for measurement of the baseline CoBF by microsphere flowmetry and simultaneous estimation of the baseline C o M R O 2. By running the cross-circulation with a Minipulse-3 tube pump at a rate of 1.2 ml/min, the microspheres (16.5 __+0.1/~m in diameter, radiolabelled with 57Co, ll3Sn, SSSr or 46Sc, NEN, Boston, MA, U.S.A.) were injected into the left cardiac ventricle through the catheter, and reference arterial blood was simultaneously sampled (1 ml over 50 s) through the femoral arterial catheter. Sinus blood (150/~1) was also sampled in parallel with the cross-circulation. An aliquot of the sampled arterial blood (150 #1) and the entire volume o f the sampled sinus blood were immediately subjected to measure their gas tensions (ABL-3, Radiometer, Copenhagen, Denmark) and oxygen content (OSM-3, Radiometer), for later calculation o f the CoMRO:. At 15-20min after the first experimental run, N M D A (1 mM, 100nl) was injected unilaterally into the potent cerebrovasodilator site within the P A G over a period of 16 s. When the peak increase in CoBF had been attained as observed by laser-Doppler flowmetry, the second experimental run was undertaken. In several rats, we repeated the same experimental protocol in order to carry out an additional pair of experimental runs. At the end of the experiment, the location of the pipette tip was marked with India ink and the site later examined histologically, as described above. The animals were killed by inducing haemorrhage. We then dissected out the
1135
parasagittal part of the bilateral cerebral cortex. The tissue specimens and reference arterial blood were weighed and processed for measuring their radioactivity to calculate the CoBF. 26 The C o M R O 2 was calculated by incorporating the CoBF and the total oxygen contents of the sampled arterial blood and sinus blood. 27 Special care was taken to ensure that the cortical area subjected to the CoBF measurement matched that subjected to C o M R O 2 measurement. 28 In nine rats of the P A G stimulation group, we subsequently evaluated the regional differences in responsiveness of the blood flow within the neocortex. Parcellation of the bilateral neocortex was carried out to obtain samples of the medial third and lateral two-thirds of the parasagittal cortex. The latter portion was further divided into frontal, intermediate and occipital thirds. Furthermore, the bilateral extra-parasagittal portion of the neocortex above the rhinal fissure was also sampled.
The flow-metabolism study undertaken in the cold exposure group The animal was laid in a prone position on the top panel of a warm/cool box, and its head was fixed on a stereotaxic frame. The interior of the box was circulated with warm water (37°C), which could be abruptly switched to cold water (0°C). The incoming water was supplied by a warm-water circulator (SP-12, Taitek, Saitama, Japan) or a cool-water circulator (CP-150F, Taitek), respectively. The box was designed so that its lumen was narrow (50 ml) and its top stainless panel was thin (0.2 mm). Sudden lowering of the temperature of the circulating water could thus be swiftly transmitted (within several seconds) to the area of shorn thoracic and belly skin. We followed the same experimental protocol as in the PAG stimulation group, except for the induction of cerebrovasodilatation by sudden exposure to cold. Data analysis The results are expressed as the means _+ S.D. Statistical analysis was performed by the paired or unpaired t-test, as appropriate. A two-way analysis of variance or a test for the overall difference over time, followed by the Bonferroni method for pairwise c o m p a r i s o n s y was also performed.
Microsphere injection & sampling of blood 20Or- ~ - 7
V
~
200f
if!
<
0
1.991~
i
Or_ Cold exposure
1 NMDA Injection 2 rain
Fig. 1. Typical recordings of the responses of the mean arterial pressure (ABP) and cortical blood flow (CoBF) in two rats with cervical cordotomy. The moment-to-moment observation of blood flow was monitored with a laser-Doppler flowmeter and is expressed in terms of its electrical output (V). The flow response was elicited by sudden exposure to cold water (0°C) or by unilateral chemical stimulation of the caudal third of the lateral longitudinal column of the PAG with microinjection of N M D A (1 mM, 100 nl, delivered over 16 s). Microsphere flowmetry and simultaneous sampling of arterial blood and dorsal sagittal sinus blood for estimation of the oxidative metabolic rate in the cortex were successfully achieved at the peak of the flow increase.
1136
M. Nakai and M. Maeda
Table 1. Responses of the neocortical regional blood flow and oxidative metabolism to cold exposure and to chemical stimulation of the caudal third of the lateral longitudinal column of the periaqueductal gray matter Cold exposure
PAG stimulation
6 8
9 10
49 _+ 19 61 __+34
114 _ 73 134 + 89
15 + 38 + 71 + 66 +
76 + 141 + 131 + 96 +
Number of rats Number of observations Neocortical blood flow (% increase) Entire bulk of the parasagittal cortex Medial third Lateral two-thirds Frontal third Intermediate third Occipital third Extra-parasagittal neocortex Parasagittal neocortical metabolic rate for oxygen (% increase)
29"s 38 57 64
38 + 21
52 99 101 59
53 + 53
The data (means __+S.D.) were obtained by sudden exposure to cold water (0°C) or by microinjecting NMDA (1 mM, 100 nl, delivered over 16 s) into the PAG subdivision (microsphere flowmetry), n~Not significantly changed from the baseline value (P > 0.05, paired t-test). All of the other changes were significant (P < 0.05~).001).
RESULTS
Basic characterization o f periaqueductal gray stimulation with N-methyl-D-aspartate to provoke a cortical blood flow increase Figure 1 shows typical records of momentto-moment changes in C o B F monitored by l a s e ~ Doppler flowmetry. It was found that unilateral P A G stimulation with 1 m M N M D A (100 nl, delivered over 16 s) provoked a bilateral increase in CoBF, without any significant changes in arterial blood pressure in rats with cervical cordotomy. The contralateral cortex was slightly less responsive, as revealed by laserDoppler flowmetry ( 9 2 + 18% of the ipsilateral increase, n = 17, P <0.05, paired t-test, 13 rats) and by microsphere flowmetry (94 + 26%, n = 33, P > 0.05, 26 rats). The cerebrovasodilator action of the P A G was widespread over the neocortical regions (Table 1; microsphere flowmetry). The cerebrovasodilator response to chemical stimulation of the P A G with 100 nl N M D A was concentration dependent, as revealed by l a s e ~ Doppler flowmetry. Employing five rats, we found that unilateral microinjection of N M D A , carried out in order of ascending concentration, changed the ipsilateral C o B F by - 2 + 3% at 0 m M (vehicle), by + 5 9 _ + 3 3 % at 0 . 3 1 6 m M , by + 9 4 _ + 2 9 % at l m M and by + 106 + 55% at 3.16 mM. These flow changes induced by N M D A were significantly different from that observed following vehicle injection (n = 5, P < 0.001 at every concentration, test for the overall difference over time, followed by the Bonferroni method for pairwise comparisons).
The reproducibility of the flow response was excellent, as investigated with 1 m M N M D A (100 nl) and employing laser-Doppler flowmetry. The second to sixth experimental runs, 10-15 min apart from each other, resulted in C o B F increases of 99___ 27, 105 +_23, 102 + 25, 97 + 26 and 92 + 28%, respectively, of the initial C o B F increase (n = 6, three rats). No significant difference was detected among any of the responses (tests for the overall difference over time). Figure 2 shows a functional map drawn for cerebrovasodilator sites within the P A G , as studied with 1 m M N M D A (30 nl, delivered over 5 s) and employing l a s e ~ D o p p l e r flowmetry. We found that cerebrovasodilatation was provoked from the intermediate and caudal thirds of the lateral longitudinal column of the PAG. A m o n g these subdivisions, however, a restricted part within the caudal third was the only region from which the greatest increase in C o B F was elicited. A reduction of the C o B F could not be elicited from any region explored within the lateral column of the PAG.
Comparison o f the cerebrovasodilator effect o f periaqueductal gray stimulation with that o f cold exposure, based on the overall data obtained by microsphere flowmetry Details of the baseline state of systemic circulatory parameters observed in the P A G stimulation group and in the cold exposure group are presented in
Rostrocaudal coordinate (mm) 10
~
+2
9
n2'
o
o,4, o
o/
o
-3
Fig. 2. Functional map of the midbrain sectioned in the sagittal plane (0.7 mm lateral to the midline) in one rat with cervical cordotomy. The dorsoventral and rostrocaudal coordinates are referred to the calamus scriptorius. Cerebrovasodilator sites were explored along five dorsoventral tracks by microinjecting 1 mM NMDA (30 nl). The magnitude of the response provoked by each site is represented by a large (6(~84% increase), medium (37 55% increase) or small solid circle (14~28% increase). Open circles represent quiescent sites. IC, inferior colliculus; PAG, periaqueductal gray; PAGD, dorsal periaqueductal gray; SC, superior colliculus.
Cerebrovasodilator action of the central gray Table 2. Systemic circulatory parameters in the cold exposure and periaqueductal gray stimulation groups
Number of rats Number of observations Mean arterial blood pressure (mmHg) Baseline Challenge Arterial blood pH Baseline Challenge Arterial blood PCO 2 (mmHg) Baseline Challenge Arterial blood P O 2 (mmHg) Baseline Challenge
Cold exposure group
PAG stimulation group
25 27
27 30
86 _+ 8 86 + 9
83 _ 7 82 +__7
7.392 __+0.053 7.390 __+0.055
7.395 _ 0.041 7.391 _ 0.044
37.4 __+2.3 37.3 __+2.7
36.6 _ 1.6 36.4 __+1.6
135 _+ 17 141 ___20
142 -4- 16 145 + 15
1137
Comparison of the cerebrovasodilator effect of periaqueductal gray stimulation with that of cold exposure, on the basis of an identical cortical oxidative metabolic state The averaged m a g n i t u d e o f e n h a n c e m e n t o f C o M R O 2 d u r i n g P A G stimulation was considerably different f r o m t h a t observed d u r i n g cold exposure, as listed in Table 3 (51% vs 41%). W e therefore t r u n c a t e d the overall plots of the pooled d a t a for each g r o u p so t h a t a quantitative c o m p a r i s o n o f the c e r e b r o v a s o d i l a t o r effect could be m a d e between the groups, only across identical ranges (from 5 % up to 6 9 % ) o f e n h a n c e m e n t of cortical oxidative m e t a b olism (Table 3; 37% vs 3 6 % o n average). Consequently, we f o u n d t h a t the c e r e b r o v a s o d i l a t o r action o f the P A G (flow increase 8 8 % ) was twice as strong as t h a t of cold exposure (45%).
The data are expressed as m e a n s _ S.D. Each of the measured variables did not differ greatly between the groups as well as between the experimental conditions (P > 0.05, two-way analysis of variance).
T a b l e 2. W e f o u n d t h a t each o f the m e a s u r e d parameters did n o t differ greatly between the groups. Typical recordings o f responses o f the arterial pressure a n d C o B F to cold exposure a n d to chemical s t i m u l a t i o n o f the P A G were d e m o n s t r a t e d in Fig. 1. As is summarized in Table 3, b o t h challenges (namely P A G s t i m u l a t i o n a n d cold exposure) effectively increased the parasagittal C o B F a n d C o M R O 2. The c e r e b r o v a s o d i l a t a t i o n elicited by cold exposure was also widespread over the neocortex (Table 1). The linear regression lines for the relationship between the percentage change in C o B F (Y) a n d t h a t in C o M R O 2 (X) were Y = 9.1 _ 1.05X (n = 27; r=0.833) for the cold exposure g r o u p a n d Y = 67.0 + 0.64X (n = 30; r = 0.412) for the P A G s t i m u l a t i o n group. T h e slopes were significantly different f r o m zero ( P < 0.001 a n d P < 0.05 in the respective groups).
DISCUSSION
Assessment of the methodology In c o n t r a s t to the previous experiments, 24 we perf o r m e d cervical c o r d o t o m y . This surgical procedure prevented the d e v e l o p m e n t o f hypertension, which would otherwise have t a k e n place d u r i n g chemical stimulation of the P A G , a n d would have m o d u l a t e d the responses of C o B F to stimulation. The l a s e r - D o p p l e r flowmetry employed by us in the present study conveniently allowed a n individually accurate a n d detailed characterization to be m a d e of the c e r e b r o v a s o d i l a t o r action o f the P A G . Consequently, we f o u n d that, in c o n t r a s t to the results o b t a i n e d previously, 23,24 a far m o r e restricted area within the P A G h a r b o u r e d the m o s t p o t e n t c e r e b r o v a s o d i l a t o r sites. The a d o p t i o n o f l a s e r - D o p p l e r flowmetry in comb i n a t i o n with microsphere flowmetry allowed us to achieve microsphere injection a n d simultaneous sampling o f arterial b l o o d a n d dorsal sagittal sinus blood, accurately at the peak o f the C o B F increase. In contrast, in the previous study in which arterial hypertension was allowed to develop, 2a we chose the
Table 3. Responses of cerebral cortical blood flow and oxidative metabolism to cold exposure and periaqueductal gray stimulation Overall data
Number of rats Number of observations Cortical blood flow Baseline (ml/min per 100 g) Challenge (% change) Cortical metabolic rate for oxygen Baseline (#mol/min per 100 g) Challenge (% change)
Truncated data
Cold exposure group
PAG stimulation group
Cold exposure group
PAG stimulation group
25 27
27 30
23 25
21 21
72___ 18 52 + 29*** 274 _ 75 41 -t- 23***
73 + 18 99 _ 41***t 273 ___77 51 _ 26***
73 _ 17 45 _ 19"** 282 _ 72 36 ___16"**
74___ 17 88 + 37"**t 292 ___80 37 _+ 17"**
The data are expressed as means __+S.D. ***Significantly different from zero (P < 0.005; paired t-test). %Significantly different from the cold exposure group (P < 0.005, unpaired t-test).
1138
M. Nakai and M. Maeda
time point for the microsphere flowmetry and simultaneous estimation of the CoMRO2 at a peak in the hypertension. We consider that the previous procedure resulted in observations that were too early to quantify well-matured metabolic responses, thereby leading to the previous conclusion that an enhancement of oxidative metabolism occurring during the PAG-induced CoBF increase was minor. Indeed, as is evident from Fig. 1 of our previous and present reports, the observations were undertaken at approximately 1 and 2min after the N M D A injection, respectively.
Basic characterization o f periaqueductal gray stimulation with N-methyl-D-aspartate to provoke a cortical blood flow increase The present results demonstrated that, in rats subjected to cervical cordotomy, chemical stimulation of the caudal third of the lateral longitudinal column of the PAG 2 with N M D A provoked a strong increase in CoBF, with little change in arterial blood pressure. The intermediate third afforded a smaller cerebrovasodilator action. The flow increase was widespread over the entire neocortical regions. The CoBF increase was a bilateral phenomenon with a slight ipsilateral predominance. The response was N M D A concentration dependent, attaining its submaximal level at 1 mM, provided that 100 nl solution was delivered. No excitotoxic effects of N M D A 34 on the PAG neurons were evident at this concentration, since an excellent reproducibility of the CoBF response was observed.
Cerebrovasodilator mechanisms attributable to the periaqueductal gray-induced cortical blood flow increase During cold exposure, the observed increase in CoBF appears to be elicited in a way that is coupled predominantly to concomitant enhancement of the cortical oxidative metabolism. Indeed, we found in the present as well as previous experiments 27 that the increase in CoBF was associated with an increase in C o M R O 2 of a compatible magnitude. This tendency was also evident when the data were examined by linear regression analysis. The correlation coefficient between the percentage change in CoBF and that in CoMRO2 was high (0.833), and the slope of the regression line (1.05) was almost unity with a very high statistical significance, thereby suggesting a very tight coupling of the flow changes to the changes in oxidative metabolism. As opposed to the previous results, 24 we found that the CoBF increase provoked by PAG stimulation was associated with a disproportionately smaller but clear-cut increase in CoMRO2. Furthermore, the cerebrosvasodilator action of the PAG was found to be twice as strong as that associated with cold exposure, provided that comparisons were made on the basis of identical levels
of enhancement of the oxidative metabolism. In view of the above observation of a tight coupling between flow and metabolism during cold exposure, our results indicate that the PAG-induced CoBF increase was twice as large as that which would be expected from the concomitant enhancement of the oxidative metabolism. We therefore conclude that the PAG-induced CoBF increase was coupled to the oxidative metabolic mechanism to the extent of about one-half, and coupled to certain other cerebrovasodilator mechanisms for the remaining half. In agreement with our previous results, 24 the above findings also indicate that a substantial proportion of the PAG-induced CoBF increase was the outcome of a concomitantly enhanced activity of certain mechanisms which are independent of the oxidative metabolism. The precise etiology of this component remains unknown, although we consider the parasympathetic nervous system 29~36 and central nitric oxide synthase system ~3'~4 as being of particular interest. This inference is based on reported findings that both systems can provoke cerebrovasodilatation with little influence on the central metabolic state, as evaluated from the cerebral glucose metabolic rate, 8,~5 and cortical neuronal activities. 9 Furthermore, recent studies have provided evidence of a tight coupling of increases in cerebral blood flow to enhancement of the non-oxidative glucose metabolism. 7'~8 The precise mechanism of this phenomenon in the generation of cerebrovasodilatation has yet to be adequately elucidated. Whatever the mechanism, we need to perform further investigations to determine whether or not the PAG stimulation enhances this non-oxidative component.
Possible contribution o f the periaqueductal gray in eliciting cortical neural activation Astrocytes, a major non-neuronal element of the brain parenchyma, can interact with neuronal cells and process the oxidative metabolism, which is coupled to glycolysis (see Ref. 19 for review). In this context, our findings for enhancement of the cortical oxidative metabolism might not point exclusively to an activation of neural elements within the cortex. Several lines of evidence reported in the literature, however, indicate an action of the PAG on cortical neural excitability. It has been suggested that the lateral longitudinal column of the PAG represents a substrate for the dense termination of putative nociceptive and thermoreceptive fibres, 3'16 and they can indeed be activated by a noxious stimulus. ~7 Further, since almost any intense sensory stimulus will cause widespread cortical activation, 32 the PAG subdivision may act in the central modulation of nociceptive and thermoreceptive sensory transmissions to enhance the cortical neural excitability and neural oxidative metabolism.
Cerebrovasodilator action of the central gray Diversity o f the cerebrovasodilator mechanism that is coupled to the oxidative metabolism
We speculate that the cerebrovasodilator neural circuitry emanating from the P A G subdivision may also subserve to provoke the cerebrovasodilatation observed during cold exposure, since, as outlined above, this subdivision is one of the preferential target sites of nociceptive and thermoreceptive fibres. We found, however, that, when compared with the C o B F increase provoked by the P A G subdivision, the flow increase occurring during cold exposure was coupled far more tightly to the oxidative metabolic mechanism. These contrasting findings could reflect a difference whereby the cold exposure tended to activate more diverse neural circuits, which would be capable of facilitating the cortical oxidative metabolism, than the P A G subdivision. Thus, during the cold exposure, the cerebrovasodilator action of the oxidative metabolism may overweigh the concomitantly involved non-oxidative component of the vasodilatation mediated by the P A G subdivision. We consider that such diverse circuits may include, at least, the following two cortical afferents (both of which are known to be nociceptive and, when stimulated, provoke a cerebrovasodilatation and cortical activation): the thalamocortical projection emanating from the midline and intralaminar thalamic nuclei, 4'1°'21 and extrathalamic cortical afferents of the basal forebrain cholinergic system. 5'2°,3~'35
1139 CONCLUSION
It was found in the present study that the increase in cortical blood flow provoked by the caudal third of the lateral column within the periaqueductal gray was dependent on the cerebrovasodilator mechanism that may be coupled to the cortical oxidative metabolism to the extent of one-half, and on certain other mechanisms for the remaining half. It is well established that this particular region serves to generate vigorous defensive reactions, which include vasodilatation of the hindlimb skeletal muscle in preparation for the muscular exertion of flight behaviour when coping with a threatening emergency. 2 In the same manner, this region should also help to meet the urgent demand for an enhanced blood supply to the cortex so as to prepare for cortical hyperactivity, which may be generated upon the execution of vigorous defensive behaviour. Such a preparatory mechanism has also been suggested in a manner in which the cerebellar fastigial nucleus may serve to couple C o B F increases in anticipation of a wide range of motivated behaviours. 22,33 Acknowledgements--This study was supported by the NCVC Research Institute (92-93), by the STA (Special Coordination Funds for Promoting Science and Technology, COE), by the MHW (Cardiovascular Diseases 6A-3) and by the MESC (Scientific Research 06670072). The authors wish to acknowledge Inter Medical Co. Ltd (3-32-16 Kasuya, Setagaya, Tokyo 157, Japan) for collaboration in fabricating the device (NTM-IR) designed for sampling cerebral sinus blood.
REFERENCES
1. Bandler R., Carrive P. and Zhang S. P. (1991) Integration of somatic and autonomic reactions within the midbrain periaqueductal gray: viscerotopic, somatotopic and functional organization. Prog. Brain Res. 87, 269-305. 2. Bandler R. and Shipley M. T. (1994) Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci. 17, 379-389. 3. Blomqvist A. and Craig A. D. (1991) Organization of spinal and trigeminal input to the PAG. In The Midbrain Periaqueductal Gray Matter (eds Depaulis A. and Bandler R.), pp.345-363. Plenum Press, New York. 4. Bullitt E. (1990) Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J. comp. Neurol. 296, 517-530. 5. Buzsaki G., Bickford R. G., Ponomareff G., Thai L. J., Mandel R. and Gage F. H. (1988) Nucleus basalis and thalamic control of neocortical activity in the freely moving rat. J. Neurosci. 8, 4007-4026. 6. Carrive P. (1993) The periaqueductal gray and defensive behaviour: functional representation and neuronal organization. Behav. Brain Res. 58, 27-47. 7. Fox P. T., Raichle M. E., Mintun M. A. and Dence C. (1988) Non-oxidative glucose consumption during focal physiologic neural activity. Science 241, 462 464. 8. Goadsby P. J. (1990) Sphenopalatine ganglion stimulation increases regional cerebral blood flow independent of glucose utilization in the cat. Brain Res. 506, 145 148. 9. Goadsby P. and Hoskin K. L. (1994) Cerebral blood flow is not coupled to neuronal activity during stimulation of the facial nerve vasodilator system. Brain Res. 647, 192-198. 10. Groenewegen H. J. and Berendse H. W. (1994) The specificity of the 'non-specific' midline and intralaminar thalamic nuclei. Trends Neurosci. 17, 52 57. 11. Heistad D. D. and Kontos H. A. (1983) Cerebral circulation. In Handbook o f Physiology, The Cardiovascular System III. Peripheral Circulation and Organ Blood Flow (eds Shepherd J. T., Abboud F. M. and Geiger S. R.), Chapter 5, pp.137-183. American Physiological Society, Bethesda, MD. 12. Hilton S. M. and Redfern W. S. (1986) A search for brain stem cell groups integrating the defence reaction in the rat. J. Physiol. 378, 213528. 13. Iadecola C. (1993) Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link? Trends Neurosci. 16, 206-214. 14. Iadecola C., Pelligrino D. A., Moskowitz M. A. and Lassen N. A. (1994) Nitric oxide synthase inhibition and cerebrovascular regulation. J. cerebr. Blood Flow Metab. 14, 175 192. 15. Iadecola C. and Xu X. (1994) Nitro-L-arginine attenuates hypercapnic cerebrovasodilation without affecting cerebral metabolism. Am. J. Physiol. 266, R518-R525. 16. Keay K. A. and Bandler R. (1992) Anatomical evidence for segregated input from the upper cervical spinal cord to functionally distinct regions of the periaqueductal gray region of the cat. Neurosci. Lett. 139, 143-148.
1140
M. Nakai and M. Maeda
17. Keay K. A. and Bandler R. (1993) Deep and superficial noxious stimulation increases Fos-like immunoreactivity in different regions of the midbrain periaqueductal grey of the rat. Neurosci. Lett. 154, 23-26. 18. Madsen P. L., Hasselbalch S. G., Hagemann L. P., Olsen K. S., Bulow J., Holm S., Wildschiodtz G., Paulson O. B. and Lassen N. A. (1995) Persistent resetting of the cerebral oxygen/glucose uptake ratio by brain activation: evidence obtained with the Kety-Schmidt technique. J. cerebr. Blood Flow Metab. 15, 485-491. 19. Magistretti P. J. (1994) Vasoactive intestinal peptide and noradrenaline regulate energy metabolism in astrocytes: a physiological function in the control of local homeostasis within the CNS. Prog. Brain Res. 100, 87 93. 20. Metherate R., Cox C. L. and Ashe J. H. (1992) Cellular bases ofneocortical activation: modulation of neural oscillations by the nucleus basalis and endogenous acetylcholine. J. Neurosci. 12, 4701 4711. 21. Mraovitch S., Feger J., Calando Y. and Seylaz J. (1989) Cardiovascular and cerebrovascular alterations elicited by cholinergic stimulation of the centromedian-parafascicular complex in the rat. In Neurotransmission and Cerebrovascular Function I (eds Seylaz J. and MacKenzie E. T.), pp.397 400. Elsevier Science, Amsterdam. 22. Nakai M., Iadecola C., Ruggiero D. A., Tucker L. W. and Reis D. J. (1983) Electrical stimulation of cerebellar fastigial nucleus increases cerebral cortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation. Brain Res. 260, 35-49. 23. Nakai M. and Maeda M. (1994) Systemic and regional haemodynamic responses elicited by microinjection of N-methyl-D-aspartate into the lateral periaqueductal gray matter in anaesthetized rats. Neuroscience 58, 777-783. 24. Nakai M. and Maeda M. (1994) Cerebrovasodilatation of metabolic and non-metabolic origin elicited by chemical stimulation of the lateral periaqueductal gray matter in anaesthetized rats. Neuroscience 58, 785-791. 25. Nakai M., Tamaki K. and Maeda M. (1991) Multiple measurement of regional blood flow in rat brain using radiolabeled microspheres. Du Pont Biotech Update 6, (4), 1-3 (with an illustration of experimental layout). 26. Nakai M., Tamaki K. and Maeda M. (1991) Hyperperfusion of the rat brain during reflex hypertension induced by sudden lowering of the cutaneous temperature. J. auton, nerv. Syst. 35, 71 82. 27. Nakai M., Tamaki K. and Maeda M. (1991) Hyperperfusion and enhanced metabolic state of the brain during sudden lowering of the cutaneous temperature in rats. J. auton, nerv. Syst. 35, 83-92. 28. Nakai M., Tamaki K. and Maeda M. (1992) Sympathetic and metabolic mechanisms of the cerebrovasomotor function of the caudal ventrolateral medulla in rats. Neuroscience 50, 655 662. 29. Nakai M., Tamaki K., Ogata J., Matsui Y. and Maeda M. (1993) Parasympathetic cerebrovasodilator center of the facial nerve. Circulation Res. 72, 470-475. 30. Nakai M., Tamaki K., Yamamoto J. and Maeda M. (1990) A minimally invasive technique for multiple measurement of regional blood flow of the rat brain using radiolabeled microspheres. Brain Res. 507, 168 171. 31. Peschanski M., Guilbaud G. and Gautron M. (1981) Posterior intralaminar region in rat: neuronal responses to noxious and nonnoxious cutaneous stimuli. Expl Neurol. 72, 226 238. 32. Pompeiano O. (1973) Reticular formation. In Handbook o f Sensory Physiology II (ed. Iggo A.), pp.381-488. Springer, Berlin. 33. Reis D. J., Doba N. and Nathan M. A. (1973) Predatory attack, grooming and consummatory behavior evoked by electrical stimulation of cerebellar nuclei in cat. Science 182, 845-847. 34. Rothman S. M. and Olney J. W. (1987) Excitotoxicity and the NMDA receptor. Trends Neurosci. 10, 299 302. 35. Sato A. and Sato Y. (1992) Regulation of regional cerebral blood flow by cholinergic fibers originating in the basal forebrain. J. Neurosci. Res. 14, 242 274. 36. Suzuki N. and Hardebo E. (1993) The cerebrovascular parasympathetic innervation. Cerebrovasc. Brain Metab. Rev. 5, 33 46. 37. Wallenstein S., Zucker C. L. and Fleiss J. L. (1980) Some statistical methods useful in circulation research. Circulation Res. 47, 1-9. (Accepted 4 January 1996)
~
Pergamon
S0306-4522(96)00013-9
Elsevier Science Ltd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00
VASODILATATION A N D E N H A N C E D OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX PROVOKED BY THE P E R I A Q U E D U C T A L GRAY MATTER IN ANAESTHETIZED RATS M. N A K A I * f and M. MAEDA:~ tNational Cardiovascular Center Research Institute, Suita, Osaka 565, Japan :~Department of Systems Physiology, University of Occupational and Environmental Health, Kita-Kyushu, Fukuoka 807, Japan Abstract--The aim of the present study was to evaluate the possible contribution of the cerebral cortical oxidative metabolism to the cortical vasodilator action of the periaqueductal gray. In 70 rats with cervical cordotomy, we found that unilateral stimulation of the caudal third of the lateral longitudinal column of the periaqueductal gray with N-methyl-D-aspartate bilaterally provoked the greatest increase in cortical blood flow (laser-Doppler flowmetry and/or microsphere flowmetry). The response was widespread over the entire neocortical regions, elicited in a dose-dependent manner, with little change in arterial blood pressure. The flow was increased effectively by a submaximal dose of the amino acid (1 mM, 100 nl), attaining a peak increase by 99 + 41% of the baseline level (mean +__S.D., n = 30), and was associated with an enhancement of the cortical metabolic rate for oxygen by 51 + 26%. We then compared the flow increase with that induced by cold exposure (by 52_ 29%, n = 27), the latter response being tightly coupled to an enhanced metabolic rate for oxygen (by 41 _ 23%). It was thus found that the increase in cortical blood flow provoked by the subdivision was dependent on the cerebrovasodilator mechanism that may be coupled to the cortical oxidative metabolism to the extent of one-half, and on certain other mechanisms for the remaining half. In view of the fact that this particular region serves to generate vigorous defence reactions that involve flight behaviour, the region should also help to meet the urgent demand for an increased cortical blood flow, so as to prepare for the possible generation of cortical hyperactivity in coping vigorously with a threatening emergency. Key words: central gray matter, cerebral blood flow, cerebral metabolic rate, cold exposure, defence
reaction, thermal stimulus.
A distinctive neuron pool within the midbrain periaqueductal gray matter (PAG) has recently been identified as a reflex centre located at the top of the central processing and organization of the natural behavioural and haemodynamic defence reactions to noxious-sensory and threatening stimuli) ,2,6,12 We recently reported for the first time that, when chemically stimulated, the neural circuitry emanating from the intermediate and caudal thirds of the lateral longitudinal column extending along the rostrocaudal axis of the P A G 2 also serves to increase the cerebral blood flow, as revealed by microsphere flowmetry in rats. 23 It has long been considered that an enhancement of the cerebral oxidative metabolism provoked by brain activation is capable of achieving a powerful and opportune cerebrovasodilatation to ensure an adequate supply of blood and substrates for energy
*Towhom correspondence should be addressed. CoBF, cortical blood flow; CoMRO2,
Abbreviations:
cortical metabolic rate for oxygen; NMDA, N-methylD-aspartate; PAG, periaqueductal gray matter.
generation to the brain region within which neurons are activated (see Ref. 11 for review). The magnitude of the cerebral blood flow could therefore be tightly coupled to the oxidative metabolic state for achieving adequate execution of brain functions. Indeed, during brain activation provoked by sudden exposure of rats to a thermal stimulus, we have demonstrated such tight coupling37 It is conceivable, therefore, that the P A G subdivision could have exerted its cerebrovasodilator action by enhancing the cortical oxidative metabolism, since the subdivision is one of the preferential target sites of nociceptive and thermoreceptive afferents. 3'16'17 We found in our subsequent study, 24 however, that the increase in cerebral cortical blood flow (CoBF) was accompanied by a minor change in oxidative metabolic state in the cortex, as estimated from the observation of the cortical metabolic rate for oxygen (CoMRO2). In this context, by devising an improved experimental approach in the present study, we attempted to reassess whether or not the cerebrovasodilator action of the P A G subdivision is really minimally
1133
1134
M. Nakai and M. Maeda
a t t r i b u t a b l e to the m e c h a n i s m t h a t is c o u p l e d to the c e r e b r a l o x i d a t i v e m e t a b o l i s m . A s a result, a clear-cut m o d e r a t e e n h a n c e m e n t o f the oxidative m e t a b o l i s m , a l t h o u g h still d i s p r o p o r t i o n a t e l y s m a l l e r t h a n t h a t o f the flow, was actually d e t e c t e d . W e t h e r e f o r e subj e c t e d a f u r t h e r s e p a r a t e g r o u p o f a n i m a l s to s u d d e n e x p o s u r e to a t h e r m a l stimulus so as to o b t a i n d a t a for t h e w e l l - e s t a b l i s h e d r e l a t i o n s h i p involving tight c o u p l i n g b e t w e e n b l o o d flow a n d o x i d a t i v e m e t a b olism. 27 A c o m p a r i s o n o f t h e results o b t a i n e d was t h e n u n d e r t a k e n to assess the e x t e n t to w h i c h the c e r e b r o v a s o d i l a t o r a c t i o n o f t h e P A G is a t t r i b u t a b l e to t h e oxidative m e t a b o l i c m e c h a n i s m .
EXPERIMENTAL PROCEDURES
General surgical procedures Experiments were conducted on 70 male Wistar rats (Oriental Bioservice, Kyoto, Japan) weighing from 240 to 350 g. The animals were divided into three groups. These groups were subjected to either a basic characterization study (the extra-PAG stimulation group, 18 rats) with chemical stimulation of the P A G with N-methyl-D-aspartate (NMDA), a flow-metabolism study with chemical stimulation of the P A G with N M D A (the P A G stimulation group, 27 rats) or a flow metabolism study with sudden exposure to cold (the cold exposure group, 25 rats). At one to three days before the experiment, the animals of each group were anaesthetized with a mixture of 2 2.5% halothane in oxygen gas blown over the nose. We cut notches along the bilateral surfaces of the rostrolateral quarter of the parietal bones with a dental drill, outlining the cranial windows which were to be opened later on the day of the experiment. This preparatory procedure served to minimize untoward cerebrovascular effects of the surgical manipulations. The exposed skull surface was covered with a sheet of bacteriocidal wound dressing (Sofratulle, Laboratories Roussel, Uxbridge, U.K.) and the skin wounds were sutured. The animals were then kept in individual cages. On the day of the experiment, the animals were anaesthetized initially with 2-2.5% halothane only during surgery, and subsequently with urethane (1.25 g/kg body weight, i.p.) during the experiments. Supplemental urethane was given at intervals of 2 h at a dose of 250 mg/kg. The adequate depth of halothane anaesthesia was maintained, as confirmed by inspection of the immobility of the animal during an earlier part of surgery. Once anaesthesia with urethane had been established, no further surgical manipulations were performed. In the animals of the cold exposure group, the chest and belly were shorn with clippers. Subsequent experimental procedures and techniques were as detailed by us in preceding reports, and will be described only briefly here. Each o f our previous reports will be cited below as appropriate. In the animals of the PAG stimulation and cold exposure groups, a left cardiac ventricular catheter for microsphere injection was threaded into place via the right brachial artery. The femoral arteries and veins were catheterized bilaterally. In the extra-PAG stimulation group, only a unilateral femoral artery and the bilateral femoral veins were catheterized. The trachea was then intubated. The animals were paralysed (D-tubocurarine, 0.3mg/kg, i.v., given at hourly intervals), artificially ventilated and maintained in a homoeothermic state at 37°C. The head was held in a stereotaxic frame with the incisor bar lowered by 17 mm from the horizontal plane passing through the interaural line. Following laminectomy, cervical cordotomy was undertaken at the C4 or C5 level in order to provide
generalized sympathectomy. For achieving cordotomy, we preferred that level to the higher cervical level, otherwise the procedure would have easily caused injury or oedema of the lower medulla. The normal level of arterial blood pressure was then maintained by continuous i.v. infusion of phenylephrine (Sigma, St Louis, MO, U.S.A.). The cranial windows were opened in the bilateral parietal bones by peeling off the pieces of bone whose outline had been notched previously. The dural surfaces so exposed were then covered with silicone oil (Medical Fluid, Dow-Corning, Asia, Tokyo, Japan). In the animals of the extra-PAG stimulation and PAG stimulation groups, the dorsal surfaces of the medulla and cerebellum were visualized by occipital craniectomy and covered with warm saline. Basic characterization of periaqueductal gray stimulation with N-methyl-D-aspartate in the extra-periaqueductal gray stimulation group The tips of a laser-Doppler flowmeter (Biomedical Science, Kanazawa, Japan) were fitted bilaterally over each of the dural surfaces. Moment-to-moment monitoring of the CoBF was commenced. The halothane anaesthesia was then replaced with urethane anaesthesia. After a subsequent resting period of 30 min, the tip of a multi-barrelled glass micropipette (Clark Electromedical Instruments, Reading, U.K.), 28 mounted on a micro-manipulator and inclined caudally at an angle of 30 ° from the vertical, was lowered into the PAG. The cerebrovasodilator sites within the PAG were explored in a series of 0.5-mm steps by microinjecting N M D A solution into the relevant brain region. N M D A had previously been dissolved (30-100 pmol as 30-100 nl of a I mM solution) in a vehicle (Dulbecco's phosphate-buffered saline, pH 7.3-7.65; Nissui, Tokyo, Japan), and delivered over 5-16 s through one lumen of the pipette using a 10-/~1 Hamilton syringe installed on a Narishige infusion pump. The tip of the micropipette was then fixed in place unilaterally within the PAG at the most potent cerebrovasodilator site so identified. The lateral (side-to-side) difference in CoBF response to unilateral PAG stimulation was evaluated. Furthermore, the relationship between N M D A concentration and the resultant CoBF response was investigated, by injecting vehicle or an N M D A solution (100nl at 0.316, 1 or 3.16mM, over 16s) into the PAG from the individual lumina of a quadruple-barrelled pipette, with a separation of 10 20 min between each injection. We also undertook functional mapping of the cerebrovasodilator sites and tests for reproducibility of the flow response. For histological confirmation, the location of the pipette tip was marked with 2% India ink solution (100 nl), delivered via another lumen of the multi-barrelled pipette into the tissue. Microsphere flowmetry was also employed to evaluate the side-to-side difference in responsiveness of the CoBF and to assess the regional difference in responsiveness of the blood flow within the neocortex. Details of these procedures are given below. Preparation for measurement of cortical blood flow and cortical metabolic rate for oxygen in the periaqueductal gray stimulation group and cold exposure group For later sampling of blood from the dorsal sagittal sinus to estimate the CoMRO2, 27 a burr hole (3 mm in diameter) was made acutely so that the dorsal surface of the stem of the sinus was exposed at its junction with the confluence of the sinuses. A device (NTM-1R, Inter Medical, Nagoya, Japan), designed by us for the sampling of sinus blood, was fitted over the burr hole and secured to the skull with dental cement. The exposed dorsal wall of the sinus was later incised, shortly before the start of the experimental runs. This procedure allowed the sinus blood (150/~1) to drain (150~tl/min) through a Minipulse-3 tube pump (Gilson, Villiers-le-Bel, France) out through the device into a glass capillary.
Cerebrovasodilator action of the central gray The halothane anaesthesia was then switched to urethane anaesthesia. During the subsequent resting period of 30 min, we injected heparin (600 U/kg, i.v.) and set up a crosscirculation circuit with a chronically catheterized donor rat, in preparation for microsphere flowmetry. 25'3° A laserDoppler flowmeter was additionally applied to the animal as described earlier, and moment-to-moment monitoring of the CoBF with the flowmeter was commenced.
The flow-metabolism study undertaken in the periaqueductal gray stimulation group The cerebrovasodilator sites within the P A G were explored by microinjecting N M D A through one lumen of a double-barrelled micropipette as described above. The tip of the pipette was fixed in place unilaterally at the most potent site. We then carried out the first experimental run for measurement of the baseline CoBF by microsphere flowmetry and simultaneous estimation of the baseline C o M R O 2. By running the cross-circulation with a Minipulse-3 tube pump at a rate of 1.2 ml/min, the microspheres (16.5 __+0.1/~m in diameter, radiolabelled with 57Co, ll3Sn, SSSr or 46Sc, NEN, Boston, MA, U.S.A.) were injected into the left cardiac ventricle through the catheter, and reference arterial blood was simultaneously sampled (1 ml over 50 s) through the femoral arterial catheter. Sinus blood (150/~1) was also sampled in parallel with the cross-circulation. An aliquot of the sampled arterial blood (150 #1) and the entire volume o f the sampled sinus blood were immediately subjected to measure their gas tensions (ABL-3, Radiometer, Copenhagen, Denmark) and oxygen content (OSM-3, Radiometer), for later calculation o f the CoMRO:. At 15-20min after the first experimental run, N M D A (1 mM, 100nl) was injected unilaterally into the potent cerebrovasodilator site within the P A G over a period of 16 s. When the peak increase in CoBF had been attained as observed by laser-Doppler flowmetry, the second experimental run was undertaken. In several rats, we repeated the same experimental protocol in order to carry out an additional pair of experimental runs. At the end of the experiment, the location of the pipette tip was marked with India ink and the site later examined histologically, as described above. The animals were killed by inducing haemorrhage. We then dissected out the
1135
parasagittal part of the bilateral cerebral cortex. The tissue specimens and reference arterial blood were weighed and processed for measuring their radioactivity to calculate the CoBF. 26 The C o M R O 2 was calculated by incorporating the CoBF and the total oxygen contents of the sampled arterial blood and sinus blood. 27 Special care was taken to ensure that the cortical area subjected to the CoBF measurement matched that subjected to C o M R O 2 measurement. 28 In nine rats of the P A G stimulation group, we subsequently evaluated the regional differences in responsiveness of the blood flow within the neocortex. Parcellation of the bilateral neocortex was carried out to obtain samples of the medial third and lateral two-thirds of the parasagittal cortex. The latter portion was further divided into frontal, intermediate and occipital thirds. Furthermore, the bilateral extra-parasagittal portion of the neocortex above the rhinal fissure was also sampled.
The flow-metabolism study undertaken in the cold exposure group The animal was laid in a prone position on the top panel of a warm/cool box, and its head was fixed on a stereotaxic frame. The interior of the box was circulated with warm water (37°C), which could be abruptly switched to cold water (0°C). The incoming water was supplied by a warm-water circulator (SP-12, Taitek, Saitama, Japan) or a cool-water circulator (CP-150F, Taitek), respectively. The box was designed so that its lumen was narrow (50 ml) and its top stainless panel was thin (0.2 mm). Sudden lowering of the temperature of the circulating water could thus be swiftly transmitted (within several seconds) to the area of shorn thoracic and belly skin. We followed the same experimental protocol as in the PAG stimulation group, except for the induction of cerebrovasodilatation by sudden exposure to cold. Data analysis The results are expressed as the means _+ S.D. Statistical analysis was performed by the paired or unpaired t-test, as appropriate. A two-way analysis of variance or a test for the overall difference over time, followed by the Bonferroni method for pairwise c o m p a r i s o n s y was also performed.
Microsphere injection & sampling of blood 20Or- ~ - 7
V
~
200f
if!
<
0
1.991~
i
Or_ Cold exposure
1 NMDA Injection 2 rain
Fig. 1. Typical recordings of the responses of the mean arterial pressure (ABP) and cortical blood flow (CoBF) in two rats with cervical cordotomy. The moment-to-moment observation of blood flow was monitored with a laser-Doppler flowmeter and is expressed in terms of its electrical output (V). The flow response was elicited by sudden exposure to cold water (0°C) or by unilateral chemical stimulation of the caudal third of the lateral longitudinal column of the PAG with microinjection of N M D A (1 mM, 100 nl, delivered over 16 s). Microsphere flowmetry and simultaneous sampling of arterial blood and dorsal sagittal sinus blood for estimation of the oxidative metabolic rate in the cortex were successfully achieved at the peak of the flow increase.
1136
M. Nakai and M. Maeda
Table 1. Responses of the neocortical regional blood flow and oxidative metabolism to cold exposure and to chemical stimulation of the caudal third of the lateral longitudinal column of the periaqueductal gray matter Cold exposure
PAG stimulation
6 8
9 10
49 _+ 19 61 __+34
114 _ 73 134 + 89
15 + 38 + 71 + 66 +
76 + 141 + 131 + 96 +
Number of rats Number of observations Neocortical blood flow (% increase) Entire bulk of the parasagittal cortex Medial third Lateral two-thirds Frontal third Intermediate third Occipital third Extra-parasagittal neocortex Parasagittal neocortical metabolic rate for oxygen (% increase)
29"s 38 57 64
38 + 21
52 99 101 59
53 + 53
The data (means __+S.D.) were obtained by sudden exposure to cold water (0°C) or by microinjecting NMDA (1 mM, 100 nl, delivered over 16 s) into the PAG subdivision (microsphere flowmetry), n~Not significantly changed from the baseline value (P > 0.05, paired t-test). All of the other changes were significant (P < 0.05~).001).
RESULTS
Basic characterization o f periaqueductal gray stimulation with N-methyl-D-aspartate to provoke a cortical blood flow increase Figure 1 shows typical records of momentto-moment changes in C o B F monitored by l a s e ~ Doppler flowmetry. It was found that unilateral P A G stimulation with 1 m M N M D A (100 nl, delivered over 16 s) provoked a bilateral increase in CoBF, without any significant changes in arterial blood pressure in rats with cervical cordotomy. The contralateral cortex was slightly less responsive, as revealed by laserDoppler flowmetry ( 9 2 + 18% of the ipsilateral increase, n = 17, P <0.05, paired t-test, 13 rats) and by microsphere flowmetry (94 + 26%, n = 33, P > 0.05, 26 rats). The cerebrovasodilator action of the P A G was widespread over the neocortical regions (Table 1; microsphere flowmetry). The cerebrovasodilator response to chemical stimulation of the P A G with 100 nl N M D A was concentration dependent, as revealed by l a s e ~ Doppler flowmetry. Employing five rats, we found that unilateral microinjection of N M D A , carried out in order of ascending concentration, changed the ipsilateral C o B F by - 2 + 3% at 0 m M (vehicle), by + 5 9 _ + 3 3 % at 0 . 3 1 6 m M , by + 9 4 _ + 2 9 % at l m M and by + 106 + 55% at 3.16 mM. These flow changes induced by N M D A were significantly different from that observed following vehicle injection (n = 5, P < 0.001 at every concentration, test for the overall difference over time, followed by the Bonferroni method for pairwise comparisons).
The reproducibility of the flow response was excellent, as investigated with 1 m M N M D A (100 nl) and employing laser-Doppler flowmetry. The second to sixth experimental runs, 10-15 min apart from each other, resulted in C o B F increases of 99___ 27, 105 +_23, 102 + 25, 97 + 26 and 92 + 28%, respectively, of the initial C o B F increase (n = 6, three rats). No significant difference was detected among any of the responses (tests for the overall difference over time). Figure 2 shows a functional map drawn for cerebrovasodilator sites within the P A G , as studied with 1 m M N M D A (30 nl, delivered over 5 s) and employing l a s e ~ D o p p l e r flowmetry. We found that cerebrovasodilatation was provoked from the intermediate and caudal thirds of the lateral longitudinal column of the PAG. A m o n g these subdivisions, however, a restricted part within the caudal third was the only region from which the greatest increase in C o B F was elicited. A reduction of the C o B F could not be elicited from any region explored within the lateral column of the PAG.
Comparison o f the cerebrovasodilator effect o f periaqueductal gray stimulation with that o f cold exposure, based on the overall data obtained by microsphere flowmetry Details of the baseline state of systemic circulatory parameters observed in the P A G stimulation group and in the cold exposure group are presented in
Rostrocaudal coordinate (mm) 10
~
+2
9
n2'
o
o,4, o
o/
o
-3
Fig. 2. Functional map of the midbrain sectioned in the sagittal plane (0.7 mm lateral to the midline) in one rat with cervical cordotomy. The dorsoventral and rostrocaudal coordinates are referred to the calamus scriptorius. Cerebrovasodilator sites were explored along five dorsoventral tracks by microinjecting 1 mM NMDA (30 nl). The magnitude of the response provoked by each site is represented by a large (6(~84% increase), medium (37 55% increase) or small solid circle (14~28% increase). Open circles represent quiescent sites. IC, inferior colliculus; PAG, periaqueductal gray; PAGD, dorsal periaqueductal gray; SC, superior colliculus.
Cerebrovasodilator action of the central gray Table 2. Systemic circulatory parameters in the cold exposure and periaqueductal gray stimulation groups
Number of rats Number of observations Mean arterial blood pressure (mmHg) Baseline Challenge Arterial blood pH Baseline Challenge Arterial blood PCO 2 (mmHg) Baseline Challenge Arterial blood P O 2 (mmHg) Baseline Challenge
Cold exposure group
PAG stimulation group
25 27
27 30
86 _+ 8 86 + 9
83 _ 7 82 +__7
7.392 __+0.053 7.390 __+0.055
7.395 _ 0.041 7.391 _ 0.044
37.4 __+2.3 37.3 __+2.7
36.6 _ 1.6 36.4 __+1.6
135 _+ 17 141 ___20
142 -4- 16 145 + 15
1137
Comparison of the cerebrovasodilator effect of periaqueductal gray stimulation with that of cold exposure, on the basis of an identical cortical oxidative metabolic state The averaged m a g n i t u d e o f e n h a n c e m e n t o f C o M R O 2 d u r i n g P A G stimulation was considerably different f r o m t h a t observed d u r i n g cold exposure, as listed in Table 3 (51% vs 41%). W e therefore t r u n c a t e d the overall plots of the pooled d a t a for each g r o u p so t h a t a quantitative c o m p a r i s o n o f the c e r e b r o v a s o d i l a t o r effect could be m a d e between the groups, only across identical ranges (from 5 % up to 6 9 % ) o f e n h a n c e m e n t of cortical oxidative m e t a b olism (Table 3; 37% vs 3 6 % o n average). Consequently, we f o u n d t h a t the c e r e b r o v a s o d i l a t o r action o f the P A G (flow increase 8 8 % ) was twice as strong as t h a t of cold exposure (45%).
The data are expressed as m e a n s _ S.D. Each of the measured variables did not differ greatly between the groups as well as between the experimental conditions (P > 0.05, two-way analysis of variance).
T a b l e 2. W e f o u n d t h a t each o f the m e a s u r e d parameters did n o t differ greatly between the groups. Typical recordings o f responses o f the arterial pressure a n d C o B F to cold exposure a n d to chemical s t i m u l a t i o n o f the P A G were d e m o n s t r a t e d in Fig. 1. As is summarized in Table 3, b o t h challenges (namely P A G s t i m u l a t i o n a n d cold exposure) effectively increased the parasagittal C o B F a n d C o M R O 2. The c e r e b r o v a s o d i l a t a t i o n elicited by cold exposure was also widespread over the neocortex (Table 1). The linear regression lines for the relationship between the percentage change in C o B F (Y) a n d t h a t in C o M R O 2 (X) were Y = 9.1 _ 1.05X (n = 27; r=0.833) for the cold exposure g r o u p a n d Y = 67.0 + 0.64X (n = 30; r = 0.412) for the P A G s t i m u l a t i o n group. T h e slopes were significantly different f r o m zero ( P < 0.001 a n d P < 0.05 in the respective groups).
DISCUSSION
Assessment of the methodology In c o n t r a s t to the previous experiments, 24 we perf o r m e d cervical c o r d o t o m y . This surgical procedure prevented the d e v e l o p m e n t o f hypertension, which would otherwise have t a k e n place d u r i n g chemical stimulation of the P A G , a n d would have m o d u l a t e d the responses of C o B F to stimulation. The l a s e r - D o p p l e r flowmetry employed by us in the present study conveniently allowed a n individually accurate a n d detailed characterization to be m a d e of the c e r e b r o v a s o d i l a t o r action o f the P A G . Consequently, we f o u n d that, in c o n t r a s t to the results o b t a i n e d previously, 23,24 a far m o r e restricted area within the P A G h a r b o u r e d the m o s t p o t e n t c e r e b r o v a s o d i l a t o r sites. The a d o p t i o n o f l a s e r - D o p p l e r flowmetry in comb i n a t i o n with microsphere flowmetry allowed us to achieve microsphere injection a n d simultaneous sampling o f arterial b l o o d a n d dorsal sagittal sinus blood, accurately at the peak o f the C o B F increase. In contrast, in the previous study in which arterial hypertension was allowed to develop, 2a we chose the
Table 3. Responses of cerebral cortical blood flow and oxidative metabolism to cold exposure and periaqueductal gray stimulation Overall data
Number of rats Number of observations Cortical blood flow Baseline (ml/min per 100 g) Challenge (% change) Cortical metabolic rate for oxygen Baseline (#mol/min per 100 g) Challenge (% change)
Truncated data
Cold exposure group
PAG stimulation group
Cold exposure group
PAG stimulation group
25 27
27 30
23 25
21 21
72___ 18 52 + 29*** 274 _ 75 41 -t- 23***
73 + 18 99 _ 41***t 273 ___77 51 _ 26***
73 _ 17 45 _ 19"** 282 _ 72 36 ___16"**
74___ 17 88 + 37"**t 292 ___80 37 _+ 17"**
The data are expressed as means __+S.D. ***Significantly different from zero (P < 0.005; paired t-test). %Significantly different from the cold exposure group (P < 0.005, unpaired t-test).
1138
M. Nakai and M. Maeda
time point for the microsphere flowmetry and simultaneous estimation of the CoMRO2 at a peak in the hypertension. We consider that the previous procedure resulted in observations that were too early to quantify well-matured metabolic responses, thereby leading to the previous conclusion that an enhancement of oxidative metabolism occurring during the PAG-induced CoBF increase was minor. Indeed, as is evident from Fig. 1 of our previous and present reports, the observations were undertaken at approximately 1 and 2min after the N M D A injection, respectively.
Basic characterization o f periaqueductal gray stimulation with N-methyl-D-aspartate to provoke a cortical blood flow increase The present results demonstrated that, in rats subjected to cervical cordotomy, chemical stimulation of the caudal third of the lateral longitudinal column of the PAG 2 with N M D A provoked a strong increase in CoBF, with little change in arterial blood pressure. The intermediate third afforded a smaller cerebrovasodilator action. The flow increase was widespread over the entire neocortical regions. The CoBF increase was a bilateral phenomenon with a slight ipsilateral predominance. The response was N M D A concentration dependent, attaining its submaximal level at 1 mM, provided that 100 nl solution was delivered. No excitotoxic effects of N M D A 34 on the PAG neurons were evident at this concentration, since an excellent reproducibility of the CoBF response was observed.
Cerebrovasodilator mechanisms attributable to the periaqueductal gray-induced cortical blood flow increase During cold exposure, the observed increase in CoBF appears to be elicited in a way that is coupled predominantly to concomitant enhancement of the cortical oxidative metabolism. Indeed, we found in the present as well as previous experiments 27 that the increase in CoBF was associated with an increase in C o M R O 2 of a compatible magnitude. This tendency was also evident when the data were examined by linear regression analysis. The correlation coefficient between the percentage change in CoBF and that in CoMRO2 was high (0.833), and the slope of the regression line (1.05) was almost unity with a very high statistical significance, thereby suggesting a very tight coupling of the flow changes to the changes in oxidative metabolism. As opposed to the previous results, 24 we found that the CoBF increase provoked by PAG stimulation was associated with a disproportionately smaller but clear-cut increase in CoMRO2. Furthermore, the cerebrosvasodilator action of the PAG was found to be twice as strong as that associated with cold exposure, provided that comparisons were made on the basis of identical levels
of enhancement of the oxidative metabolism. In view of the above observation of a tight coupling between flow and metabolism during cold exposure, our results indicate that the PAG-induced CoBF increase was twice as large as that which would be expected from the concomitant enhancement of the oxidative metabolism. We therefore conclude that the PAG-induced CoBF increase was coupled to the oxidative metabolic mechanism to the extent of about one-half, and coupled to certain other cerebrovasodilator mechanisms for the remaining half. In agreement with our previous results, 24 the above findings also indicate that a substantial proportion of the PAG-induced CoBF increase was the outcome of a concomitantly enhanced activity of certain mechanisms which are independent of the oxidative metabolism. The precise etiology of this component remains unknown, although we consider the parasympathetic nervous system 29~36 and central nitric oxide synthase system ~3'~4 as being of particular interest. This inference is based on reported findings that both systems can provoke cerebrovasodilatation with little influence on the central metabolic state, as evaluated from the cerebral glucose metabolic rate, 8,~5 and cortical neuronal activities. 9 Furthermore, recent studies have provided evidence of a tight coupling of increases in cerebral blood flow to enhancement of the non-oxidative glucose metabolism. 7'~8 The precise mechanism of this phenomenon in the generation of cerebrovasodilatation has yet to be adequately elucidated. Whatever the mechanism, we need to perform further investigations to determine whether or not the PAG stimulation enhances this non-oxidative component.
Possible contribution o f the periaqueductal gray in eliciting cortical neural activation Astrocytes, a major non-neuronal element of the brain parenchyma, can interact with neuronal cells and process the oxidative metabolism, which is coupled to glycolysis (see Ref. 19 for review). In this context, our findings for enhancement of the cortical oxidative metabolism might not point exclusively to an activation of neural elements within the cortex. Several lines of evidence reported in the literature, however, indicate an action of the PAG on cortical neural excitability. It has been suggested that the lateral longitudinal column of the PAG represents a substrate for the dense termination of putative nociceptive and thermoreceptive fibres, 3'16 and they can indeed be activated by a noxious stimulus. ~7 Further, since almost any intense sensory stimulus will cause widespread cortical activation, 32 the PAG subdivision may act in the central modulation of nociceptive and thermoreceptive sensory transmissions to enhance the cortical neural excitability and neural oxidative metabolism.
Cerebrovasodilator action of the central gray Diversity o f the cerebrovasodilator mechanism that is coupled to the oxidative metabolism
We speculate that the cerebrovasodilator neural circuitry emanating from the P A G subdivision may also subserve to provoke the cerebrovasodilatation observed during cold exposure, since, as outlined above, this subdivision is one of the preferential target sites of nociceptive and thermoreceptive fibres. We found, however, that, when compared with the C o B F increase provoked by the P A G subdivision, the flow increase occurring during cold exposure was coupled far more tightly to the oxidative metabolic mechanism. These contrasting findings could reflect a difference whereby the cold exposure tended to activate more diverse neural circuits, which would be capable of facilitating the cortical oxidative metabolism, than the P A G subdivision. Thus, during the cold exposure, the cerebrovasodilator action of the oxidative metabolism may overweigh the concomitantly involved non-oxidative component of the vasodilatation mediated by the P A G subdivision. We consider that such diverse circuits may include, at least, the following two cortical afferents (both of which are known to be nociceptive and, when stimulated, provoke a cerebrovasodilatation and cortical activation): the thalamocortical projection emanating from the midline and intralaminar thalamic nuclei, 4'1°'21 and extrathalamic cortical afferents of the basal forebrain cholinergic system. 5'2°,3~'35
1139 CONCLUSION
It was found in the present study that the increase in cortical blood flow provoked by the caudal third of the lateral column within the periaqueductal gray was dependent on the cerebrovasodilator mechanism that may be coupled to the cortical oxidative metabolism to the extent of one-half, and on certain other mechanisms for the remaining half. It is well established that this particular region serves to generate vigorous defensive reactions, which include vasodilatation of the hindlimb skeletal muscle in preparation for the muscular exertion of flight behaviour when coping with a threatening emergency. 2 In the same manner, this region should also help to meet the urgent demand for an enhanced blood supply to the cortex so as to prepare for cortical hyperactivity, which may be generated upon the execution of vigorous defensive behaviour. Such a preparatory mechanism has also been suggested in a manner in which the cerebellar fastigial nucleus may serve to couple C o B F increases in anticipation of a wide range of motivated behaviours. 22,33 Acknowledgements--This study was supported by the NCVC Research Institute (92-93), by the STA (Special Coordination Funds for Promoting Science and Technology, COE), by the MHW (Cardiovascular Diseases 6A-3) and by the MESC (Scientific Research 06670072). The authors wish to acknowledge Inter Medical Co. Ltd (3-32-16 Kasuya, Setagaya, Tokyo 157, Japan) for collaboration in fabricating the device (NTM-IR) designed for sampling cerebral sinus blood.
REFERENCES
1. Bandler R., Carrive P. and Zhang S. P. (1991) Integration of somatic and autonomic reactions within the midbrain periaqueductal gray: viscerotopic, somatotopic and functional organization. Prog. Brain Res. 87, 269-305. 2. Bandler R. and Shipley M. T. (1994) Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci. 17, 379-389. 3. Blomqvist A. and Craig A. D. (1991) Organization of spinal and trigeminal input to the PAG. In The Midbrain Periaqueductal Gray Matter (eds Depaulis A. and Bandler R.), pp.345-363. Plenum Press, New York. 4. Bullitt E. (1990) Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J. comp. Neurol. 296, 517-530. 5. Buzsaki G., Bickford R. G., Ponomareff G., Thai L. J., Mandel R. and Gage F. H. (1988) Nucleus basalis and thalamic control of neocortical activity in the freely moving rat. J. Neurosci. 8, 4007-4026. 6. Carrive P. (1993) The periaqueductal gray and defensive behaviour: functional representation and neuronal organization. Behav. Brain Res. 58, 27-47. 7. Fox P. T., Raichle M. E., Mintun M. A. and Dence C. (1988) Non-oxidative glucose consumption during focal physiologic neural activity. Science 241, 462 464. 8. Goadsby P. J. (1990) Sphenopalatine ganglion stimulation increases regional cerebral blood flow independent of glucose utilization in the cat. Brain Res. 506, 145 148. 9. Goadsby P. and Hoskin K. L. (1994) Cerebral blood flow is not coupled to neuronal activity during stimulation of the facial nerve vasodilator system. Brain Res. 647, 192-198. 10. Groenewegen H. J. and Berendse H. W. (1994) The specificity of the 'non-specific' midline and intralaminar thalamic nuclei. Trends Neurosci. 17, 52 57. 11. Heistad D. D. and Kontos H. A. (1983) Cerebral circulation. In Handbook o f Physiology, The Cardiovascular System III. Peripheral Circulation and Organ Blood Flow (eds Shepherd J. T., Abboud F. M. and Geiger S. R.), Chapter 5, pp.137-183. American Physiological Society, Bethesda, MD. 12. Hilton S. M. and Redfern W. S. (1986) A search for brain stem cell groups integrating the defence reaction in the rat. J. Physiol. 378, 213528. 13. Iadecola C. (1993) Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link? Trends Neurosci. 16, 206-214. 14. Iadecola C., Pelligrino D. A., Moskowitz M. A. and Lassen N. A. (1994) Nitric oxide synthase inhibition and cerebrovascular regulation. J. cerebr. Blood Flow Metab. 14, 175 192. 15. Iadecola C. and Xu X. (1994) Nitro-L-arginine attenuates hypercapnic cerebrovasodilation without affecting cerebral metabolism. Am. J. Physiol. 266, R518-R525. 16. Keay K. A. and Bandler R. (1992) Anatomical evidence for segregated input from the upper cervical spinal cord to functionally distinct regions of the periaqueductal gray region of the cat. Neurosci. Lett. 139, 143-148.
1140
M. Nakai and M. Maeda
17. Keay K. A. and Bandler R. (1993) Deep and superficial noxious stimulation increases Fos-like immunoreactivity in different regions of the midbrain periaqueductal grey of the rat. Neurosci. Lett. 154, 23-26. 18. Madsen P. L., Hasselbalch S. G., Hagemann L. P., Olsen K. S., Bulow J., Holm S., Wildschiodtz G., Paulson O. B. and Lassen N. A. (1995) Persistent resetting of the cerebral oxygen/glucose uptake ratio by brain activation: evidence obtained with the Kety-Schmidt technique. J. cerebr. Blood Flow Metab. 15, 485-491. 19. Magistretti P. J. (1994) Vasoactive intestinal peptide and noradrenaline regulate energy metabolism in astrocytes: a physiological function in the control of local homeostasis within the CNS. Prog. Brain Res. 100, 87 93. 20. Metherate R., Cox C. L. and Ashe J. H. (1992) Cellular bases ofneocortical activation: modulation of neural oscillations by the nucleus basalis and endogenous acetylcholine. J. Neurosci. 12, 4701 4711. 21. Mraovitch S., Feger J., Calando Y. and Seylaz J. (1989) Cardiovascular and cerebrovascular alterations elicited by cholinergic stimulation of the centromedian-parafascicular complex in the rat. In Neurotransmission and Cerebrovascular Function I (eds Seylaz J. and MacKenzie E. T.), pp.397 400. Elsevier Science, Amsterdam. 22. Nakai M., Iadecola C., Ruggiero D. A., Tucker L. W. and Reis D. J. (1983) Electrical stimulation of cerebellar fastigial nucleus increases cerebral cortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation. Brain Res. 260, 35-49. 23. Nakai M. and Maeda M. (1994) Systemic and regional haemodynamic responses elicited by microinjection of N-methyl-D-aspartate into the lateral periaqueductal gray matter in anaesthetized rats. Neuroscience 58, 777-783. 24. Nakai M. and Maeda M. (1994) Cerebrovasodilatation of metabolic and non-metabolic origin elicited by chemical stimulation of the lateral periaqueductal gray matter in anaesthetized rats. Neuroscience 58, 785-791. 25. Nakai M., Tamaki K. and Maeda M. (1991) Multiple measurement of regional blood flow in rat brain using radiolabeled microspheres. Du Pont Biotech Update 6, (4), 1-3 (with an illustration of experimental layout). 26. Nakai M., Tamaki K. and Maeda M. (1991) Hyperperfusion of the rat brain during reflex hypertension induced by sudden lowering of the cutaneous temperature. J. auton, nerv. Syst. 35, 71 82. 27. Nakai M., Tamaki K. and Maeda M. (1991) Hyperperfusion and enhanced metabolic state of the brain during sudden lowering of the cutaneous temperature in rats. J. auton, nerv. Syst. 35, 83-92. 28. Nakai M., Tamaki K. and Maeda M. (1992) Sympathetic and metabolic mechanisms of the cerebrovasomotor function of the caudal ventrolateral medulla in rats. Neuroscience 50, 655 662. 29. Nakai M., Tamaki K., Ogata J., Matsui Y. and Maeda M. (1993) Parasympathetic cerebrovasodilator center of the facial nerve. Circulation Res. 72, 470-475. 30. Nakai M., Tamaki K., Yamamoto J. and Maeda M. (1990) A minimally invasive technique for multiple measurement of regional blood flow of the rat brain using radiolabeled microspheres. Brain Res. 507, 168 171. 31. Peschanski M., Guilbaud G. and Gautron M. (1981) Posterior intralaminar region in rat: neuronal responses to noxious and nonnoxious cutaneous stimuli. Expl Neurol. 72, 226 238. 32. Pompeiano O. (1973) Reticular formation. In Handbook o f Sensory Physiology II (ed. Iggo A.), pp.381-488. Springer, Berlin. 33. Reis D. J., Doba N. and Nathan M. A. (1973) Predatory attack, grooming and consummatory behavior evoked by electrical stimulation of cerebellar nuclei in cat. Science 182, 845-847. 34. Rothman S. M. and Olney J. W. (1987) Excitotoxicity and the NMDA receptor. Trends Neurosci. 10, 299 302. 35. Sato A. and Sato Y. (1992) Regulation of regional cerebral blood flow by cholinergic fibers originating in the basal forebrain. J. Neurosci. Res. 14, 242 274. 36. Suzuki N. and Hardebo E. (1993) The cerebrovascular parasympathetic innervation. Cerebrovasc. Brain Metab. Rev. 5, 33 46. 37. Wallenstein S., Zucker C. L. and Fleiss J. L. (1980) Some statistical methods useful in circulation research. Circulation Res. 47, 1-9. (Accepted 4 January 1996)