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Local cerebral impedance and blood flow during sleep and arousal
EXPERIMENTAL
Local
NEUROLOGY
Cerebral
LUCY Stanford
9,
269-285
(1964)
Impedance Sleep and BIRZIS Research Received
AND
and Blood Arousal
SHUNRO
Institute, Sovembev
Flow
during
TACHIBANA’
Menlo
Park,
Calijomia
18, 1963
Local impedance fluctuations in cerebral cortex, midbrain reticular formation, dorsal hippocampus and hypothalamus were recorded simultaneously and continuously in unrestrained cats with permanently implanted electrodes. Distinct patterns of impedance recordings were correlated with behavioral and EEG signs of sleep, wakefulness and arousal by various stimuli. The most striking response to alerting stimuli was the marked, selective increase in impedance pulse in a specific area of the posterior hypothalamus, The exquisite sensitivity of this site is consistent with its affective and autonomic roles. Startle stimuli elicited tachycardia and a general, widespread reduction in cerebral impedance pulse amplitude. These two responses were most frequently seen in combination, particularly following the intravenous injection of 1-2 Bg/kg epinephrine. Hippocampal impedance changes in response to alerting stimuli were also observed, but less consistently. The local impedance patterns in different stages of sleep are also described; patterns seen during “paradoxical” sleep were very similar to those of the a!rrt state. Evidence is presented to support the hypothesis that the amplitude of the impedance pulse provides an index of the relative blood flow changes in the vicinity of the electrode. Introduction
Innumerable methods have been introduced for the assessment of cerebral circulation (3, 7, 12, 14, 15) and these have been applied in both lower animals and man in an attempt to correlate electrical activity of the brain with blood flow. Often such techniques tacitly assume the brain to have a completely homogeneous circulation, since they do not permit 1 This work was supported in part by research grants NB 03323 and NB 04210 from the National Institutes of Health and in part by contract Nonr-2993(00) between the Office of Naval Research and Stanford Research Institute. Reproduction in whole or part is permitted for any purpose of the United States Government. The authors thank Dr. Eva and Dr. Keith Killam for critical review of the manuscript. Dr. Tachibana’s present address is Department of Physiology, Kurume Medical School, Kurume. Japan. 269
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study of flow in discrete regions but simply measure total regional (3, 12) or even total brain perfusion (14). Again, many such methods are limited in that they do not offer a continuous picture of flow changes such as is needed to compare with the dynamic electrical changes of the brain (14). Others are unsuited for use in the chronic animal (7, 12) and some are potentially destructive of excitable tissues ( 15). An attempt was made in the present study to obtain estimates of relative blood flow within small, well-defined regions of the brain by extrapolation from measures of tissue electrical impedance. This work stemmed from preliminary studies (4) in which such an extrapolation was justified by in vitro and in viva experiments. The method of measuring impedance is generally similar to that reported in other recent studies (1, 11)) in that an alternating current bridge was used and the current density kept low to avoid possible stimulation of, or injury to, brain tissue. However, considerably higher frequencies were employed to emphasize those rhythmic changes which correlate with the cardiac cycle. Particular interest centered around simultaneous recording of impedance changes in those parts of the brain closely implicated in sleep, wakefulness and attention in the unanesthetized, unrestrained cat. Methods
In each of fifteen cats with permanently implanted electrodes, data were obtained from five to thirty recording sessionson each cat over periods up to 8 months. Recordings were also taken in ten acute experiments on spinal cats immobilized with gallamine triethiodide; in the latter preparations, pressure points and wound margins were infiltrated with lidocaine to prevent pain. Electrode placementsincluded “association” cortex, midbrain reticular formation (RF), dorsal hippocampus, medial thalamic nuclei, and posterior hypothalamus. Somecats had pairs of bilaterally symmetrical hippocampal and hypothalamic electrodes. These electrodes were used for recording electrical activity (EEG) or impedance, or for electrical stimulation. All placements were histologically verified from frozen, thioninestained brain sections. Chronic experiments were conducted with the cat in a sound-deadened Faraday chamber fitted with a one-way window. Impedance fluctuations, together with EEG and other physiological parameters, were recorded on an eight-channel Grass electroencephalograph.Four a-c impedance circuits (4) were used, each operating on a different generator frequency ( 10, 12.5, 15, and 17.5 kc) to enable impedance recordings to be made from four brain areassimultaneously.
CEREBRAL
IMPEDANCE
271
PATTERNS
Relation Between Impedance and Circulation. To test the hypothesis that cerebral impedancereflected changesin blood flow, cerebral impedance fluctuations were recorded in Cl spinal cats before and after irreversible brain damage from occlusion of the cranial blood supply for 1 hour. The impedance changesbefore cerebral asphyxia were compared with changes recorded following restoration of circulation. This is illustrated in Fig. 1. The upper trace (BP) represents the femoral arterial pressure and the
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---.,ric---x---
‘J~~~“~~~~~.~l~~ FIG.
campus record blood linear these femoral
___.. .-,
..
. --...~,.
I,.vLL? * ”“,F\
I.
Local cerebral impedance (IMP) in reticular formation (RF) and hippo(HP) of Cl spinal cat. Control-record before asphyxia. After asphyxiataken following cerebral asphyxia of I-hour duration, after reinstatement of flow, which was regulated by epinephrine infusion. Curves on the right show relationship between impedance amplitude and systemic arterial pressure for same two areas. EEG recorded from cortex. Blood pressure recorded from artery.
secondis the EEG record from a pair of cortical electrodes.The two lower tracings represent the impedance fluctuations in RF and hippocampus. After cerebral asphyxia, the EEG tracing is flat, but the impedancerecordings are similar to those of the control period. The peak-to-peak amplitude of the impedancepulse increasesas the systemic blood pressureis raised by epinephrine infusion and falls when the blood pressurefalls. The relationship is linear (as seenfrom the curves on the right), and the slopesare identical at the two recording sites over the pressurerange studied. Impedance Recording in Chronic Preparation. The impedancerecordings were stable throughout the experimental day and reproducible from day to
272
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day. The 20-ohm calibration signal in the four channels was reduced by only 2-10s of initial values after 6 hours of continuous operation. Although continuous application of the low-intensity, high-frequency bridge current (1 pa, 10-17.5 kc) had no visible effect on the EEG recordings or behavior, it was applied only during the short periods of recording. The brain impedance amplitudes at each site were also very constant (except for functional changes) for at least 8 months. Since the pulsatile impedance amplitudes were independent of absolute impedance level, gradual changes in interelectrode resistance were unimportant. Local cerebral impedance patterns differed according to electrode placement, state of animal activity and quality and quantity of applied stimuli. During sleep or quiet rest, the tracings were remarkably regular and stable for long periods. One or more tracings sometimes showed periodic or aperiodic spontaneous variations. Spindle-shaped waxing and waning of amplitude was a characteristic pattern observed frequently in hippocampus, lessfrequently in hypothalamus, but rarely in cortex, RF, or thalamus. In all eight cats showing hippocampal spindles, these occurred in rather irregular bursts of 4 to 6 per min. They were not correlated with respiratory or other movements,and, in fact, were not invariably present. Usually they were associatedwith sleep or relaxation and were out of phase with spindlesin other channels,except that bilaterally placed hippocampal leads were often synchronous (Fig. 2D). Responses to Stimulation. When cats were subjected to a variety of alerting stimuli, three distinct patterns of transient change in the impedance tracings were observed. The pattern dependedpartly on the stimulus and partly on the behavioral responsivenessof the cat. The first type of response(Fig. 2A) consistedof an immediate, generalized reduction in impedance pulse amplitude associatedwith tachycardia
FIG. 2. Patterns of cerebral impedance responses recorded from unrestrained, intact cats. A, B and C-responses to opening of cage door (indicated by bar) ; in C cat petted at arrow, D-bilateral comparison of responses to clicks in hippocampus and hypothalamus, E-responses to puffs of cigarette smoke (at arrows), F-out-ofphase responses in hippocampus and hypothalamus to opening of door. In all figures, IMP-impedance, HP-hippocampus, HY-hypothalamus, RF-reticular formation, C-cortex, VA-ventralis anterior thalamic nucleus, MD-medialis dorsalis thalamic nucleus, PH-posterior hypothalamic nucleus, F-fornix, OT-optic tract, CPcerebral peduncle, MB-mamillary body, VM-ventromedial hypothalamic nucleus. EEC calibration = 100 pv and time calibration = 1 set; absolute calibrations of impedance vary according to electrode placement and are not meaningful.
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after a sudden, strong stimulus. This could be elicited in all cats, but the adequate stimulus varied from a simple opening of the chamber door to a very loud, sudden noise or a painful pinch of the tail. Some cats showed this pattern spontaneously on awakening from sleep. The second type of response (Fig. 2B) was a selective and highly sensitive increase in impedance pulse in a specific hypothalamic region. Characteristically, this response could be elicited by alerting the cat with clicks, tones, fish smell, smoke, mild RF stimulation, or by otherwise attracting the cat’s attention. The most effective stimuli were those eliciting purring (i.e., petting, reassurance, etc.), The response involved a gradual increase and then a decrease in amplitude of hypothalamic impedance with slight, if any, change in other brain areas. The latency varied from 2 to 6 set and the duration was about 10 to 40 set in all cats. The degree and duration of the response were related to the stimulus intensity and to the apparent significance of the stimulus to the cat, and it could be shown to disappear or attenuate after repeated trials. For example, fish presented to a hungry cat caused a high amplitude, prolonged response, and the animal would eat. Later presentation, when the cat was sated, gave only a small, brief response and no eating. A characteristic response to excessive cigarette smoke blown into the cage was a prolonged increase in the hypothalamic impedance pulse. In certain cats the increase later spread to other brain areas and was accompanied by progressive and often profound cardiac slowing (Fig. 2E). With RF stimulation, illustrated in Fig. 3, the degree (A) and duration (B) of increase in hypothalamic impedance pulse were directly related to the intensity of stimulation. The hypothalamic responses, such as those described, were consistently obtained in nine cats, present to some extent in five cats, and never seen in one cat. In cat 11, which was tested repeatedly for nearly 8 months, it was elicited at least 100 times. The special sensitivity demonstrated in the second type of response resided in a very circumscribed region of the hypothalamus, and in cats with bilateral placements, one side might be consistently reactive, while the other side showed less or no response. Two such divergent bilateral placements (HY) and their responses are illustrated in Fig. 4. For all cats, most marked responses were obtained from the ventral hypothalamus, between the cerebral peduncle and mamillary body (or fornix), as shown in the composite brain-stem cross sections of Fig. 5. In two cats the placement closer to the peduncle was more responsive than that close to the mamillary body. The reactive site seemed to coincide with the area through which
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PATTERNS
600
2
4
6 V
2
4
6 V
FIG 3. The relationship of stimulating voltage (v) to the amplitude (A) and duration (B) of the hypothalamic impedance response to suprathreshold RF stimulation. Amplitude is expressed as a percentage of control amplitude and duration is in seconds. Each curve represents responses in a different animal.
HP
HY
c RHP LHP RHY L HY
FIG. 4. Hippocampal and hypothalamic impedance records obtained therefrom. Abbreviations as in Fig. 2.
electrode placements in cat 16. with actual RF stimulation (5 volt, lOO/sec) at bar.
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pass the fibers of the medial forebrain bundle, an area containing many large cell bodies among the fibers. Although the brain sections were not specially stained for blood vessels, it was apparent from the thioninestained sections that reactivity in the hypothalamus cannot be related to capillary density nor to the presence of large blood vessels near the electrode
FIG.
sentative response response.
tip.
5.
Hypothalamic cross sections to stimulation, Abbreviations
electrode placements in all 15 cats, plotted on two reprethrough hypothalamus. Large black dots-clear-cut impedance small black dots-slight response, and open circles-no as in Fig. 2.
The third type of response, a combination of the first two types, is illustrated in Fig. 2C. Opening the door caused an immediate and pronounced tachycardia and decreased impedance amplitude in all leads (least in hypothalamus), followed seconds later by the selective wave of hypothalamic increase. In all areas, recovery was gradual and impedance tracing and heart rate eventually returned to normal (not illustrated). In many cats, marked increase in the impedance amplitude of the dorsal hippocampus also occurred. The response was variable in incidence, degree, and phase relationship to the hypothalamic increase. It could not be shown to fall into the above classification with any consistency. An example is shown in Fig. 2F. Five cats had at least one active hippocampal placement, four had some hippocampal activity, and six had none. In two cats, the right and left hippocampal patterns were synchronous (Fig. ZD), but in others each varied independently, or one side was inactive (Fig. 4). No correlations between reactivity and electrode localization within dorsal hippocampus could be made. However, one placement found to lie in white matter just outside the hippocampus was inactive.
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In the remaining brain areas studied, alerting was usually accompanied by a decreased impedance pulse amplitude. The association cortex showed either a decrease with alerting, or there was no appreciable change. The midbrain RF and medial thalamic nuclei were also surprisingly inactive, except for small and variable increases in some cats. Yet all RF sites elicited electrocortical arousal when electrically stimulated.
FIG. 6. Effect of epinephrine (iv) on cerebral impedance in chronic (B) and acute (A) experiments on the same cat. The bottom records in A and B are continuous with the respective top records. Needle inserted at arrow in B and injection indicated by bar. In B, note the similarity between the response to epinephrine and to handling of the cat. Abbreviations as in Fig. 2.
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Eflect of Intravenous Epinephrine. Injection of Lepinephrine bitartrate ( 1-2 yg/kg, iv) in cats with good hypothalamic responses produced alterations comparable to alerting. Figure 6B illustrates one such experiment. Handling the cat caused the typical attention response, but this attenuated before injection began. After a slight initial decrease (6 set after start of injection), the hypothalamic impedance amplitude and the heart rate increased (at 12.5 set) to be followed by bradycardia and a further increase in amplitude at about 30 sec. Similar patterns were obtained in this cat with repeated trials and in other cats. The latency in all cats ranged between 8 and 14 set, and the duration, 20-70 sec. Acute Experiments. It was much more difficult to elicit cerebral impedance responses in immobilized cats, but hypothalamic increases were observed with both sound and direct RF stimulation. Variable hippocampal responses could also be obtained. Cats with Cl sections were more responsive than those paralyzed with gallamine triethiodide. In the former, responses had a lower amplitude and longer latency than in intact cats and tended to appear after cessation of stimulation. In terminal experiments on cat with electrodes implanted, paralysis with gallamine did not alter the sequence of changes after epinephrine found in chronic experiments on these cats, but responses were spread out over a longer time period and there were minimal or no heart-rate changes (Fig. 6A). The blood pressure rose, with accompanying reduction in hypothalamic impedance amplitude at lo-12 set, and the characteristic increase began about 54 set after injection, during the subsequent phase of slowly falling blood pressure. The hypothalamic response could not be elicited at all in anesthetized cats. Sleep and Wakefulness. In contrast to the clear and reproducible responses to alerting stimuli, the changes in impedance pulse with various spontaneous states of alertness were complex and variable. However, certain general trends were distinguishable. In the lightly sleeping state, there was an increase in the amplitude of the impedance pulsations from the cortex and from RF (especially in cats which responded to stimuli by decreases in the same areas) whereas hypothalamic impedance waves were significantly decreased. In the hippocampus the pattern was very variable, often having the appearance of spindles. In deeper, continued behavioral sleep, during which high-voltage, slow waves dominated the EEG, the amplitude of the impedance pulse tended to increase in all areas studied. In several cats, “paradoxical” sleep (8) with low-voltage EEG was observed, and under these conditions there was a clear similarity of the im-
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IMPEDANCE
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PATTERNS
EM‘
FIG. 7. Cerebral impedance patterns during alert wakefulness (A), high-voltage sleep (B), and low-voltage (paradoxical) sleep (C). Note similarity of hypothalamic records in A and C and of RF records in B and C. Abbreviations as in Fig. 2.
FIG.
parison
8. Hippocampal and RF synchrony with EEG tracings (A). RF electrode
of impedance tracings (B), placement shown in diagram.
and
com-
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AND
TACHIBANA
pedance patterns with those seen in the alert animal. With onset of paradoxical sleep, hypothalamic amplitude increased to the alert value (Fig. 7). Cortical and hippocampal amplitudes decreased,and that in RF either increased or decreased.Heart rates were usually lower than in the alert state. Impedance and EEG. Simultaneous recordings of EEG and impedance from the same site could not be made because of interference from the bridge current. However, some comparisons were possible by sequential recording of EEG and impedance under similar conditions, or by simultaneous recording of EEG from one site and impedance from the contralateral placement. Although arousal from sleep was associated with the immediate appearance of low-voltage fast electrical activity at all sites (except for theta activity at limbic sites), impedance changeswere much more variable, as described earlier. An interesting sidelight was the finding that, in one cat, the hippocampal and RF impedance traces were nearly identical in all records examined (e.g., Fig. 8B). The EEG tracings from these sites (Fig. 8A) are not exactly alike, since the hippocampushas more theta and the RF more fast activity. The location of the RF electrode (Fig. 8: RF) was found to be in the dorsal midbrain tegmentum, in the region where hippocampalentorhinal pathways are thought to terminate (2). In two other cats, with electrodesplaced slightly more ventromedially in the RF, hippocampal and RF impedance tracings were very similar but not identical. The RF placements of all other cats were outside the confines of this region, and their impedancepatterns did not lock with those of the hippocampus. Discussion
The variations in electrical impedance recorded from localized brain areas can be separated into two dynamic components: an aperiodic, comparatively slow change in total impedance level; and a fast, rhythmic fluctuation about the slowly changing baseline. With the usual low-frequency bridges of O-1000 cycle/‘sec, only the first component predominates, but at frequencies in the range of 15 kc and by the use of a suitably sensitive bridge circuit, the pulsatile component is also revealed. This latter component, which has the cardiac periodicity, is of interest because of indications that it may be related to rate of blood flow locally. The evidence for this supposition has been given in a previous publication (4). With the electrodes in a simulated circulatory system filled with saline
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solution, the amplitude of the impedance fluctuations was shown to be a linear function of the rate of flow of the ionic solution and independent of total impedance level in the physiological range; the slope of the function was found to be dependent on the ion concentration of the solution. In the living brain, ion movements arise not only from circulation of the blood, but from functionally related ion transfer across cell membranes. Although it is unlikely that the latter movements would be synchronous with the circulatory pulse, the possibility of their contaminating the blood flow recording was investigated in control experiments in which brain function was abolished by prolonged cerebral asphyxia. Under these conditions, reinstatement of circulation through the dead brain restored the impedance pulsations. Their amplitude was linearly related to the head of pressure and thus to flow, since vasomotor tone was also abolished by asphyxia and the circulatory hydraulic resistance remained constant. Conversely, in the dead or living brain, when the circulation was interrupted momentarily, no impedance fluctuations were recorded. On the basis of this evidence, the local cerebral impedance pulsations have been interrupted as reflecting relative blood flow at the electrode site. Relative changes in blood flow were shown to be comparable from one site to another, since the curves had parallel slopes. Correlations with actual volume flow could not be made because the pulse amplitude is also a function of the orientation of the electrodes with respect to direction of flow, maximum amplitudes being obtained when the flow is perpendicular to the plane of the electrode dipole. This dependence on direction may present a complication in evaluating cerebral blood flow changes by this method, because of the possibility of confusing changes in direction with changes in magnitude of flow. However, in view of the consistency of results from repeated trials in a given animal or from similar placements in different animals, this factor does not seem to be important. Brain activity has been shown to be accompanied by complex patterns of transient and prolonged local impedance changes, which we interpret as local vascular changes. Such changes are much more pronounced and site-specific than are visually observed changes in EEG. This is especially true of the hypothalamic increase elicited by alerting, which was more sensitive than electrocortical desynchronization and did not require a drowsy or sleepy baseline against which to be recognized. Since this impedance response appears to be related to the significance of the stimulus to the animal, it may be a useful parameter for studying the acquisi-
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tion of conditional responses or for evaluating the relative emotional values of various stimuli. In a small series of experiments, this same response was also observed from the hypothalamus of the dog (unpublished data). The reactive points were concentrated in the ventral part of the posterolateral hypothalamus, in the region containing the fibers and cell bodies of the medial forebrain bundle, although one reactive placement was located more medially in the posterior hypothalamic area near the fornix. Since this response was not easily elicited in acute preparations, no systematic exploration of the hypothalamus was made, but there was evidence of site-specificity from the chronic experiments. With small shifts away from the critical region in the same or different cats, the response was absent or much diminished. It may be significant that the responsive site lies within the hypothalamic area concerned with the regulation of sympathetic outflow and emotional expression. This part of the brain has a moderate, but not exceptional blood supply compared to other hypothalamic areas, such as the supraoptic and paraventricular nuclei, or with other parts of the brain (9). Within the sensitive region, in our cats, the best placements were not associated with greater capillary density. Thus the large increase in hypothalamic impedance waves with activity is not related to excessive vascularity per se, but appears to have a more dynamic origin. It is apparent that two different processes may be involved in the impedance response to stimulation. The immediate tachycardia and generalized reduction in impedance pulse, which we equated to vasoconstriction, appear to be neurally mediated, as part of the peripheral-sympathetic reaction to sudden, intense, or threatening stimuli. The delayed increase in impedance pulse (“vasodilatation”) in the hypothalamus unaccompanied by heart-rate changes, on the other hand, is probably initiated by humoral agents released either locally or systematically. This is reminiscent of the biphasic mechanism of electrocortical arousal postulated by Bonvallet (5). From the similarity between the effects of such stimulation and of intravenous epinephrine, one is tempted to attribute the entire arousal response to the effects of systemic epinephrine release. The injection of epinephrine shows the complete sequence of impedance events seen in the third type of response to alerting, i.e., an initial reduction in impedance pulse and tachycardia, followed by a hypothalamic increase, with bradycardia supervening. Since the animals were used to handling, insertion of the needle caused no effect. The response always occurred later, during the
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actual intravenous injection of the drug and was, therefore, not attributable to a pain reflex. The actual mechanism of the hypothalamic response, whether related to systemic epinephrine or not, remains to be considered. If the increased hypothalamic impedance represents a local increase in blood flow, the possible causes of this increase are a rise in blood pressure, an increase in local pCOZ, or a local hormone release. The influence of blood pressure can only be inferred from acute experiments. In these, there was usually an inverse relationship between blood pressure and cerebral blood flow, especially in the hypothalamus. If this relationship holds in the intact cat, then a generalized reflex vasoconstriction might explain the initial response, but the reason for the selective delayed increase in hypothalamic blood flow is not so clear. In some acute experiments, alerting stimuli or RF stimulation caused an immediate or a poststimulatory fall in arterial pressure, and the hypothalamic blood flow increase occurred during the falling phase. However, similar increases in blood flow have been inferred from the impedance records when there was no apparent pressure change or even during a rise in pressure in cats with Cl sections. The increase in hypothalamic flow with anoxia, though it begins just after the pressure has reached its peak, exceeds in amount and duration the increases seen with comparable spontaneous or mechanically induced hypotension. Thus, although the hypothalamic vasomotor response may be modified by systemic blood pressure changes, it is more probably generated locally through the accumulation of either CO, or a neurohormone. The latter deserves particular consideration because of the correlation between specificity of the hypothalamic response and the selective distribution of transmitter substances in hypothalamus and other brain areas (16). One such substance present in the hypothalamus is acetylcholine, a known vasodilator, whose release during neuronal activity might account for local vasodilatation. In addition, the catecholamines also concentrated in this region may be released in small amounts and facilitate the vasodilatation by acetylcholine. Low concentrations of epinephrine, for example, are thought to facilitate the action of acetylcholine in the nervous system (6). The occurrence of vasodilatation with hippocampal activity could also be explained on similar grounds, although the particular transmitter or facilitatory substances are not necessarily identical to those in the hypothalamus. Lack of response in association cortex and thalamus would also be consistent with this view. The low incidence of vasodilatation with activity in RF is difficult to understand, since it is also well supplied with
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neurohumors. It is possible that vasodilatation is masked by an overriding reflex or tonic vasoconstriction, or that the concentration of simultaneously active neurons is not as great as in the hypothalamic site. It is also possible that a response to stimuli with emotional significance involves greater activity in the hypothalamic-paleocortical activating circuit (13) than in the reticular-neocortical circuit. The question arises whether increased impedance pulse, and hence increased blood flow, at a site can be used as an index of local neuronal activity. The answer appears to be that in general it cannot. Transient increases may indeed signify local activity, but their absence need not imply lack of activity, and prolonged increases may have a different significance. Although increases in cortical or total cerebral blood flow with arousal have been reported (3, 12), the results are not comparable with ours, since they were obtained on acutely anesthetized animals, and the blood flow from large brain areas was measured. It is possible that sensorimotor or other cortical areas might show local vasodilatation with arousal, but these were not explored in our study, Blood-flow changes in these and other brain areas will be explored in future experiments. The data that have been obtained on blood flow during sleep also require a broader interpretation of circulatory events in relation to behavioral activity levels. With the onset of drowsiness and sleep, hypothalamic blood flow (or more strictly, impedance pulse) typically fell to basal levels, but that in cortex and RF was unchanged or slightly higher and tended to increase progressively with continued sleep. This increase may be due to a gradual relaxation in vasomotor tone, especially in areas such as RF which had previously been high. The increased circulation would remove metabolic COZ, to which the RF is especially sensitive (IO) and thus further reduce its excitability. The finding of an ‘(active” cerebrovascular pattern in paradoxical sleep, however, is consistent with other evidence that this low-voltage stage of sleep is associated with activity in the ascending reticular system (8, 17). References 1. 2.
3.
ADEY, W. R., R. T. KADO, and J. Dmro. 1962. Impedance measurements in brain tissue of animals using microvolt signals. Exptl. Neuvol. 5: 47-66. ADEY, W. R., S. SUNDERLAND, and C. W. DUNLOP. 1957. The entorhinal area; electrophysiological studies of its interrelations with rhinencephalic structures and the brainstem. Electroencephalog. Clin. Neurophysiol. 9: 309-324. BENETATO, G., I. HAULICA, V. NOST~ANU, E. BTJBUIANU, M. GARDEV, E. GHUARI, S. DIMITRIU, A. BADESCU, and M. CRIGORIU. 1963. Cerebral metabolic altera-
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5. 6. 7.
8.
9. 10. 11. 12.
13.
14.
15. 16.
17.
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tions occurring during the arousal reaction obtained by stimulation of the mesencephalic reticular formation in dogs, Electroencephalog. Clin. Neurophysioz. 16: 530. BIRZIS, L., and S. TACHIBA~A. 1962. Measurement of local cerebral blood flow by impedance changes. Life Sci. 11: 587-598. 1954. Tonus sympathique et activite BONVALLET, M., P. DELL, and G. HIEBEL. Clectrique corticale. Electroencephalog. Clin. Neurophysiol. 6: 119-144. BURN, J. H. 1945. The relation of adrenaline to acetylcholine in the nervous system. Physiol. Rev. 25: 377-394. DARROW, C. W. and C. G. GRAF. 1945. Relation of electroencephalogram to photometrically observed vasomotor changes in the brain. J. Neurophysiol. 8: 449-461. DEMENT, W. 1958. The occurrence of low voltage, fast electroencephalogram patterns during behavioral sleep in the cat. Electroencephalog. Clin. Neurophysiol. 10: 291-296. FINLEY, K. H. 1940. Angio-architecture of the hypotha!amus and its peculiarities. Res. Pwbl. Assoc. Res. A%‘ervous Mental Disease 20: 286-309. GELLHORN, E. 1960. “Physiological Foundations of Neurology and Psychiatry.” Univ. of Minnesota Press, Minneapolis, Minnesota. YAN HARREVELD, A., and S. OCHS. 1956. Cerebral impedance changes after circulatory arrest. .4m. J. Physiol. 187: 180-192. INC~AR, D. H. 1958. Cortical state of excitability and cortical circulation, pp. 381-412. In “Reticular Formation of the Brain.” Little, Brown, Boston, Massachusetts. KAWAMURA, H., Y. NAKAMIJRA, and T. TO~IZANE. 1961. Effect of acute brain stem lesions on the electrical activities of the limbic system and neocortex. Japan. J. Physiol. 11: 564-575. KETY, S. S. 1960. The cerebral circulation, pp. 1751.1760. In “Handbook of Physiology, Sect. I. Neurophysiology” 3. American Physiological Society, Washington, D.C. SEROTA, H., and R. W. GERARD. 19.18. Localized thermal changes in the cat’s brain. J. ,VezrrophysioZ. 1: 115-124. VOGT, M. 1954. The concentration of sympathin in different parts of the CNS under normal conditions and after the administration of drugs. J. Physiol. London 123: 451-481. WIXTERS, W. D. 1963. Click responses in cortical and subcortical structures during various stages of wakefulness and sleep, Pharmacologist 5: 266.
Local
NEUROLOGY
Cerebral
LUCY Stanford
9,
269-285
(1964)
Impedance Sleep and BIRZIS Research Received
AND
and Blood Arousal
SHUNRO
Institute, Sovembev
Flow
during
TACHIBANA’
Menlo
Park,
Calijomia
18, 1963
Local impedance fluctuations in cerebral cortex, midbrain reticular formation, dorsal hippocampus and hypothalamus were recorded simultaneously and continuously in unrestrained cats with permanently implanted electrodes. Distinct patterns of impedance recordings were correlated with behavioral and EEG signs of sleep, wakefulness and arousal by various stimuli. The most striking response to alerting stimuli was the marked, selective increase in impedance pulse in a specific area of the posterior hypothalamus, The exquisite sensitivity of this site is consistent with its affective and autonomic roles. Startle stimuli elicited tachycardia and a general, widespread reduction in cerebral impedance pulse amplitude. These two responses were most frequently seen in combination, particularly following the intravenous injection of 1-2 Bg/kg epinephrine. Hippocampal impedance changes in response to alerting stimuli were also observed, but less consistently. The local impedance patterns in different stages of sleep are also described; patterns seen during “paradoxical” sleep were very similar to those of the a!rrt state. Evidence is presented to support the hypothesis that the amplitude of the impedance pulse provides an index of the relative blood flow changes in the vicinity of the electrode. Introduction
Innumerable methods have been introduced for the assessment of cerebral circulation (3, 7, 12, 14, 15) and these have been applied in both lower animals and man in an attempt to correlate electrical activity of the brain with blood flow. Often such techniques tacitly assume the brain to have a completely homogeneous circulation, since they do not permit 1 This work was supported in part by research grants NB 03323 and NB 04210 from the National Institutes of Health and in part by contract Nonr-2993(00) between the Office of Naval Research and Stanford Research Institute. Reproduction in whole or part is permitted for any purpose of the United States Government. The authors thank Dr. Eva and Dr. Keith Killam for critical review of the manuscript. Dr. Tachibana’s present address is Department of Physiology, Kurume Medical School, Kurume. Japan. 269
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study of flow in discrete regions but simply measure total regional (3, 12) or even total brain perfusion (14). Again, many such methods are limited in that they do not offer a continuous picture of flow changes such as is needed to compare with the dynamic electrical changes of the brain (14). Others are unsuited for use in the chronic animal (7, 12) and some are potentially destructive of excitable tissues ( 15). An attempt was made in the present study to obtain estimates of relative blood flow within small, well-defined regions of the brain by extrapolation from measures of tissue electrical impedance. This work stemmed from preliminary studies (4) in which such an extrapolation was justified by in vitro and in viva experiments. The method of measuring impedance is generally similar to that reported in other recent studies (1, 11)) in that an alternating current bridge was used and the current density kept low to avoid possible stimulation of, or injury to, brain tissue. However, considerably higher frequencies were employed to emphasize those rhythmic changes which correlate with the cardiac cycle. Particular interest centered around simultaneous recording of impedance changes in those parts of the brain closely implicated in sleep, wakefulness and attention in the unanesthetized, unrestrained cat. Methods
In each of fifteen cats with permanently implanted electrodes, data were obtained from five to thirty recording sessionson each cat over periods up to 8 months. Recordings were also taken in ten acute experiments on spinal cats immobilized with gallamine triethiodide; in the latter preparations, pressure points and wound margins were infiltrated with lidocaine to prevent pain. Electrode placementsincluded “association” cortex, midbrain reticular formation (RF), dorsal hippocampus, medial thalamic nuclei, and posterior hypothalamus. Somecats had pairs of bilaterally symmetrical hippocampal and hypothalamic electrodes. These electrodes were used for recording electrical activity (EEG) or impedance, or for electrical stimulation. All placements were histologically verified from frozen, thioninestained brain sections. Chronic experiments were conducted with the cat in a sound-deadened Faraday chamber fitted with a one-way window. Impedance fluctuations, together with EEG and other physiological parameters, were recorded on an eight-channel Grass electroencephalograph.Four a-c impedance circuits (4) were used, each operating on a different generator frequency ( 10, 12.5, 15, and 17.5 kc) to enable impedance recordings to be made from four brain areassimultaneously.
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Relation Between Impedance and Circulation. To test the hypothesis that cerebral impedancereflected changesin blood flow, cerebral impedance fluctuations were recorded in Cl spinal cats before and after irreversible brain damage from occlusion of the cranial blood supply for 1 hour. The impedance changesbefore cerebral asphyxia were compared with changes recorded following restoration of circulation. This is illustrated in Fig. 1. The upper trace (BP) represents the femoral arterial pressure and the
\//*,p
---.,ric---x---
‘J~~~“~~~~~.~l~~ FIG.
campus record blood linear these femoral
___.. .-,
..
. --...~,.
I,.vLL? * ”“,F\
I.
Local cerebral impedance (IMP) in reticular formation (RF) and hippo(HP) of Cl spinal cat. Control-record before asphyxia. After asphyxiataken following cerebral asphyxia of I-hour duration, after reinstatement of flow, which was regulated by epinephrine infusion. Curves on the right show relationship between impedance amplitude and systemic arterial pressure for same two areas. EEG recorded from cortex. Blood pressure recorded from artery.
secondis the EEG record from a pair of cortical electrodes.The two lower tracings represent the impedance fluctuations in RF and hippocampus. After cerebral asphyxia, the EEG tracing is flat, but the impedancerecordings are similar to those of the control period. The peak-to-peak amplitude of the impedancepulse increasesas the systemic blood pressureis raised by epinephrine infusion and falls when the blood pressurefalls. The relationship is linear (as seenfrom the curves on the right), and the slopesare identical at the two recording sites over the pressurerange studied. Impedance Recording in Chronic Preparation. The impedancerecordings were stable throughout the experimental day and reproducible from day to
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day. The 20-ohm calibration signal in the four channels was reduced by only 2-10s of initial values after 6 hours of continuous operation. Although continuous application of the low-intensity, high-frequency bridge current (1 pa, 10-17.5 kc) had no visible effect on the EEG recordings or behavior, it was applied only during the short periods of recording. The brain impedance amplitudes at each site were also very constant (except for functional changes) for at least 8 months. Since the pulsatile impedance amplitudes were independent of absolute impedance level, gradual changes in interelectrode resistance were unimportant. Local cerebral impedance patterns differed according to electrode placement, state of animal activity and quality and quantity of applied stimuli. During sleep or quiet rest, the tracings were remarkably regular and stable for long periods. One or more tracings sometimes showed periodic or aperiodic spontaneous variations. Spindle-shaped waxing and waning of amplitude was a characteristic pattern observed frequently in hippocampus, lessfrequently in hypothalamus, but rarely in cortex, RF, or thalamus. In all eight cats showing hippocampal spindles, these occurred in rather irregular bursts of 4 to 6 per min. They were not correlated with respiratory or other movements,and, in fact, were not invariably present. Usually they were associatedwith sleep or relaxation and were out of phase with spindlesin other channels,except that bilaterally placed hippocampal leads were often synchronous (Fig. 2D). Responses to Stimulation. When cats were subjected to a variety of alerting stimuli, three distinct patterns of transient change in the impedance tracings were observed. The pattern dependedpartly on the stimulus and partly on the behavioral responsivenessof the cat. The first type of response(Fig. 2A) consistedof an immediate, generalized reduction in impedance pulse amplitude associatedwith tachycardia
FIG. 2. Patterns of cerebral impedance responses recorded from unrestrained, intact cats. A, B and C-responses to opening of cage door (indicated by bar) ; in C cat petted at arrow, D-bilateral comparison of responses to clicks in hippocampus and hypothalamus, E-responses to puffs of cigarette smoke (at arrows), F-out-ofphase responses in hippocampus and hypothalamus to opening of door. In all figures, IMP-impedance, HP-hippocampus, HY-hypothalamus, RF-reticular formation, C-cortex, VA-ventralis anterior thalamic nucleus, MD-medialis dorsalis thalamic nucleus, PH-posterior hypothalamic nucleus, F-fornix, OT-optic tract, CPcerebral peduncle, MB-mamillary body, VM-ventromedial hypothalamic nucleus. EEC calibration = 100 pv and time calibration = 1 set; absolute calibrations of impedance vary according to electrode placement and are not meaningful.
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after a sudden, strong stimulus. This could be elicited in all cats, but the adequate stimulus varied from a simple opening of the chamber door to a very loud, sudden noise or a painful pinch of the tail. Some cats showed this pattern spontaneously on awakening from sleep. The second type of response (Fig. 2B) was a selective and highly sensitive increase in impedance pulse in a specific hypothalamic region. Characteristically, this response could be elicited by alerting the cat with clicks, tones, fish smell, smoke, mild RF stimulation, or by otherwise attracting the cat’s attention. The most effective stimuli were those eliciting purring (i.e., petting, reassurance, etc.), The response involved a gradual increase and then a decrease in amplitude of hypothalamic impedance with slight, if any, change in other brain areas. The latency varied from 2 to 6 set and the duration was about 10 to 40 set in all cats. The degree and duration of the response were related to the stimulus intensity and to the apparent significance of the stimulus to the cat, and it could be shown to disappear or attenuate after repeated trials. For example, fish presented to a hungry cat caused a high amplitude, prolonged response, and the animal would eat. Later presentation, when the cat was sated, gave only a small, brief response and no eating. A characteristic response to excessive cigarette smoke blown into the cage was a prolonged increase in the hypothalamic impedance pulse. In certain cats the increase later spread to other brain areas and was accompanied by progressive and often profound cardiac slowing (Fig. 2E). With RF stimulation, illustrated in Fig. 3, the degree (A) and duration (B) of increase in hypothalamic impedance pulse were directly related to the intensity of stimulation. The hypothalamic responses, such as those described, were consistently obtained in nine cats, present to some extent in five cats, and never seen in one cat. In cat 11, which was tested repeatedly for nearly 8 months, it was elicited at least 100 times. The special sensitivity demonstrated in the second type of response resided in a very circumscribed region of the hypothalamus, and in cats with bilateral placements, one side might be consistently reactive, while the other side showed less or no response. Two such divergent bilateral placements (HY) and their responses are illustrated in Fig. 4. For all cats, most marked responses were obtained from the ventral hypothalamus, between the cerebral peduncle and mamillary body (or fornix), as shown in the composite brain-stem cross sections of Fig. 5. In two cats the placement closer to the peduncle was more responsive than that close to the mamillary body. The reactive site seemed to coincide with the area through which
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600
2
4
6 V
2
4
6 V
FIG 3. The relationship of stimulating voltage (v) to the amplitude (A) and duration (B) of the hypothalamic impedance response to suprathreshold RF stimulation. Amplitude is expressed as a percentage of control amplitude and duration is in seconds. Each curve represents responses in a different animal.
HP
HY
c RHP LHP RHY L HY
FIG. 4. Hippocampal and hypothalamic impedance records obtained therefrom. Abbreviations as in Fig. 2.
electrode placements in cat 16. with actual RF stimulation (5 volt, lOO/sec) at bar.
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pass the fibers of the medial forebrain bundle, an area containing many large cell bodies among the fibers. Although the brain sections were not specially stained for blood vessels, it was apparent from the thioninestained sections that reactivity in the hypothalamus cannot be related to capillary density nor to the presence of large blood vessels near the electrode
FIG.
sentative response response.
tip.
5.
Hypothalamic cross sections to stimulation, Abbreviations
electrode placements in all 15 cats, plotted on two reprethrough hypothalamus. Large black dots-clear-cut impedance small black dots-slight response, and open circles-no as in Fig. 2.
The third type of response, a combination of the first two types, is illustrated in Fig. 2C. Opening the door caused an immediate and pronounced tachycardia and decreased impedance amplitude in all leads (least in hypothalamus), followed seconds later by the selective wave of hypothalamic increase. In all areas, recovery was gradual and impedance tracing and heart rate eventually returned to normal (not illustrated). In many cats, marked increase in the impedance amplitude of the dorsal hippocampus also occurred. The response was variable in incidence, degree, and phase relationship to the hypothalamic increase. It could not be shown to fall into the above classification with any consistency. An example is shown in Fig. 2F. Five cats had at least one active hippocampal placement, four had some hippocampal activity, and six had none. In two cats, the right and left hippocampal patterns were synchronous (Fig. ZD), but in others each varied independently, or one side was inactive (Fig. 4). No correlations between reactivity and electrode localization within dorsal hippocampus could be made. However, one placement found to lie in white matter just outside the hippocampus was inactive.
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In the remaining brain areas studied, alerting was usually accompanied by a decreased impedance pulse amplitude. The association cortex showed either a decrease with alerting, or there was no appreciable change. The midbrain RF and medial thalamic nuclei were also surprisingly inactive, except for small and variable increases in some cats. Yet all RF sites elicited electrocortical arousal when electrically stimulated.
FIG. 6. Effect of epinephrine (iv) on cerebral impedance in chronic (B) and acute (A) experiments on the same cat. The bottom records in A and B are continuous with the respective top records. Needle inserted at arrow in B and injection indicated by bar. In B, note the similarity between the response to epinephrine and to handling of the cat. Abbreviations as in Fig. 2.
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Eflect of Intravenous Epinephrine. Injection of Lepinephrine bitartrate ( 1-2 yg/kg, iv) in cats with good hypothalamic responses produced alterations comparable to alerting. Figure 6B illustrates one such experiment. Handling the cat caused the typical attention response, but this attenuated before injection began. After a slight initial decrease (6 set after start of injection), the hypothalamic impedance amplitude and the heart rate increased (at 12.5 set) to be followed by bradycardia and a further increase in amplitude at about 30 sec. Similar patterns were obtained in this cat with repeated trials and in other cats. The latency in all cats ranged between 8 and 14 set, and the duration, 20-70 sec. Acute Experiments. It was much more difficult to elicit cerebral impedance responses in immobilized cats, but hypothalamic increases were observed with both sound and direct RF stimulation. Variable hippocampal responses could also be obtained. Cats with Cl sections were more responsive than those paralyzed with gallamine triethiodide. In the former, responses had a lower amplitude and longer latency than in intact cats and tended to appear after cessation of stimulation. In terminal experiments on cat with electrodes implanted, paralysis with gallamine did not alter the sequence of changes after epinephrine found in chronic experiments on these cats, but responses were spread out over a longer time period and there were minimal or no heart-rate changes (Fig. 6A). The blood pressure rose, with accompanying reduction in hypothalamic impedance amplitude at lo-12 set, and the characteristic increase began about 54 set after injection, during the subsequent phase of slowly falling blood pressure. The hypothalamic response could not be elicited at all in anesthetized cats. Sleep and Wakefulness. In contrast to the clear and reproducible responses to alerting stimuli, the changes in impedance pulse with various spontaneous states of alertness were complex and variable. However, certain general trends were distinguishable. In the lightly sleeping state, there was an increase in the amplitude of the impedance pulsations from the cortex and from RF (especially in cats which responded to stimuli by decreases in the same areas) whereas hypothalamic impedance waves were significantly decreased. In the hippocampus the pattern was very variable, often having the appearance of spindles. In deeper, continued behavioral sleep, during which high-voltage, slow waves dominated the EEG, the amplitude of the impedance pulse tended to increase in all areas studied. In several cats, “paradoxical” sleep (8) with low-voltage EEG was observed, and under these conditions there was a clear similarity of the im-
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EM‘
FIG. 7. Cerebral impedance patterns during alert wakefulness (A), high-voltage sleep (B), and low-voltage (paradoxical) sleep (C). Note similarity of hypothalamic records in A and C and of RF records in B and C. Abbreviations as in Fig. 2.
FIG.
parison
8. Hippocampal and RF synchrony with EEG tracings (A). RF electrode
of impedance tracings (B), placement shown in diagram.
and
com-
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pedance patterns with those seen in the alert animal. With onset of paradoxical sleep, hypothalamic amplitude increased to the alert value (Fig. 7). Cortical and hippocampal amplitudes decreased,and that in RF either increased or decreased.Heart rates were usually lower than in the alert state. Impedance and EEG. Simultaneous recordings of EEG and impedance from the same site could not be made because of interference from the bridge current. However, some comparisons were possible by sequential recording of EEG and impedance under similar conditions, or by simultaneous recording of EEG from one site and impedance from the contralateral placement. Although arousal from sleep was associated with the immediate appearance of low-voltage fast electrical activity at all sites (except for theta activity at limbic sites), impedance changeswere much more variable, as described earlier. An interesting sidelight was the finding that, in one cat, the hippocampal and RF impedance traces were nearly identical in all records examined (e.g., Fig. 8B). The EEG tracings from these sites (Fig. 8A) are not exactly alike, since the hippocampushas more theta and the RF more fast activity. The location of the RF electrode (Fig. 8: RF) was found to be in the dorsal midbrain tegmentum, in the region where hippocampalentorhinal pathways are thought to terminate (2). In two other cats, with electrodesplaced slightly more ventromedially in the RF, hippocampal and RF impedance tracings were very similar but not identical. The RF placements of all other cats were outside the confines of this region, and their impedancepatterns did not lock with those of the hippocampus. Discussion
The variations in electrical impedance recorded from localized brain areas can be separated into two dynamic components: an aperiodic, comparatively slow change in total impedance level; and a fast, rhythmic fluctuation about the slowly changing baseline. With the usual low-frequency bridges of O-1000 cycle/‘sec, only the first component predominates, but at frequencies in the range of 15 kc and by the use of a suitably sensitive bridge circuit, the pulsatile component is also revealed. This latter component, which has the cardiac periodicity, is of interest because of indications that it may be related to rate of blood flow locally. The evidence for this supposition has been given in a previous publication (4). With the electrodes in a simulated circulatory system filled with saline
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solution, the amplitude of the impedance fluctuations was shown to be a linear function of the rate of flow of the ionic solution and independent of total impedance level in the physiological range; the slope of the function was found to be dependent on the ion concentration of the solution. In the living brain, ion movements arise not only from circulation of the blood, but from functionally related ion transfer across cell membranes. Although it is unlikely that the latter movements would be synchronous with the circulatory pulse, the possibility of their contaminating the blood flow recording was investigated in control experiments in which brain function was abolished by prolonged cerebral asphyxia. Under these conditions, reinstatement of circulation through the dead brain restored the impedance pulsations. Their amplitude was linearly related to the head of pressure and thus to flow, since vasomotor tone was also abolished by asphyxia and the circulatory hydraulic resistance remained constant. Conversely, in the dead or living brain, when the circulation was interrupted momentarily, no impedance fluctuations were recorded. On the basis of this evidence, the local cerebral impedance pulsations have been interrupted as reflecting relative blood flow at the electrode site. Relative changes in blood flow were shown to be comparable from one site to another, since the curves had parallel slopes. Correlations with actual volume flow could not be made because the pulse amplitude is also a function of the orientation of the electrodes with respect to direction of flow, maximum amplitudes being obtained when the flow is perpendicular to the plane of the electrode dipole. This dependence on direction may present a complication in evaluating cerebral blood flow changes by this method, because of the possibility of confusing changes in direction with changes in magnitude of flow. However, in view of the consistency of results from repeated trials in a given animal or from similar placements in different animals, this factor does not seem to be important. Brain activity has been shown to be accompanied by complex patterns of transient and prolonged local impedance changes, which we interpret as local vascular changes. Such changes are much more pronounced and site-specific than are visually observed changes in EEG. This is especially true of the hypothalamic increase elicited by alerting, which was more sensitive than electrocortical desynchronization and did not require a drowsy or sleepy baseline against which to be recognized. Since this impedance response appears to be related to the significance of the stimulus to the animal, it may be a useful parameter for studying the acquisi-
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tion of conditional responses or for evaluating the relative emotional values of various stimuli. In a small series of experiments, this same response was also observed from the hypothalamus of the dog (unpublished data). The reactive points were concentrated in the ventral part of the posterolateral hypothalamus, in the region containing the fibers and cell bodies of the medial forebrain bundle, although one reactive placement was located more medially in the posterior hypothalamic area near the fornix. Since this response was not easily elicited in acute preparations, no systematic exploration of the hypothalamus was made, but there was evidence of site-specificity from the chronic experiments. With small shifts away from the critical region in the same or different cats, the response was absent or much diminished. It may be significant that the responsive site lies within the hypothalamic area concerned with the regulation of sympathetic outflow and emotional expression. This part of the brain has a moderate, but not exceptional blood supply compared to other hypothalamic areas, such as the supraoptic and paraventricular nuclei, or with other parts of the brain (9). Within the sensitive region, in our cats, the best placements were not associated with greater capillary density. Thus the large increase in hypothalamic impedance waves with activity is not related to excessive vascularity per se, but appears to have a more dynamic origin. It is apparent that two different processes may be involved in the impedance response to stimulation. The immediate tachycardia and generalized reduction in impedance pulse, which we equated to vasoconstriction, appear to be neurally mediated, as part of the peripheral-sympathetic reaction to sudden, intense, or threatening stimuli. The delayed increase in impedance pulse (“vasodilatation”) in the hypothalamus unaccompanied by heart-rate changes, on the other hand, is probably initiated by humoral agents released either locally or systematically. This is reminiscent of the biphasic mechanism of electrocortical arousal postulated by Bonvallet (5). From the similarity between the effects of such stimulation and of intravenous epinephrine, one is tempted to attribute the entire arousal response to the effects of systemic epinephrine release. The injection of epinephrine shows the complete sequence of impedance events seen in the third type of response to alerting, i.e., an initial reduction in impedance pulse and tachycardia, followed by a hypothalamic increase, with bradycardia supervening. Since the animals were used to handling, insertion of the needle caused no effect. The response always occurred later, during the
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actual intravenous injection of the drug and was, therefore, not attributable to a pain reflex. The actual mechanism of the hypothalamic response, whether related to systemic epinephrine or not, remains to be considered. If the increased hypothalamic impedance represents a local increase in blood flow, the possible causes of this increase are a rise in blood pressure, an increase in local pCOZ, or a local hormone release. The influence of blood pressure can only be inferred from acute experiments. In these, there was usually an inverse relationship between blood pressure and cerebral blood flow, especially in the hypothalamus. If this relationship holds in the intact cat, then a generalized reflex vasoconstriction might explain the initial response, but the reason for the selective delayed increase in hypothalamic blood flow is not so clear. In some acute experiments, alerting stimuli or RF stimulation caused an immediate or a poststimulatory fall in arterial pressure, and the hypothalamic blood flow increase occurred during the falling phase. However, similar increases in blood flow have been inferred from the impedance records when there was no apparent pressure change or even during a rise in pressure in cats with Cl sections. The increase in hypothalamic flow with anoxia, though it begins just after the pressure has reached its peak, exceeds in amount and duration the increases seen with comparable spontaneous or mechanically induced hypotension. Thus, although the hypothalamic vasomotor response may be modified by systemic blood pressure changes, it is more probably generated locally through the accumulation of either CO, or a neurohormone. The latter deserves particular consideration because of the correlation between specificity of the hypothalamic response and the selective distribution of transmitter substances in hypothalamus and other brain areas (16). One such substance present in the hypothalamus is acetylcholine, a known vasodilator, whose release during neuronal activity might account for local vasodilatation. In addition, the catecholamines also concentrated in this region may be released in small amounts and facilitate the vasodilatation by acetylcholine. Low concentrations of epinephrine, for example, are thought to facilitate the action of acetylcholine in the nervous system (6). The occurrence of vasodilatation with hippocampal activity could also be explained on similar grounds, although the particular transmitter or facilitatory substances are not necessarily identical to those in the hypothalamus. Lack of response in association cortex and thalamus would also be consistent with this view. The low incidence of vasodilatation with activity in RF is difficult to understand, since it is also well supplied with
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neurohumors. It is possible that vasodilatation is masked by an overriding reflex or tonic vasoconstriction, or that the concentration of simultaneously active neurons is not as great as in the hypothalamic site. It is also possible that a response to stimuli with emotional significance involves greater activity in the hypothalamic-paleocortical activating circuit (13) than in the reticular-neocortical circuit. The question arises whether increased impedance pulse, and hence increased blood flow, at a site can be used as an index of local neuronal activity. The answer appears to be that in general it cannot. Transient increases may indeed signify local activity, but their absence need not imply lack of activity, and prolonged increases may have a different significance. Although increases in cortical or total cerebral blood flow with arousal have been reported (3, 12), the results are not comparable with ours, since they were obtained on acutely anesthetized animals, and the blood flow from large brain areas was measured. It is possible that sensorimotor or other cortical areas might show local vasodilatation with arousal, but these were not explored in our study, Blood-flow changes in these and other brain areas will be explored in future experiments. The data that have been obtained on blood flow during sleep also require a broader interpretation of circulatory events in relation to behavioral activity levels. With the onset of drowsiness and sleep, hypothalamic blood flow (or more strictly, impedance pulse) typically fell to basal levels, but that in cortex and RF was unchanged or slightly higher and tended to increase progressively with continued sleep. This increase may be due to a gradual relaxation in vasomotor tone, especially in areas such as RF which had previously been high. The increased circulation would remove metabolic COZ, to which the RF is especially sensitive (IO) and thus further reduce its excitability. The finding of an ‘(active” cerebrovascular pattern in paradoxical sleep, however, is consistent with other evidence that this low-voltage stage of sleep is associated with activity in the ascending reticular system (8, 17). References 1. 2.
3.
ADEY, W. R., R. T. KADO, and J. Dmro. 1962. Impedance measurements in brain tissue of animals using microvolt signals. Exptl. Neuvol. 5: 47-66. ADEY, W. R., S. SUNDERLAND, and C. W. DUNLOP. 1957. The entorhinal area; electrophysiological studies of its interrelations with rhinencephalic structures and the brainstem. Electroencephalog. Clin. Neurophysiol. 9: 309-324. BENETATO, G., I. HAULICA, V. NOST~ANU, E. BTJBUIANU, M. GARDEV, E. GHUARI, S. DIMITRIU, A. BADESCU, and M. CRIGORIU. 1963. Cerebral metabolic altera-
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tions occurring during the arousal reaction obtained by stimulation of the mesencephalic reticular formation in dogs, Electroencephalog. Clin. Neurophysioz. 16: 530. BIRZIS, L., and S. TACHIBA~A. 1962. Measurement of local cerebral blood flow by impedance changes. Life Sci. 11: 587-598. 1954. Tonus sympathique et activite BONVALLET, M., P. DELL, and G. HIEBEL. Clectrique corticale. Electroencephalog. Clin. Neurophysiol. 6: 119-144. BURN, J. H. 1945. The relation of adrenaline to acetylcholine in the nervous system. Physiol. Rev. 25: 377-394. DARROW, C. W. and C. G. GRAF. 1945. Relation of electroencephalogram to photometrically observed vasomotor changes in the brain. J. Neurophysiol. 8: 449-461. DEMENT, W. 1958. The occurrence of low voltage, fast electroencephalogram patterns during behavioral sleep in the cat. Electroencephalog. Clin. Neurophysiol. 10: 291-296. FINLEY, K. H. 1940. Angio-architecture of the hypotha!amus and its peculiarities. Res. Pwbl. Assoc. Res. A%‘ervous Mental Disease 20: 286-309. GELLHORN, E. 1960. “Physiological Foundations of Neurology and Psychiatry.” Univ. of Minnesota Press, Minneapolis, Minnesota. YAN HARREVELD, A., and S. OCHS. 1956. Cerebral impedance changes after circulatory arrest. .4m. J. Physiol. 187: 180-192. INC~AR, D. H. 1958. Cortical state of excitability and cortical circulation, pp. 381-412. In “Reticular Formation of the Brain.” Little, Brown, Boston, Massachusetts. KAWAMURA, H., Y. NAKAMIJRA, and T. TO~IZANE. 1961. Effect of acute brain stem lesions on the electrical activities of the limbic system and neocortex. Japan. J. Physiol. 11: 564-575. KETY, S. S. 1960. The cerebral circulation, pp. 1751.1760. In “Handbook of Physiology, Sect. I. Neurophysiology” 3. American Physiological Society, Washington, D.C. SEROTA, H., and R. W. GERARD. 19.18. Localized thermal changes in the cat’s brain. J. ,VezrrophysioZ. 1: 115-124. VOGT, M. 1954. The concentration of sympathin in different parts of the CNS under normal conditions and after the administration of drugs. J. Physiol. London 123: 451-481. WIXTERS, W. D. 1963. Click responses in cortical and subcortical structures during various stages of wakefulness and sleep, Pharmacologist 5: 266.