Journal of Cerebral Blood Flow and Metabolism 10:375-382 © 1990 Raven Press, Ltd., New York
Dissociation by Chloralose of the Cardiovascular and Cerebrovascular Responses Evoked from the Cerebellar Fastigial Nucleus
Costantino ladecola, Mary E. Springston, and Donald J. Reis Division of Neurobiology, Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York, U.S.A.
Summary: We studied the effects of chloralose anesthe sia on the elevation in arterial pressure (AP), heart rate (HR), and regional CBF (rCBF) elicited by stimulation of the cerebellar fastigial nucleus (FN). Rats were anesthe tized with an initial dose of chloralose (40 mg/kg s.c.), paralyzed, and artificially ventilated. The FN was stimu lated (50-100 fLA, 50 Hz, I s onll s off) with microelec trodes stereotaxically implanted. During the stimulation AP was carefully maintained within cerebrovascular au toregulation. CBF was measured by the [14C]iodoanti pyrine technique with regional dissection. In rats that re ceived only the initial dose of chloralose, FN stimulation elevated rCBF in brain and spinal cord, up to 209 ± 13% of control in frontal cortex (n = 5; p < 0.0 1, analysis of variance). Administration of additional chloralose ( 10 mg/
kg i.v., 30 min prior to measurement of CBF) did not affect resting rCBF (n = 5), the EEG, or the elevation in AP and HR elicited by FN stimulation (n = 4). However, the additional chloralose abolished the elevations in rCBF (n = 5; p > 0.05). Thus, the cerebrovasodilation elicited from the FN is more susceptible to the effects of addi tional anesthesia than the elevation in AP and HR. These results indicate that the cerebrovascular and cardiovas cular responses elicited from the FN are functionally dis tinct and provide additional evidence for the notion that these responses are mediated by different neural path ways and transmitters. Key Words: Anesthesia Cerebellum-Cerebral blood flow-e4C]Iodoantipy rine-Spinal cord blood flow.
Electrical stimulation of the rostral ventromedial portion of the cerebellar fastigial nucleus (FN) in rat, as in cat, rabbit, and monkey, elicits a global increase in regional CBF (rCBF) (Doba and Reis, 1972; McKee et aI., 1976; Nakai et aI., 1982; Reis et aI., 1982; Mraovitch et aI., 1986; Maeda, 1988; Goadsby and Lambert, 1989; Iadecola and Reis, 1989). The cerebrovasodilation is greatest in the ce rebral cortex (up to 2 15% of control), wherein the rCBF increases are largely independent of changes in local metabolism (Nakai et aI., 1983) and are me diated by neural pathways contained entirely within the brain (Nakai et aI., 1982; Iadecola et aI., 1983b,
1987a). In addition to its actions on the cerebral circulation, stimulation of the FN produces power ful effects on the systemic circulation in several species. These include an elevation in arterial pres sure (AP) and heart rate (HR) (Miura and Reis, 1970; Dormer and Stone, 1976; McKee et aI., 1976; Nakai et aI., 1982; Reis et aI., 1982) and release of renin (Koyama et aI., 1980), vasopressin (Del Bo et aI., 1982b), and adrenal catecholamines (Del Bo et aI., 1982a). These responses have been collectively termed the fastigial pressor response (FPR) (Miura and Reis, 1970). The neural substrates underlying the cardiovas cular and cerebrovascular components of the FPR are unknown. While it has been proposed that they are initiated by different neuronal pools (Mraovitch et aI., 1986), recent studies suggest that the path ways mediating both components are closely asso ciated at some sites and may go through a common relay station within the C l area of the rostral vent rolateral medulla (Chida et aI., 1989). The latter
Received September 1 1, 1989; revised November 23, 1989; accepted November 27, 1989. Address correspondence and reprint requests to Dr. C. lade cola at Laboratory of Neurobiology, 4 1 1 E. 69 St., New York, NY 10021, U.S.A. Abbreviations used: AP, arterial pressure; FN, fastigial nu cleus; FPR, fastigial pressor response; HR, heart rate; lAP, io doantipyrine; rCBF, regional CBF.
375
C. IADECOLA ET AL.
376
may indicate that the effects on systemic and cere bral circulation are mediated through the same ba sic neuronal circuitry. However, a recent study in cats anesthetized with doses of chloralose larger than previously used has failed to demonstrate the vasodilation in the cerebral cortex, while the changes in AP and HR were preserved (Williams et al., 1987, 1989). These findings are of interest as they raise the possibility that chloralose, above a critical threshold, may se lectively affect the cerebrovasodilation, sparing the systemic cardiovascular changes elicited from the FN. This would not be surprising as chloralose does not depress cardiovascular reflexes (Cox and Bag shaw, 1979) while it suppresses spontaneous and evoked neuronal activity (Collins et al., 1983). In this study we have therefore investigated whether chloralose is able to dissociate the cardio vascular and cerebrovascular effects of FN stimu lation. We shall demonstrate that administration of an additional dose of chloralose abolishes the va sodilation evoked from the FN in brain and in spinal cord without affecting the elevations in AP and HR. These findings indicate that the cardiovascular and cerebrovascular components of the FPR can be dis sociated and probably are mediated through distinct neural mechanisms. METHODS
Methods for surgical preparation of rats, electrical stimulation of the FN with controlled AP, and measure ment of rCBF have been described in detail in earlier publications (Nakai et aI., 1982; ladecola et aI., 1983a, 1987a,b) and will only be summarized here. General procedures
Studies were performed on 24 male Sprague-Dawley rats weighing 320-390 g maintained in a light-cycled (0700 h oni1900 h off), thermally controlled (27°C) environment and fed lab chow ad libitum. A 1% solution of chloralose in 0. 9% NaCI was prepared. After adding chloralose to prewarmed 0. 9% NaCI, the mixture was stirred and heated until the anesthetic was completely solubilized. The solution was not allowed to boil. Rats were anesthetized with chloralose (40 mg/kg, s.c.) after induction with 5% isoflurane (Anaquest) in 100% oxygen, administered through a facial mask. During sur gery isoflurane was administered at a reduced rate (2%). Catheters were inserted in both femoral arteries and veins and the trachea was cannulated. Animals were then placed on a stereotaxic frame (Kopf), paralyzed with tu bocurarine (2 mglkg i.m.), and artificially ventilated with 100% oxygen by a mechanical ventilator (rodent respira tor; Harvard Apparatus). One of the arterial catheters was connected to a pressure transducer (Gould) fed into a polygraph (Grass) for continuous recording of AP, mean AP, and HR. Body temperature was maintained at 37 ± O.soC using a heating lamp thermostatically controlled by a rectal probe (YSI). The midline portion of the caudal
J
Cereb Blood Flow Metab. Vol. 10, No.3. 1990
occipital bone was removed to expose the cerebellar ver mis and the dorsal medulla. A stainless-steel screw was inserted through the left parietal bone to lie on the dura at a site 2 mm lateral to the midline and 2 mm caudal to bregma for monopolar recording of the EEG. At the end of the surgery, isoflurane anesthesia was discontinued. Arterial Pcoz, POz, and pH were measured on 0. 2 ml of blood by using a blood gas analyzer (Micro 13 System; Instrumentation Laboratory). Stimulation of the FN
The FN was stimulated monopolarly with negative square-wave pulses delivered through a stimulus isolation unit from a constant-current stimulator (Grass S88). Elec trodes were fabricated from Teflon-coated stainless-steel wires (diameter 150 /lorn) with the cut tip exposed. The ground was a metal clip attached to the animal's scalp. The stimulus current was measured and displayed on an oscilloscope as previously described (Nakai et aI., 1982). Measurement of CBF
CBF was measured in brain homogenates by using e4C]iodoantipyrine ([14C]lAP) as diffusible indicator (Sakurada et aI., 1978; Ohno et aI., 1979). As described in detail elsewhere (Nakai et aI., 1982; ladecola et aI., 1983a), lAP (spec. act. 40-60 mCilmmol; NEN) was dis solved in -1 ml of saline after elimination of ethanol and infused intravenously at a constant rate for 30 s by using an infusion pump (model 940; Harvard Apparatus). Prior to the start of the infusion, 500 units of heparin (Elkins Sinn) was administered intravenously. During the lAP infusion, timed arterial samples were collected at -2- to 5-s intervals to determine the arterial concentration-time course of the isotope (Iadecola et aI., 1983a). Toward the end of the infusion, during the collection of the last blood sample, the heart was stopped by an intravenous bolus injection of 1 ml of saturated KCI. The brain and the upper portion of the cervical cord were rapidly removed and placed on a cooled glass plate. Large vessels on the brain surface were removed and 14 regions were dis sected out. These included upper cervical cord (CI-C3), medulla, cerebellum, pons, inferior colliculus, superior colliculus, hypothalamus, thalamus, hippocampus, cau date nucleus, frontal, occipital, and parietal cortices, and corpus callosum. Tissue samples were placed in pre weighed vials that were then reweighed for determination of the sample's weight. After adding 1 ml of Protosol (NEN) to each sample, the vials were placed in a shaking water bath at 60°C for 6 h to allow for tissue solubiliza tion. The radioactivity of the samples (dpm) was deter mined by a liquid scintillation spectrophotometer (Beck man) using procedures described in earlier pUblications (Nakai et aI., 1982; ladecola et aI., 1987b). For determi nation of lAP concentration in blood, 40-/lol aliquots of arterial blood were transferred in vials containing 1 ml of Protosol-ethanol (NEN) and processed for determination of radioactivity (dpm) by liquid scintillation counting (Ia decola et aI., 1987a,b). CBF (mUloo glmin) was calcu lated from the arterial time course of lAP concentration (nCi/g) and tissue concentration of lAP (nCilg) using the equation described by Kety (see Nakai et aI., 1982). The lAP partition coefficient was set at 0. 8 (Sakurada et aI., 1978). Details on the accuracy of the computer program and on the computational resolution at high flow rates have been published (Nakai et aI., 1982).
CHLORALOSE AND CBF INCREASE FROM CEREBELLUM Experimental protocol
After establishment of the anesthesia, insertion of the catheters, artificial ventilation, and exposure of the cau dal brainstem, an electrode mounted on a micromanipu lator with a 10° posterior inclination was positioned on the calamus scriptorius and the stereotaxic coordinates re corded as stereotaxic zero (Nakai et al., 1982). The elec trode was then moved 5 mm rostral and 0.8 mm lateral from zero, lowered into the cerebellum until the vertical zero was reached, and left at that site until the exploration for the FN was begun (Nakai et al., 1982). Blood gases were carefully adjusted: Arterial Pco2 was maintained between 33 and 38 mm Hg by varying the stroke volume of the ventilator and Po, was maintained above 100 mm Hg by ventilating the animals with 100% 02' The latter was necessary to counteract the effects of atelectasis occurring in artificially ventilated rats (Nathan and Reis, 1975) and to compensate for any loss of oxygen carrying capacity resulting from blood sampling and con trolled hemorrhage (see below). As a consequence of the high Fio2, the P02 of the animals was elevated (Table 1). However, the effects of this degree of hyperoxia on CBF are negligible (Reivich, 1968). After the blood gases were adjusted, the procedure for localization of the FN was started. As described else where (Nakai et al., 1982), the electrode was withdrawn in 0.5-mm steps and at each step exploratory stimuli, con sisting of 8-s trains of 0.5-ms pulses at 50 Hz and with a current intensity of 10-20 f..lA, were delivered. An active site in the FN was defined as one in which stimulation resulted in a stimulus-locked 10- to 20-mm Hg AP eleva tion. These small increases in AP are within the range of cerebrovascular autoregulation (Hernandez et al., 1978) and have no effect on CBF (C. Iadecola, unpublished observations). Once the most active site in FN was local ized, the electrode was left in place at that site. Blood gases were measured again and FN stimulation begun. The FN was stimulated with intermittent trains of stimuli (1 s onll s off, 50 Hz). During the first 2-4 min, the cur rent intensity was gradually increased up to five times the threshold current (i.e., the stimulus current producing a lO-mm Hg elevation in AP, usually 10-20 f..lA). At the same time, the evoked elevations in AP were offset by slow removal of arterial blood. By this procedure during FN stimulation AP never exceeded 150 mm Hg, a value that is within the autoregulated range of CBF in rats un der identical experimental conditions (Iadecola et al.,
377
1983b). Blood gases were measured, and, if necessary, adjusted. After 10-15 min of stimulation, at a time when AP and blood gases had been in a steady state for at least 5 min, the infusion of lAP for CBF measurement was begun. Thirty seconds later the heart was stopped by in travenous injection of KCl, the brain and cervical cord removed, and CBF determined as described above. In animals receiving supplemental anesthesia, 10 mg/kg of chloralose was administered by slow intravenous infusion 30 min prior to the measurement of CBF. In unstimulated animals surgical procedures and conduct of the experi ments were identical to those of rats in which the FN was stimulated with the exception that an electrode was low ered in the cerebellar vermis but no stimuli were deliv ered. Experiments were timed as follows. The time of the injection of chloralose at the beginning of the experiment (40 mglkg s.c.) was considered time zero. In all animals, with and without FN stimulation or supplemental anes thesia, CBF was measured within or near 3 h after the administration of chloralose (Table 1). This was neces sary to ensure stability of the level of anesthesia as es tablished in preliminary experiments. The state of anes thesia of the animals was assessed by EEG criteria (Win ters and Spooner, 1966). As shown in Fig. 1, the EEG 1 h after chloralose administration exhibits the low frequency (3-5 Hz) hypersynchronous burst activity (0.2OA mY) typical of chloralose anesthesia (Winters and Spooner, 1966). This distinctive EEG pattern persisted 2 and 3 h after the initial administration of chloralose and was not altered by stimulation of the FN (Fig. 1). That the animals were anesthetized is also demonstrated by the fact that their rCBF was low in comparison with that of awake animals (see Table 3; Ohno et al., 1979). Particu larly, rCBF was depressed in regions such as thalamus and cortex where the greatest increases occur during dis comfort and stress (Lacombe and Seylaz, 1984). Thus, during the experimental period, including the phase in which the FN was stimulated, rats were in a stable anes thetized state. Statistical analysis
Data were evaluated by the one-way analysis of vari ance and differences among multiple groups were as sessed by the Newman-Keuls test as a post hoc multiple comparison procedure (Tables 1 and 3). Two-group com parisons were analyzed by the paired t test (Table 2). For
5) in rats with and without chloralose supplementation or stimulation of the fastigial nucleus (FN)
TABLE 1. Arterial pressure (AP) and blood gases (mean ± SEM, n
=
Unstimulated: chloralose dose
FN stimulation: chloralose dose
40 mg/kg (control) --�--m - ---------------m �A ( g 126 H )
Pc02 (mm Hg) P02 (mm Hg)
pH Exp. timeb (min)
36 413 7.43 183
40 mg/kg 40 + 10 mg/kg (control) 40 + 10 mg/kg � - ---- - -- - C -------------- - - --- ---------± ---------------1 26 ± 5 ±4 ±5 116 2 134 ± 0 .6 36.6 ± 05 . 36.4 ± 1 .0 34.8 ± 0.7 392 ±8 413 ± 25 ± 33 377 ± 36 7 3 . 2 ± O.Old 7 4 . 0 ± 0.02 ± 0 .02 7.41 ± 0 .01 178 ±6 186 ± 8 ±5 185 ± 4
a During CBF measurement. b From chloralose administration at the beginning of the experiment. c p < 00 . 5 from FN stimulation control group.
dp
< 00 .1
(analysis of variance and Newman-Keuls test).
J
Cereb BLood FLow Metab. VoL. 10, No.3, 1990
C. IADECOLA ET AL.
378
1 hr
2 h'r
3 hr
]
FN stim
0.2 mV
(50 �A, 50 Hz) on
off 1
sec
FIG. 1. EEG in paralyzed, artificially ventilated rats 1, 2, and 3 h after administration of 40 mg/kg s.c. of chloralose and during
stimulation of the fastigial nucleus (FN stim). Note that the EEG exhibits the hypersynchronous burst pattern typical of chloralose anesthesia. This pattern is present throughout the 3-h period and during FN stimulation, which was carried out slightly over 3 h after the administration of chloralose. Thus, throughout the experimental period, rats are in a stable state of chloralose anesthesia.
both statistical procedures, differences were considered significant for probability values of < 0.05. RESULTS Effect of additional chloralose on FPR
The effects of supplemental anesthesia on the cardiovascular changes elicited by FN stimulation were studied in four rats. Before chloralose supple mentation, FN stimulation elicited the well established increases in AP and HR, i.e., the FPR. The minimal current required to produce a lO-mm Hg AP elevation (threshold current) ranged be tween lO and 20 !-LA (Fig. 2; Table 2). Administra tion of additional chloralose ( 10 mg/kg i.v.) did not affect resting AP and HR or the amplitude of the FPR (p > 0.05, paired t test; Table 2; Fig. 2). The threshold current was also unchanged (Table 2). Thus, additional chloralose does not affect the changes in AP and HR elicited by FN stimulation. Effect of additional chloralose on increase in CBF
Stimulation of the FN in untreated animals in creased rCBF in all regions studied (p < 0.0 1, anal ysis of variance and Newman-Keuls test; Table 3). The magnitude and regional distribution of the changes were similar to those previously reported from this laboratory (Nakai et aI., 1982, 1983; Iade cola et aI., 1983b, 1986, 1987a; Americ et aI., 1987), the greatest changes occurring in cerebral cortex J
Cereb Blood Flow Metab. Vol. 10. No. 3, 1990
(up to 209% of control in frontal cortex) and thala mus ( 190%). Substantial increases were also ob served in hippocampus ( 176%), caudate nucleus ( 180%), and, interestingly, cervical cord, where rCBF rose to 153% of control (p < 0.01). FN stim ulation had no effect on the EEG (Fig. 1). Administration of an additional dose of chloral ose ( 10 mg/kg i.v.) 30 min prior to CBF measure ment did not affect EEG activity (Fig. 3) or resting rCBF (p > 0.05; Table 3). However, the elevations in rCBF elicited by FN stimulation were virtually abolished throughout the entire brain and in the cer vical cord (p > 0.05) (Table 3). The small residual increases observed in medulla ( 1 19%) and superior colliculus ( 126%) (Table 3) did not reach statistical significance (p > 0.05). These small increases may reflect metabolical activation of sites receiving di rect (monosynaptic) projections from the FN (Del Bo et aI., 1982; Nakai et aI., 1983). DISCUSSION
We have demonstrated that the global cerebro vascular vasodilation elicited by electrical stimula tion of the FN is more sensitive to chloralose than the FPR. Thus, when chloralose is given at an initial dose of 40 mg/kg, FN stimulation increases AP, HR, and rCBF. However, supplementing this initial dose with additional chloralose abolishes the cere-
379
CHLORALOSE AND CBF INCREASE FROM CEREBELLUM 4) on cardiovascular changes elicited by electrical stimulation of the fastigial nucleus (FN stim)
TABLE 2. Effect of chloralose supplementation (mean ± SEM, n
=
Before chloralose
Arterial pressure (mm Hg) Heart rate (beats/min)
Mter chloralosea
Resting
FN stirn
Il
116 ± 6 324 ± 43
166 ± 7 424 ± 22
100 ± 36
50 ± 4
Resting
FN stirn
Il
114 ± 5
166 ± 5 406 ± 20 12 ± 1
106 ± 40
300 ± 47
11 ± 1
Threshold current (f.LA)
52 ± 5
Stimulation parameters: 8-s trains, 50 Hz, current intensity set at three times threshold current. Differences before and after chloralose were not significant (p > 0.05, paired t test). a 30 min after chloralose supplementation (10 mg/kg).
of additional chloralose did not produce further de creases in rCBF, a variable closely "coupled" to cerebral glucose utilization even during chloralose anesthesia (Nakai et aI., 1983), and did not influ ence EEG activity. A more likely scenario is that chloralose, above a specific dose, impairs and/or modifies the functional characteristics of a critical group of neurons that is essential to the expression of the cerebrovasodilation but not of the FPR. The effects on CBF of changes in activity of this, pre sumably small, neuronal population must be be yond the relatively limited resolution of the tissue sampling technique used in this study to measure
brovasodilation without affecting the hypertension and tachycardia. These results indicate that this agent within the appropriate dose range dissociates the cardiovascular and cerebrovascular compo nents of the response. The mechanisms whereby chloralose supplemen tation abolishes the cerebrovasodilation elicited from the FN are unknown. It is unlikely that the effect of chloralose on the cerebrovasodilation is a nonspecific consequence of global depression of ce rebral metabolic activity. While this agent lowers cerebral glucose utilization throughout the brain (Dudley et aI., 1982), we found that administration
200 AP
(mm Hg)
125 50 20 0
MAP
(mm Hg)
125 50
HR
5 00
(beats/min) 300
l l
-
[
Stimulus (50 Hz)
wi\,.
.A.
r -1.
...
J\,.
�
A
�
�
R-
-----
-----v--
0
0
10 IlA
151lA
0 45 IlA
-
1
Chloralose 10
0 10 IJA
mg/kg.
�
0 15 IlA
--'\...--
0 45 IJA
Lv.
min FIG. 2. Effect of administration of a supplemental dose of chloralose (10 mg/kg Lv.) on the elevation in arterial pressure (AP). mean AP (MAP). and heart rate (HR) elicited by electrical stimulation of the cerebellar fastigial nucleus (FN) in rat. Animals were anesthetized with an initial dose of chloralose (40 mg/kg s.c.). paralyzed, and artificially ventilated. Left: Effect of FN stimulation (8 s, 50 Hz, 10-45 f.LA) on AP, MAP, and HR. The response appears at 15 f.LA (threshold current) and stimulation at three times threshold produces MAP elevations of -50 mm Hg. Right: Thirty minutes after administration of supplemental chloralose, the threshold current and the amplitude of the pressor response remain unchanged.
J
Cereb Blood Flow Metab, Vol. 10, No.3, 1990
C. IADECOLA ET AL.
380
(10 mg/kg; mean ± SEM, n 5) on increase in CBF elicited by stimulation of the fastigial nucleus (FN) in rat
TABLE 3. Effect of chloralose supplementation
=
CBF (mI/100 g/min) Unstimulated:
FN stimulation:
chloralose dose
chloralose dose
40 mg/kg (control) Cervical cord Medulla Cerebellum Pons Inf. colliculus Sup. colliculus Hypothalamus Thalamus Hippocampus Caudate nucleus Frontal cortex Parietal cortex Occipital cortex Corpus callosum
40 + 10 mg/kg
40 mg/kg (control)
4 5 4 5 7 7
86 139 126 142 156 148
± ± ± ± ± ±
6 13 10 13 13 13
89 ± 7 71 ± 6
75 ± 6 66 ± 4
65 ± 6
62 70 69 77 67 49
122 136 114 131
± ± ± ±
10 13 11 10
56 82 74 82 92 88
± ± ± ± ± ±
5 10 7 7 1O
II
73 ± 6 72 ± 6 80 ± 6 68 ± 5 49 ± 6
55 76 79 83 95 85
± ± ± ± ± ±
± ± ± ± ± ±
4 5 3 4 4 4
151 ± 10 153 ± 8 135 ± 12 84 ± 8
% of
control 153b 169b l70b 173b 170b 167b 137b 191b 176b 180b 209b 190b 197b 171b
40 + 10 mg/kg 60 90 85 93 104 107
± ±
± ± ±
±
% of controla 109 119 108 112 110 126 106 110
5 7 6 7 4 8
79 ± 5 73 ± 7 62 ± 6 66 ± 5 69 ± 6
100 95 100 106 97
82 ± 7 65 ± 5 47 ± 3
96
a Differences from respective control were not significant (p > 0.05). b P < 0.01 (analysis of variance and Newman-Keuls test).
rCBF (Iadecola et al., 1983c). Another possibility is that chloralose acts directly at the neurovascular effector level by interfering with the mechanisms governing the interaction between neurons and ce rebral microvasculature or, less likely, by affecting vascular smooth muscle and/or endothelial func tion. There is no experimental evidence to support the latter possibilities. Irrespective of the mecha-
nisms, this effect of chloralose occurs above a threshold concentration that is reached with the ad ministration of an additional dose 30 min prior to measuring rCBF. In contrast to its dramatic action on the cerebral circulation, chloralose supplementation does not af fect the FPR. Thus, the cerebrovascular and car diovascular actions exerted by the FN are distinct
0 min
t
Chloralose 10 mg/kg. i.v.
5 min
15 min
30 min
JO.2
mV
L-.J 1 sec FIG. 3. Effect of administration of a supplemental dose of chloralose (10 mg/kg Lv.) on the EEG. Rats were anesthetized with an initial dose of chloralose (40 mg/kg s.c.). paralyzed, and artificially ventilated. Administration of chloralose did not affect EEG.
J
Cereb Blood Flow Metab, Vol. 10. No.3, 1990
CHLORALOSE AND CBF INCREASE FROM CEREBELLUM
with respect to the action of chloralose. The biolog ical basis of this distinction must reside in differ ences in the neural substrates mediating cardiovas cular and cerebrovascular responses evoked from the FN, as well as in a selectivity in the action of chloralose, which spares (Cox and Bagshaw, 1979) or even enhances (Stephenson and Donald, 1980) reflex cardiovascular responses. A dissociation be tween cardiovascular and cerebrovascular effects occurs also after systemic administration of atro pine, a treatment that abolishes the increase in rCBF without affecting the FPR (Iadecola et aI., 1986). Thus, while the cerebrovasodilative pathway involves at least one cholinergic synapse, the path way mediating the FPR does not. In addition, elec trical stimulation of the caudal portion of the FN produces increases in cortical CBF that are not as sociated with changes in AP or HR (Mraovitch et aI., 1986). This finding has been interpreted to indi cate that cerebrovascular and cardiovascular re sponses may originate from separate neuronal pools within the FN (Mraovitch et aI., 1986). However, this issue is complicated by the recent discovery that the pathways mediating both the FPR and the cerebrovascular effects originate from fibers pro tecting into or passing through the area and not from FN neurons (Chida et aI., 1986, 1989). It is thus conceivable that these pathways arise from separate brainstem sites and then converge while terminating into or passing through the FN on their course to their next relay station. The present results indicate that the recent failure to confirm the cortical cerebrovasodilation elicited from the FN in cats using the microsphere tech nique to measure rCBF is probably a consequence of the use of higher initial doses of chloralose (70 mg/kg) followed by multiple supplementations throughout the experiment (Williams et aI., 1987, 1989). Interestingly, these authors found small «30%) increases in brainstem regions that corre spond to sites receiving primary projections from the FN (Del Bo et aI., 1982). This observation sup ports the contention, suggested also by the present study, that monosynaptic connections may be more resistant to the effects of chloralose on neurogenic vasodilation. In any event, it is unlikely that species differences account for the inability to reproduce the response because large increases in CBF have been reported in cats during stimulation of the FN by independent laboratories and using different techniques to measure CBF (Doba and Reis, 1972; Maeda, 1988; P. J. Goadsby, personal communica tion). The effects of chloralose on the cerebrovasodila tion elicited by FN stimulation are not specific to
381
this agent but are seen also with other general an esthetics. Using a laser-Doppler perfusion probe to monitor changes in CBF over the parietal cortex of spinal cord-transected rats anesthetized with iso flurane, we have observed that the vasodilation is abolished by a cumulative dose of pentobarbital of 30 mg/kg i.v. (Iadecola and Reis, 1989). Similarly, an increase in concentration of the volatile anes thetic isoflurane from 1 to 2% significantly attenu ates the vasodilation (C. Iadecola, unpublished ob servations). The finding that the cerebrovasodila tion is exquisitely sensitive to agents with an inhibitory effect on neuronal and synaptic activity provides strong support for the notion that the in crease in CBF elicited from the FN is neurally me diated. However, it is of interest that the increase in cortical CBF elicited by somatosensory stimulation is preserved at doses of chloralose of 80 mg/kg (Ueki et aI., 1988), while the flow increase elicited from the FN is abolished at a cumulative dose of 50 mg/kg. This difference may indicate that the neural systems mediating the "primary" vasodilation evoked from the FN are distinct, with respect to the action of anesthetics, from those involved in the "metabolic" vasodilation associated with cortical activation. Another finding of the present study is that FN stimulation produces substantial increases in spinal cord blood flow. This new observation demon strates that the effects of FN are not restricted to the brain but rather involve the circulation of the entire neuraxis. Whether neurogenic factors are ca pable of influencing the circulation of the spinal cord is not well established (Marcus et aI., 1977). We have recently shown that stimulation of the dor sal medullary reticular formation can increase flow in all segments of the spinal cord, an effect that is attenuated by removal of the adrenal glands (Iade cola et aI., 1989). Thus, in the spinal cord, as in brain, the increases in blood flow elicited by stim ulation of the dorsal medullary reticular formation depend in part on adrenal catecholamines and are probably mediated by an increase in local metabo lism (Iadecola et aI., 1987b). Whether the increases in spinal cord blood flow elicited by FN stimulation are metabolically mediated or whether, as in most of the cerebral cortex, they are independent of local metabolic activity remains to be determined. In conclusion, we have shown that the wide spread cardiovascular and cerebrovascular changes elicited by FN stimulation can be dissociated by the use of a critical dose of chloralose. This finding, while supporting the notion that the neural sub strates mediating the cardiovascular and cerebro vascular responses evoked from the FN are funcJ
Cereb Blood Flow Metab, Vol. ]0, No.3, 1990
382
C. IADECOLA ET AL.
tionally distinct, provides further evidence for the concept that the circulation of the brain and spinal cord is subject to substantial neurogenic influences. The integrated neurovascular mechanisms deter mining these effects, their ultimate cellular and mo lecular mediators, and, most importantly, their bi ological significance remain to be unraveled.
Role of local neurons in the cerebrocortical vasodilation elic ited from cerebellum. Am J Physiol 252:RI082-RI091 Iadecola C, Lacombe PM, Ishitsuka T, Underwood MD, Reis DJ (I987b) Role of adrenal catecholamines in the cerebrova sodilation evoked from the brain stem. Am J Physiol 252: H1183-H1191
Iadecola C, Chida K, Lacombe PM, Underwood MD, Reis DJ
vasodilation elicited by focal electrical stimulation within the dorsal medullary reticular formation in anesthetized rat. J Cereb Blood Flow Metab 3:270-279
(1989) Global increase in spinal cord blood flow elicited from the dorsal medullary reticular formation in rat. J Cereb Blood Flow Metab 9:S480 Koyama S, Ammons WS, Manning JW (1980) Altered renal vas cular tone and plasma renin activity due to fastigial and baro receptor activation. Am J Physiol 239:H 232-H237 LaCombe PM, Seylaz J (1984) Significance of the cerebrovascu lar effects of immobilization stress in the rabbit. J Cereb Blood Flow Metab 4:397-406 Maeda M (1988) Changes in intracranial pressure elicited by elec trical stimulation of the brain stem reticular formation in spinal cats with vagotomy. J Auton Nerv Syst 25:155-164 Marcus ML, Heistad DD, Ehrhardt JC, Abboud FM (1977) Reg ulation of total and regional spinal cord blood flow. Circ Res 41:128-134 McKee JC, Denn MJ, Stone HL (1976) Neurogenic cerebral va sodilation from electrical stimulation of the cerebellum in the monkey. Stroke 7: 179-186 Miura M, Reis DJ (1970) A blood pressure response from fasti gial nucleus and its relay pathways in the brainstem. Am J Physiol 219:1330-1336 Mraovitch S, Pinard E, Seylaz J (1986) Two neural mechanisms in rat fastigial nucleus regulating systemic and cerebral cir culation. Am J PhysioI 251:HI53-HI63 Nakai M, Iadecola C, Reis DJ (1982) Global cerebral vasodila tion by stimulation of rat fastigial cerebellar nucleus. Am J Physiol 243:H 2 26-H235 Nakai M, Iadecola C, Ruggiero DA, Tucker LW, Reis DJ (1983) Electrical stimulation of cerebellar fastigial nucleus in creases cerebrocortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation. Brain Res 260:35-49 Nathan MA, Reis DJ (1975) Hypoxemia, atelectasis and the el evation of arterial pressure and heart rate in paralyzed arti ficially ventilated rats. Life Sci 16:1103-1120 Ohno K, Pettigrew KD, Rapoport SI (1979) Local cerebral blood flow in the conscious rat as measured with CI4-iodoanti pyrine and H3-nicotine. Stroke 10:62-67 Reis DJ, Iadecola C, MacKenzie E, Mori M, Nakai M, Tucker LW (1982) Primary and metabolically coupled cerebrovas cular dilation elicited by stimulation of two intrinsic systems of brain. In: Cerebral Blood Flow: Effects of Nerves and Neurotransmitters (Heistad DD, Marcus ML, eds), New York, Elsevier/North Holland, pp 475-484 Reivich M (1968) Cerebral circulatory responses to respiratory influences. In: Cerebral Vascular Diseases (Toole JF, Siekert RG, Wishnant JP, eds), New York, Grune and Strat ton, pp 91-100 Sakurada 0, Kennedy C, Jehle J, Brown JD, Carbin GL, Sokoloff L (1978) Measurement of local cerebral blood flow with iodo(C14)antipyrine. Am J Physiol 234:H59-H66 Stephenson RB, Donald DE (1980) Reflexes from isolated ca rotid sinuses of intact and vagotomized conscious dogs. Am J Physiol 238:H815-H8 2 2
Iadecola C, LeDoux JE, Thompson ME, Tucker LW, Reis DJ (l983c) Focal increases in cerebral blood flow during brief
Williams JL, Heistad DD, Siems JL, Talman WT (1987) Effect of stimulation of fastigial nucleus on cerebral blood flow in cats. Soc Neurosci Abstr 13:283
REFERENCES Arneric SP, ladecola C, Underwood MD, Reis DJ (1987) Local cholinergic mechanisms participate in the increase in corti cal cerebral blood flow elicited by electrical stimulation of the fastigial nucleus in rat. Brain Res 411: 212-225 Chida K, Iadecola C, Underwood M, Reis DJ (1986) A novel vasodepressor response elicited from the rat cerebellar fas tigial nucleus: the fastigial depressor response. Brain Res
370:378-382 Chida K, ladecola C, Reis DJ (1990) Lesions of rostral ventro lateral medulla abolish some cardio- and cerebrovascular components of the cerebellar pressor and depressor re sponses. Brain Res (in press) Collins JG, Kawahara M, Homma E, Kitahata LM (1983) Alpha chloralose suppression of neuronal activity. Life Sci 32: 2995-2999 Cox RH, Bagshaw DRJ (1979) Influence of anesthesia on the response to carotid sinus hypotension in dogs. Am J Physiol 237:H424-H432 Del Bo A, Ruggiero DA, Ross CA, Wiley R, Reis DJ (1982) Pathways from the fastigial pressor area in rat. Soc Neurosci Abstr 8:76
Del Bo A, Ross Ca, Pardal JF, Saavedra JM, Reis DJ (l983a) Fastigial stimulation in rats releases adrenomedullary cate cholamines. Am J Physiol 244:R801-R809 Del Bo A, Sved AF, Reis DJ (I983b) Fastigial stimulation re leases vasopressin in amounts which elevate arterial pres sure. Am J Physiol 244:H687-H694
Doba N, Reis DJ (1972) Changes in regional blood flow and cardiodynamics evoked by electrical stimulation of the fas tigial nucleus in cat and their similarity to orthostatic re flexes. J Physiol (Lond) 227:7 29-747
Dormer KJ, Stone HL (1976) Cerebellar pressor response in the dog. Am J Physiol 41:574-580
Dudley RE, Nelson SR, Samson F (1982) Influence of chlora lose on brain regional glucose utilization. Brain Res 233:173180
Goadsby PJ, Lambert GA (1990) Electrical stimulation of the fastigial nucleus increases total cerebral blood flow in the monkey. Neurosci Lett 107:141-144 Hernandez MJ, Brennan RW, Bowman GS (1978) Cerebral blood flow autoregulation in the rat. Stroke 9:150-155
Iadecola C, Reis DJ (1990) Cerebral blood flow by laser-Doppler flowmetry during stimulation of the cerebellum. J Cereb
Blood Flow Metab (in press)
Iadecola C, Nakai M, Arbit E, Reis DJ (I983a) Global cerebral
Iadecola C, Mraovitch S, Meeley MP, Reis DJ (l983b) Lesions of the basal forebrain in rat selectively impair the cortical vasodilation elicited from cerebellar fastigial nucleus. Brain Res 279:41-52
acoustic stimulation in conscious rat. Fed Proc 45:1346
Iadecola C, Underwood MD, Reis DJ (1986) Muscarinic cholin
ergic receptors mediate the cerebrovasodilation elicited by stimulation of the cerebellar fastigial nucleus in rat. Brain Res 368:375-379
Iadecola C, Arneric SP, Baker H, Tucker LW, Reis DJ (l987a)
J
Cereb Blood Flow Metab, Vol. 10, No.3, 1990
Ueki M , Linn F , Hossmann K-A (1988) Functional activation of cerebral blood flow and metabolism before and after global ischemia of rat brain. J Cereb Blood Flow Metab 8:486-494
Williams JL, Heistad DD, Siems JL, Talman WT (1989) Effects of stimulation of fastigial nucleus on cerebral blood flow in cats. Am J Physiol 257:H297-H304
Winters WD, Spooner CE (1966) A neurophysiological compar ison of alpha-chloralose with gamma-hydroxybutyrate in cats. Electroencephalogr Clin Neurophysiol 20:83-90
Dissociation by Chloralose of the Cardiovascular and Cerebrovascular Responses Evoked from the Cerebellar Fastigial Nucleus
Costantino ladecola, Mary E. Springston, and Donald J. Reis Division of Neurobiology, Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York, U.S.A.
Summary: We studied the effects of chloralose anesthe sia on the elevation in arterial pressure (AP), heart rate (HR), and regional CBF (rCBF) elicited by stimulation of the cerebellar fastigial nucleus (FN). Rats were anesthe tized with an initial dose of chloralose (40 mg/kg s.c.), paralyzed, and artificially ventilated. The FN was stimu lated (50-100 fLA, 50 Hz, I s onll s off) with microelec trodes stereotaxically implanted. During the stimulation AP was carefully maintained within cerebrovascular au toregulation. CBF was measured by the [14C]iodoanti pyrine technique with regional dissection. In rats that re ceived only the initial dose of chloralose, FN stimulation elevated rCBF in brain and spinal cord, up to 209 ± 13% of control in frontal cortex (n = 5; p < 0.0 1, analysis of variance). Administration of additional chloralose ( 10 mg/
kg i.v., 30 min prior to measurement of CBF) did not affect resting rCBF (n = 5), the EEG, or the elevation in AP and HR elicited by FN stimulation (n = 4). However, the additional chloralose abolished the elevations in rCBF (n = 5; p > 0.05). Thus, the cerebrovasodilation elicited from the FN is more susceptible to the effects of addi tional anesthesia than the elevation in AP and HR. These results indicate that the cerebrovascular and cardiovas cular responses elicited from the FN are functionally dis tinct and provide additional evidence for the notion that these responses are mediated by different neural path ways and transmitters. Key Words: Anesthesia Cerebellum-Cerebral blood flow-e4C]Iodoantipy rine-Spinal cord blood flow.
Electrical stimulation of the rostral ventromedial portion of the cerebellar fastigial nucleus (FN) in rat, as in cat, rabbit, and monkey, elicits a global increase in regional CBF (rCBF) (Doba and Reis, 1972; McKee et aI., 1976; Nakai et aI., 1982; Reis et aI., 1982; Mraovitch et aI., 1986; Maeda, 1988; Goadsby and Lambert, 1989; Iadecola and Reis, 1989). The cerebrovasodilation is greatest in the ce rebral cortex (up to 2 15% of control), wherein the rCBF increases are largely independent of changes in local metabolism (Nakai et aI., 1983) and are me diated by neural pathways contained entirely within the brain (Nakai et aI., 1982; Iadecola et aI., 1983b,
1987a). In addition to its actions on the cerebral circulation, stimulation of the FN produces power ful effects on the systemic circulation in several species. These include an elevation in arterial pres sure (AP) and heart rate (HR) (Miura and Reis, 1970; Dormer and Stone, 1976; McKee et aI., 1976; Nakai et aI., 1982; Reis et aI., 1982) and release of renin (Koyama et aI., 1980), vasopressin (Del Bo et aI., 1982b), and adrenal catecholamines (Del Bo et aI., 1982a). These responses have been collectively termed the fastigial pressor response (FPR) (Miura and Reis, 1970). The neural substrates underlying the cardiovas cular and cerebrovascular components of the FPR are unknown. While it has been proposed that they are initiated by different neuronal pools (Mraovitch et aI., 1986), recent studies suggest that the path ways mediating both components are closely asso ciated at some sites and may go through a common relay station within the C l area of the rostral vent rolateral medulla (Chida et aI., 1989). The latter
Received September 1 1, 1989; revised November 23, 1989; accepted November 27, 1989. Address correspondence and reprint requests to Dr. C. lade cola at Laboratory of Neurobiology, 4 1 1 E. 69 St., New York, NY 10021, U.S.A. Abbreviations used: AP, arterial pressure; FN, fastigial nu cleus; FPR, fastigial pressor response; HR, heart rate; lAP, io doantipyrine; rCBF, regional CBF.
375
C. IADECOLA ET AL.
376
may indicate that the effects on systemic and cere bral circulation are mediated through the same ba sic neuronal circuitry. However, a recent study in cats anesthetized with doses of chloralose larger than previously used has failed to demonstrate the vasodilation in the cerebral cortex, while the changes in AP and HR were preserved (Williams et al., 1987, 1989). These findings are of interest as they raise the possibility that chloralose, above a critical threshold, may se lectively affect the cerebrovasodilation, sparing the systemic cardiovascular changes elicited from the FN. This would not be surprising as chloralose does not depress cardiovascular reflexes (Cox and Bag shaw, 1979) while it suppresses spontaneous and evoked neuronal activity (Collins et al., 1983). In this study we have therefore investigated whether chloralose is able to dissociate the cardio vascular and cerebrovascular effects of FN stimu lation. We shall demonstrate that administration of an additional dose of chloralose abolishes the va sodilation evoked from the FN in brain and in spinal cord without affecting the elevations in AP and HR. These findings indicate that the cardiovascular and cerebrovascular components of the FPR can be dis sociated and probably are mediated through distinct neural mechanisms. METHODS
Methods for surgical preparation of rats, electrical stimulation of the FN with controlled AP, and measure ment of rCBF have been described in detail in earlier publications (Nakai et aI., 1982; ladecola et aI., 1983a, 1987a,b) and will only be summarized here. General procedures
Studies were performed on 24 male Sprague-Dawley rats weighing 320-390 g maintained in a light-cycled (0700 h oni1900 h off), thermally controlled (27°C) environment and fed lab chow ad libitum. A 1% solution of chloralose in 0. 9% NaCI was prepared. After adding chloralose to prewarmed 0. 9% NaCI, the mixture was stirred and heated until the anesthetic was completely solubilized. The solution was not allowed to boil. Rats were anesthetized with chloralose (40 mg/kg, s.c.) after induction with 5% isoflurane (Anaquest) in 100% oxygen, administered through a facial mask. During sur gery isoflurane was administered at a reduced rate (2%). Catheters were inserted in both femoral arteries and veins and the trachea was cannulated. Animals were then placed on a stereotaxic frame (Kopf), paralyzed with tu bocurarine (2 mglkg i.m.), and artificially ventilated with 100% oxygen by a mechanical ventilator (rodent respira tor; Harvard Apparatus). One of the arterial catheters was connected to a pressure transducer (Gould) fed into a polygraph (Grass) for continuous recording of AP, mean AP, and HR. Body temperature was maintained at 37 ± O.soC using a heating lamp thermostatically controlled by a rectal probe (YSI). The midline portion of the caudal
J
Cereb Blood Flow Metab. Vol. 10, No.3. 1990
occipital bone was removed to expose the cerebellar ver mis and the dorsal medulla. A stainless-steel screw was inserted through the left parietal bone to lie on the dura at a site 2 mm lateral to the midline and 2 mm caudal to bregma for monopolar recording of the EEG. At the end of the surgery, isoflurane anesthesia was discontinued. Arterial Pcoz, POz, and pH were measured on 0. 2 ml of blood by using a blood gas analyzer (Micro 13 System; Instrumentation Laboratory). Stimulation of the FN
The FN was stimulated monopolarly with negative square-wave pulses delivered through a stimulus isolation unit from a constant-current stimulator (Grass S88). Elec trodes were fabricated from Teflon-coated stainless-steel wires (diameter 150 /lorn) with the cut tip exposed. The ground was a metal clip attached to the animal's scalp. The stimulus current was measured and displayed on an oscilloscope as previously described (Nakai et aI., 1982). Measurement of CBF
CBF was measured in brain homogenates by using e4C]iodoantipyrine ([14C]lAP) as diffusible indicator (Sakurada et aI., 1978; Ohno et aI., 1979). As described in detail elsewhere (Nakai et aI., 1982; ladecola et aI., 1983a), lAP (spec. act. 40-60 mCilmmol; NEN) was dis solved in -1 ml of saline after elimination of ethanol and infused intravenously at a constant rate for 30 s by using an infusion pump (model 940; Harvard Apparatus). Prior to the start of the infusion, 500 units of heparin (Elkins Sinn) was administered intravenously. During the lAP infusion, timed arterial samples were collected at -2- to 5-s intervals to determine the arterial concentration-time course of the isotope (Iadecola et aI., 1983a). Toward the end of the infusion, during the collection of the last blood sample, the heart was stopped by an intravenous bolus injection of 1 ml of saturated KCI. The brain and the upper portion of the cervical cord were rapidly removed and placed on a cooled glass plate. Large vessels on the brain surface were removed and 14 regions were dis sected out. These included upper cervical cord (CI-C3), medulla, cerebellum, pons, inferior colliculus, superior colliculus, hypothalamus, thalamus, hippocampus, cau date nucleus, frontal, occipital, and parietal cortices, and corpus callosum. Tissue samples were placed in pre weighed vials that were then reweighed for determination of the sample's weight. After adding 1 ml of Protosol (NEN) to each sample, the vials were placed in a shaking water bath at 60°C for 6 h to allow for tissue solubiliza tion. The radioactivity of the samples (dpm) was deter mined by a liquid scintillation spectrophotometer (Beck man) using procedures described in earlier pUblications (Nakai et aI., 1982; ladecola et aI., 1987b). For determi nation of lAP concentration in blood, 40-/lol aliquots of arterial blood were transferred in vials containing 1 ml of Protosol-ethanol (NEN) and processed for determination of radioactivity (dpm) by liquid scintillation counting (Ia decola et aI., 1987a,b). CBF (mUloo glmin) was calcu lated from the arterial time course of lAP concentration (nCi/g) and tissue concentration of lAP (nCilg) using the equation described by Kety (see Nakai et aI., 1982). The lAP partition coefficient was set at 0. 8 (Sakurada et aI., 1978). Details on the accuracy of the computer program and on the computational resolution at high flow rates have been published (Nakai et aI., 1982).
CHLORALOSE AND CBF INCREASE FROM CEREBELLUM Experimental protocol
After establishment of the anesthesia, insertion of the catheters, artificial ventilation, and exposure of the cau dal brainstem, an electrode mounted on a micromanipu lator with a 10° posterior inclination was positioned on the calamus scriptorius and the stereotaxic coordinates re corded as stereotaxic zero (Nakai et al., 1982). The elec trode was then moved 5 mm rostral and 0.8 mm lateral from zero, lowered into the cerebellum until the vertical zero was reached, and left at that site until the exploration for the FN was begun (Nakai et al., 1982). Blood gases were carefully adjusted: Arterial Pco2 was maintained between 33 and 38 mm Hg by varying the stroke volume of the ventilator and Po, was maintained above 100 mm Hg by ventilating the animals with 100% 02' The latter was necessary to counteract the effects of atelectasis occurring in artificially ventilated rats (Nathan and Reis, 1975) and to compensate for any loss of oxygen carrying capacity resulting from blood sampling and con trolled hemorrhage (see below). As a consequence of the high Fio2, the P02 of the animals was elevated (Table 1). However, the effects of this degree of hyperoxia on CBF are negligible (Reivich, 1968). After the blood gases were adjusted, the procedure for localization of the FN was started. As described else where (Nakai et al., 1982), the electrode was withdrawn in 0.5-mm steps and at each step exploratory stimuli, con sisting of 8-s trains of 0.5-ms pulses at 50 Hz and with a current intensity of 10-20 f..lA, were delivered. An active site in the FN was defined as one in which stimulation resulted in a stimulus-locked 10- to 20-mm Hg AP eleva tion. These small increases in AP are within the range of cerebrovascular autoregulation (Hernandez et al., 1978) and have no effect on CBF (C. Iadecola, unpublished observations). Once the most active site in FN was local ized, the electrode was left in place at that site. Blood gases were measured again and FN stimulation begun. The FN was stimulated with intermittent trains of stimuli (1 s onll s off, 50 Hz). During the first 2-4 min, the cur rent intensity was gradually increased up to five times the threshold current (i.e., the stimulus current producing a lO-mm Hg elevation in AP, usually 10-20 f..lA). At the same time, the evoked elevations in AP were offset by slow removal of arterial blood. By this procedure during FN stimulation AP never exceeded 150 mm Hg, a value that is within the autoregulated range of CBF in rats un der identical experimental conditions (Iadecola et al.,
377
1983b). Blood gases were measured, and, if necessary, adjusted. After 10-15 min of stimulation, at a time when AP and blood gases had been in a steady state for at least 5 min, the infusion of lAP for CBF measurement was begun. Thirty seconds later the heart was stopped by in travenous injection of KCl, the brain and cervical cord removed, and CBF determined as described above. In animals receiving supplemental anesthesia, 10 mg/kg of chloralose was administered by slow intravenous infusion 30 min prior to the measurement of CBF. In unstimulated animals surgical procedures and conduct of the experi ments were identical to those of rats in which the FN was stimulated with the exception that an electrode was low ered in the cerebellar vermis but no stimuli were deliv ered. Experiments were timed as follows. The time of the injection of chloralose at the beginning of the experiment (40 mglkg s.c.) was considered time zero. In all animals, with and without FN stimulation or supplemental anes thesia, CBF was measured within or near 3 h after the administration of chloralose (Table 1). This was neces sary to ensure stability of the level of anesthesia as es tablished in preliminary experiments. The state of anes thesia of the animals was assessed by EEG criteria (Win ters and Spooner, 1966). As shown in Fig. 1, the EEG 1 h after chloralose administration exhibits the low frequency (3-5 Hz) hypersynchronous burst activity (0.2OA mY) typical of chloralose anesthesia (Winters and Spooner, 1966). This distinctive EEG pattern persisted 2 and 3 h after the initial administration of chloralose and was not altered by stimulation of the FN (Fig. 1). That the animals were anesthetized is also demonstrated by the fact that their rCBF was low in comparison with that of awake animals (see Table 3; Ohno et al., 1979). Particu larly, rCBF was depressed in regions such as thalamus and cortex where the greatest increases occur during dis comfort and stress (Lacombe and Seylaz, 1984). Thus, during the experimental period, including the phase in which the FN was stimulated, rats were in a stable anes thetized state. Statistical analysis
Data were evaluated by the one-way analysis of vari ance and differences among multiple groups were as sessed by the Newman-Keuls test as a post hoc multiple comparison procedure (Tables 1 and 3). Two-group com parisons were analyzed by the paired t test (Table 2). For
5) in rats with and without chloralose supplementation or stimulation of the fastigial nucleus (FN)
TABLE 1. Arterial pressure (AP) and blood gases (mean ± SEM, n
=
Unstimulated: chloralose dose
FN stimulation: chloralose dose
40 mg/kg (control) --�--m - ---------------m �A ( g 126 H )
Pc02 (mm Hg) P02 (mm Hg)
pH Exp. timeb (min)
36 413 7.43 183
40 mg/kg 40 + 10 mg/kg (control) 40 + 10 mg/kg � - ---- - -- - C -------------- - - --- ---------± ---------------1 26 ± 5 ±4 ±5 116 2 134 ± 0 .6 36.6 ± 05 . 36.4 ± 1 .0 34.8 ± 0.7 392 ±8 413 ± 25 ± 33 377 ± 36 7 3 . 2 ± O.Old 7 4 . 0 ± 0.02 ± 0 .02 7.41 ± 0 .01 178 ±6 186 ± 8 ±5 185 ± 4
a During CBF measurement. b From chloralose administration at the beginning of the experiment. c p < 00 . 5 from FN stimulation control group.
dp
< 00 .1
(analysis of variance and Newman-Keuls test).
J
Cereb BLood FLow Metab. VoL. 10, No.3, 1990
C. IADECOLA ET AL.
378
1 hr
2 h'r
3 hr
]
FN stim
0.2 mV
(50 �A, 50 Hz) on
off 1
sec
FIG. 1. EEG in paralyzed, artificially ventilated rats 1, 2, and 3 h after administration of 40 mg/kg s.c. of chloralose and during
stimulation of the fastigial nucleus (FN stim). Note that the EEG exhibits the hypersynchronous burst pattern typical of chloralose anesthesia. This pattern is present throughout the 3-h period and during FN stimulation, which was carried out slightly over 3 h after the administration of chloralose. Thus, throughout the experimental period, rats are in a stable state of chloralose anesthesia.
both statistical procedures, differences were considered significant for probability values of < 0.05. RESULTS Effect of additional chloralose on FPR
The effects of supplemental anesthesia on the cardiovascular changes elicited by FN stimulation were studied in four rats. Before chloralose supple mentation, FN stimulation elicited the well established increases in AP and HR, i.e., the FPR. The minimal current required to produce a lO-mm Hg AP elevation (threshold current) ranged be tween lO and 20 !-LA (Fig. 2; Table 2). Administra tion of additional chloralose ( 10 mg/kg i.v.) did not affect resting AP and HR or the amplitude of the FPR (p > 0.05, paired t test; Table 2; Fig. 2). The threshold current was also unchanged (Table 2). Thus, additional chloralose does not affect the changes in AP and HR elicited by FN stimulation. Effect of additional chloralose on increase in CBF
Stimulation of the FN in untreated animals in creased rCBF in all regions studied (p < 0.0 1, anal ysis of variance and Newman-Keuls test; Table 3). The magnitude and regional distribution of the changes were similar to those previously reported from this laboratory (Nakai et aI., 1982, 1983; Iade cola et aI., 1983b, 1986, 1987a; Americ et aI., 1987), the greatest changes occurring in cerebral cortex J
Cereb Blood Flow Metab. Vol. 10. No. 3, 1990
(up to 209% of control in frontal cortex) and thala mus ( 190%). Substantial increases were also ob served in hippocampus ( 176%), caudate nucleus ( 180%), and, interestingly, cervical cord, where rCBF rose to 153% of control (p < 0.01). FN stim ulation had no effect on the EEG (Fig. 1). Administration of an additional dose of chloral ose ( 10 mg/kg i.v.) 30 min prior to CBF measure ment did not affect EEG activity (Fig. 3) or resting rCBF (p > 0.05; Table 3). However, the elevations in rCBF elicited by FN stimulation were virtually abolished throughout the entire brain and in the cer vical cord (p > 0.05) (Table 3). The small residual increases observed in medulla ( 1 19%) and superior colliculus ( 126%) (Table 3) did not reach statistical significance (p > 0.05). These small increases may reflect metabolical activation of sites receiving di rect (monosynaptic) projections from the FN (Del Bo et aI., 1982; Nakai et aI., 1983). DISCUSSION
We have demonstrated that the global cerebro vascular vasodilation elicited by electrical stimula tion of the FN is more sensitive to chloralose than the FPR. Thus, when chloralose is given at an initial dose of 40 mg/kg, FN stimulation increases AP, HR, and rCBF. However, supplementing this initial dose with additional chloralose abolishes the cere-
379
CHLORALOSE AND CBF INCREASE FROM CEREBELLUM 4) on cardiovascular changes elicited by electrical stimulation of the fastigial nucleus (FN stim)
TABLE 2. Effect of chloralose supplementation (mean ± SEM, n
=
Before chloralose
Arterial pressure (mm Hg) Heart rate (beats/min)
Mter chloralosea
Resting
FN stirn
Il
116 ± 6 324 ± 43
166 ± 7 424 ± 22
100 ± 36
50 ± 4
Resting
FN stirn
Il
114 ± 5
166 ± 5 406 ± 20 12 ± 1
106 ± 40
300 ± 47
11 ± 1
Threshold current (f.LA)
52 ± 5
Stimulation parameters: 8-s trains, 50 Hz, current intensity set at three times threshold current. Differences before and after chloralose were not significant (p > 0.05, paired t test). a 30 min after chloralose supplementation (10 mg/kg).
of additional chloralose did not produce further de creases in rCBF, a variable closely "coupled" to cerebral glucose utilization even during chloralose anesthesia (Nakai et aI., 1983), and did not influ ence EEG activity. A more likely scenario is that chloralose, above a specific dose, impairs and/or modifies the functional characteristics of a critical group of neurons that is essential to the expression of the cerebrovasodilation but not of the FPR. The effects on CBF of changes in activity of this, pre sumably small, neuronal population must be be yond the relatively limited resolution of the tissue sampling technique used in this study to measure
brovasodilation without affecting the hypertension and tachycardia. These results indicate that this agent within the appropriate dose range dissociates the cardiovascular and cerebrovascular compo nents of the response. The mechanisms whereby chloralose supplemen tation abolishes the cerebrovasodilation elicited from the FN are unknown. It is unlikely that the effect of chloralose on the cerebrovasodilation is a nonspecific consequence of global depression of ce rebral metabolic activity. While this agent lowers cerebral glucose utilization throughout the brain (Dudley et aI., 1982), we found that administration
200 AP
(mm Hg)
125 50 20 0
MAP
(mm Hg)
125 50
HR
5 00
(beats/min) 300
l l
-
[
Stimulus (50 Hz)
wi\,.
.A.
r -1.
...
J\,.
�
A
�
�
R-
-----
-----v--
0
0
10 IlA
151lA
0 45 IlA
-
1
Chloralose 10
0 10 IJA
mg/kg.
�
0 15 IlA
--'\...--
0 45 IJA
Lv.
min FIG. 2. Effect of administration of a supplemental dose of chloralose (10 mg/kg Lv.) on the elevation in arterial pressure (AP). mean AP (MAP). and heart rate (HR) elicited by electrical stimulation of the cerebellar fastigial nucleus (FN) in rat. Animals were anesthetized with an initial dose of chloralose (40 mg/kg s.c.). paralyzed, and artificially ventilated. Left: Effect of FN stimulation (8 s, 50 Hz, 10-45 f.LA) on AP, MAP, and HR. The response appears at 15 f.LA (threshold current) and stimulation at three times threshold produces MAP elevations of -50 mm Hg. Right: Thirty minutes after administration of supplemental chloralose, the threshold current and the amplitude of the pressor response remain unchanged.
J
Cereb Blood Flow Metab, Vol. 10, No.3, 1990
C. IADECOLA ET AL.
380
(10 mg/kg; mean ± SEM, n 5) on increase in CBF elicited by stimulation of the fastigial nucleus (FN) in rat
TABLE 3. Effect of chloralose supplementation
=
CBF (mI/100 g/min) Unstimulated:
FN stimulation:
chloralose dose
chloralose dose
40 mg/kg (control) Cervical cord Medulla Cerebellum Pons Inf. colliculus Sup. colliculus Hypothalamus Thalamus Hippocampus Caudate nucleus Frontal cortex Parietal cortex Occipital cortex Corpus callosum
40 + 10 mg/kg
40 mg/kg (control)
4 5 4 5 7 7
86 139 126 142 156 148
± ± ± ± ± ±
6 13 10 13 13 13
89 ± 7 71 ± 6
75 ± 6 66 ± 4
65 ± 6
62 70 69 77 67 49
122 136 114 131
± ± ± ±
10 13 11 10
56 82 74 82 92 88
± ± ± ± ± ±
5 10 7 7 1O
II
73 ± 6 72 ± 6 80 ± 6 68 ± 5 49 ± 6
55 76 79 83 95 85
± ± ± ± ± ±
± ± ± ± ± ±
4 5 3 4 4 4
151 ± 10 153 ± 8 135 ± 12 84 ± 8
% of
control 153b 169b l70b 173b 170b 167b 137b 191b 176b 180b 209b 190b 197b 171b
40 + 10 mg/kg 60 90 85 93 104 107
± ±
± ± ±
±
% of controla 109 119 108 112 110 126 106 110
5 7 6 7 4 8
79 ± 5 73 ± 7 62 ± 6 66 ± 5 69 ± 6
100 95 100 106 97
82 ± 7 65 ± 5 47 ± 3
96
a Differences from respective control were not significant (p > 0.05). b P < 0.01 (analysis of variance and Newman-Keuls test).
rCBF (Iadecola et al., 1983c). Another possibility is that chloralose acts directly at the neurovascular effector level by interfering with the mechanisms governing the interaction between neurons and ce rebral microvasculature or, less likely, by affecting vascular smooth muscle and/or endothelial func tion. There is no experimental evidence to support the latter possibilities. Irrespective of the mecha-
nisms, this effect of chloralose occurs above a threshold concentration that is reached with the ad ministration of an additional dose 30 min prior to measuring rCBF. In contrast to its dramatic action on the cerebral circulation, chloralose supplementation does not af fect the FPR. Thus, the cerebrovascular and car diovascular actions exerted by the FN are distinct
0 min
t
Chloralose 10 mg/kg. i.v.
5 min
15 min
30 min
JO.2
mV
L-.J 1 sec FIG. 3. Effect of administration of a supplemental dose of chloralose (10 mg/kg Lv.) on the EEG. Rats were anesthetized with an initial dose of chloralose (40 mg/kg s.c.). paralyzed, and artificially ventilated. Administration of chloralose did not affect EEG.
J
Cereb Blood Flow Metab, Vol. 10. No.3, 1990
CHLORALOSE AND CBF INCREASE FROM CEREBELLUM
with respect to the action of chloralose. The biolog ical basis of this distinction must reside in differ ences in the neural substrates mediating cardiovas cular and cerebrovascular responses evoked from the FN, as well as in a selectivity in the action of chloralose, which spares (Cox and Bagshaw, 1979) or even enhances (Stephenson and Donald, 1980) reflex cardiovascular responses. A dissociation be tween cardiovascular and cerebrovascular effects occurs also after systemic administration of atro pine, a treatment that abolishes the increase in rCBF without affecting the FPR (Iadecola et aI., 1986). Thus, while the cerebrovasodilative pathway involves at least one cholinergic synapse, the path way mediating the FPR does not. In addition, elec trical stimulation of the caudal portion of the FN produces increases in cortical CBF that are not as sociated with changes in AP or HR (Mraovitch et aI., 1986). This finding has been interpreted to indi cate that cerebrovascular and cardiovascular re sponses may originate from separate neuronal pools within the FN (Mraovitch et aI., 1986). However, this issue is complicated by the recent discovery that the pathways mediating both the FPR and the cerebrovascular effects originate from fibers pro tecting into or passing through the area and not from FN neurons (Chida et aI., 1986, 1989). It is thus conceivable that these pathways arise from separate brainstem sites and then converge while terminating into or passing through the FN on their course to their next relay station. The present results indicate that the recent failure to confirm the cortical cerebrovasodilation elicited from the FN in cats using the microsphere tech nique to measure rCBF is probably a consequence of the use of higher initial doses of chloralose (70 mg/kg) followed by multiple supplementations throughout the experiment (Williams et aI., 1987, 1989). Interestingly, these authors found small «30%) increases in brainstem regions that corre spond to sites receiving primary projections from the FN (Del Bo et aI., 1982). This observation sup ports the contention, suggested also by the present study, that monosynaptic connections may be more resistant to the effects of chloralose on neurogenic vasodilation. In any event, it is unlikely that species differences account for the inability to reproduce the response because large increases in CBF have been reported in cats during stimulation of the FN by independent laboratories and using different techniques to measure CBF (Doba and Reis, 1972; Maeda, 1988; P. J. Goadsby, personal communica tion). The effects of chloralose on the cerebrovasodila tion elicited by FN stimulation are not specific to
381
this agent but are seen also with other general an esthetics. Using a laser-Doppler perfusion probe to monitor changes in CBF over the parietal cortex of spinal cord-transected rats anesthetized with iso flurane, we have observed that the vasodilation is abolished by a cumulative dose of pentobarbital of 30 mg/kg i.v. (Iadecola and Reis, 1989). Similarly, an increase in concentration of the volatile anes thetic isoflurane from 1 to 2% significantly attenu ates the vasodilation (C. Iadecola, unpublished ob servations). The finding that the cerebrovasodila tion is exquisitely sensitive to agents with an inhibitory effect on neuronal and synaptic activity provides strong support for the notion that the in crease in CBF elicited from the FN is neurally me diated. However, it is of interest that the increase in cortical CBF elicited by somatosensory stimulation is preserved at doses of chloralose of 80 mg/kg (Ueki et aI., 1988), while the flow increase elicited from the FN is abolished at a cumulative dose of 50 mg/kg. This difference may indicate that the neural systems mediating the "primary" vasodilation evoked from the FN are distinct, with respect to the action of anesthetics, from those involved in the "metabolic" vasodilation associated with cortical activation. Another finding of the present study is that FN stimulation produces substantial increases in spinal cord blood flow. This new observation demon strates that the effects of FN are not restricted to the brain but rather involve the circulation of the entire neuraxis. Whether neurogenic factors are ca pable of influencing the circulation of the spinal cord is not well established (Marcus et aI., 1977). We have recently shown that stimulation of the dor sal medullary reticular formation can increase flow in all segments of the spinal cord, an effect that is attenuated by removal of the adrenal glands (Iade cola et aI., 1989). Thus, in the spinal cord, as in brain, the increases in blood flow elicited by stim ulation of the dorsal medullary reticular formation depend in part on adrenal catecholamines and are probably mediated by an increase in local metabo lism (Iadecola et aI., 1987b). Whether the increases in spinal cord blood flow elicited by FN stimulation are metabolically mediated or whether, as in most of the cerebral cortex, they are independent of local metabolic activity remains to be determined. In conclusion, we have shown that the wide spread cardiovascular and cerebrovascular changes elicited by FN stimulation can be dissociated by the use of a critical dose of chloralose. This finding, while supporting the notion that the neural sub strates mediating the cardiovascular and cerebro vascular responses evoked from the FN are funcJ
Cereb Blood Flow Metab, Vol. ]0, No.3, 1990
382
C. IADECOLA ET AL.
tionally distinct, provides further evidence for the concept that the circulation of the brain and spinal cord is subject to substantial neurogenic influences. The integrated neurovascular mechanisms deter mining these effects, their ultimate cellular and mo lecular mediators, and, most importantly, their bi ological significance remain to be unraveled.
Role of local neurons in the cerebrocortical vasodilation elic ited from cerebellum. Am J Physiol 252:RI082-RI091 Iadecola C, Lacombe PM, Ishitsuka T, Underwood MD, Reis DJ (I987b) Role of adrenal catecholamines in the cerebrova sodilation evoked from the brain stem. Am J Physiol 252: H1183-H1191
Iadecola C, Chida K, Lacombe PM, Underwood MD, Reis DJ
vasodilation elicited by focal electrical stimulation within the dorsal medullary reticular formation in anesthetized rat. J Cereb Blood Flow Metab 3:270-279
(1989) Global increase in spinal cord blood flow elicited from the dorsal medullary reticular formation in rat. J Cereb Blood Flow Metab 9:S480 Koyama S, Ammons WS, Manning JW (1980) Altered renal vas cular tone and plasma renin activity due to fastigial and baro receptor activation. Am J Physiol 239:H 232-H237 LaCombe PM, Seylaz J (1984) Significance of the cerebrovascu lar effects of immobilization stress in the rabbit. J Cereb Blood Flow Metab 4:397-406 Maeda M (1988) Changes in intracranial pressure elicited by elec trical stimulation of the brain stem reticular formation in spinal cats with vagotomy. J Auton Nerv Syst 25:155-164 Marcus ML, Heistad DD, Ehrhardt JC, Abboud FM (1977) Reg ulation of total and regional spinal cord blood flow. Circ Res 41:128-134 McKee JC, Denn MJ, Stone HL (1976) Neurogenic cerebral va sodilation from electrical stimulation of the cerebellum in the monkey. Stroke 7: 179-186 Miura M, Reis DJ (1970) A blood pressure response from fasti gial nucleus and its relay pathways in the brainstem. Am J Physiol 219:1330-1336 Mraovitch S, Pinard E, Seylaz J (1986) Two neural mechanisms in rat fastigial nucleus regulating systemic and cerebral cir culation. Am J PhysioI 251:HI53-HI63 Nakai M, Iadecola C, Reis DJ (1982) Global cerebral vasodila tion by stimulation of rat fastigial cerebellar nucleus. Am J Physiol 243:H 2 26-H235 Nakai M, Iadecola C, Ruggiero DA, Tucker LW, Reis DJ (1983) Electrical stimulation of cerebellar fastigial nucleus in creases cerebrocortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation. Brain Res 260:35-49 Nathan MA, Reis DJ (1975) Hypoxemia, atelectasis and the el evation of arterial pressure and heart rate in paralyzed arti ficially ventilated rats. Life Sci 16:1103-1120 Ohno K, Pettigrew KD, Rapoport SI (1979) Local cerebral blood flow in the conscious rat as measured with CI4-iodoanti pyrine and H3-nicotine. Stroke 10:62-67 Reis DJ, Iadecola C, MacKenzie E, Mori M, Nakai M, Tucker LW (1982) Primary and metabolically coupled cerebrovas cular dilation elicited by stimulation of two intrinsic systems of brain. In: Cerebral Blood Flow: Effects of Nerves and Neurotransmitters (Heistad DD, Marcus ML, eds), New York, Elsevier/North Holland, pp 475-484 Reivich M (1968) Cerebral circulatory responses to respiratory influences. In: Cerebral Vascular Diseases (Toole JF, Siekert RG, Wishnant JP, eds), New York, Grune and Strat ton, pp 91-100 Sakurada 0, Kennedy C, Jehle J, Brown JD, Carbin GL, Sokoloff L (1978) Measurement of local cerebral blood flow with iodo(C14)antipyrine. Am J Physiol 234:H59-H66 Stephenson RB, Donald DE (1980) Reflexes from isolated ca rotid sinuses of intact and vagotomized conscious dogs. Am J Physiol 238:H815-H8 2 2
Iadecola C, LeDoux JE, Thompson ME, Tucker LW, Reis DJ (l983c) Focal increases in cerebral blood flow during brief
Williams JL, Heistad DD, Siems JL, Talman WT (1987) Effect of stimulation of fastigial nucleus on cerebral blood flow in cats. Soc Neurosci Abstr 13:283
REFERENCES Arneric SP, ladecola C, Underwood MD, Reis DJ (1987) Local cholinergic mechanisms participate in the increase in corti cal cerebral blood flow elicited by electrical stimulation of the fastigial nucleus in rat. Brain Res 411: 212-225 Chida K, Iadecola C, Underwood M, Reis DJ (1986) A novel vasodepressor response elicited from the rat cerebellar fas tigial nucleus: the fastigial depressor response. Brain Res
370:378-382 Chida K, ladecola C, Reis DJ (1990) Lesions of rostral ventro lateral medulla abolish some cardio- and cerebrovascular components of the cerebellar pressor and depressor re sponses. Brain Res (in press) Collins JG, Kawahara M, Homma E, Kitahata LM (1983) Alpha chloralose suppression of neuronal activity. Life Sci 32: 2995-2999 Cox RH, Bagshaw DRJ (1979) Influence of anesthesia on the response to carotid sinus hypotension in dogs. Am J Physiol 237:H424-H432 Del Bo A, Ruggiero DA, Ross CA, Wiley R, Reis DJ (1982) Pathways from the fastigial pressor area in rat. Soc Neurosci Abstr 8:76
Del Bo A, Ross Ca, Pardal JF, Saavedra JM, Reis DJ (l983a) Fastigial stimulation in rats releases adrenomedullary cate cholamines. Am J Physiol 244:R801-R809 Del Bo A, Sved AF, Reis DJ (I983b) Fastigial stimulation re leases vasopressin in amounts which elevate arterial pres sure. Am J Physiol 244:H687-H694
Doba N, Reis DJ (1972) Changes in regional blood flow and cardiodynamics evoked by electrical stimulation of the fas tigial nucleus in cat and their similarity to orthostatic re flexes. J Physiol (Lond) 227:7 29-747
Dormer KJ, Stone HL (1976) Cerebellar pressor response in the dog. Am J Physiol 41:574-580
Dudley RE, Nelson SR, Samson F (1982) Influence of chlora lose on brain regional glucose utilization. Brain Res 233:173180
Goadsby PJ, Lambert GA (1990) Electrical stimulation of the fastigial nucleus increases total cerebral blood flow in the monkey. Neurosci Lett 107:141-144 Hernandez MJ, Brennan RW, Bowman GS (1978) Cerebral blood flow autoregulation in the rat. Stroke 9:150-155
Iadecola C, Reis DJ (1990) Cerebral blood flow by laser-Doppler flowmetry during stimulation of the cerebellum. J Cereb
Blood Flow Metab (in press)
Iadecola C, Nakai M, Arbit E, Reis DJ (I983a) Global cerebral
Iadecola C, Mraovitch S, Meeley MP, Reis DJ (l983b) Lesions of the basal forebrain in rat selectively impair the cortical vasodilation elicited from cerebellar fastigial nucleus. Brain Res 279:41-52
acoustic stimulation in conscious rat. Fed Proc 45:1346
Iadecola C, Underwood MD, Reis DJ (1986) Muscarinic cholin
ergic receptors mediate the cerebrovasodilation elicited by stimulation of the cerebellar fastigial nucleus in rat. Brain Res 368:375-379
Iadecola C, Arneric SP, Baker H, Tucker LW, Reis DJ (l987a)
J
Cereb Blood Flow Metab, Vol. 10, No.3, 1990
Ueki M , Linn F , Hossmann K-A (1988) Functional activation of cerebral blood flow and metabolism before and after global ischemia of rat brain. J Cereb Blood Flow Metab 8:486-494
Williams JL, Heistad DD, Siems JL, Talman WT (1989) Effects of stimulation of fastigial nucleus on cerebral blood flow in cats. Am J Physiol 257:H297-H304
Winters WD, Spooner CE (1966) A neurophysiological compar ison of alpha-chloralose with gamma-hydroxybutyrate in cats. Electroencephalogr Clin Neurophysiol 20:83-90
