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Norepinephrine depletion alters cerebral oxidative metabolism in the ‘active’ state
Brain Research, 204 (1981) 8%101 © Elsevier/North-Holland Biomedical Press
87
N O R E P I N E P H R I N E D E P L E T I O N ALTERS C E R E B R A L OXIDATIVE METABOLISM IN T H E 'ACTIVE' STATE
JOSEPH C. LaMANNA, SAMI I. HARIK, ANDREW I. LIGHT and MYRON ROSENTHAL Department of Neurology, University of Miami School of Medicine, Miami, Fla. 33101 (U.S.A.)
(Accepted June 19th, 1980) Key words: norepinephrine - - locus coeruleus - - oxidative metabolism - - cytochrome oxidase --
NAD
-
-
evoked potential
SUMMARY Unilateral chemical lesion o f the nucleus locus coeruleus in rats produced unilateral depletion of ipsilatetal cortical norepinephrine. Norepinephrine depletion was not associated with changes in 'resting' metabolic balance of the cerebral cortex, as determined by in situ reflection spectrophotometry of the redox state of cytochrome oxidase. Norepinephrine depletion, however, caused slowing of the transient metabolic response to sudden increases in energy demand produced by direct cortical electrical stimulation. The effect was apparent in the redox state of both cytochrome oxidase and NAD, the latter being measured, also in situ, by reflection microfluorometry. These results demonstrate that norepinephrine has a role in modulating the response to increased metabolic demand inthe cerebral cortex. Norepinephrine may mediate its effect by potentiating Na +, K+-ATPase or through its effects on vascular reactivity, or both.
INTRODUCTION Norepinephrine (NE) is a putative central nervous system neurotransmitter or modulator which is suspected of playing an important role in several physiological processes and pathological conditions. Among its suggested functions is the regulation of cerebral blood flow a,a0 and capillary permeability a°. Since cerebral metabolism and tissue perfusion are closely linked, it would be expected that if effects of NE on flow and permeability are physiologically significant, then these effects would be expressed through changes in regional cerebral oxidative metabolism. In this report, we directly explore the role of N E on cerebral oxidative energy metabolism. This was done by comparing the relationship between energy utilization
88 and the cellular capability to maintain oxidative metabolic homeostasis under 'resting' and 'active' conditions in the presence and absence of NE. Oxidative metabolism was monitored non-invasively from the surface of rat cerebral hemispheres in situ in two ways: by continuous dual-wavelength reflection spectrophotometry oflocal changes in the reduction/oxidation (redox) state of cytochrome oxidase and blood volumela; and by fluorometrically recording redox changes of pyridine nucleotide4,19. Since pyridine nucleotide is the initial coenzyme of the mitochondrial respiratory chain and cytochrome oxidase (cytochrome a,aa) is its terminal member, changes in the redox ratio of each of these directly indicate intracellular oxidative metabolic activities 6,17. Comparisons were made between hemispheres with normal NE content and those that were NE-depleted by unilateral 6-hydroxydopamine (6-OHDA) lesions of the locus coeruleus (LC). These investigations take advantage of the fact that almost all cerebral NE arises from neurons in the LC through an ipsilateral pathway20,a9. Unilateral 6OHDA lesion of the LC produces marked depletion of NE in the ipsilateral cortex while leaving the contralateral cortex with normal NE content thereby serving as an internal control 11,~6. We report here that the redox ratio of cytochrome oxidase was unchanged under 'resting' conditions in NE-depleted cortical hemispheres and conclude that the relationship of energy demand and supply is not affected by NE depletion when energy demand is low. However, marked changes occurred in NE-depleted hemispheres when energy demand was increased by electrical stimulation. Preliminary reports of these results have been published 2a,31. METHODS Locus coeruleus lesion Male Wistar rats (175-200 g) were allowed 5-7 days of acclimation during which time they were kept under diurnal light conditions with free access to laboratory chow and water. The LC was lesioned unilaterally by the local infusion of 2.5/~1 of 6OHDA solution containing 5/~g of 6-OHDA base under chloral hydrate (400 mg/kg, i.p.) anesthesia. Pargyline (50 mg/kg, i.p.) was given 15-30 min before the infusion of 6-OHDA to produce a more effective lesionzl. 6-OHDA was freshly dissolved in 0.9 NaCI solution containing 1 mg/ml ascorbic acid, adjusted to a pH of approximately 5.5 and kept on ice until used. The 6-OHDA solution was slowly infused through a glass micropipet (tip diameter 40-60 #m) attached to a Hamilton syringe, over a period of 4-5 min. The stereotaxic coordinates were 1.1 mm posterior, 1.3 mm lateral and 7.5 mm ventral to the lambda. Approximately equal numbers of right and left side lesions were made. The animals were returned to their cages when awake and allowed to recover for a period of two weeks before they were used in the metabolic experiments. The operative mortality of the unilateral LC lesion procedure was less than 10 ~. In preliminary experiments, the LC was infused with vehicle solution. This did not result in any significant changes in the NE content of the ipsilateral cerebral cortex in comparison to the non-infused side. For this reason, non-lesioned rats that were anesthetized with chloral hydrate and given pargyline served as additional controls for
89 the unilaterally lesioned rats so that NE-depleted hemispheres could be compared to non-depleted hemispheres contralateral to the unilateral LC lesion and to those of non-lesioned control rats.
Preparation of animals for metabolic studies Two weeks after LC lesions were made, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The femoral artery and vein were cannulated for monitoring of arterial blood pressure, for sampling of arterial blood gas parameters (paO2, p aCO2 and pH) and for intravenous infusion of supplemental pentobarbital to maintain light anesthesia. Tracheostomy was performed and the rats were paralyzed with tubocurarine. They were immediately connected to a positive pressure respirator and ventilated with a gas mixture containing 30 ~ Oz and 70 % N2 at approximately 55 strokes/min and 3 ml stroke volume. Stroke volume and respiratory rate were adjusted to maintain blood gas values within the normal range: pH approximately 7.44 (7.41-7.47); p aCO2 approximately 38 mmHg (36-42); and paO2 above 110 mmHg. A rectal thermocouple was inserted and body temperature maintained at 37 °C by a heating pad. The bone overlying the frontoparietal region on each side of the midline suture was thert thinned by drilling at slow speed with a burr. Each area was about 8 × 5 mm. The thinned portion was hooked with a curved needle and lifted carefully away from the intact dura. The optical equipment was focused on either the left or right side field, alternating from experiment to experiment. An Ag-AgC1 recording electrode was placed on the dura in the center of the area to be monitored optically. This electrode was used to record both the electrocorticogram (ECoG) and the DC-coupled electrical potential (the so-called 'cortical steady potential'), and was referenced to an Ag-AgC1 electrode inserted into the neck musculature. A forked stimulating electrode (tips 0.1 mm in diameter, 0.5 mm apart) was placed about 1 mm from the recording electrode, near the edge of the optical field. Blood pressure, ECoG, and the cortical steady potential were displayed on a strip chart recorder.
Measurement of cytochrome oxidase redox state by reflection spectrophotometry The reflectance spectrophotometric monitoring system for cytochrome oxidase redox changes and changes in blood volume is based on the light absorption properties of the cytochrome components of tissue mitochondria. Since cytochromes absorb light more strongly in the reduced form than in the oxidized form, changes in redox ratios can be determined by measuring changes in reflected light intensity at a wavelength of maximal absorption difference between the reduced and the oxidized species of each of these mitochondrial components. The 605 nm wavelength has been well worked out for cytochrome oxidase in the intact cerebral cortex. A detailed description of the method has been published is. In brief, light from a 150 W xenon short-arc lamp was divided mechanically into two beams and alternately directed (at 30 I-Iz) through two monochromators, one of these was set at 605 nm (the 'sample' beam for cytochrome oxidase), the other at 590 nm to provide reference compensation for changes in blood oxygenation and volume. These monochromatic light beams were directed by fiber optic light guides onto the cerebral cortex.
90 Microscope optics were used to gather the reflected light scattered back from the tissue and focus it on a photomultiplier tube housed in the microscope barrel. Both the light guides and the microscope optics were set at an angle to minimize specular reflection from the tissue surface. The signals representing the intensity of light reflected at the reference wavelength and the difference between the intensity of light at the sample and reference wavelengths were displayed on a strip chart recorder. The first of these signals, compensated for and provided a useful indicator of blood volume shifts. The instrument was calibrated by making the dark level equal to zero and the reference intensity equal to a full scale deflection of the strip chart recorder pen. Sample light was then adjusted to be equal to the reference intensity. All data is then recorded as per cent of full scale.
Measurement of pyridine nucleotide redox state by microfluorometry The fluorometric measurement of the intramitochondrial pyridine nucleotide redox state is derived from two findings: (1) the reduced form of the coenzyme fluoresces at 450 nm when excited by light at 366 nm while the oxidized form does not; and (2) the bound enzyme shows enhanced fluorescence over the unbound cytoplasmic form z,9,2s. The technique was first developed by Chance and Jfbsis 5 and adapted to the intact cerebrum by Chance et al. 4. Light at 366 nm from a 1000 W mercury arc lamp was directed onto the cortical surface of the rat. Reflected and fluorescent light from the cortical surface were collected by microscope optics, separated by a beam-splitter and filters, and measured by separate photomultiplier tubes. The reflected excitation light was subtracted from fluorescent light as a compensation for non-specific changes in optical properties of the preparation 19. The signals representing fluorescence, reflectance and the corrected difference between these, were displayed on a strip chart recorder. Full scale refers to the initial intensities of each of these from the cortical surface with zero being no light and 1O0 being those initial values.
Responses to inspired gas changes After a period of stabilization, during which the rats were respired with 30 ~o 02/70 ~ N2, the ventilation mixture was changed either to 100 ~ O2 or to 95 ~ 02/5 COz in experiments to monitor redox changes of cytochrome oxidase by reflection spectrophotometry. After approximately 5 min, the animals again were respired with 30 9/0002/70 ~o N2 until stabilized and then were ventilated with the other hyperoxic gas for an approximately equal period. This was followed by brief periods of ventilation with 100 ~ N2. Changes in the pattern of presentation of these gases did not produce noticeable alteration in the results. These procedures allowed a reproducible calculation of the redox ratio of cytochrome oxidase. This ratio was calculated as the sum of the magnitudes of the pen deflections with high oxygen mixtures and the deflection with nitrogen. The magnitude of the redox changes produced by hyperoxia were then divided by that number to obtain the per cent changes as described by Sylvia and
91 Rosenthal as. Since previous studies and preliminary observations have demonstrated only reduction of NAD with 100~o Nz but little affect of transition to hyperoxia, fluorometric studies of changes in inspired gases were not pursued.
Cortical electrical stimulation The cortex was stimulated by electrical pulses applied directly to the exposed dura. The usual protocol was to apply pulses of 0.5 msec duration at 20 Hz for 2 see with increasing stimulus voltages, beginning at 8 V, until a negative shift of the cortical steady potential (SP shift) was observed. Stimulus voltage was then increased until a total of 7-12 responses were recorded for SP shifts from 0.5-5.0 mV, or until the maximal response of the SP shift was achieved, or until the cortex was driven into spreading depression. This procedure was then repeated on the other side of the brain. With few exceptions, cortical stimulation of this type did not result in a change in blood pressure or pulse rate. When such cardiovascular effects were observed, the animal was discarded. Norepinephrine assay At the end of each experiment, rats were killed by decapitation. Samples (30--60 mg) from both sides of the parietal cortex were quickly obtained and frozen in liquid nitrogen. These samples were stored at ----60 °C until assayed for NE content by the radioenzymatic assay of Henry et al.16. Since the LC contributes most, if not all, the NE innervation of the ipsilateral cerebral cortex, the success of the LC lesion was ascertained by the degree of NE depletion induced in the ipsilateral hemisphere. Data analysis Data generated by cortical stimulation was entered into a Tektronix 4051 graphic system by tracing over each strip chart record with a stylus connected to a digitizing tablet. The program performed a baseline correct!on by normalizing the data to a line calculated by least-squares regression from an indicated prestimulation and poststimulation portion of that response. For the signals representing changes in the redox states of NAD or cytochrome oxidase and that representing changes in blood volume, the program calculated: (1) amplitude of response (Pmax); (2) time from stimulus onset to peak of response (tPm ax); and (3) time from peak of response to return to half of Pmax (tt off). For the SP shift signal, only the amplitude was calculated. For non-lesioned, control rats, data from right hemispheres were compared with data from left hemispheres. For each animal, the average tPraax, ttoff, and the ABV/Acytochrome oxidase redox ratio was compared from side to side by the Student's t-test (two-tailed). The means for these measurements from each rat were then grouped by right or left side of all controls, and differences analyzed by the Student's t-test (two-tailed). Similar statistical analyses were performed for the rats with unilateral LC lesions, comparing the NE-depleted hemispheres ipsilateral to the lesion, to the non-depleted contralateral hemispheres, as well as to results obtained from the non-lesioned control group.
92 RESULTS Efficacy o f L C lesion
Throughout these experiments, the LC was considered to be successfully lesioned if the NE content of the cerebral hemisphere ipsilateral to the lesion was less than 30 % of that of the contralateral hemisphere, providing that the N E content of the contralateral hemisphere was within 2 standard deviations from the mean N E content of the non-lesioned control group. O f the 40 animals which underwent unilateral LC lesion and from which data were recorded, 34 had ipsilateral N E contents of less than 30 % of the contralateral. The average depletion of N E was 87 % (Table I). Of the remaining 6 animals, 4~vjere depleted of N E in both hemispheres, presumably due to spillage of 6 - O H D A into the fourth ventricle. One rat had no depletion of N E in either hemisphere, and in the remaining rat, the ipsilateral hemisphere was NE-depleted to 35 % of the contralateral side. Results obtained from these 6 rats were excluded from data analysis. The NE content of the contralateral hemisphere of the successfully lesioned rats was significantly different from the ipsilateral hemisphere at P < 0.001 but was not significantly different from the N E content o f hemispheres from non-lesioned control animals. To study the effect of electrical stimulation of the cerebral cortex on NE content, comparisons were made of N E levels in stimulated control, non-stimulated control hemispheres and hemispheres contralateral to LC lesions. There were no significant differences among these groups (Table I). There was no significant difference in body weight between lesioned and control rats.
TABLE I Cerebral norepinephrine ( NE) content in rats with unilateral locus coeruleus (LC) lesions and of nonlesioned controls
Values are expressed as means q- S.E.M.; n is number of observations. Both groups of non-lesioned control rats underwent anesthesia, arterial and venous cannulation, tracheostomy, curarization and positive pressure ventilation. Group
Unilateral LC lesion Ipsilateral Contralateral Non-lesioned controls with cortical stimulation Non-lesioned controls without cortical stimulation
n
NE (ng/g)
34 34
34 ± 4* 276 ~: 19"*
10
296 ± 26**
13
245 ± 15" *
* Different from all other groups at P < 0.001. No significant differences among the contralateral hemisphere results and those of the control groups.
**
93 CYTOCHROME A,A3 (SOS - 59O NM)
I
RIGHT
02
5% CO 2
I
30% 02 70'& Ns
LEFT
30% 02 7 0 % N2
I
I
11°° °21
9s~ o 2
CO2 I
I 100% 02 [
1 MIN
! MIN
Fig. 1. Local cerebral responses to changes of the impired gas mixture from 30 % Os/70 % N~ to 95 % Os/5% COs, to 100% Os and to Ns. In this and the subsequent figure, a decrease in light reflected at 605 nm is presented as an upward deflection of the trace signalling an increase in reduced cytochrome a, aa.In this example, the right hemisphere was contra]atcra] to the LC lesion and had an NE content of 324 ng/g. The left hemisphere, ipsilatera] to the LC lesion had an NE content of 36 ng/g.
Cytochrome oxidase in the resting state
When the level of inspired oxygen was increased from the normal 30 % value (with 70 % N2) there occurred a decrease in the reduction/oxidation ratio of cytochrome oxidase. This oxidative response was always obtained from cortical hemispheres of control animals and from NE-depleted (ipsilateral) and non-depleted (contralateral) hemispheres of unilateral LC-lesioned rats. Oxidative responses of even greater amplitude were recorded in all hemispheres when a gas mixture containing 95 % 02 with 5 % COs was substituted although paO2 was similar in both instances. When oxygen availability was decreased by inhalation of 100 % N~, the response, in all cases, was an increased level of reduced cytochrome oxidase. Examples of these TABLE II Cerebral cytochrome oxidase redox ratio at 'rest' andnorepinephrine ( NE) content in rats with unilateral locus coeruleus (LC) lesions
Values denote means 4- S.E.M. The number of observations (n) is in parentheses. Cerebral h e m i s p h e r e
Contralateral to LC lesion Ipsilateral to LC lesion
Cytochrome oxidase ( % reduced at peak oxidation) 100 % 02
95 % Os/5 % COs
11.7 4- 1.2 (9) 11.2 4- 0.7* (9)
30.5 :k 3.1 (12) 30.0 ± 2.3* (12)
* Not significantlydifferent from the contralateral hemisphere. ** Different from the contralateral hemisphere at P < 0.001.
NE (ng/g)
329 ± 35 (14) 32 4- 7** (14)
94 changes recorded from hemisheres ipsilateral and contralateral to an LC lesion are presented in Fig. 1. These changes are similar to those obtained from non-lesioned control rats. Although no qualitative effects of NE depletion were apparent in these responses, it was still to be determined whether NE depletion had any quantitative effects which could document an N E influence upon cerebral oxidative metabolism 'at rest'. To accomplish this, the amplitudes of oxidation obtained with inspiration of either 100 K 02 or 95~o Oz with 5 ~ CO2 were compared to the total 'labile' signal defined as the total signal change between peak oxidation and peak reduction produced by 100 ~ N2 respiration. The results are presented in Table II. N o significant differences in the percentage of reduced cytochrome oxidase were evident between contralateral and lesioned hemispheres when cytochrome oxidation was produced by inspiration of either 100K Oz or 95 ~ 02 with 5 ~ COz. Non-lesioned control rats exhibited redox ratios on either hemisphere that were not significantly different from these. In all groups, the percentage of the reduced species of cytochrome oxidase was
LEFT HEMISPHERE BL. VOL.
STIM
I ~__j~'~--~
(590 NM)
....
CYT A,A 3 (605 - 590 N
SP
i
I2% F.S.
~
M
)
~
4
"F' MV I
20 SEC
I
RIGHT HEMISPHERE BL.VOL.
I
(590 NM)
CYT A,A
3
I
(605 - 590 N M ) ~
SP
_17_~_~ -
Fig. 2. Optical and electrical responses to stimulation of the cerebral surface of hemispheres contralateral (left) and ipsilateral (right) to LC lesion. Stimulation was presented at the time mark for 2 sec, pulses at 0.5 msec duration 20 Hz with intensity at approximately 10 V. Decreased reflectance at the reference (590 nrn) wavelength is plotted as an upward deflection indicating increased blood volume. Negative shifts in the cortical steady potential are recorded as downward doflections, NE contents of the left and right hemispheres were 152 ng/g and 42 ng/g, respectively.
95 TABLE III Stimulus-induced responses of cytochrome oxidase ( Cyt a, as) and blood volume (BV) and cerebral norepinephrine ( NE) content in non-lesioned control rats Cerebral hemisphere
n
Cyt a, a8 tPmax ( sec)
Cyt a, a8 q o f f ( sec)
B V Pmax/Cyt a, aa Pmax (ratio)
N E (ng/g)
Left Right Both
7 7 14
7.05 :t: 0.31 7.41 -4- 0.20 7.23 ± 0.17
6.52 -4- 0.87 7.14 -4- 0.60 6.83 ± 0.48
1.37 ± 0.18 1.56 -4- 0.33 1.45 ± 0.16
269 ± 41 322 ± 33 295 4- 29
The values denote means ± S.E.M. For detailed explanation of the stimulus-induced responses see text. There were no significant differences between the two hemispheres in any of the parameters mentioned. higher with 9 5 % 0 2 / 5 % CO2 t h a n with 100% 03. This is because t h e extent o f o x i d a t i o n was always g r e a t e r w h e n COs was present. It should be noted, however, t h a t these values are relative because the degree o f c y t o c h r o m e o x i d a t i o n w i t h either h y p e r o x i c gas was n o t at its a b s o l u t e p e a k as d e m o n s t r a t e d by the very high degree o f o x i d a t i o n r e c o r d e d whert a n i m a l s were r e s p i r e d with high 02 a n d CO2 mixtures u n d e r h y p e r b a r i c c o n d i t i o n s 15. Likewise, r e d u c t i o n was n o t at its a b s o l u t e m a x i m u m since the d u r a t i o n o f 100 % N2 r e s p i r a t i o n was k e p t s h o r t t o p r e v e n t h y p o x i c b r a i n injury. C y t o c h r o m e oxidase in an 'active state'
W h e n the c e r e b r u m was s t i m u l a t e d to increased activity b y electrical pulses a p p l i e d to the d u r a , there was a negative shift o f the c o r t i c a l steady p o t e n t i a l as m o n i t o r e d b y D C - c o u p l e d A g - A g C 1 electrodes. This o c c u r r e d in c o n t r o l rats a n d in r a t s two weeks following L C lesion. A s s o c i a t e d with this shift, there was a t r a n s i e n t o x i d a t i o n o f c y t o c h r o m e oxidase a n d a decrease in the a m o u n t o f light reflected at 590 nm. I n previous studies, this decrease was i n t e r p r e t e d to indicate a n increase in the TABLE IV Stimulus-induced responses of cytochrome oxidase ( Cyt a, as) and blood volume ( BV) and cerebral norepinephrine ( NE) content in rats with unilateral locus coeruleus (LC) lesions
The values denote means 4- S.E.M. of the number of observations enclosed in parentheses. Explanation of the various stimulus-induced parameters is detailed in the text. Cerebral hemisphere
Cyt a, as tPraax (sec)
Contralateral to LClesion 6.82 -4- 0.31 (13) Ipsilateral toLClesion 7.45 ± 0.28* (13)
Cyt a, a8 ttoff (sec)
B V Pmax/Cyt a, a3Pmax (ratio)
N E (ng/g)
5.73 ± 0.79 (12)
1.68 -4- 0.26 (12)
247 ± 25 (13)
8.92 ± 1.I1"* (12) 0.99 4- 0.14"* (12)
* Not significantly different from the contralateral hemisphere. ** Different from the contralateral hemisphere at P < 0.05. *** Different from the contralateral hemisphere at P < 0.001.
36 -4- 5"**(13)
96 local volume of hemoglobin is,a4. Fig. 2 shows examples of these responses recorded from cerebral hemispheres ipsilateral and contralateral to an LC lesion. To determine whether NE depletion influences the rates of the metabolic responses, the times from stimulus onset to peak oxidation and blood volume increase (tPmax) and the time from peak oxidation to half recovery (ttoff) were compared in NE-depleted and non-depleted hemispheres of LC-lesioned rats and in controls. Table III summarizes these data. In non-lesioned control rats, there were no differences between hemispheres in either tPraax or ttoff. Also, the rates of cytochrome oxidase redox changes and blood volume responses recorded from contralateral hemispheres of unilateral LC-lesioned rats were very s milar to those of non-lesioned controls (Tables III and IV). Also, tPmax of cytochrome oxidase recorded from the NEdepleted was similar to that of the contrateral hemisphere. However, the t~off in NE-depleted cerebral hemispheres was 70 ~ slower than that of contralateral hemispheres (P < 0.05; Table IV). To determine if N E depletion affects the relationship between changes in the redox state of cytochrome oxidase and local blood volume, the amplitudes of the response to stimulation of each of these were compared. This provided a ratio which we assumed to be independent of stimulus intensity. In control rats, the ~ cytochrome oxidation/z~ blood volume increase was 1.46 4- 0,18 (mean 4- S.E.M.). The ratio obtained from contralateral hemispheres of LC-lesioned rats was 1.68 4- 0,26 which was not significantly different from non-lesioned controls. However, the ratio obtained from the ipsilateral hemispheres of LC-lesioned rats was 0.99 4- 0.14, which was significantly lower than contralateral hemispheres or that from control rats (P < 0.05; Table IV). The lowered ratio of stimulus-induced cytochrome oxidase to blood volume changes occurred in 10 of 12 rats. In these 10 rats, the average ratio of the responses in the ipsilateral hemisphere was decreased to about half that of the contralateral hemisphere.
FL - REFL ( 4 6 o - m me) RIGHT
IllM_ ~.2 t
LEFT
..,. i
.~..L'~.. ~:~.~-~
.,,
~..~
-
Fig. 3. Responses of pyridine nucleotide fluorescenceto direct cortical stimulation (2-sectrain, 20 Hz, 0.5 msec pulses at approximately 6 V). Corrected fluorescence signals were averaged for 8 stimulus presentations for each hemisphere by computer. Decreased fluorescence(NAD oxidation) is recorded as a downward deflection. NE content of the right hemispherecontralateral to LC lesion was 302 ng/g. NE content of the left hemisphere ipsilateral to LC lesion was 9 ng/g.
97 TABLE V Stimulus-induced response of pyridine nucleotide ( NADH) and cerebral norepinephrine ( NE) content in rats with unilateral locus coeruleus (LCJ lesions
The values denote means 4- S.E.M. of 8 observations. Explanation of the stimulus-induced parameters is detailed in the text. Cerebral hemisphere
NADH tPraax (sec)
NA DH t~off (sec)
NE (ng/g)
Contralateral to LC lesion Ipsilateral to LC lesion
7.22 4- 0.40 10.4 4- 1.50"
6.01 4- 0.45 11.34 4- 1.90"
258 4- 25 35 4- 9**
* Different from the eontralateral hemisphere at P < 0.05. ** Different from the eontralateral hemisphere at P < 0.001. Pyridine nucleotide in an 'active" state
The response, monitored by surface fluorometry, to direct electrical stimulation of cerebral cortex in both NE-depleted and non-depleted hemispheres consisted of a transient decrease in fluorescence which was interpreted as an oxidation of intramitochondrial NAD. An example of signal-averaged responses recorded in hemispheres contralateral and ipsilateral to an LC lesion is shown in Fig. 3. Although the magnitude and the direction of stimulus-induced N A D responses were unaffected by NE-depletion, both the rates of oxidation and re-reduction of N A D were slowed in NE-depleted tissue. The average time to peak oxidation of N A D was 7.22 see and 10.40 sec, respectively (P < 0.05, Table V). The mean time to half-recovery on the ipsilateral side was 11.34 sec and that on the contralateral side was 6.01 see (P < 0.05; Table V). DISCUSSION These data demonstrate that lesion of the LC, and the resultant depletion of NE, influences cerebral oxidative metabolism in situ. This influence was not observed under conditions of low energy demand, during a 'resting' state, but was seen during increased metabolic demand induced by direct cortical electrical stimulation. The role, if any, of the noradrenergic system in the control o f cerebral circulation and metabolism has been unclear. Anatomic evidence, from light and electron microscopy and histofluorescence microscopy, indicates a dense noradrenergic innervation of cerebral vessels ~7. Yet, the physiological function of this innervation and its importance to circulatory control remains controversial (see e.g. refs. 13, 14 and 29). Since the suggestion by Hartman et al. 12 that N E innervation of the intraparenchymal cerebral vasculature arises in the LC, interest has shifted from peripheral sympathetic innervation of cerebral vessels through the superior cervical ganglion and has focused on the LC 8. Electrical stimulation of the LC has been reported to decrease cerebral blood flow 7, and to result in a mild decrease in 2-deoxyglucose uptake in some brain areas 1. Chemical activation o f the LC has been reported to decrease cerebral blood
98 flow and to increase capillary permeabilityz0. Conversely, LC ablation has been reported to cause increased cerebral blood flow and loss of COg reactivity in cats a. However, no significant changes in cerebral cortical metabolism could be demonstrated after LC ablation by 2-deoxyglucose autoradiography35. It is difficult to reconcile these reports with studies which show an association between increased activity of LC neurons during stress and increased alertness in unanesthetized animals x°, since these conditions are generally associated with increased cerebral blood flow and metabolic rate ~7. We have shown that the relative redox state of cerebral cytochrome oxidase remains unchanged in the absence of NE. These data, however, must be interpreted in a very limited manner. In the aged brain, for example, no differences were observed in the relative redox state of cytochrome oxidase in the 'resting state', but deficiencies in metabolic capability were detected when cerebral energy demand was increased39. An unchanged cytochrome oxidase redox ratio in NE-depleted hemispheres under 'resting' conditions only indicates that energy demand and energy conservation are in balance independent of NE content. Such data cannot take into account whether NE has an influence upon energy turnover or energy demand. These, and other data on the influence of NE on metabolism, may be misleading if based only on static measurements from 'resting' brain. In both NE-depleted and control hemispheres, the oxidation of cytochrome oxidase produced by inspiration of 95 ~ O~/5 ~ COs was greater than that produced by inspiration of 100 ~ Oe. This COs effect has been reported previously3a and has been correlated with a greater increase in tissue oxygen tension, as measured polarographically2L Such a CO~ effect could be due to the known vasodilatation produced by hypercapnea. However, Bates et al. 3 showed that, in cats, the CO~ reactivity of cerebral vasculature was lost in cortical hemispheres ipsilateral to LC lesion. If this is true in rats, then the large cytochrome oxidation is likely due to a COz effect upon cellular metabolism (see e.g. ref. 26). Whatever the mechanism underlying this CO2 influence, these data demonstrate that it is unaffected by NE depletion. If the noradrenergic fibers from the LC are involved in the response to stress, then it is most appropriate to look for an effect of NE depletion on metabolic changes associated with increased energy demand. Our results show that NE depletion is associated with a slowed rate of recovery of NAD and cytochrome oxidase, each of which had been oxidized in response to increased energy demand produced by direct cortical stimulation. Stimulation of this type had previously been shown to produce oxidative responses ofpyridine nucleotide and cytochrome a,a31S,zL Such stimulation also evokes negative shifts of the cortical steady potential and increased extracellular potassium activity in proportion to the oxidation of the mitochrondrial components2L Some caution was necessary since barbiturates had previously been show to produce a s!milar slowing of these metabolic responses to stimulation28. However, such a barbiturate effect would tend to decrease differences between the NE-depleted and control hemispheres. Nevertheless, we considered it important to maintain a similar depth of anesthesia for both hemispheres and to vary the order of experimental recording.
99 A slowed rate of re-reduction of NAD or cytochrome oxidase likely indicates a decreased rate of ATP generation and a decreased capacity of cortical tissue to respond to increased metabolic demand. There are several possible explanations for this effect. One is that NE has an influence within the respiratory chain, such that its absence results in either uncoupling of oxidation from phosphorylation or a block in the chain itself. Uncoupling is unlikely since this would be expected to result in a loss of respiratory control and a higher resting ratio of oxidized respiratory components, as occurs in vitro. Such a response was not demonstrated. Uncoupling of oxidation from phosphorytation would also be expected to be accompanied by greater oxidation of each respiratory component for a given stimulus because of decreased efficiency of the oxidative apparatus. This did not occur. The possibility of a block in the chain is also unlikely because such a block would be associated with an altered relationship between respiratory chain members on each side of the block (i.e. a crossover point). The absence of NE produced no apparent differences in the responses of NAD and cytochrome oxidase to stimulation. Another possibility is that the slowed reduction of NAD and cytochrome oxidase results from prolonged ion displacement following increased electrical activity. Such a prolongation of potassium ion clearance from the extracellular space was associated with the slow re-reduction seen with phenobarbital ~3. With NE depletion, however, the rate of NAD oxidation was slowed consistent with a slower generation of ADP from decreased Na+-K+-ATPase, but preliminary studies show that potassium clearance is not prolonged in the absence of NE after stimulation (T. Sick, personal communication). A third possibility is that availability of substrate for re-reduction of the oxidized mitochondrial components may be affected by NE-depletion. However, this effect should be observed as a more oxidized resting state in a manner similar to the state 2 condition of isolated mitochondria6. It is possible, nevertheless, that there is a mechanism which responds to increased energy demand with an increase in substrate provision, and that the regulation of substrate availability to increased demand is altered when NE is absent. A fourth possibility is that the NE influence on cerebral metabolism is mediated through an effect on local cerebral circulation. If the increase in local blood volume in response to stimulation reflects a local mechanism for increasingsubstrate and oxygen delivery, then our finding of a decrease in this response after NE-depletion is consistent with transient inadequate provision of reducing equivalents to the respiratory chain. No matter how they are mediated, these results are consistent with a role for NE in the regulation of cerebral oxidative metabolism in response to sudden increases in energy requirements. ACKNOWLEDGEMENT Supported by PHS Grants NS-05820, NS-14319 and NS-14325. We thank M. Ganapathi for excellent technical assistance. J.C.L. is a recipient of NIH Research Career Development Award NS-00399,
100 REFERENCES 1 Abraham, W. C., Delanoy, R. L., Dunn, A. J. and Zornetzer, S. F., Locus coeruleus stimulation decreases deoxyglucose uptake in ipsilateral mouse cerebral cortex, Brain Research, 172 (1979) 387-392. 2 Avi-Dor, J. J., Olson, M., Doherty, M. D. and Kaplan, N. O., Fluorescence of pyridine nucleotides in mitochondria, J. bioL Chem., 237 (1962) 2377-2383. 3 Bates, D., Weinshilboum, R. M., Campbell, R. J. and Sundt, T. M., The effects of lesions in the locus ceruleus on the physiological responses of the cerebral blood vessels in cats, Brain Research, 136 (1977) 431--443. 4 Chance, B., Cohen, P., J/Sbsis, F. F. and SchiSner, B., Intracellular oxidation-reduction in vivo, Science, 137 (1962) 499-508. 5 Chance, B. and J6bsis, F. F., Changes in fluorescence in a frog sartorius muscle following a twitch, Nature (Lond.), 184 (1959) 195-196. 6 Chance, B. and Williams, G. R., The respiratory chain and oxidative phosphorylation, Advanc. EnzymoL, 17 (1956) 65-134. 7 De la Torre, J. C., Surgeon, J. W. and Walker, R. H., Effects of locus coeruleus stimulation on cerebral blood flow in selected brain regions, Acta neuroL scand., Suppl., 64 (1977) 104-105. 8 Edvinsson, L., Lindvall, M., Nielsen, K. C. and Owman, C. H,, Are brain vessels innervated also by central (non-sympathetic) adrenergic neurons, Brain Research, 63 (1973) 496--499. 9 Estabrook, R. W., Fluorometric measurements of reduced pyridine nucleotide in cellular and subcellular particles, Analyt. Biochem., 4 (1962) 231-245. 10 Foote, S. L. and Bloom, F. E., Activity of norepinephrine-containinglocus ceruleus neurons in the unanesthetized squirrel monkey. In Usdin, Kopin and Barchas, (Eds.), Catecholamines: Basic and Clinical Frontiers, Pergamon, New York, 1979. 11 Harik, S. I., LaMarma, J. C., Light, A. I. and Rosenthal, M., Cerebral norepinephrine: influence on cortical oxidative metabolism in situ, Science, 206 (1979) 69-71. 12 Hartman, B. K., Zide, D. and Udenfriend, S., The use of dopamine beta hydroxylase as a marker for the central noradrenergic nervous system in rat brain, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2722-2726. 13 Heistad, D. D. and Marcus, M. L., Evidence that neural mechanisms do not have important effects on cerebral blood flow, Circulat. Res., 42 (1978) 295-302. 14 Heistad, D. D. and Marcus, M. L., Response to'Do vasomotor nerves significantly regulate cerebral blood flow?' Circulat. Res., 43 (1978) 494-495. 15 Hempel, F. G., JSbsis, F. F., LaManna, J. C., Rosenthal, M. and Saltzman, H. A., Redox levels of cytoehrome a, aa in cat cerebral cortex during hyperbaric oxygenation, J. appt. PhysioL, 43 (1977) 873-879. 16 Henry, D. P., Statman, B. J., Johnson, D. G. and Williams, R. H., A sensitive radioenzymatic assay for norepinephrine in tissue and plasma, Life ScL, 16 (1975) 375-384. 17 J~Sbsis,F. F., Basic processes in cellular respiration. In Fenn and Rahn (Eds.), Handbook of Physiology, Amer. Physiol. Soc., Washington, D.C., 1964, pp. 63-124. 18 J6bsis, F. F., Keizer, L, LaManna, J. C. and Rosenthal, M., Reflectance spectrophotometry of cytochrome a, as in vivo, J. appL Physiol., 43 (1977) 858-872. 19 JSbsis, F. F., O'Connor, M. J., Vitale, A. and Vreman, H., Intracellularredox changes in functioning cerebral cortex. I. Metabolic effects of epileptiforrn activity, J. NeurophysioL, 34 (1971) 735-749. 20 Kobayashi, R. M., Palkovits, M., Jacobowitz, D. M. and Kopin, I. J., Biochemical mapping of the noradrenergic projection from the locus ceruleus, Neurology, 25 (1975) 223-233. 21 Koda, L. Y., Schulman, J. A. and Bloom, F. E., Ultrastructural identification of noradrenergic terminalsin rat hippocampus: Unilateral destruction of the locus ceruleus with 6-hydroxydopamine, Brain Research, 145 (1978) 190--195. 22 Kreisman, N. R., Sick, T. J., LaManna, J. C. and Rosenthal, M., Local tissue oxygen tensioncytochrome al, az redox relationships in rat cerebral cortex in vivo, Brain Research, submitted. 23 LaManna, J. C., Cordingley, G. and Rosenthal, M., Phenobarbital actions in vivo: Effects on extracellular potassium activity and oxidative metabolism in cat cerebral cortex, J. PharmacoL exp. Ther., 200 (1977) 560--569. 24 LaManna, J. C., Light, A. I., Harik, S. I. and Rosenthal, M., Locus coeruleus lesions: effects on cerebral oxidative metabolism in vivo. II. The coupling between oxygen delivery and energy demand, Neurosci. dbstr., 5 (1979) 340.
101 25 Lothman, E., LaManna, J. C., Cordingley, G., Rosenthal, M. and Somjen, G., Responses of electrical potential, potassium levels and oxidative metabolic activity of the cerebral neocortex of cats, Brain Research, 88 (1975) 15-36. 26 Miller, A. L., Hawkins, R. A. and Veech, R. L., Decreased rate of glucose utilization by rat brain in vivo after exposures to atmospheres containing high concentrations of CO2, J. Neurochem., 25 (1975) 553-558. 27 Nielsen, K. C. and Owman, C. H., Adrenergic innervation of pial arteries related to the circle of Willis in the cat, Brain Research, 6 (1967) 773-776. 28 O'Connor, M. J., Origin of labile NADH tissue fluorescence, In J6bsis, (Ed.), Oxygen andPhysiological Function, Professional Information Library, Dallas, 1977, pp. 90-99. 29 Purves, M. J., Do vasomotor nerves significantly regulate cerebral blood flow? Circulat. Res., 43 (1978) 485-493. 30 Raichle, M. E., Hartman, B. K., Eichling, J. O. and Sharpe, L. G., Central noradrenergic regulation of cerebral blood flow and vascular permeability, Proc. nat. Acad. ScL (Lond.), 72 (1975) 3726-3730. 31 Rosenthal, M., Harik, S. I., LaManna, J. C. and Light, A. I., Locus coeruleus lesions: effects on cerebral oxidative metabolism in vivo. I. Cytochrome oxidase in the steady state, NeuroscL Abstr., 5 (1979) 350. 32 Rosenthal, M. and J6bsis, F. F., Intracellular redox changes in the functioningcerebral cortex. II. Effects of direct cortical stimulation, J. Neurophysiol., 32 (1971) 750-761. 33 Rosenthal, M., LaManna, J. C., J6bsis, F. F., Levasseur, J., Kontos, H. and Patterson, J., Effects of respiratory gases on cytochrome a in intact cerebral cortex: Is there a critical PO~? Brain Research, 108 (1976) 143-154. 34 Rosenthal, M., LaManna, J. C., Yamada, S. ,Younts, B. W. and Somjen, G., Oxidative metabolism, extracellular potassium and sustained potential shifts in cat spinal cord in situ, Brain Research, 162 (1979) 113-127. 35 Schwartz, W. J., 6-hydroxydopamine lesions of rat locus ceruleus alter brain glucose consumption, as measured by the 2-deoxy-D-[14C]glucosetracer technique, Neurosci. Lett., 7 (1978) 141-150. 36 Sharma, V. K., Harik, S. I., Ganapathi, M., Busto, R. and Banerjee, S. P., Locus ceruleus lesion and chronic reserpine treatment: effects on adrenergic and cholinergic receptors in cerebral cortex and hippocampus, Exp. Neurol., 65 (1979) 685-690. 37 Siesj6, B. K., Brain Energy Metabolism, J. Wiley, New York, 1978, pp. 607. 38 Sylvia, A. L. and Rosenthal, M., The effect of age and lung pathology on cytochrome a, a3 redox levels in rat cerebral cortex, Brain Research, 146 (1978) 109-122. 39 Sylvia, A. L. and Rosenthal, M., Effects of age on brain oxidative metabolism in vivo, Brain Research, 165 (1979) 235-248. 40 Ungerstedt, V., Stereotaxic mapping of monoamine pathways in the rat brain, ,4ctaphysiol. scand., Suppl., 367 (1971) 1-48.
87
N O R E P I N E P H R I N E D E P L E T I O N ALTERS C E R E B R A L OXIDATIVE METABOLISM IN T H E 'ACTIVE' STATE
JOSEPH C. LaMANNA, SAMI I. HARIK, ANDREW I. LIGHT and MYRON ROSENTHAL Department of Neurology, University of Miami School of Medicine, Miami, Fla. 33101 (U.S.A.)
(Accepted June 19th, 1980) Key words: norepinephrine - - locus coeruleus - - oxidative metabolism - - cytochrome oxidase --
NAD
-
-
evoked potential
SUMMARY Unilateral chemical lesion o f the nucleus locus coeruleus in rats produced unilateral depletion of ipsilatetal cortical norepinephrine. Norepinephrine depletion was not associated with changes in 'resting' metabolic balance of the cerebral cortex, as determined by in situ reflection spectrophotometry of the redox state of cytochrome oxidase. Norepinephrine depletion, however, caused slowing of the transient metabolic response to sudden increases in energy demand produced by direct cortical electrical stimulation. The effect was apparent in the redox state of both cytochrome oxidase and NAD, the latter being measured, also in situ, by reflection microfluorometry. These results demonstrate that norepinephrine has a role in modulating the response to increased metabolic demand inthe cerebral cortex. Norepinephrine may mediate its effect by potentiating Na +, K+-ATPase or through its effects on vascular reactivity, or both.
INTRODUCTION Norepinephrine (NE) is a putative central nervous system neurotransmitter or modulator which is suspected of playing an important role in several physiological processes and pathological conditions. Among its suggested functions is the regulation of cerebral blood flow a,a0 and capillary permeability a°. Since cerebral metabolism and tissue perfusion are closely linked, it would be expected that if effects of NE on flow and permeability are physiologically significant, then these effects would be expressed through changes in regional cerebral oxidative metabolism. In this report, we directly explore the role of N E on cerebral oxidative energy metabolism. This was done by comparing the relationship between energy utilization
88 and the cellular capability to maintain oxidative metabolic homeostasis under 'resting' and 'active' conditions in the presence and absence of NE. Oxidative metabolism was monitored non-invasively from the surface of rat cerebral hemispheres in situ in two ways: by continuous dual-wavelength reflection spectrophotometry oflocal changes in the reduction/oxidation (redox) state of cytochrome oxidase and blood volumela; and by fluorometrically recording redox changes of pyridine nucleotide4,19. Since pyridine nucleotide is the initial coenzyme of the mitochondrial respiratory chain and cytochrome oxidase (cytochrome a,aa) is its terminal member, changes in the redox ratio of each of these directly indicate intracellular oxidative metabolic activities 6,17. Comparisons were made between hemispheres with normal NE content and those that were NE-depleted by unilateral 6-hydroxydopamine (6-OHDA) lesions of the locus coeruleus (LC). These investigations take advantage of the fact that almost all cerebral NE arises from neurons in the LC through an ipsilateral pathway20,a9. Unilateral 6OHDA lesion of the LC produces marked depletion of NE in the ipsilateral cortex while leaving the contralateral cortex with normal NE content thereby serving as an internal control 11,~6. We report here that the redox ratio of cytochrome oxidase was unchanged under 'resting' conditions in NE-depleted cortical hemispheres and conclude that the relationship of energy demand and supply is not affected by NE depletion when energy demand is low. However, marked changes occurred in NE-depleted hemispheres when energy demand was increased by electrical stimulation. Preliminary reports of these results have been published 2a,31. METHODS Locus coeruleus lesion Male Wistar rats (175-200 g) were allowed 5-7 days of acclimation during which time they were kept under diurnal light conditions with free access to laboratory chow and water. The LC was lesioned unilaterally by the local infusion of 2.5/~1 of 6OHDA solution containing 5/~g of 6-OHDA base under chloral hydrate (400 mg/kg, i.p.) anesthesia. Pargyline (50 mg/kg, i.p.) was given 15-30 min before the infusion of 6-OHDA to produce a more effective lesionzl. 6-OHDA was freshly dissolved in 0.9 NaCI solution containing 1 mg/ml ascorbic acid, adjusted to a pH of approximately 5.5 and kept on ice until used. The 6-OHDA solution was slowly infused through a glass micropipet (tip diameter 40-60 #m) attached to a Hamilton syringe, over a period of 4-5 min. The stereotaxic coordinates were 1.1 mm posterior, 1.3 mm lateral and 7.5 mm ventral to the lambda. Approximately equal numbers of right and left side lesions were made. The animals were returned to their cages when awake and allowed to recover for a period of two weeks before they were used in the metabolic experiments. The operative mortality of the unilateral LC lesion procedure was less than 10 ~. In preliminary experiments, the LC was infused with vehicle solution. This did not result in any significant changes in the NE content of the ipsilateral cerebral cortex in comparison to the non-infused side. For this reason, non-lesioned rats that were anesthetized with chloral hydrate and given pargyline served as additional controls for
89 the unilaterally lesioned rats so that NE-depleted hemispheres could be compared to non-depleted hemispheres contralateral to the unilateral LC lesion and to those of non-lesioned control rats.
Preparation of animals for metabolic studies Two weeks after LC lesions were made, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The femoral artery and vein were cannulated for monitoring of arterial blood pressure, for sampling of arterial blood gas parameters (paO2, p aCO2 and pH) and for intravenous infusion of supplemental pentobarbital to maintain light anesthesia. Tracheostomy was performed and the rats were paralyzed with tubocurarine. They were immediately connected to a positive pressure respirator and ventilated with a gas mixture containing 30 ~ Oz and 70 % N2 at approximately 55 strokes/min and 3 ml stroke volume. Stroke volume and respiratory rate were adjusted to maintain blood gas values within the normal range: pH approximately 7.44 (7.41-7.47); p aCO2 approximately 38 mmHg (36-42); and paO2 above 110 mmHg. A rectal thermocouple was inserted and body temperature maintained at 37 °C by a heating pad. The bone overlying the frontoparietal region on each side of the midline suture was thert thinned by drilling at slow speed with a burr. Each area was about 8 × 5 mm. The thinned portion was hooked with a curved needle and lifted carefully away from the intact dura. The optical equipment was focused on either the left or right side field, alternating from experiment to experiment. An Ag-AgC1 recording electrode was placed on the dura in the center of the area to be monitored optically. This electrode was used to record both the electrocorticogram (ECoG) and the DC-coupled electrical potential (the so-called 'cortical steady potential'), and was referenced to an Ag-AgC1 electrode inserted into the neck musculature. A forked stimulating electrode (tips 0.1 mm in diameter, 0.5 mm apart) was placed about 1 mm from the recording electrode, near the edge of the optical field. Blood pressure, ECoG, and the cortical steady potential were displayed on a strip chart recorder.
Measurement of cytochrome oxidase redox state by reflection spectrophotometry The reflectance spectrophotometric monitoring system for cytochrome oxidase redox changes and changes in blood volume is based on the light absorption properties of the cytochrome components of tissue mitochondria. Since cytochromes absorb light more strongly in the reduced form than in the oxidized form, changes in redox ratios can be determined by measuring changes in reflected light intensity at a wavelength of maximal absorption difference between the reduced and the oxidized species of each of these mitochondrial components. The 605 nm wavelength has been well worked out for cytochrome oxidase in the intact cerebral cortex. A detailed description of the method has been published is. In brief, light from a 150 W xenon short-arc lamp was divided mechanically into two beams and alternately directed (at 30 I-Iz) through two monochromators, one of these was set at 605 nm (the 'sample' beam for cytochrome oxidase), the other at 590 nm to provide reference compensation for changes in blood oxygenation and volume. These monochromatic light beams were directed by fiber optic light guides onto the cerebral cortex.
90 Microscope optics were used to gather the reflected light scattered back from the tissue and focus it on a photomultiplier tube housed in the microscope barrel. Both the light guides and the microscope optics were set at an angle to minimize specular reflection from the tissue surface. The signals representing the intensity of light reflected at the reference wavelength and the difference between the intensity of light at the sample and reference wavelengths were displayed on a strip chart recorder. The first of these signals, compensated for and provided a useful indicator of blood volume shifts. The instrument was calibrated by making the dark level equal to zero and the reference intensity equal to a full scale deflection of the strip chart recorder pen. Sample light was then adjusted to be equal to the reference intensity. All data is then recorded as per cent of full scale.
Measurement of pyridine nucleotide redox state by microfluorometry The fluorometric measurement of the intramitochondrial pyridine nucleotide redox state is derived from two findings: (1) the reduced form of the coenzyme fluoresces at 450 nm when excited by light at 366 nm while the oxidized form does not; and (2) the bound enzyme shows enhanced fluorescence over the unbound cytoplasmic form z,9,2s. The technique was first developed by Chance and Jfbsis 5 and adapted to the intact cerebrum by Chance et al. 4. Light at 366 nm from a 1000 W mercury arc lamp was directed onto the cortical surface of the rat. Reflected and fluorescent light from the cortical surface were collected by microscope optics, separated by a beam-splitter and filters, and measured by separate photomultiplier tubes. The reflected excitation light was subtracted from fluorescent light as a compensation for non-specific changes in optical properties of the preparation 19. The signals representing fluorescence, reflectance and the corrected difference between these, were displayed on a strip chart recorder. Full scale refers to the initial intensities of each of these from the cortical surface with zero being no light and 1O0 being those initial values.
Responses to inspired gas changes After a period of stabilization, during which the rats were respired with 30 ~o 02/70 ~ N2, the ventilation mixture was changed either to 100 ~ O2 or to 95 ~ 02/5 COz in experiments to monitor redox changes of cytochrome oxidase by reflection spectrophotometry. After approximately 5 min, the animals again were respired with 30 9/0002/70 ~o N2 until stabilized and then were ventilated with the other hyperoxic gas for an approximately equal period. This was followed by brief periods of ventilation with 100 ~ N2. Changes in the pattern of presentation of these gases did not produce noticeable alteration in the results. These procedures allowed a reproducible calculation of the redox ratio of cytochrome oxidase. This ratio was calculated as the sum of the magnitudes of the pen deflections with high oxygen mixtures and the deflection with nitrogen. The magnitude of the redox changes produced by hyperoxia were then divided by that number to obtain the per cent changes as described by Sylvia and
91 Rosenthal as. Since previous studies and preliminary observations have demonstrated only reduction of NAD with 100~o Nz but little affect of transition to hyperoxia, fluorometric studies of changes in inspired gases were not pursued.
Cortical electrical stimulation The cortex was stimulated by electrical pulses applied directly to the exposed dura. The usual protocol was to apply pulses of 0.5 msec duration at 20 Hz for 2 see with increasing stimulus voltages, beginning at 8 V, until a negative shift of the cortical steady potential (SP shift) was observed. Stimulus voltage was then increased until a total of 7-12 responses were recorded for SP shifts from 0.5-5.0 mV, or until the maximal response of the SP shift was achieved, or until the cortex was driven into spreading depression. This procedure was then repeated on the other side of the brain. With few exceptions, cortical stimulation of this type did not result in a change in blood pressure or pulse rate. When such cardiovascular effects were observed, the animal was discarded. Norepinephrine assay At the end of each experiment, rats were killed by decapitation. Samples (30--60 mg) from both sides of the parietal cortex were quickly obtained and frozen in liquid nitrogen. These samples were stored at ----60 °C until assayed for NE content by the radioenzymatic assay of Henry et al.16. Since the LC contributes most, if not all, the NE innervation of the ipsilateral cerebral cortex, the success of the LC lesion was ascertained by the degree of NE depletion induced in the ipsilateral hemisphere. Data analysis Data generated by cortical stimulation was entered into a Tektronix 4051 graphic system by tracing over each strip chart record with a stylus connected to a digitizing tablet. The program performed a baseline correct!on by normalizing the data to a line calculated by least-squares regression from an indicated prestimulation and poststimulation portion of that response. For the signals representing changes in the redox states of NAD or cytochrome oxidase and that representing changes in blood volume, the program calculated: (1) amplitude of response (Pmax); (2) time from stimulus onset to peak of response (tPm ax); and (3) time from peak of response to return to half of Pmax (tt off). For the SP shift signal, only the amplitude was calculated. For non-lesioned, control rats, data from right hemispheres were compared with data from left hemispheres. For each animal, the average tPraax, ttoff, and the ABV/Acytochrome oxidase redox ratio was compared from side to side by the Student's t-test (two-tailed). The means for these measurements from each rat were then grouped by right or left side of all controls, and differences analyzed by the Student's t-test (two-tailed). Similar statistical analyses were performed for the rats with unilateral LC lesions, comparing the NE-depleted hemispheres ipsilateral to the lesion, to the non-depleted contralateral hemispheres, as well as to results obtained from the non-lesioned control group.
92 RESULTS Efficacy o f L C lesion
Throughout these experiments, the LC was considered to be successfully lesioned if the NE content of the cerebral hemisphere ipsilateral to the lesion was less than 30 % of that of the contralateral hemisphere, providing that the N E content of the contralateral hemisphere was within 2 standard deviations from the mean N E content of the non-lesioned control group. O f the 40 animals which underwent unilateral LC lesion and from which data were recorded, 34 had ipsilateral N E contents of less than 30 % of the contralateral. The average depletion of N E was 87 % (Table I). Of the remaining 6 animals, 4~vjere depleted of N E in both hemispheres, presumably due to spillage of 6 - O H D A into the fourth ventricle. One rat had no depletion of N E in either hemisphere, and in the remaining rat, the ipsilateral hemisphere was NE-depleted to 35 % of the contralateral side. Results obtained from these 6 rats were excluded from data analysis. The NE content of the contralateral hemisphere of the successfully lesioned rats was significantly different from the ipsilateral hemisphere at P < 0.001 but was not significantly different from the N E content o f hemispheres from non-lesioned control animals. To study the effect of electrical stimulation of the cerebral cortex on NE content, comparisons were made of N E levels in stimulated control, non-stimulated control hemispheres and hemispheres contralateral to LC lesions. There were no significant differences among these groups (Table I). There was no significant difference in body weight between lesioned and control rats.
TABLE I Cerebral norepinephrine ( NE) content in rats with unilateral locus coeruleus (LC) lesions and of nonlesioned controls
Values are expressed as means q- S.E.M.; n is number of observations. Both groups of non-lesioned control rats underwent anesthesia, arterial and venous cannulation, tracheostomy, curarization and positive pressure ventilation. Group
Unilateral LC lesion Ipsilateral Contralateral Non-lesioned controls with cortical stimulation Non-lesioned controls without cortical stimulation
n
NE (ng/g)
34 34
34 ± 4* 276 ~: 19"*
10
296 ± 26**
13
245 ± 15" *
* Different from all other groups at P < 0.001. No significant differences among the contralateral hemisphere results and those of the control groups.
**
93 CYTOCHROME A,A3 (SOS - 59O NM)
I
RIGHT
02
5% CO 2
I
30% 02 70'& Ns
LEFT
30% 02 7 0 % N2
I
I
11°° °21
9s~ o 2
CO2 I
I 100% 02 [
1 MIN
! MIN
Fig. 1. Local cerebral responses to changes of the impired gas mixture from 30 % Os/70 % N~ to 95 % Os/5% COs, to 100% Os and to Ns. In this and the subsequent figure, a decrease in light reflected at 605 nm is presented as an upward deflection of the trace signalling an increase in reduced cytochrome a, aa.In this example, the right hemisphere was contra]atcra] to the LC lesion and had an NE content of 324 ng/g. The left hemisphere, ipsilatera] to the LC lesion had an NE content of 36 ng/g.
Cytochrome oxidase in the resting state
When the level of inspired oxygen was increased from the normal 30 % value (with 70 % N2) there occurred a decrease in the reduction/oxidation ratio of cytochrome oxidase. This oxidative response was always obtained from cortical hemispheres of control animals and from NE-depleted (ipsilateral) and non-depleted (contralateral) hemispheres of unilateral LC-lesioned rats. Oxidative responses of even greater amplitude were recorded in all hemispheres when a gas mixture containing 95 % 02 with 5 % COs was substituted although paO2 was similar in both instances. When oxygen availability was decreased by inhalation of 100 % N~, the response, in all cases, was an increased level of reduced cytochrome oxidase. Examples of these TABLE II Cerebral cytochrome oxidase redox ratio at 'rest' andnorepinephrine ( NE) content in rats with unilateral locus coeruleus (LC) lesions
Values denote means 4- S.E.M. The number of observations (n) is in parentheses. Cerebral h e m i s p h e r e
Contralateral to LC lesion Ipsilateral to LC lesion
Cytochrome oxidase ( % reduced at peak oxidation) 100 % 02
95 % Os/5 % COs
11.7 4- 1.2 (9) 11.2 4- 0.7* (9)
30.5 :k 3.1 (12) 30.0 ± 2.3* (12)
* Not significantlydifferent from the contralateral hemisphere. ** Different from the contralateral hemisphere at P < 0.001.
NE (ng/g)
329 ± 35 (14) 32 4- 7** (14)
94 changes recorded from hemisheres ipsilateral and contralateral to an LC lesion are presented in Fig. 1. These changes are similar to those obtained from non-lesioned control rats. Although no qualitative effects of NE depletion were apparent in these responses, it was still to be determined whether NE depletion had any quantitative effects which could document an N E influence upon cerebral oxidative metabolism 'at rest'. To accomplish this, the amplitudes of oxidation obtained with inspiration of either 100 K 02 or 95~o Oz with 5 ~ CO2 were compared to the total 'labile' signal defined as the total signal change between peak oxidation and peak reduction produced by 100 ~ N2 respiration. The results are presented in Table II. N o significant differences in the percentage of reduced cytochrome oxidase were evident between contralateral and lesioned hemispheres when cytochrome oxidation was produced by inspiration of either 100K Oz or 95 ~ 02 with 5 ~ COz. Non-lesioned control rats exhibited redox ratios on either hemisphere that were not significantly different from these. In all groups, the percentage of the reduced species of cytochrome oxidase was
LEFT HEMISPHERE BL. VOL.
STIM
I ~__j~'~--~
(590 NM)
....
CYT A,A 3 (605 - 590 N
SP
i
I2% F.S.
~
M
)
~
4
"F' MV I
20 SEC
I
RIGHT HEMISPHERE BL.VOL.
I
(590 NM)
CYT A,A
3
I
(605 - 590 N M ) ~
SP
_17_~_~ -
Fig. 2. Optical and electrical responses to stimulation of the cerebral surface of hemispheres contralateral (left) and ipsilateral (right) to LC lesion. Stimulation was presented at the time mark for 2 sec, pulses at 0.5 msec duration 20 Hz with intensity at approximately 10 V. Decreased reflectance at the reference (590 nrn) wavelength is plotted as an upward deflection indicating increased blood volume. Negative shifts in the cortical steady potential are recorded as downward doflections, NE contents of the left and right hemispheres were 152 ng/g and 42 ng/g, respectively.
95 TABLE III Stimulus-induced responses of cytochrome oxidase ( Cyt a, as) and blood volume (BV) and cerebral norepinephrine ( NE) content in non-lesioned control rats Cerebral hemisphere
n
Cyt a, a8 tPmax ( sec)
Cyt a, a8 q o f f ( sec)
B V Pmax/Cyt a, aa Pmax (ratio)
N E (ng/g)
Left Right Both
7 7 14
7.05 :t: 0.31 7.41 -4- 0.20 7.23 ± 0.17
6.52 -4- 0.87 7.14 -4- 0.60 6.83 ± 0.48
1.37 ± 0.18 1.56 -4- 0.33 1.45 ± 0.16
269 ± 41 322 ± 33 295 4- 29
The values denote means ± S.E.M. For detailed explanation of the stimulus-induced responses see text. There were no significant differences between the two hemispheres in any of the parameters mentioned. higher with 9 5 % 0 2 / 5 % CO2 t h a n with 100% 03. This is because t h e extent o f o x i d a t i o n was always g r e a t e r w h e n COs was present. It should be noted, however, t h a t these values are relative because the degree o f c y t o c h r o m e o x i d a t i o n w i t h either h y p e r o x i c gas was n o t at its a b s o l u t e p e a k as d e m o n s t r a t e d by the very high degree o f o x i d a t i o n r e c o r d e d whert a n i m a l s were r e s p i r e d with high 02 a n d CO2 mixtures u n d e r h y p e r b a r i c c o n d i t i o n s 15. Likewise, r e d u c t i o n was n o t at its a b s o l u t e m a x i m u m since the d u r a t i o n o f 100 % N2 r e s p i r a t i o n was k e p t s h o r t t o p r e v e n t h y p o x i c b r a i n injury. C y t o c h r o m e oxidase in an 'active state'
W h e n the c e r e b r u m was s t i m u l a t e d to increased activity b y electrical pulses a p p l i e d to the d u r a , there was a negative shift o f the c o r t i c a l steady p o t e n t i a l as m o n i t o r e d b y D C - c o u p l e d A g - A g C 1 electrodes. This o c c u r r e d in c o n t r o l rats a n d in r a t s two weeks following L C lesion. A s s o c i a t e d with this shift, there was a t r a n s i e n t o x i d a t i o n o f c y t o c h r o m e oxidase a n d a decrease in the a m o u n t o f light reflected at 590 nm. I n previous studies, this decrease was i n t e r p r e t e d to indicate a n increase in the TABLE IV Stimulus-induced responses of cytochrome oxidase ( Cyt a, as) and blood volume ( BV) and cerebral norepinephrine ( NE) content in rats with unilateral locus coeruleus (LC) lesions
The values denote means 4- S.E.M. of the number of observations enclosed in parentheses. Explanation of the various stimulus-induced parameters is detailed in the text. Cerebral hemisphere
Cyt a, as tPraax (sec)
Contralateral to LClesion 6.82 -4- 0.31 (13) Ipsilateral toLClesion 7.45 ± 0.28* (13)
Cyt a, a8 ttoff (sec)
B V Pmax/Cyt a, a3Pmax (ratio)
N E (ng/g)
5.73 ± 0.79 (12)
1.68 -4- 0.26 (12)
247 ± 25 (13)
8.92 ± 1.I1"* (12) 0.99 4- 0.14"* (12)
* Not significantly different from the contralateral hemisphere. ** Different from the contralateral hemisphere at P < 0.05. *** Different from the contralateral hemisphere at P < 0.001.
36 -4- 5"**(13)
96 local volume of hemoglobin is,a4. Fig. 2 shows examples of these responses recorded from cerebral hemispheres ipsilateral and contralateral to an LC lesion. To determine whether NE depletion influences the rates of the metabolic responses, the times from stimulus onset to peak oxidation and blood volume increase (tPmax) and the time from peak oxidation to half recovery (ttoff) were compared in NE-depleted and non-depleted hemispheres of LC-lesioned rats and in controls. Table III summarizes these data. In non-lesioned control rats, there were no differences between hemispheres in either tPraax or ttoff. Also, the rates of cytochrome oxidase redox changes and blood volume responses recorded from contralateral hemispheres of unilateral LC-lesioned rats were very s milar to those of non-lesioned controls (Tables III and IV). Also, tPmax of cytochrome oxidase recorded from the NEdepleted was similar to that of the contrateral hemisphere. However, the t~off in NE-depleted cerebral hemispheres was 70 ~ slower than that of contralateral hemispheres (P < 0.05; Table IV). To determine if N E depletion affects the relationship between changes in the redox state of cytochrome oxidase and local blood volume, the amplitudes of the response to stimulation of each of these were compared. This provided a ratio which we assumed to be independent of stimulus intensity. In control rats, the ~ cytochrome oxidation/z~ blood volume increase was 1.46 4- 0,18 (mean 4- S.E.M.). The ratio obtained from contralateral hemispheres of LC-lesioned rats was 1.68 4- 0,26 which was not significantly different from non-lesioned controls. However, the ratio obtained from the ipsilateral hemispheres of LC-lesioned rats was 0.99 4- 0.14, which was significantly lower than contralateral hemispheres or that from control rats (P < 0.05; Table IV). The lowered ratio of stimulus-induced cytochrome oxidase to blood volume changes occurred in 10 of 12 rats. In these 10 rats, the average ratio of the responses in the ipsilateral hemisphere was decreased to about half that of the contralateral hemisphere.
FL - REFL ( 4 6 o - m me) RIGHT
IllM_ ~.2 t
LEFT
..,. i
.~..L'~.. ~:~.~-~
.,,
~..~
-
Fig. 3. Responses of pyridine nucleotide fluorescenceto direct cortical stimulation (2-sectrain, 20 Hz, 0.5 msec pulses at approximately 6 V). Corrected fluorescence signals were averaged for 8 stimulus presentations for each hemisphere by computer. Decreased fluorescence(NAD oxidation) is recorded as a downward deflection. NE content of the right hemispherecontralateral to LC lesion was 302 ng/g. NE content of the left hemisphere ipsilateral to LC lesion was 9 ng/g.
97 TABLE V Stimulus-induced response of pyridine nucleotide ( NADH) and cerebral norepinephrine ( NE) content in rats with unilateral locus coeruleus (LCJ lesions
The values denote means 4- S.E.M. of 8 observations. Explanation of the stimulus-induced parameters is detailed in the text. Cerebral hemisphere
NADH tPraax (sec)
NA DH t~off (sec)
NE (ng/g)
Contralateral to LC lesion Ipsilateral to LC lesion
7.22 4- 0.40 10.4 4- 1.50"
6.01 4- 0.45 11.34 4- 1.90"
258 4- 25 35 4- 9**
* Different from the eontralateral hemisphere at P < 0.05. ** Different from the eontralateral hemisphere at P < 0.001. Pyridine nucleotide in an 'active" state
The response, monitored by surface fluorometry, to direct electrical stimulation of cerebral cortex in both NE-depleted and non-depleted hemispheres consisted of a transient decrease in fluorescence which was interpreted as an oxidation of intramitochondrial NAD. An example of signal-averaged responses recorded in hemispheres contralateral and ipsilateral to an LC lesion is shown in Fig. 3. Although the magnitude and the direction of stimulus-induced N A D responses were unaffected by NE-depletion, both the rates of oxidation and re-reduction of N A D were slowed in NE-depleted tissue. The average time to peak oxidation of N A D was 7.22 see and 10.40 sec, respectively (P < 0.05, Table V). The mean time to half-recovery on the ipsilateral side was 11.34 sec and that on the contralateral side was 6.01 see (P < 0.05; Table V). DISCUSSION These data demonstrate that lesion of the LC, and the resultant depletion of NE, influences cerebral oxidative metabolism in situ. This influence was not observed under conditions of low energy demand, during a 'resting' state, but was seen during increased metabolic demand induced by direct cortical electrical stimulation. The role, if any, of the noradrenergic system in the control o f cerebral circulation and metabolism has been unclear. Anatomic evidence, from light and electron microscopy and histofluorescence microscopy, indicates a dense noradrenergic innervation of cerebral vessels ~7. Yet, the physiological function of this innervation and its importance to circulatory control remains controversial (see e.g. refs. 13, 14 and 29). Since the suggestion by Hartman et al. 12 that N E innervation of the intraparenchymal cerebral vasculature arises in the LC, interest has shifted from peripheral sympathetic innervation of cerebral vessels through the superior cervical ganglion and has focused on the LC 8. Electrical stimulation of the LC has been reported to decrease cerebral blood flow 7, and to result in a mild decrease in 2-deoxyglucose uptake in some brain areas 1. Chemical activation o f the LC has been reported to decrease cerebral blood
98 flow and to increase capillary permeabilityz0. Conversely, LC ablation has been reported to cause increased cerebral blood flow and loss of COg reactivity in cats a. However, no significant changes in cerebral cortical metabolism could be demonstrated after LC ablation by 2-deoxyglucose autoradiography35. It is difficult to reconcile these reports with studies which show an association between increased activity of LC neurons during stress and increased alertness in unanesthetized animals x°, since these conditions are generally associated with increased cerebral blood flow and metabolic rate ~7. We have shown that the relative redox state of cerebral cytochrome oxidase remains unchanged in the absence of NE. These data, however, must be interpreted in a very limited manner. In the aged brain, for example, no differences were observed in the relative redox state of cytochrome oxidase in the 'resting state', but deficiencies in metabolic capability were detected when cerebral energy demand was increased39. An unchanged cytochrome oxidase redox ratio in NE-depleted hemispheres under 'resting' conditions only indicates that energy demand and energy conservation are in balance independent of NE content. Such data cannot take into account whether NE has an influence upon energy turnover or energy demand. These, and other data on the influence of NE on metabolism, may be misleading if based only on static measurements from 'resting' brain. In both NE-depleted and control hemispheres, the oxidation of cytochrome oxidase produced by inspiration of 95 ~ O~/5 ~ COs was greater than that produced by inspiration of 100 ~ Oe. This COs effect has been reported previously3a and has been correlated with a greater increase in tissue oxygen tension, as measured polarographically2L Such a CO~ effect could be due to the known vasodilatation produced by hypercapnea. However, Bates et al. 3 showed that, in cats, the CO~ reactivity of cerebral vasculature was lost in cortical hemispheres ipsilateral to LC lesion. If this is true in rats, then the large cytochrome oxidation is likely due to a COz effect upon cellular metabolism (see e.g. ref. 26). Whatever the mechanism underlying this CO2 influence, these data demonstrate that it is unaffected by NE depletion. If the noradrenergic fibers from the LC are involved in the response to stress, then it is most appropriate to look for an effect of NE depletion on metabolic changes associated with increased energy demand. Our results show that NE depletion is associated with a slowed rate of recovery of NAD and cytochrome oxidase, each of which had been oxidized in response to increased energy demand produced by direct cortical stimulation. Stimulation of this type had previously been shown to produce oxidative responses ofpyridine nucleotide and cytochrome a,a31S,zL Such stimulation also evokes negative shifts of the cortical steady potential and increased extracellular potassium activity in proportion to the oxidation of the mitochrondrial components2L Some caution was necessary since barbiturates had previously been show to produce a s!milar slowing of these metabolic responses to stimulation28. However, such a barbiturate effect would tend to decrease differences between the NE-depleted and control hemispheres. Nevertheless, we considered it important to maintain a similar depth of anesthesia for both hemispheres and to vary the order of experimental recording.
99 A slowed rate of re-reduction of NAD or cytochrome oxidase likely indicates a decreased rate of ATP generation and a decreased capacity of cortical tissue to respond to increased metabolic demand. There are several possible explanations for this effect. One is that NE has an influence within the respiratory chain, such that its absence results in either uncoupling of oxidation from phosphorylation or a block in the chain itself. Uncoupling is unlikely since this would be expected to result in a loss of respiratory control and a higher resting ratio of oxidized respiratory components, as occurs in vitro. Such a response was not demonstrated. Uncoupling of oxidation from phosphorytation would also be expected to be accompanied by greater oxidation of each respiratory component for a given stimulus because of decreased efficiency of the oxidative apparatus. This did not occur. The possibility of a block in the chain is also unlikely because such a block would be associated with an altered relationship between respiratory chain members on each side of the block (i.e. a crossover point). The absence of NE produced no apparent differences in the responses of NAD and cytochrome oxidase to stimulation. Another possibility is that the slowed reduction of NAD and cytochrome oxidase results from prolonged ion displacement following increased electrical activity. Such a prolongation of potassium ion clearance from the extracellular space was associated with the slow re-reduction seen with phenobarbital ~3. With NE depletion, however, the rate of NAD oxidation was slowed consistent with a slower generation of ADP from decreased Na+-K+-ATPase, but preliminary studies show that potassium clearance is not prolonged in the absence of NE after stimulation (T. Sick, personal communication). A third possibility is that availability of substrate for re-reduction of the oxidized mitochondrial components may be affected by NE-depletion. However, this effect should be observed as a more oxidized resting state in a manner similar to the state 2 condition of isolated mitochondria6. It is possible, nevertheless, that there is a mechanism which responds to increased energy demand with an increase in substrate provision, and that the regulation of substrate availability to increased demand is altered when NE is absent. A fourth possibility is that the NE influence on cerebral metabolism is mediated through an effect on local cerebral circulation. If the increase in local blood volume in response to stimulation reflects a local mechanism for increasingsubstrate and oxygen delivery, then our finding of a decrease in this response after NE-depletion is consistent with transient inadequate provision of reducing equivalents to the respiratory chain. No matter how they are mediated, these results are consistent with a role for NE in the regulation of cerebral oxidative metabolism in response to sudden increases in energy requirements. ACKNOWLEDGEMENT Supported by PHS Grants NS-05820, NS-14319 and NS-14325. We thank M. Ganapathi for excellent technical assistance. J.C.L. is a recipient of NIH Research Career Development Award NS-00399,
100 REFERENCES 1 Abraham, W. C., Delanoy, R. L., Dunn, A. J. and Zornetzer, S. F., Locus coeruleus stimulation decreases deoxyglucose uptake in ipsilateral mouse cerebral cortex, Brain Research, 172 (1979) 387-392. 2 Avi-Dor, J. J., Olson, M., Doherty, M. D. and Kaplan, N. O., Fluorescence of pyridine nucleotides in mitochondria, J. bioL Chem., 237 (1962) 2377-2383. 3 Bates, D., Weinshilboum, R. M., Campbell, R. J. and Sundt, T. M., The effects of lesions in the locus ceruleus on the physiological responses of the cerebral blood vessels in cats, Brain Research, 136 (1977) 431--443. 4 Chance, B., Cohen, P., J/Sbsis, F. F. and SchiSner, B., Intracellular oxidation-reduction in vivo, Science, 137 (1962) 499-508. 5 Chance, B. and J6bsis, F. F., Changes in fluorescence in a frog sartorius muscle following a twitch, Nature (Lond.), 184 (1959) 195-196. 6 Chance, B. and Williams, G. R., The respiratory chain and oxidative phosphorylation, Advanc. EnzymoL, 17 (1956) 65-134. 7 De la Torre, J. C., Surgeon, J. W. and Walker, R. H., Effects of locus coeruleus stimulation on cerebral blood flow in selected brain regions, Acta neuroL scand., Suppl., 64 (1977) 104-105. 8 Edvinsson, L., Lindvall, M., Nielsen, K. C. and Owman, C. H,, Are brain vessels innervated also by central (non-sympathetic) adrenergic neurons, Brain Research, 63 (1973) 496--499. 9 Estabrook, R. W., Fluorometric measurements of reduced pyridine nucleotide in cellular and subcellular particles, Analyt. Biochem., 4 (1962) 231-245. 10 Foote, S. L. and Bloom, F. E., Activity of norepinephrine-containinglocus ceruleus neurons in the unanesthetized squirrel monkey. In Usdin, Kopin and Barchas, (Eds.), Catecholamines: Basic and Clinical Frontiers, Pergamon, New York, 1979. 11 Harik, S. I., LaMarma, J. C., Light, A. I. and Rosenthal, M., Cerebral norepinephrine: influence on cortical oxidative metabolism in situ, Science, 206 (1979) 69-71. 12 Hartman, B. K., Zide, D. and Udenfriend, S., The use of dopamine beta hydroxylase as a marker for the central noradrenergic nervous system in rat brain, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2722-2726. 13 Heistad, D. D. and Marcus, M. L., Evidence that neural mechanisms do not have important effects on cerebral blood flow, Circulat. Res., 42 (1978) 295-302. 14 Heistad, D. D. and Marcus, M. L., Response to'Do vasomotor nerves significantly regulate cerebral blood flow?' Circulat. Res., 43 (1978) 494-495. 15 Hempel, F. G., JSbsis, F. F., LaManna, J. C., Rosenthal, M. and Saltzman, H. A., Redox levels of cytoehrome a, aa in cat cerebral cortex during hyperbaric oxygenation, J. appt. PhysioL, 43 (1977) 873-879. 16 Henry, D. P., Statman, B. J., Johnson, D. G. and Williams, R. H., A sensitive radioenzymatic assay for norepinephrine in tissue and plasma, Life ScL, 16 (1975) 375-384. 17 J~Sbsis,F. F., Basic processes in cellular respiration. In Fenn and Rahn (Eds.), Handbook of Physiology, Amer. Physiol. Soc., Washington, D.C., 1964, pp. 63-124. 18 J6bsis, F. F., Keizer, L, LaManna, J. C. and Rosenthal, M., Reflectance spectrophotometry of cytochrome a, as in vivo, J. appL Physiol., 43 (1977) 858-872. 19 JSbsis, F. F., O'Connor, M. J., Vitale, A. and Vreman, H., Intracellularredox changes in functioning cerebral cortex. I. Metabolic effects of epileptiforrn activity, J. NeurophysioL, 34 (1971) 735-749. 20 Kobayashi, R. M., Palkovits, M., Jacobowitz, D. M. and Kopin, I. J., Biochemical mapping of the noradrenergic projection from the locus ceruleus, Neurology, 25 (1975) 223-233. 21 Koda, L. Y., Schulman, J. A. and Bloom, F. E., Ultrastructural identification of noradrenergic terminalsin rat hippocampus: Unilateral destruction of the locus ceruleus with 6-hydroxydopamine, Brain Research, 145 (1978) 190--195. 22 Kreisman, N. R., Sick, T. J., LaManna, J. C. and Rosenthal, M., Local tissue oxygen tensioncytochrome al, az redox relationships in rat cerebral cortex in vivo, Brain Research, submitted. 23 LaManna, J. C., Cordingley, G. and Rosenthal, M., Phenobarbital actions in vivo: Effects on extracellular potassium activity and oxidative metabolism in cat cerebral cortex, J. PharmacoL exp. Ther., 200 (1977) 560--569. 24 LaManna, J. C., Light, A. I., Harik, S. I. and Rosenthal, M., Locus coeruleus lesions: effects on cerebral oxidative metabolism in vivo. II. The coupling between oxygen delivery and energy demand, Neurosci. dbstr., 5 (1979) 340.
101 25 Lothman, E., LaManna, J. C., Cordingley, G., Rosenthal, M. and Somjen, G., Responses of electrical potential, potassium levels and oxidative metabolic activity of the cerebral neocortex of cats, Brain Research, 88 (1975) 15-36. 26 Miller, A. L., Hawkins, R. A. and Veech, R. L., Decreased rate of glucose utilization by rat brain in vivo after exposures to atmospheres containing high concentrations of CO2, J. Neurochem., 25 (1975) 553-558. 27 Nielsen, K. C. and Owman, C. H., Adrenergic innervation of pial arteries related to the circle of Willis in the cat, Brain Research, 6 (1967) 773-776. 28 O'Connor, M. J., Origin of labile NADH tissue fluorescence, In J6bsis, (Ed.), Oxygen andPhysiological Function, Professional Information Library, Dallas, 1977, pp. 90-99. 29 Purves, M. J., Do vasomotor nerves significantly regulate cerebral blood flow? Circulat. Res., 43 (1978) 485-493. 30 Raichle, M. E., Hartman, B. K., Eichling, J. O. and Sharpe, L. G., Central noradrenergic regulation of cerebral blood flow and vascular permeability, Proc. nat. Acad. ScL (Lond.), 72 (1975) 3726-3730. 31 Rosenthal, M., Harik, S. I., LaManna, J. C. and Light, A. I., Locus coeruleus lesions: effects on cerebral oxidative metabolism in vivo. I. Cytochrome oxidase in the steady state, NeuroscL Abstr., 5 (1979) 350. 32 Rosenthal, M. and J6bsis, F. F., Intracellular redox changes in the functioningcerebral cortex. II. Effects of direct cortical stimulation, J. Neurophysiol., 32 (1971) 750-761. 33 Rosenthal, M., LaManna, J. C., J6bsis, F. F., Levasseur, J., Kontos, H. and Patterson, J., Effects of respiratory gases on cytochrome a in intact cerebral cortex: Is there a critical PO~? Brain Research, 108 (1976) 143-154. 34 Rosenthal, M., LaManna, J. C., Yamada, S. ,Younts, B. W. and Somjen, G., Oxidative metabolism, extracellular potassium and sustained potential shifts in cat spinal cord in situ, Brain Research, 162 (1979) 113-127. 35 Schwartz, W. J., 6-hydroxydopamine lesions of rat locus ceruleus alter brain glucose consumption, as measured by the 2-deoxy-D-[14C]glucosetracer technique, Neurosci. Lett., 7 (1978) 141-150. 36 Sharma, V. K., Harik, S. I., Ganapathi, M., Busto, R. and Banerjee, S. P., Locus ceruleus lesion and chronic reserpine treatment: effects on adrenergic and cholinergic receptors in cerebral cortex and hippocampus, Exp. Neurol., 65 (1979) 685-690. 37 Siesj6, B. K., Brain Energy Metabolism, J. Wiley, New York, 1978, pp. 607. 38 Sylvia, A. L. and Rosenthal, M., The effect of age and lung pathology on cytochrome a, a3 redox levels in rat cerebral cortex, Brain Research, 146 (1978) 109-122. 39 Sylvia, A. L. and Rosenthal, M., Effects of age on brain oxidative metabolism in vivo, Brain Research, 165 (1979) 235-248. 40 Ungerstedt, V., Stereotaxic mapping of monoamine pathways in the rat brain, ,4ctaphysiol. scand., Suppl., 367 (1971) 1-48.