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Circadian changes in extracellular ascorbate in rat cortex, accumbens, striatum and hippocampus: Correlations with motor activity
Neuroscience Letters, 60 (1985) 331-336
331
Elsevier Scientific Publishers Ireland Ltd.
NSL 03556
CIRCADIAN CHANGES IN EXTRACELLULAR ASCORBATE IN RAT CORTEX, ACCUMBENS, STRIATUM AND H I P P O C A M P U S : CORRELATIONS WITH M O T O R ACTIVITY
ROBERT D. O'NEILL* and MARIANNE FILLENZ
University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT (U.K.) (Received June 6th, 1985; Revised version received and accepted July 10th, 1985)
Key words: ascorbate - excitatory amino acid - motor activity - circadian - voltammetry - rat
We have used linear sweep voltammetry with carbon-paste electrodes to monitor changes in the ascorbate signal simultaneously in rat frontal cortex, nucleus accumbens, striatum and hippocampus together with motor activity. The relative amplitude of the ascorbate signal recorded in the four regions corresponded to the relative density of excitatory amino acid (EAA) transmission determined by other methods; this result provides further evidence that the ascorbate signal may be used as an index of EAA release. Changes in motor activity were associated with changes in the ascorbate signal; linear regression analysis for motor activity versus the ascorbate signal revealed differences between the release of ascorbate in the four brain regions.
The ability of glutamate to stimulate almost all neurones, which suggested that it was a non-specific excitant, was invoked in the past as evidence against its role as a neurotransmitter. However, neurochemical studies in the 25 years since the demonstration by Curtis et al. [4] of its excitatory effects have established it as one of the main transmitters within the mammalian central nervous system (CNS). Ascorbic acid is also found throughout the CNS and, although its role is unclear, there is evidence that ascorbate may modulate binding to a variety of neurotransmitter receptors [12, 13]. Although it has been suggested that the extracellular concentration of ascorbate is under homeostatic control [18], a number of groups have observed both spontaneous and drug-induced changes in extracellular ascorbate levels [2, 5, 8, 18]. However, distinct from the problem of the role of ascorbate in the extracellular fluid is the question of the mechanism of ascorbate release. The finding that L-glutamate (L-GIu) and aspartate (Asp) cause release of ascorbate from synaptosomes through a carrier-mediated hetero-exchange mechanism (i.e. it is not receptor mediated) [10], led us to suggest that changes in extracellular brain ascorbate may be linked to excitatory amino acid (EAA) release. This is supported by the finding that L-Glu and Asp, when injected systemically or locally into the *Author for correspondence at present address: Department of Chemistry, University College, Belfield, Dublin 4, Eire. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
332
brain, increase the extracellular concentration of brain ascorbate [16]. Lesions of the glutamatergic cortico-striatal projection [18], but not kainate lesions of the striatum or 6-hydroxydopamine lesions of the substantia nigra [9], substantially reduce extracellular ascorbate in the striatum. Finally, electrical stimulation of the perforant path, which sends glutamatergic fibers to the dentate gyrus of the hippocampus, increases the ascorbate signal only when the sensor is near the dentate [16]. On the basis of these findings we have suggested that changes in extracellular ascorbate concentration may be used as an index of EAA release [6]. In this study we have recorded extracellular ascorbate in 4 brain regions where there is evidence for the presence of EAA-releasing terminals. The identification of neurons releasing EAAs depends on a number of criteria. Among these are the demonstration of a reduction in high-affi•;ty uptake and endogenous tissue levels of Glu or Asp after selective lesions of specific pathways, and studies of release of endogenous or exogenous Glu, or Asp, after selective activation of nerve fibers [7]. As a result of these methods cortico-fugal pathways, including cortico-striatal and cortico-accumbens projections, have been shown to be glutamatergic [20]. The accumbens and striatum, in addition to the neocortical input, also receive an allocortical projection via the fornix/fimbria, which is much larger in the accumbens than in the striatum [20]. In the hippocampus, the perforant path (which is an input from the entorhinal cortex to the dentate gyrus), the commissural interhippocampal fibers and the Shaffer collaterals, all fulfill some of the criteria for EAA-releasing pathways [3]. Studies on high-affinity Glu binding and uptake in vitro provide evidence that there is glutamatergic transmission in the frontal cortex [11]. The study of Glu release, even in vitro, has proved difficult because there are several pools of Glu within the nerve terminal; some of these have a purely metabolic role, whereas others are involved in neurotransmission, being released by a Ca2+-de pendent mechanism [7]. There are, at present, few measurements of the release of EAAs in vivo. Abdul-Ghani et al. [1], using a cortical-cup technique, have reported spontaneous and stimulus-evoked release of Glu from awake, behaviourally normal rats. The authors emphasize that changes in the Glu content of the perfusate represent the balance between release and the subsequent uptake, which means that small changes in release may not be detected. The aim of this study was to provide further evidence that ascorbate can be used as an index of EAA release and to investigate the relationship between this release and motor activity. We have used microcomputer-based linear sweep voltammetry with carbon-paste electrodes to monitor simultaneously circadian changes in rat motor activity and in the ascorbate signal in frontal cortex, accumbens, striatum and hippocampus. Ten male Sprague-Dawley rats were stereotaxically implanted, under chloral hydrate anesthesia (7 ml/kg 5~o solution i.p.), with 4 carbon-paste electrodes (300 ~m o.d.) as described previously [17]. The coordinates, with the head level between bregma and lambda, were: frontal cortex, AP 3.0 (from bregma), L 1.5 (from bregma), and DV 1.5 (from skull); accumbens, 1.5, 1.5 and 6.5; striatum, - 0 . 5 , 3.0 and 4.8; and hippocampus, - 3 . 6 , 1.8 and 4.4. The rats were given at least two days to recover before being placed in the recording cages (25 x 25 x 25 cm) and connected
333
to the microcomputer-based equipment [15]. Linear sweep voltammograms were recorded at 12-min intervals at a scan rate of 5 mV/s between 0 and 650 mV with respect to a Ag reference electrode. Peak 1 obtained using this technique is due to the oxidation of ascorbic acid [15, 17]; changes in the height of this peak reflect changes in the extracellular concentration of ascorbate. Up to 8 electrodes were scanned simultaneously over recording periods of 24 h. Mean__+ S.E.M. are quoted; n is the number of determinations. The significance of differences in between the 4 brain regions was calculated using paired t-tests on data from each animal. The average day-time (08.00 to 20.00 h) value of the ascorbate peak height was: frontal c o r t e x = 5 . 8 + 0 . 9 nA; accumbens =2.5___0.4 nA; striatum =4.1 +0.6 nA; hipp o c a m p u s = 1.7+0.2 nA; n = 2 0 . The order of the relative sizes was: frontal cortex > striatum (P < 0.05); striatum > accumbens (P < 0.05); and accumbens > hippocampus (P < 0.02). High-affinity glutamate binding, whether to receptors or to the uptake sites, has been used as an index of innervation density [11]. The density of such binding sites show the same rank order as the size of the ascorbate peak in these areas, providing further evidence that the ascorbate signal may be used as an index of EAA release. Circadian changes in signals were calculated by comparing the average value for the light-on period (08.00 to 20.00 h, 60 points) with that for the light-off period (20.00 to 08.00 h, 60 points). The nocturnal increase, expressed as a percentage of the day-time value was: frontal c o r t e x = 6 3 + 7 ~ ; a c c u m b e n s = 4 1 + 5 ~ ; striat u m = 4 9 + 6 ~ o ; hippocampus= 14+3~o; n = 2 0 , P<0.001 for all increases. Here the sequence was: frontal cortex>striatum (P<0.01); striatum=accumbens; and accumbens > hippocampus (P < 0.001). Total motor activity of each rat was monitored between electrochemical recordings (ca. 9 min) using a Doppler-shift microwave device [14]. The average level of motor activity in a given 24-h period was the same for implanted and unimplanted animals [14]. To investigate the relationship between motor activity and EAA release, we calculated correlation coefficients for spontaneous changes in the ascorbate signal in each region with changes in motor activity. An example which represents continuous recording over a 10-h period is shown in Fig. 1. The analysis consisted of calculations of correlation coefficients on the weighted moving average of the original data [14] for each day's recording (120 points). In order to find the time when maximum correlation occurred, cross-correlations were calculated by comparing, for example, m(t + 1) with hvcx(t), where hvcx(t) is the value of ascorbate signal at time t in frontal cortex, and m(t+ 1) is the value of the motor activity counter one scan (12 min) later; cross-correlations were calculated in the range t - 5 to t + 10. Cross-correlation analysis of the data in Fig. 1 shows that maximum correlation occurs when the motoractivity data is shifted by 12 rain (1 scan), and falls off steeply with shifts in either direction. The nocturnal rise in the ascorbate signal in the 4 brain regions over a 24-h period shows a progressive decrease in the size, and an increase in the delay of onset, from frontal cortex to striatum to accumbens to hippocampus. A quantitative assessment of these differences was made by calculating regression and correlation coefficients
334
60
4.5 -500
nA
•t
¢-
._m oo
3.0
0 0 co
motor counts
-250
].5
> 0
2
4
6
8
]o
time/h
OBJ
J
0,6
04
g o
.~
02-
0
-02-
~@
-24 shift/min
Fig. 1. Upper section: time-course, at 12-min intervals, over a lO-h period for changes in the ascorbate signal (O), recorded with a single carbon-paste electrode in the striatum, and total motor activity (O). Start time was 12.00 h. Lower section: cross-correlation analysis of the ascorbate and motor-activity data shown above. Maximum correlation (0.86) was obtained when the movement data were shifted by + 1 scan (12 min) with respect to the ascorbate data.
335 TABLE I REGRESSION ANALYSIS FOR MOTOR ACTIVITY VERSUS ASCORBATE SIGNAL Regression analysis of the circadian changes in motor activity versus the ascorbate signal recorded in frontal cortex, nucleus accumbens, striatum and hippocampus giving the maximum correlation coefficient (r) from the cross-correlation analysis (see Fig. 1), time shift (s) needed for maximum correlation and the slope of the regression line (m) which corresponded to maximum correlation. There was no significant difference between r values for frontal cortex, nucleus accumbens and striatum; that for hippocampus was significantly smaller (P < 0.02). The s value was smallest in frontal cortex (P< 0.01 compared with striatum) and greatest in hippocampus (P<0.01 compared with nucleus accumbens). The rank order of the m values was frontal cortex > striatum (P < 0.01) = nucleus accumbens > hippocampus (P < 0.001). Brain region
Frontal cortex (n = 18) Nucleus accumbens (n=32) Striatum (n = 30) Hippocampus ( n - 18)
Correlation coefficient
Timeshift (min)
Regression slope (pA/move)
0.82+0.03
32+3
12+2
0.78+0.03
66+6
7+ 1
0.82+0.02
61 +5
7_+ 1
0.46+0.10
100_+7
2.5_+0.8
between circadian changes in m o t o r activity and the ascorbate signals. Table I shows that the mean correlation coefficient for m o t o r activity versus ascorbate is the same for frontal cortex, accumbens and striatum, and is significantly smaller for the hippocampus. The cross-correlation analysis shows that the time-shift needed for maxim u m correlation is lowest in frontal cortex and greatest in hippocampus. The regression slopes calculated at the time-shift for m a x i m u m correlation had a rank order o f frontal cortex > striatum = accumbens > hippocampus. In view o f the rapid rise (latency < l min) and fall in the ascorbate signal following brief electrical stimulation (2.5 s) [16], it is perhaps surprising that m a x i m u m correlation is obtained when the circadian motor-activity data are shifted by between 30 and 100 min with respect to ascorbate signal (Table I). The time-course and crosscorrelation analysis illustrated in Fig. 1 shows that when there are relatively short bursts ( < l h) o f activity interspersed with resting periods, the time shift for maxim u m correlation is one scan (12 min) which is the m i n i m u m delay that can be resolved. The longer shifts observed for the normal circadian pattern o f sustained nocturnal activity m a y be due to the saturation o f a removal process for ascorbate leading to its progressive accumulation in the extracellular fluid which obscures the intrinsically rapid response o f the signal. The relative size o f the ascorbate signal recorded in frontal cortex, nucleus accumbens, striatum and h i p p o c a m p u s provides further support for the hypothesis that changes in the extracellular concentration o f brain ascorbate serve as an index o f E A A release. Furthermore, regression analysis for m o t o r activity versus the ascorbate signal reveals differences between ascorbate release in the 4 brain regions.
T h i s w o r k w a s f u n d e d in p a r t b y t h e M e d i c a l R e s e a r c h C o u n c i l . R . D . O . is a Beit M e m o r i a l Research Fellow. We are grateful for a grant t o w a r d s the e q u i p m e n t f r o m t h e E.P. A b r a h a m s C e p h a l o s p o r i n F u n d . W e t h a n k L e s l e y Sellars f o r a r t w o r k . I Abdul-Ghani, A.-S., Coutinho-Netto, J. and Bradford, H.F., In vivo superfusion methods and the re-
2
3
4 5
6
7
8 9 10 1t 12
13 14
15
16
17 18
19 20
lease of glutamate. In P.J. Roberts, J. Storm-Mathisen and G.A.R. Johnston (Eds.), Glutamate: Transmitter in the Central Nerw)us System, J. Wiley & Sons, Chichester, 1981, pp. 155 204. Clemens, J.A. and Phebus, L.A., Changes in brain chemistry produced by dopaminergic agents: in vivo electrochemical monitoring reveals opposite changes in anaesthetised vs unanaesthetised rats, Brain Res., 267 (1983) 183- 186. Cotman, C.W. and Nadler, J.V., Glutamate and aspartate as hippocampal transmitters: biochemical and pharmacological evidencc. In P.J. Roberts, J. Storm-Mathisen and G.A.R. Johnston (Eds.), Glutamate: Transmitter in the Central Nervous System, J. Wiley & Sons, Chichester, 1981, pp. 117 154. Curtis, D.R., Phillis, J.W. and Watkins, J.C., The chemical excitation of spinal neurones by certain acidic amino acids, J. Physiol. (Lond.), 150 (1961)) 656 682. Ewing, A.G., Alloway. K.I)., Curtis, S.I)., Dayton, M .A., Wightman, R.M. and Curtis, G.V., Simultaneous electrochemical and unit recording measurements: characterization of the effects of d-amphetamine and ascorbic acid on neostriatal neurons, Brain Rcs., 261 (1983) 101 108. Fillenz, M., O'Neill. R.I). and Grunewald, R.A., Changes in extracellular brain ascorbate concentration as an index ot excitatory amino acid release. In M.H. Joseph, M. Fillenz, I.A. Macdonald and ('.A. Marsden (l'.ds.), Monitoring Neurolransmiuer Release during Behaviour, Ellis Horwood, Chichester, in press. Fonnum, F. and Malthe-Sorenssen, D., Localisation of glutamate neurons. In P.J. Roberts, J. StormMathisen and G.A.R. Johnston (Eds.), Glutamate: Transmitter in the Central Nervous System, J. Wiley & Sons, Chichester, 1981, pp. 205 222. Gonon, F., Buda. M., Cespuglio. R., Jouvet, M. and Pujol, M.-F., Voltammetry in the striatum of chronic freely moving rats: detection of catechols and ascorbic acid, Brain Res., 223 (1981) 69 80. Grunewald, R.A., Ascorbic Acid in The Brain, D. Phil. Thesis, Oxford University, 1983. Grunewald. R.A. and Fillenz, M., Release of ascorbate from synaptosomal fraction of rat brain, Neurochem. Int., 6 (1984) 491 500. Halpin, S., Wieczorek, C.M. and Rainbow, T.C., Localization of L-glutamate receptors in rat brain by quantitative autoradiography, J. Neurosci., 4 (1984) 2247-2258. Heikkila, R.E.. Cabbat, F.S. and Manzino, L., Inhibitory effects of ascorbic acid on the binding of [~tt]dopamine antagonists to neostriatal membrane preparations: relationship to lipid peroxidation, J. Neurochem., 38 (1982) 1000~ 1006. Leslie, F.M., Dunlap, C.E. and Cox, B.M., Ascorbate decreases ligand binding to neurotransmitter receptors, J. Neurochem., 34 (1980) 219--221. O'Neill, R.D. and Fillenz, M., Detection of homovanillic acid in vivo using microcomputer-controlled voltammetry: simultaneous monitoring of rat motor activity and striatal dopamine release, Neuroscience, 14 (1985) 753- 763. O'Neill, R.D., Fillenz, M., Albery, W.J. and Goddard, N.J., The monitoring of ascorbate and monoamine transmitter metabolites in the striatum of the unanaesthetised rat using microprocessor-controlled voltammetry, Neuroscience, 9 (1983) 87-93. O'Neill, R.D., Fillenz, M., Sundstrom, L. and Rawlins, J.N.P., Voltammetrically monitored brain ascorbate as an index of excitatory amino acid release in the unrestrained rat, Neurosci. Lett., 52 (1984) 227-.233. O'Neill, R.D., Grunewald, R.A., Fillenz, M. and Albery, W.J., Linear sweep voltammetry with carbon-paste electrodes in the rat striatum, Neuroscience, 7 (1982) 1945 1954. O'Neill, R.D., Grunewald, R.A., Fillenz, M. and Albery, W.J., The effect of unilateral cortical lesions on the circadian changes in rat striatal ascorbate and homovanillic acid levels measured in vivo using voltammetry, Neurosci. Lett., 42 (1983) 105 110. Schenk, J.O., Miller, E., Gaddis, R. and Adams, R.N., Homeostatic control of ascorbate concentration in the CNS extracellular fluid, Brain Res., 253 (1982) 353-356. Walaas, I., Biochemical evidence for overlapping neocortical and allocortical glutamate projections to the nucleus accumbens and rostral caudate putamen in the rat brain, Neuroscience, 6 (1981) 399 405
331
Elsevier Scientific Publishers Ireland Ltd.
NSL 03556
CIRCADIAN CHANGES IN EXTRACELLULAR ASCORBATE IN RAT CORTEX, ACCUMBENS, STRIATUM AND H I P P O C A M P U S : CORRELATIONS WITH M O T O R ACTIVITY
ROBERT D. O'NEILL* and MARIANNE FILLENZ
University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT (U.K.) (Received June 6th, 1985; Revised version received and accepted July 10th, 1985)
Key words: ascorbate - excitatory amino acid - motor activity - circadian - voltammetry - rat
We have used linear sweep voltammetry with carbon-paste electrodes to monitor changes in the ascorbate signal simultaneously in rat frontal cortex, nucleus accumbens, striatum and hippocampus together with motor activity. The relative amplitude of the ascorbate signal recorded in the four regions corresponded to the relative density of excitatory amino acid (EAA) transmission determined by other methods; this result provides further evidence that the ascorbate signal may be used as an index of EAA release. Changes in motor activity were associated with changes in the ascorbate signal; linear regression analysis for motor activity versus the ascorbate signal revealed differences between the release of ascorbate in the four brain regions.
The ability of glutamate to stimulate almost all neurones, which suggested that it was a non-specific excitant, was invoked in the past as evidence against its role as a neurotransmitter. However, neurochemical studies in the 25 years since the demonstration by Curtis et al. [4] of its excitatory effects have established it as one of the main transmitters within the mammalian central nervous system (CNS). Ascorbic acid is also found throughout the CNS and, although its role is unclear, there is evidence that ascorbate may modulate binding to a variety of neurotransmitter receptors [12, 13]. Although it has been suggested that the extracellular concentration of ascorbate is under homeostatic control [18], a number of groups have observed both spontaneous and drug-induced changes in extracellular ascorbate levels [2, 5, 8, 18]. However, distinct from the problem of the role of ascorbate in the extracellular fluid is the question of the mechanism of ascorbate release. The finding that L-glutamate (L-GIu) and aspartate (Asp) cause release of ascorbate from synaptosomes through a carrier-mediated hetero-exchange mechanism (i.e. it is not receptor mediated) [10], led us to suggest that changes in extracellular brain ascorbate may be linked to excitatory amino acid (EAA) release. This is supported by the finding that L-Glu and Asp, when injected systemically or locally into the *Author for correspondence at present address: Department of Chemistry, University College, Belfield, Dublin 4, Eire. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
332
brain, increase the extracellular concentration of brain ascorbate [16]. Lesions of the glutamatergic cortico-striatal projection [18], but not kainate lesions of the striatum or 6-hydroxydopamine lesions of the substantia nigra [9], substantially reduce extracellular ascorbate in the striatum. Finally, electrical stimulation of the perforant path, which sends glutamatergic fibers to the dentate gyrus of the hippocampus, increases the ascorbate signal only when the sensor is near the dentate [16]. On the basis of these findings we have suggested that changes in extracellular ascorbate concentration may be used as an index of EAA release [6]. In this study we have recorded extracellular ascorbate in 4 brain regions where there is evidence for the presence of EAA-releasing terminals. The identification of neurons releasing EAAs depends on a number of criteria. Among these are the demonstration of a reduction in high-affi•;ty uptake and endogenous tissue levels of Glu or Asp after selective lesions of specific pathways, and studies of release of endogenous or exogenous Glu, or Asp, after selective activation of nerve fibers [7]. As a result of these methods cortico-fugal pathways, including cortico-striatal and cortico-accumbens projections, have been shown to be glutamatergic [20]. The accumbens and striatum, in addition to the neocortical input, also receive an allocortical projection via the fornix/fimbria, which is much larger in the accumbens than in the striatum [20]. In the hippocampus, the perforant path (which is an input from the entorhinal cortex to the dentate gyrus), the commissural interhippocampal fibers and the Shaffer collaterals, all fulfill some of the criteria for EAA-releasing pathways [3]. Studies on high-affinity Glu binding and uptake in vitro provide evidence that there is glutamatergic transmission in the frontal cortex [11]. The study of Glu release, even in vitro, has proved difficult because there are several pools of Glu within the nerve terminal; some of these have a purely metabolic role, whereas others are involved in neurotransmission, being released by a Ca2+-de pendent mechanism [7]. There are, at present, few measurements of the release of EAAs in vivo. Abdul-Ghani et al. [1], using a cortical-cup technique, have reported spontaneous and stimulus-evoked release of Glu from awake, behaviourally normal rats. The authors emphasize that changes in the Glu content of the perfusate represent the balance between release and the subsequent uptake, which means that small changes in release may not be detected. The aim of this study was to provide further evidence that ascorbate can be used as an index of EAA release and to investigate the relationship between this release and motor activity. We have used microcomputer-based linear sweep voltammetry with carbon-paste electrodes to monitor simultaneously circadian changes in rat motor activity and in the ascorbate signal in frontal cortex, accumbens, striatum and hippocampus. Ten male Sprague-Dawley rats were stereotaxically implanted, under chloral hydrate anesthesia (7 ml/kg 5~o solution i.p.), with 4 carbon-paste electrodes (300 ~m o.d.) as described previously [17]. The coordinates, with the head level between bregma and lambda, were: frontal cortex, AP 3.0 (from bregma), L 1.5 (from bregma), and DV 1.5 (from skull); accumbens, 1.5, 1.5 and 6.5; striatum, - 0 . 5 , 3.0 and 4.8; and hippocampus, - 3 . 6 , 1.8 and 4.4. The rats were given at least two days to recover before being placed in the recording cages (25 x 25 x 25 cm) and connected
333
to the microcomputer-based equipment [15]. Linear sweep voltammograms were recorded at 12-min intervals at a scan rate of 5 mV/s between 0 and 650 mV with respect to a Ag reference electrode. Peak 1 obtained using this technique is due to the oxidation of ascorbic acid [15, 17]; changes in the height of this peak reflect changes in the extracellular concentration of ascorbate. Up to 8 electrodes were scanned simultaneously over recording periods of 24 h. Mean__+ S.E.M. are quoted; n is the number of determinations. The significance of differences in between the 4 brain regions was calculated using paired t-tests on data from each animal. The average day-time (08.00 to 20.00 h) value of the ascorbate peak height was: frontal c o r t e x = 5 . 8 + 0 . 9 nA; accumbens =2.5___0.4 nA; striatum =4.1 +0.6 nA; hipp o c a m p u s = 1.7+0.2 nA; n = 2 0 . The order of the relative sizes was: frontal cortex > striatum (P < 0.05); striatum > accumbens (P < 0.05); and accumbens > hippocampus (P < 0.02). High-affinity glutamate binding, whether to receptors or to the uptake sites, has been used as an index of innervation density [11]. The density of such binding sites show the same rank order as the size of the ascorbate peak in these areas, providing further evidence that the ascorbate signal may be used as an index of EAA release. Circadian changes in signals were calculated by comparing the average value for the light-on period (08.00 to 20.00 h, 60 points) with that for the light-off period (20.00 to 08.00 h, 60 points). The nocturnal increase, expressed as a percentage of the day-time value was: frontal c o r t e x = 6 3 + 7 ~ ; a c c u m b e n s = 4 1 + 5 ~ ; striat u m = 4 9 + 6 ~ o ; hippocampus= 14+3~o; n = 2 0 , P<0.001 for all increases. Here the sequence was: frontal cortex>striatum (P<0.01); striatum=accumbens; and accumbens > hippocampus (P < 0.001). Total motor activity of each rat was monitored between electrochemical recordings (ca. 9 min) using a Doppler-shift microwave device [14]. The average level of motor activity in a given 24-h period was the same for implanted and unimplanted animals [14]. To investigate the relationship between motor activity and EAA release, we calculated correlation coefficients for spontaneous changes in the ascorbate signal in each region with changes in motor activity. An example which represents continuous recording over a 10-h period is shown in Fig. 1. The analysis consisted of calculations of correlation coefficients on the weighted moving average of the original data [14] for each day's recording (120 points). In order to find the time when maximum correlation occurred, cross-correlations were calculated by comparing, for example, m(t + 1) with hvcx(t), where hvcx(t) is the value of ascorbate signal at time t in frontal cortex, and m(t+ 1) is the value of the motor activity counter one scan (12 min) later; cross-correlations were calculated in the range t - 5 to t + 10. Cross-correlation analysis of the data in Fig. 1 shows that maximum correlation occurs when the motoractivity data is shifted by 12 rain (1 scan), and falls off steeply with shifts in either direction. The nocturnal rise in the ascorbate signal in the 4 brain regions over a 24-h period shows a progressive decrease in the size, and an increase in the delay of onset, from frontal cortex to striatum to accumbens to hippocampus. A quantitative assessment of these differences was made by calculating regression and correlation coefficients
334
60
4.5 -500
nA
•t
¢-
._m oo
3.0
0 0 co
motor counts
-250
].5
> 0
2
4
6
8
]o
time/h
OBJ
J
0,6
04
g o
.~
02-
0
-02-
~@
-24 shift/min
Fig. 1. Upper section: time-course, at 12-min intervals, over a lO-h period for changes in the ascorbate signal (O), recorded with a single carbon-paste electrode in the striatum, and total motor activity (O). Start time was 12.00 h. Lower section: cross-correlation analysis of the ascorbate and motor-activity data shown above. Maximum correlation (0.86) was obtained when the movement data were shifted by + 1 scan (12 min) with respect to the ascorbate data.
335 TABLE I REGRESSION ANALYSIS FOR MOTOR ACTIVITY VERSUS ASCORBATE SIGNAL Regression analysis of the circadian changes in motor activity versus the ascorbate signal recorded in frontal cortex, nucleus accumbens, striatum and hippocampus giving the maximum correlation coefficient (r) from the cross-correlation analysis (see Fig. 1), time shift (s) needed for maximum correlation and the slope of the regression line (m) which corresponded to maximum correlation. There was no significant difference between r values for frontal cortex, nucleus accumbens and striatum; that for hippocampus was significantly smaller (P < 0.02). The s value was smallest in frontal cortex (P< 0.01 compared with striatum) and greatest in hippocampus (P<0.01 compared with nucleus accumbens). The rank order of the m values was frontal cortex > striatum (P < 0.01) = nucleus accumbens > hippocampus (P < 0.001). Brain region
Frontal cortex (n = 18) Nucleus accumbens (n=32) Striatum (n = 30) Hippocampus ( n - 18)
Correlation coefficient
Timeshift (min)
Regression slope (pA/move)
0.82+0.03
32+3
12+2
0.78+0.03
66+6
7+ 1
0.82+0.02
61 +5
7_+ 1
0.46+0.10
100_+7
2.5_+0.8
between circadian changes in m o t o r activity and the ascorbate signals. Table I shows that the mean correlation coefficient for m o t o r activity versus ascorbate is the same for frontal cortex, accumbens and striatum, and is significantly smaller for the hippocampus. The cross-correlation analysis shows that the time-shift needed for maxim u m correlation is lowest in frontal cortex and greatest in hippocampus. The regression slopes calculated at the time-shift for m a x i m u m correlation had a rank order o f frontal cortex > striatum = accumbens > hippocampus. In view o f the rapid rise (latency < l min) and fall in the ascorbate signal following brief electrical stimulation (2.5 s) [16], it is perhaps surprising that m a x i m u m correlation is obtained when the circadian motor-activity data are shifted by between 30 and 100 min with respect to ascorbate signal (Table I). The time-course and crosscorrelation analysis illustrated in Fig. 1 shows that when there are relatively short bursts ( < l h) o f activity interspersed with resting periods, the time shift for maxim u m correlation is one scan (12 min) which is the m i n i m u m delay that can be resolved. The longer shifts observed for the normal circadian pattern o f sustained nocturnal activity m a y be due to the saturation o f a removal process for ascorbate leading to its progressive accumulation in the extracellular fluid which obscures the intrinsically rapid response o f the signal. The relative size o f the ascorbate signal recorded in frontal cortex, nucleus accumbens, striatum and h i p p o c a m p u s provides further support for the hypothesis that changes in the extracellular concentration o f brain ascorbate serve as an index o f E A A release. Furthermore, regression analysis for m o t o r activity versus the ascorbate signal reveals differences between ascorbate release in the 4 brain regions.
T h i s w o r k w a s f u n d e d in p a r t b y t h e M e d i c a l R e s e a r c h C o u n c i l . R . D . O . is a Beit M e m o r i a l Research Fellow. We are grateful for a grant t o w a r d s the e q u i p m e n t f r o m t h e E.P. A b r a h a m s C e p h a l o s p o r i n F u n d . W e t h a n k L e s l e y Sellars f o r a r t w o r k . I Abdul-Ghani, A.-S., Coutinho-Netto, J. and Bradford, H.F., In vivo superfusion methods and the re-
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