Brain Research, 111 (1976)95-108
95
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
CATECHOLAMINES RELEASED FROM CEREBRAL CORTEX IN THE CAT; DECREASE D U R I N G SENSORY STIMULATION
TOMAS A. READER*, JACQUES DE CHAMPLAIN and HERBERT JASPER Groupe de Recherche en Sciences Neurologiques du Conseil de Recherche M~dicale du Canada, D~partement de physiologie, Universitd de Montreal, C.P. 6208, Succursale A, Montreal, Quebec H3C 3T8 (Canada)
(Accepted December 1st, 1975)
SUMMARY In an attempt to determine the functional role of catecholamine (CA) nerve terminals in cerebral cortex the release of endogenous norepinephrine (NE) and dopamine (DA) into superfusates from visual and somatosensory cortex of the cat have been measured by a sensitive radiometric enzymatic assay based on the methylation of CA by catechol-O-methyltransferase (COMT) in the presence of a [all]methyl donor and followed by resolution of aH derivatives through a series of organic extractions. In the flaxedilized animal maintained under local anaesthesia with artificial respiration the concentration of CA measured in 30-min superfusates was fairly constant in a given experiment under basal conditions without sensory stimulation, but varied widely from one experiment to another. Variations in NE were often independent of those for DA. For visual cortex the average basal release of NE in experiments was 20.09 + 3.64 pg/min/sq.cm while the average for DA was 34.01 ± 7.62 pg/min/sq.cm. In all experiments intermittent visual stimulation (15/sec) produced a significant reduction in release rate averaging about 42 % for NE and 64 % for DA in visual cortex. The reduction was relatively non-specific since visual or somatic sensory stimulation produced a decrease in release from both visual and somatic sensory cortical areas. Since it has been shown that there is a relatively non-specific increase in acetylcholine (ACh) release from sensory cortex during stimulation, it is proposed that ACh may regulate CA release at presynaptic CA terminals in the cortex as it does in the periphery. A marked increase in CA release observed on perfusing with nicotine or atropine is consistent with this hypothesis.
* Post-doctoral Fellow, Medical Research Council of Canada.
96 INTRODUCTION
The presence of significant numbers of axonal terminals containing the biogenic amines norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) in cerebral cortex has now been clearly demonstrated by histofluorescent light microscopy as well as by electron microscopic autoradiography3,13,16,30,3L The fact that such terminals are relatively few in number, and rarely associated with a specialized postsynaptic membrane as in the classical electron microscopic picture of a synapse, raises interesting questions regarding their mode of action and physiological significance. The importance of biogenic amines in the regulation of states of consciousness, in the sleep and waking cycles, in affective disorders and in mental and motor behaviour TM ZT,2S,40,41 gives added impetus to the search for their functional mechanisms at the level of the cerebral cortex, as well as in subcortical brain structures. By the use of a radioactively labelled precursor (such as tyrosine) to preload the tissue with labelled DA, the liberation of DA from the caudate nucleus has been measured in the cat 5, the rat 4 and in the monkey6. The use of a recently developed highly specific and sensitive method for the measurement of catechotamines (CA) in picogram quantities lo,u has made it possible for us to measure the small quantities of the endogenous transmitter released from cerebral cortex under physiological conditions without preloading with a radioactive precursor. The technique of cortical superfusion has been used by numerous investigators to demonstrate important changes in the release of acetylcholine (ACh) and amino acids from cerebral cortex during different states of sleep and waking and during stimulation of specific and non-specific cortical afferent pathwayss,9,14,24-26,3a,aS-37. Of relevance to the present studies, the rate of ACh release has shown to be increased during specific sensory stimulation35-37 and during states of arousal 8,z6,37. In the present study using the technique of superfusion the effect of specific sensory stimulation (visual and somatic) upon the release of endogenous NE and DA from the surface of sensory cortex has been studied in the cat under local anaesthesia and immobilization with Flaxedil. The possible role of ACh in regulating the release of catecholamines has been studied by means of drugs which may mimic or block specific actions of ACh. The results of these studies have suggested an interaction between ACh and CA release mechanisms, probably at the presynaptic level.
MATERIALS AND METHODS
(a) Preparation of the animals The experiments were performed on 25 adult cats (weighing 2.5-3.0 kg) of either sex that were anaesthetized with sodium methohexital (Brietal), with an initial dose of 10 mg/kg (i.v.). In all experiments all pressure points and incisions were infiltrated with a solution of Efocaine. A polyethylene cannula was inserted in a rear limb vein to facilitate the periodic administration of small amounts of gallamine triethiodide (Flaxedil) while providing constant artificial respiration through a tracheal
97 cannula. The end-tidal COg percentage was maintained at 3.2-3.8 % and the arterial blood pressure was monitored by means of a cannula inserted in the femoral artery. The animals tended to fall asleep when not stimulated, as judged by the appearance of the pupils and by the continuous recording of the EEG. After trephining the skull over the striate cortex or over both the striate and the postcruciate cortical areas, the dura was carefully removed and nylon chambers (cylinders or cups) 12 mm in diameter were placed on the cortical surface as previously described s,9,z6,as,aS. Each chamber covered a surface of approximately 1.10 sq.cm and the centres of the chambers were determined by the following stereotaxic coordinates2a: for the striate cortex at A +2.0 mm and L 6.0 mm and for the postcruciate cortex at A +26.0 mm and L 8.0 mm. The bottom of the chambers were carefully fixed to the skull with dental cement.
(b) Perfusion techniques As soon as the chambers were in place the cortical surfaces were flushed with Elliott's Solution B (see ref. 14) and the perfusion was started. Elliott's Solution B contained NaC1, 112.5 mM; NaCOa, 21.7 mM; KC1, 4.0 mM; Na2HPO4 • 7H~O, 1.0 mM; CaC1, 1.44 mM; MgCI2, 0.39 mM; dextrose, 7.39 mM; ascorbic acid, 0.1 mM and Na~EDTA, 0.004 mM, unless otherwise specified. The perfusing solutions were stabilized by bubbling them with 5 ~ CO2, kept on crushed ice and before using them they were gently warmed to 37 °C. The cortical tissues were exposed to the perfusing solutions for at least 30 min prior to the collection of the first sample and the solutions used to bath the cortical tissues over this 30-min period were discarded. The perfusion was continuous by means of a Harvard pump at a flow rate of 6 ml/h. Each sample was collected over a 30-min period in tubes placed on crushed ice. Between each period there was a 10-min interval to allow a steady state in the release and diffusion of the transmitters into the chambers. The superfusates once collected were acidified with 0.1 vol. (v/v) of a solution of 2 N perchloric acid containing 1 ~ EGTA and 70 mM of MgClz (see refs. 11, 47) and maintained frozen until the assay was performed.
(c) The biochemical assay of CA The CA content of the samples was determined by means of a sensitive and specific radiometric assay based on the modification of the technique published by Coyle and Henry10 as reported by De Champlain et al. 11. One of the improvements of great relevance was the use of perchloric acid containing EGTA and Mg z+ to acidify the samples as soon as they were collected thus avoiding the inhibitory effect of free Ca 2+ on the activity of the enzyme catechol-O-methyltransferase (COMT) (see ref. 11) The use of duplicates and internal standards for each sample in order to check the efficiency of the COMT and of the separation steps allowed us to rule out errors in the assay technique10,11,47. This method is based on the methylation of CA by COMT (E.C. 2.1.1.11) in the presence of [aH]methyl-S-adenosylmethionine as a methyl donor. The all-derivatives are thereafter isolated by selective organic extraction. COMT was prepared and purified from rat liver by the method of Axelrod and Tomchick 1. In order to avoid contamination, blanks for every perfusing solution and for
98 every pharmacological agent were assayed and the counts of this blank were subtracted from the total counts of the sample. The internal standards used for the superfusates were made of 0.5 ng of L-NE (free base) and of 0.5 ng of L-DA (free base). At the end of the perfusions, samples of brain tissue were quickly removed, weighed and homogenized in 100 vol. (w/v) of 0.1 N cold perchloric acid in a glass homogenizer with a glass pestle. The homogenates were centrifuged at low speed and the supernatant fluid was decanted for assay. The assay followed the same procedure as for the superfusates except that the internal standards used were made of 2.0 ng of L-NE (free base) and of 2.0 ng of L-DA (free base).
(d) Electrophysiological techniques Intermittent brief light flashes of the entire visual field, using a Grass PS-20 Photostimulator were used at a frequency of 15.0/sec for activation of the visual system. The pupils were dilated with 2 ~o atropine sulphate and the nictitating membrane contracted with 1 ~ ephedrine hydrochloride. Care was taken to keep the corneas moist with saline solution. In order to avoid possible complications due to the retina electrical stimulation of the optic nerve with steel bipolar electrodes (0.5 mm separation and 0.5 mm exposed tips) placed stereotaxically was also used in some of the experiments. The electrodes were placed in the optic nerve at A + 13.5 mm; L 2.0 mm and H --6.0 mm (ref. 23). In some experiments stimulation of the forelimb was carried out with a bipolar steel needle electrode in the paw in order to compare the effects of somatic and visual sensory systems, and to determine the specificity of changes. The electrical stimuli were rectangular pulses of 0.5 msec duration for the optic nerve and 1.0 msec duration for the forelimb obtained from a Grass $44 stimulator at a frequency of 0.5/sec or 15.0/sec and at an intensity of 3 times the threshold for the evoked potential. Evoked potentials after adequate amplification were either displayed on the screen of a dual beam Tektronix oscilloscope 565 and averaged on-line 32-64 times by a signal averager-memory control unit (Ortec models 4623 and 4620) or stored in Ampex FM magnetic tape for further analysis. Bipolar surface silver electrodes were used to record the activity of the cortex. In all experiments the EEGs were obtained from the exposed cortical areas and after suitable amplification displayed on paper with a 6-channel Brush recorder. RESULTS
Basal release of CA under 'resting' conditions Under the conditions of these experiments conducted in a relatively quiet room with adequate local anaesthesia the immobilized animals usually became drowsy or lightly asleep as judged by pupiltary constriction with retraction of the nictitating membrane and by persistent spindles and a few slow waves in the EEG. Basal release of CA was measured only in experiments in which satisfactory superfusates were obtained during stable EEG records, constant arterial blood pressure and with artificial respiration adjusted to maintain constant end-tidal CO2 concentration at about 3.8 ~ . Results are expressed in pg/min/sq.cm although each collection period was
99 TABLE I Basal release of cateeholamines from the visual cortex of the cat
Values expressed in pg/min/sq.cm, n = 18. Experiment
Norepinephrine
Dopamine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
32.60 22.75 45.96 61.96 39.60 8.76 20.41 19.92 9.80 11.10 7.56 15.14 9.45 9.86 19.22 13.16 7.80 6.61
35.99 98.84 72.21 107.85 37.60 6.16 8.08 70.90 20.30 10.05 40.98 8.74 31.26 20.02 6.48 5.71 26.30 7.03
Mean ± S.E.
20.094- 3.64
34.01 4- 7.62
30 min in duration in order to provide a sufficient volume for assays to be performed in duplicate with one or two internal standards for each sample 1°,11,47. With these precautions it was possible to obtain fairly constant values for both N E and D A over periods of several hours in some experiments in which sensory stimulation was not carried out (see Fig. 4 for an example). However, resting values varied widely from one experiment to another as shown in Table I. For visual cortex the range of values for N E was between 6.61 and 61.96 pg/min/sq.cm with an average of 20.09 -4- 3.64 pg/min/sq.cm (n ---- 18). F o r D A the range was 5.71-107.85 pg/min/ sq.cm with an average of 34.01 q- 7.62 pg/min/sq.cm (n = 18). Variations in N E and D A were relatively independent in most instances, the basal levels for D A being higher than for N E in 11 out of 18 experiments and higher for N E in 7 experiments. This would suggest that these variations in basal release rate can not be attributed only to the technical variation in superfusion. That diffusion barriers may play some role was shown, however, in two experiments in which the basal values were increased by about 30 ~o following multiple punctures of the piarachnoid membranes with a glass micropipette. Average basal release of CA from somatosensory cortex was about twice the values obtained for visual cortex in 6 experiments in which superfusion of both cortical areas was carried out simultaneously. In these experiments the average release of N E f r o m visual cortex was 14.53 -4- 5.78 pg/min/sq.cm as compared to 25.80 -q- 8.91 pg/min/sq.cm f r o m somatosensory cortex. F o r D A the average basal release of
100
40-
~CONTROL ~ STIM.
30
•~
20-
O.
10"
. Fig. 1. The effect of stimulating the visual system on the release of norepinephrine (NE) and dopamine (DA) from the visual cortex. All experiments were performed using a stimulating frequency of 15.0/ see; in 8 animals photic stimulation of the visual field was used and in 3 clerical stimulation of the optic nerve at 3 times the evoked potential threshold (see Material and Methods). Each bar represents the mean 4- S.E. of 11 animals.
visual cortex was 21.50 ~ 5.58 as c o m p a r e d to 55.52 i 12.28 pg/min/sq,cm for somatosensory cortex. We were unable to establish any consistent correlation between the E E G pattern and the differences in release o f N E or D A in the resting state.
Endogenous cortical tissue concentration of CA The endogenous C A were measured in the contralateral non-perfused cortical areas. The values showed an average o f 0.202 -4- 0.027 # g / g (n = 13) for N E and o f 0.199 ~: 0.024 #g/g (n = 13) for D A . I n 6 animals basal release values o f C A were c o m p a r e d with the endogenous content in the tissues but no correlation was f o u n d either for D A or for NE. These values are similar to those obtained by Gessa et al. 17, using a fluorometric m e t h o d o f measurement.
Effects of sensory stimulation During stimulation o f the visual system at a frequency o f 15.0/sec (Fig. 1) a
101
6o-
1CONTROL ~O.N. STIM.
._c
lid_ NOREPINEPHRINE
DOPAMINE
Fig. 2. Recovery following reduction in release of CA from visual cortex by optic nerve stimulation (O.N. STIM.) at 15.0/secat intensity 3 times threshold for visual evoked potentials. Each bar represents the mean -4- S.E. of 7 animals for NE and 6 animals for DA.
decrease in the release of both N E and DA was observed in 11 successful experiments. In these animals the average basal release of NE was of 24.13 ± 5.56 pg/min/sq.cm (n = I 1) and during stimulation the release dropped to 11.75 -4- 2.43 pg/min/sq.cm (n = 11). The release of DA was also decreased during the periods of stimulation from 37.86 4- 11.49 pg/min/sq.cm (n = 11) to 12.61 4- 4.17 pg/min/sq.cm (n = 11). This decrease in the release of CA from the visual cortex was obtained either with photic stimulation or with electrical stimulation of the optic nerve. This effect of visual stimulation on CA release was reversible. Fig. 2 shows the recovery periods in 6 animals after stimulating the optic nerve (electrical stimulation). The control (basal) levels of CA returned to their previous levels and in the case of D A there was a slight increase as compared to the first control period. In some animals the reduction in CA release persisted for 30 min to 1 h after the period of stimulation, while in others there seemed to be a rebound increase above the basal level following stimulation. In order to study the specificity of the decrease in CA release induced by visual stimulation in 6 animals, two chambers were placed: one over the visual cortex and the other over the postcruciate cortex (somatosensory) (see Materials and Methods). Either somatic or visual stimulation decreased the release of DA and N E in both the somatosensory and visual cortical areas (Fig. 3). Somatic stimulation seemed to be somewhat more effective than visual stimulation, in general, for either visual or somatosensory areas and for both N E and DA. From these results it would appear that the effect of sensory stimulation is non-specific and not clearly related to local projecting sensory pathways. These results may be compared with the non-specific increase in the release of ACh with sensory stimulation under similar physiological conditions. This raises the question of some form of interaction between ACh and catecholamine release mechanisms which could in some way explain the reduction in CA release under conditions which cause an increase in release of ACh ~,9,aa,aS-aT.
102 30-
VISUAL CORTEX
SOMATOSENSORY CORTEX
VISUAL CORTEX 80-
m
SOMATOSENSORY CORTEX
CONTROL
]VISUAL STIMULATION [-~'1 SOMATICSTIMULATION 20 -
60-
lo 2O
0
0
NOREPINEPHRINE
DOPAMINE
Fig. 3. The effects of sensory stimulation on the release of CA from the visual and somatosensory cortical areas. Observe that either visual stimulation or somatic stimulation reduced the release of CA from both the visual and the somatosensory cortical areas. These experiments were performed with two chambers simultaneously perfused in the same animal. Both optic nerve and paw were stimulated at 15.0/sec with intensities 3 times threshold for cortical evoked potentials. Each bar represents the mean S: S.E. of 6 animals.
Effects of nicotine and atropine Perfusion with nicotine in Elliott's Solution at a concentration of 5 × 10-3 M caused a large increase in the release of both NE and D A as shown in Fig. 4. In this experiment evoked potentials in response to photic stimulation were sampled at 2-sec intervals throughout the entire experiment. Nicotine at the concentration of 10-8 M caused only a 1 0 ~ increase in CA release but at a concentration of 5 × 10-a M, CA release was increased 4-5 times the previous basal value. The increase was maximum during the first 30-min collection period and tended to fall to a lower value during the second 30-min period while still remaining well above the control levels before nicotine, which levels remained relatively constant for about 5 h previous to perfusion with the higher concentration of nicotine. In another experiment (Fig. 5) a moderate increase in CA release was obtained by perfusion with 10-3M nicotine solution and more importantly stimulation of the optic nerve after nicotine caused an increase rather than a decrease in DA. Although there was a slight reduction in NE during optic nerve stimulation after nicotine, these values remained well above the control value before nicotine. Atropine perfused in a concentration of 10-4M also caused an increase in basal release of both N E and D A as shown in Fig. 5, Stimulation of the optic nerve following atropine was found to cause an increase rather than a decrease in DA, while NE returned to its basal control level in this experiment during optic nerve stimulation. The stimulating effect of nicotine upon CA release is similar to that which has
103
~CONTROL 260
I
IO0t~NICOTINE' 220
180t
30 20
Ii]1
! 6O
,:.~
J 20
120 240 360 rain. NOREPINEPHRINE
120 240 360 rain. DOPAMINE
Fig. 4. The effects of nicotine on the release of CA from the visual cortex. In this experiment all collections were performed while stimulating photically the visual field at a frequency of 0.5/sec. Nicotine (free base) was employed at a concentration of 10-aM for the first period and in the two last collection periods at a concentration of 5 x 10-3M. Observe the relative stability of the basal release values (control periods) throughout the length of the experiment. The higher dose of nicotine (5 x 10-aM) induced a very important release of both CA, but in a second period of collection this release declined although it still remained well above the control values.
been observed in the periphery and in slices of brain tissue in vitro 1s,g2,44,46,4s. This suggests the presence of nicotinic ACh receptors capable of increasing CA release and of modifying the effects of sensory stimulation. The effect of atropine may be due to blocking muscarinic inhibitory ACh receptors thus also modifying the effects of sensory stimulation. The site of action of both of these effects are presumed to be upon presynaptic CA terminals. DISCUSSION
In the present studies we have shown that the visual cortex of the cat contains
104 ATROPINE 20
NICOTINE
m CONTROL ]
20- NE
ON.STtM
ATROPINE
- - O.NTST,M~ ~0 []
NICOTINE ~
[]
NICOTINE O.N~TIM. 0 60- DA
DA
I
~ 40E
~,o-
20-
0 "
~
~
~
0
N
Fig. 5. The effects of optic nerve stimulation, nicotine and atropine on the release of CA from the visual cortex. The concentrations of the agents employed were 10-4M for atropine sulphate and lO-ZM for nicotine. Observe that optic nerve stimulation (O.N.STIM.) reduced the release of both DA and NE. The release of CA was increased by both atropine and nicotine. During O.Ni stimulation while continuing nicotine perfusion the release of NE was reduced but remained above the control level. O.N. stimulation produced an increase in DA release in both atropine and nicotine treated cortex. about equal concentrations of endogenous N E and D A (0.2 #g/g). Preliminary studies using the histofluorescent method of Falck-Hillarp 18 have shown that the visual cortex of the cat is supplied with characteristic CA axonal terminals which disappear following perfusion of the cortical surface with 6-hydroxydopamine (unpublished results). Such terminals are relatively few in number and this is consistent with the relative sparsity of such terminals found in the cortex of the rat by the quantitative electron microscopic autoradiographic studies of Descarries and Lapierre 13 and Lapierre et al. 3°. It has been pointed out recently by Descarries et al. 12, that CA and 5-HT terminals in the cerebral cortex are rarely found to be associated with postsynaptic membrane specializations as seen in the electron microscope. This pattern of innervation is compatible with the suggestion that these biogenic amine terminals may not have specific punctate or local synaptic action as has been postulated for classical synapses. These findings and interpretations raise important questions regarding the functional significance of CA and 5-HT terminals in the cerebral cortex, and the mechanisms governing their release. By means of a recently developed highly sensitive enzymatic assay method 1°, 11,47 it has been possible to measure N E and D A concentrations in samples of extracellular fluid obtained by superfusion of a limited area of cortical surface, a method previously found useful in studies on the liberation of other putative neurotransmitters
105
in the intact animal under a variety of physiological conditions (including specific sensory stimulation, sleep and waking, and stimulation of the brain stem) s,9,za-2o,aa, 85,86. The quantities of NE and DA measured in the superfusates in our experiments are smaller by 2 and 3 orders of magnitude than those reported for ACh s,26,3a,35 and for glutamic acid2~ respectively and using the same superfusion techniques. However, these low values of CA are not surprising since DA and NE fibers seem to be rather few in number and on the other hand re-uptake and degradative mechanisms may account for a great reduction in the overflow. It is worth pointing out here that no pharmacological agents known to inhibit the activities of monoamine oxydase or catechol-O-methyltransferase or to block the re-uptake mechanisms were used in the experiments here reported in order to keep the animals as near to the physiological state as possible. Therefore the actual 'real' or 'true' amount of CA released by the nerve terminals and acting in the central nervous system at a specific site may be several times greater than what is measured with the superfusion method. This fact does not invalidate the method but shows that it is only an indirect measurement of the total release of free CA which have escaped metabolic degradation or re-uptake. Such a sampling may be of particular importance if biogenic amines act as local neurohormones affecting a large population of neurones by diffusion into extracellular spaces distant from their site of liberation. With regard to the specificity of the changes in CA release induced by sensory stimulation it was apparent from our experiments (in single or double chamber experiments) that suitable somatic stimulation was capable of altering the release of CA in visual cortex and stimulation of the visual system was able to reduce CA liberation in the somatosensory cortex and vice versa. This suggests a non-specific effect of sensory stimulation and opposite in direction to that which has been observed for the release of ACh s,9,26,35-3:. Recently the hypothesis has been proposed that ACh released from cholinergic fibres might cause an inhibition of NE fibres and a decrease in the release of NE by an action upon presynaptic receptors. Such an effect of ACh on CA release has been documented in the periphery. A muscarinic inhibitory effect of ACh on the release of NE for the cat heart 19, the rabbit heart al, the perfused rat mesenteric arterya4 and the central ear artery of the rabbit 89 has been reported. That such an inhibitory effect may be also present in the cerebral cortex is suggested by the increase in the release of both DA and NE when muscarinic ACh receptors are blocked with atropine (Fig. 5). Furthermore, visual stimulation in atropinized visual cortex caused an increase rather than a decrease in the release of DA, and reduced the usual effect of stimulation on NE release. This reversal of the effect of stimulation by atropine would suggest the possibility that there may be also facilitatory actions of ACh on CA release unmasked when the inhibitory mechanism is blocked by atropine. This facilitatory action would be magnified by the fact that atropine has been shown to increase extracellular ACh concentrations in the cerebral cortex s,35,42. Nicotine has been shown to cause a direct release of CA from peripheral adrenergic neurones81, 45, to lower the CA content of the rat diencephalon and the
106 mouse brain 4°, to show amphetamine-like effects releasing NE from central neurones 7,22,48 and to release [14C]NE and [14C]DA from rat brain striatum and hypothalamus slices 18. In a recent article Westfal144 has reported that nicotine produces a significant increase in the release of [aH]NE from incubated slices of rat cortex, hypothalamus and cerebellum. He presents further pharmacological evidence suggesting that ACh may control the release of CA from central neurones by means of nicotinic presynaptic receptors on the terminals of CA fibres, similar to the mechanism proposed for N E in peripheral adrenergic neuroneslL The marked increase in DA and NE release observed in the present studies when superfusing with nicotine (Figs. 4 and 5) is consistent with its effect on peripheral CA terminals and brain slices. These results suggest that the release of CA in cerebral cortex may be increased by similar nicotinic presynaptic facilitatory mechanisms which become apparent when muscarinic receptors are blocked with atropine. Further speculation regarding ACh-CA interaction is not warranted at the present time since non-specific effects of atropine and nicotine on cerebral cortex cannot be entirely ruled out 29,a8. We do not mean to imply that the observed effects of sensory stimulation upon CA release in cerebral cortex are all mediated by interactions with ACh. There are very probably direct effects of sensory stimulation upon CA neuronal systems of the brain stem which are known to project diffusely to the cerebral cortex. The relation of cortical CA release and metabolism to states of sleep, waking, and arousal has yet to be determined since it was not possible to make such observations in the above experiments. Such studies are presently being undertaken, as well as studies of the action of CA upon cortical synaptic function at the unitary level in order to gain further insight into the functional significance of CA terminals in the cerebral cortex. ACKNOWLEDGEMENTS This work was supported by the Medical Research Council Group in Neurological Sciences at the Universit6 de Montr6al (Drs. H. H. Jasper and J. de Champlain) and by a Post-doctoral Fellowship from the Medical Research Council (Canada) to Dr. T. A. Reader. The authors also acknowledge the skilful technical assistance of Miss Lise Farley.
REFERENCES 1 Axelrod, J. and Tomchick, R., Enzymatic O-methylation of epinephrine and other catechols, Biol. Chem., 233 (1958) 702-705. 2 Bartolini, A., Weisenthal, L. and Domino, E. F., Effect of photic stimulation on acetylcholine release from cerebral cortex, Neuropharmacology, 11 (1972) 113-122. 3 Berger, B., Tassin, J. P., Blanc, G., Moyne, M. A. and Thierry, A. M., Histochemical confirmation for dopaminergic innervation of the rat cerebral cortex after destruction of the noradrenergic ascending pathways, Brain Research, 81 (1974) 332-337. 4 Besson, M. J., Cheramy, A:, Feltz, P. and Glowinski, J., Release of newly synthesized dopamine
107 from dopamine-containingterminals in the striatum of the rat, Proc. nat. Acad. Sci. (Wash.), 62 (1969) 741-748. 5 Besson, M. J., Cheramy, A., Feltz, P. and Glowinski, J., Dopamine: spontaneous and drug indueext release from the caudate nucleus in the cat, Brain Research, 32 (1971) 407-424. 6 Bioulac, B., Gauchy, C., Cheramy, A., Besson, M. J., Glowinski, J. and Vincent, J.-D., Chronic estimation of DA release from the caudate nucleus of the Macaca mulatta, Fed. Proc., 32, No. 3 (1973) Abstract 3372. 7 Carr, L. A. and Moore, K. E., Effects of amphetamine on the contents of norepinephrine and its metabolites in the effluent of perfused cerebral ventricles of the cat, Biochem. Pharmacol., 19 (1970) 2361-2374. 8 Celesia, G. G. and Jasper, H. H., Acetylcholine released from cerebral cortex in relation to state of activation, Neurology (Minneap.), 16 (1966) 1053-1064. 9 Collier, B. and Mitchell, J. F., The central release of acetylcholine during stimulation of the visual pathway, J. Physiol. (Lond.), 184 (1966) 239-254. 10 Coyle, J. T. and Henry, D., Catecholamines in fetal and newbom rat brain, J. Neurochem., 21 (1973) 61-67. 11 De Champlain, J., Farley, L., Cousineau, D. and van Ameringen, M.-R., Circulating catecholamine levels in human and experimental hypertension, Circular. Res., 38 (1976) 109-114. 12 Descarries, L., Beaudet, A. and Lapierre, Y., Personal communication, 1975. 13 Descarries, L. and Lapierre, Y., Noradrenergic axon terminals in the cerebral cortex of rat. I. Radioautographic visualization after topical application of [aH]DL-norepinephrine, Brain Research, 51 (1973) 141-160. 14 Elliott, K. A. C. and Jasper, H. H., Physiological salt solutions for brain surgery. Studies on local pH and pial vessels reaction to buffered and unbuffered isotonic solutions, J. Neurosurg., 6 (1949) 140-152. 15 Enero, M. A., Langer, S. Z., Rothlin, R. P. and Stefano, F. J. E., Role of the a-adrenoceptor in regulating noradrenaline overflow by nerve stimulation, Brit. J. Pharmacol., 44 (1972) 672-688. 16 Falck, B., Hillarp, N. A., Thieme, G. and Torp, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 17 Gessa, G. L., Biggio, G., Vargiul, L., Napoleone, F. and Tagliamonte, A., Norepinephrine and dopamine concentrations in the cerebral cortex of man, monkeys and other mammals, Experientia (Basel), 30 (1974) 1295-1296. 18 Goodman, F. R., Effects of nicotine on distribution and release of 14C-norepinephrine and 14Cdopamine in rat brain striatum and hypothalamic slices, Neuropharmacology, 13 (1974) 1025-1032. 19 Haeusler, G., Thoenen, H., Haefely, W., and Huerlimann, N., Electrical events in cardiac adrenergic nerves and noradrenalinerelease from the heart induced by acetylcholine and KC1, NaunynSchmiedeberg's Arch. exp. Path. Pharmak., 261 (1968) 389-411. 20 H/~kfelt, T., Ljungdahl, A., Fuxe, A., and Johansson, O., Dopamine nerve terminals in the rat limbic cortex: aspects of the dopaminergic hypothesis of schizophrenia, Science, 184 (1973) 177-179. 21 Iversen, L. L., Dopamine receptors in the brain, Science, 188 (1975) 1084-1089. 22 Izquierdo, I. and Izquierdo, J. A., Effects of drugs on deep brain centers, Ann. Rev. Pharmacol. 11 (1971) 189-208. 23 Jasper, H. H. and Ajmone-Marsan, C., A Stereotaxic Atlas of the Diencephalon of the Cat, National Research Council of Canada, Ottawa, 1954. 24 Jasper, H. H., Khan, R. T. and Elliott, K. A. C., Amino acids released from the cerebral cortex in relation to the state of activation, Science, 147 (1965) 1448-1449. 25 Jasper, H. H. and Koyama, I., Rate of release of amino acids from the cerebral cortex in the cat as affected by brainstem and thalamic stimulation, Canad. J. Physiol. PharmacoL, 47 (1969) 889-905. 26 Jasper, H. H. and Tessier, J., Acetylcholine liberation from cerebral cortex during paradoxical (R.E.M.) sleep, Science, 172 (1971) 601-602. 27 Jouvet, M., Role of catecholamines in the control of the sleep waking cycle. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, pp. 751757. 28 Kety, S. A., Norepinephrine in the central nervous system and its correlation with behavior. In A. G. Karczmar and J. C. Eccles (Eds.), Brain and Human Behavior, Springer, New York, 1972, pp. 115-129. 29 Krnjevi6, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540.
108 30 Lapierre, Y., Beaudet, A., Demianczuk, N. and Descarries, L., Noradrenergic axon terminals in the cerebral cortex of rat. II. Quantitative data revealed by light and electron microscope radioautography of the frontal cortex, Brain Research, 63 (1973) 175-182. 31 Lidmar, R., Loffelholz, K. and Muscholl, E., A muscarinic mechanism inhibiting the release of noradrenaline from peripheral adrenergic nerve fibers by nicotinic drugs, Brit. J. PharmacoL, 32 (1968) 280--294. 32 Lindvall, O., BjOrklund, A., Moore, R. Y., and Stenevi, V., Mesencephalic dopamine neurons projecting to neocortex, Brain Research, 81 (1974) 325-331. 33 Macintosh, F. C., and Oborin, P. E., Release of acetylcholine from intact cerebral cortex. In Int. Physiol. Congr., XIXth, 1953, pp. 580-581, abstract. 34 Malik, K. U. and Ling, G. M., Modifications by acetylcholine on the response of rat mesenteric arteries to sympathetic stimulation, Circulat. Res., 25 (1969) 1-9. 35 Mitchell, J. F., The spontaneous and evoked release of acetylcholine from the cerebral cortex, J. PhysioL (Lond.), 165 (1963) 98-116. 36 Mullin, W. J. and Phillis, J. W., The effect of graded forelimb afferent volleys on acetylcholine release from cat sensorimotor cortex, J. PhysioL (Lond.), 244 (1975) 741-756. 37 Pepeu, G., The release of acetylcholine from the brain. An approach to the study of the central cholinergic mechanisms, Progr. NeurobioL, 2 (1973) 257-288. 38 Portig, P. J. and Vogt, M., Release into the cerebral ventricles of substances with possible transmitter function in the caudate nucleus, J. Physiol. (Lond.), 204 (1969) 687-715. 39 Rand, M. J. and Varma, B., The effects of cholinomimetic drugs on the response to sympathetic nerve stimulation and noradrenaline in the rabbit ear artery, Brit. J. PharmacoL, 38 (1970) 758-770. 40 Schildkraut, J. J. and Kety, S. S., Biogenic amines and emotion, Science, 156 (1967) 21-30. 41 Seeman, P. and Lee, T., Antipsychotic drugs: direct correlation between clinical potency and presynaptic action on dopamine neurons, Science, 188 (1975) 1217-1219. 42 Szerb, J. C., The effect of tertiary and quaternary atropine on cortical acetylcholine output and on the electroencephalogram in cats, Canad. J. Physiol. PharmacoL, 42 (1964) 303-314. 43 Thierry, A. M., Blanc, G., Sobel, A., Stinus, L. and Glowinski, J., Dopaminergic terminals in the rat cortex, Science, 182 (1973) 499-501. 44 Westfall, T. C., Effect of nicotine and other drugs on the release of ZH-norepinephrine and 3Hdopamine from rat brain slices, Neuropharmacology, 13 (1974) 693-700. 45 Westfall, T. C. and Brasted, M., The mechanism of nicotine on adrenergic neurons in the perfused guinea-pig heart, J. Pharmacol. exp. Ther., 182 (1972) 409418. 46 Westfall, T. C., Fleming, R. M., Fudger, M. F. and Clark, W. G., Effect of nicotine and related substances upon amine levels in the brain, Ann. N. Y. Acad. Sci., 142 (1967) 83-100. 47 Yamaguchi, Y., De Champlain, J. and Nadeau, R., Correlation between the response of the heart to sympathetic stimulation and the release of endogenous catecholamines into the coronary sinus of the dog, Circulat. Res., 36 (1975) 662-668. 48 Ziance, R. J. and Rutledge, C. O., A comparison of the effects of fenfluramine and amphetamine on the uptake, release and catabolism of norepinephrine in rat brain, J. PharmacoL exp. Ther., 180 (1972) 118-126.
95
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
CATECHOLAMINES RELEASED FROM CEREBRAL CORTEX IN THE CAT; DECREASE D U R I N G SENSORY STIMULATION
TOMAS A. READER*, JACQUES DE CHAMPLAIN and HERBERT JASPER Groupe de Recherche en Sciences Neurologiques du Conseil de Recherche M~dicale du Canada, D~partement de physiologie, Universitd de Montreal, C.P. 6208, Succursale A, Montreal, Quebec H3C 3T8 (Canada)
(Accepted December 1st, 1975)
SUMMARY In an attempt to determine the functional role of catecholamine (CA) nerve terminals in cerebral cortex the release of endogenous norepinephrine (NE) and dopamine (DA) into superfusates from visual and somatosensory cortex of the cat have been measured by a sensitive radiometric enzymatic assay based on the methylation of CA by catechol-O-methyltransferase (COMT) in the presence of a [all]methyl donor and followed by resolution of aH derivatives through a series of organic extractions. In the flaxedilized animal maintained under local anaesthesia with artificial respiration the concentration of CA measured in 30-min superfusates was fairly constant in a given experiment under basal conditions without sensory stimulation, but varied widely from one experiment to another. Variations in NE were often independent of those for DA. For visual cortex the average basal release of NE in experiments was 20.09 + 3.64 pg/min/sq.cm while the average for DA was 34.01 ± 7.62 pg/min/sq.cm. In all experiments intermittent visual stimulation (15/sec) produced a significant reduction in release rate averaging about 42 % for NE and 64 % for DA in visual cortex. The reduction was relatively non-specific since visual or somatic sensory stimulation produced a decrease in release from both visual and somatic sensory cortical areas. Since it has been shown that there is a relatively non-specific increase in acetylcholine (ACh) release from sensory cortex during stimulation, it is proposed that ACh may regulate CA release at presynaptic CA terminals in the cortex as it does in the periphery. A marked increase in CA release observed on perfusing with nicotine or atropine is consistent with this hypothesis.
* Post-doctoral Fellow, Medical Research Council of Canada.
96 INTRODUCTION
The presence of significant numbers of axonal terminals containing the biogenic amines norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) in cerebral cortex has now been clearly demonstrated by histofluorescent light microscopy as well as by electron microscopic autoradiography3,13,16,30,3L The fact that such terminals are relatively few in number, and rarely associated with a specialized postsynaptic membrane as in the classical electron microscopic picture of a synapse, raises interesting questions regarding their mode of action and physiological significance. The importance of biogenic amines in the regulation of states of consciousness, in the sleep and waking cycles, in affective disorders and in mental and motor behaviour TM ZT,2S,40,41 gives added impetus to the search for their functional mechanisms at the level of the cerebral cortex, as well as in subcortical brain structures. By the use of a radioactively labelled precursor (such as tyrosine) to preload the tissue with labelled DA, the liberation of DA from the caudate nucleus has been measured in the cat 5, the rat 4 and in the monkey6. The use of a recently developed highly specific and sensitive method for the measurement of catechotamines (CA) in picogram quantities lo,u has made it possible for us to measure the small quantities of the endogenous transmitter released from cerebral cortex under physiological conditions without preloading with a radioactive precursor. The technique of cortical superfusion has been used by numerous investigators to demonstrate important changes in the release of acetylcholine (ACh) and amino acids from cerebral cortex during different states of sleep and waking and during stimulation of specific and non-specific cortical afferent pathwayss,9,14,24-26,3a,aS-37. Of relevance to the present studies, the rate of ACh release has shown to be increased during specific sensory stimulation35-37 and during states of arousal 8,z6,37. In the present study using the technique of superfusion the effect of specific sensory stimulation (visual and somatic) upon the release of endogenous NE and DA from the surface of sensory cortex has been studied in the cat under local anaesthesia and immobilization with Flaxedil. The possible role of ACh in regulating the release of catecholamines has been studied by means of drugs which may mimic or block specific actions of ACh. The results of these studies have suggested an interaction between ACh and CA release mechanisms, probably at the presynaptic level.
MATERIALS AND METHODS
(a) Preparation of the animals The experiments were performed on 25 adult cats (weighing 2.5-3.0 kg) of either sex that were anaesthetized with sodium methohexital (Brietal), with an initial dose of 10 mg/kg (i.v.). In all experiments all pressure points and incisions were infiltrated with a solution of Efocaine. A polyethylene cannula was inserted in a rear limb vein to facilitate the periodic administration of small amounts of gallamine triethiodide (Flaxedil) while providing constant artificial respiration through a tracheal
97 cannula. The end-tidal COg percentage was maintained at 3.2-3.8 % and the arterial blood pressure was monitored by means of a cannula inserted in the femoral artery. The animals tended to fall asleep when not stimulated, as judged by the appearance of the pupils and by the continuous recording of the EEG. After trephining the skull over the striate cortex or over both the striate and the postcruciate cortical areas, the dura was carefully removed and nylon chambers (cylinders or cups) 12 mm in diameter were placed on the cortical surface as previously described s,9,z6,as,aS. Each chamber covered a surface of approximately 1.10 sq.cm and the centres of the chambers were determined by the following stereotaxic coordinates2a: for the striate cortex at A +2.0 mm and L 6.0 mm and for the postcruciate cortex at A +26.0 mm and L 8.0 mm. The bottom of the chambers were carefully fixed to the skull with dental cement.
(b) Perfusion techniques As soon as the chambers were in place the cortical surfaces were flushed with Elliott's Solution B (see ref. 14) and the perfusion was started. Elliott's Solution B contained NaC1, 112.5 mM; NaCOa, 21.7 mM; KC1, 4.0 mM; Na2HPO4 • 7H~O, 1.0 mM; CaC1, 1.44 mM; MgCI2, 0.39 mM; dextrose, 7.39 mM; ascorbic acid, 0.1 mM and Na~EDTA, 0.004 mM, unless otherwise specified. The perfusing solutions were stabilized by bubbling them with 5 ~ CO2, kept on crushed ice and before using them they were gently warmed to 37 °C. The cortical tissues were exposed to the perfusing solutions for at least 30 min prior to the collection of the first sample and the solutions used to bath the cortical tissues over this 30-min period were discarded. The perfusion was continuous by means of a Harvard pump at a flow rate of 6 ml/h. Each sample was collected over a 30-min period in tubes placed on crushed ice. Between each period there was a 10-min interval to allow a steady state in the release and diffusion of the transmitters into the chambers. The superfusates once collected were acidified with 0.1 vol. (v/v) of a solution of 2 N perchloric acid containing 1 ~ EGTA and 70 mM of MgClz (see refs. 11, 47) and maintained frozen until the assay was performed.
(c) The biochemical assay of CA The CA content of the samples was determined by means of a sensitive and specific radiometric assay based on the modification of the technique published by Coyle and Henry10 as reported by De Champlain et al. 11. One of the improvements of great relevance was the use of perchloric acid containing EGTA and Mg z+ to acidify the samples as soon as they were collected thus avoiding the inhibitory effect of free Ca 2+ on the activity of the enzyme catechol-O-methyltransferase (COMT) (see ref. 11) The use of duplicates and internal standards for each sample in order to check the efficiency of the COMT and of the separation steps allowed us to rule out errors in the assay technique10,11,47. This method is based on the methylation of CA by COMT (E.C. 2.1.1.11) in the presence of [aH]methyl-S-adenosylmethionine as a methyl donor. The all-derivatives are thereafter isolated by selective organic extraction. COMT was prepared and purified from rat liver by the method of Axelrod and Tomchick 1. In order to avoid contamination, blanks for every perfusing solution and for
98 every pharmacological agent were assayed and the counts of this blank were subtracted from the total counts of the sample. The internal standards used for the superfusates were made of 0.5 ng of L-NE (free base) and of 0.5 ng of L-DA (free base). At the end of the perfusions, samples of brain tissue were quickly removed, weighed and homogenized in 100 vol. (w/v) of 0.1 N cold perchloric acid in a glass homogenizer with a glass pestle. The homogenates were centrifuged at low speed and the supernatant fluid was decanted for assay. The assay followed the same procedure as for the superfusates except that the internal standards used were made of 2.0 ng of L-NE (free base) and of 2.0 ng of L-DA (free base).
(d) Electrophysiological techniques Intermittent brief light flashes of the entire visual field, using a Grass PS-20 Photostimulator were used at a frequency of 15.0/sec for activation of the visual system. The pupils were dilated with 2 ~o atropine sulphate and the nictitating membrane contracted with 1 ~ ephedrine hydrochloride. Care was taken to keep the corneas moist with saline solution. In order to avoid possible complications due to the retina electrical stimulation of the optic nerve with steel bipolar electrodes (0.5 mm separation and 0.5 mm exposed tips) placed stereotaxically was also used in some of the experiments. The electrodes were placed in the optic nerve at A + 13.5 mm; L 2.0 mm and H --6.0 mm (ref. 23). In some experiments stimulation of the forelimb was carried out with a bipolar steel needle electrode in the paw in order to compare the effects of somatic and visual sensory systems, and to determine the specificity of changes. The electrical stimuli were rectangular pulses of 0.5 msec duration for the optic nerve and 1.0 msec duration for the forelimb obtained from a Grass $44 stimulator at a frequency of 0.5/sec or 15.0/sec and at an intensity of 3 times the threshold for the evoked potential. Evoked potentials after adequate amplification were either displayed on the screen of a dual beam Tektronix oscilloscope 565 and averaged on-line 32-64 times by a signal averager-memory control unit (Ortec models 4623 and 4620) or stored in Ampex FM magnetic tape for further analysis. Bipolar surface silver electrodes were used to record the activity of the cortex. In all experiments the EEGs were obtained from the exposed cortical areas and after suitable amplification displayed on paper with a 6-channel Brush recorder. RESULTS
Basal release of CA under 'resting' conditions Under the conditions of these experiments conducted in a relatively quiet room with adequate local anaesthesia the immobilized animals usually became drowsy or lightly asleep as judged by pupiltary constriction with retraction of the nictitating membrane and by persistent spindles and a few slow waves in the EEG. Basal release of CA was measured only in experiments in which satisfactory superfusates were obtained during stable EEG records, constant arterial blood pressure and with artificial respiration adjusted to maintain constant end-tidal CO2 concentration at about 3.8 ~ . Results are expressed in pg/min/sq.cm although each collection period was
99 TABLE I Basal release of cateeholamines from the visual cortex of the cat
Values expressed in pg/min/sq.cm, n = 18. Experiment
Norepinephrine
Dopamine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
32.60 22.75 45.96 61.96 39.60 8.76 20.41 19.92 9.80 11.10 7.56 15.14 9.45 9.86 19.22 13.16 7.80 6.61
35.99 98.84 72.21 107.85 37.60 6.16 8.08 70.90 20.30 10.05 40.98 8.74 31.26 20.02 6.48 5.71 26.30 7.03
Mean ± S.E.
20.094- 3.64
34.01 4- 7.62
30 min in duration in order to provide a sufficient volume for assays to be performed in duplicate with one or two internal standards for each sample 1°,11,47. With these precautions it was possible to obtain fairly constant values for both N E and D A over periods of several hours in some experiments in which sensory stimulation was not carried out (see Fig. 4 for an example). However, resting values varied widely from one experiment to another as shown in Table I. For visual cortex the range of values for N E was between 6.61 and 61.96 pg/min/sq.cm with an average of 20.09 -4- 3.64 pg/min/sq.cm (n ---- 18). F o r D A the range was 5.71-107.85 pg/min/ sq.cm with an average of 34.01 q- 7.62 pg/min/sq.cm (n = 18). Variations in N E and D A were relatively independent in most instances, the basal levels for D A being higher than for N E in 11 out of 18 experiments and higher for N E in 7 experiments. This would suggest that these variations in basal release rate can not be attributed only to the technical variation in superfusion. That diffusion barriers may play some role was shown, however, in two experiments in which the basal values were increased by about 30 ~o following multiple punctures of the piarachnoid membranes with a glass micropipette. Average basal release of CA from somatosensory cortex was about twice the values obtained for visual cortex in 6 experiments in which superfusion of both cortical areas was carried out simultaneously. In these experiments the average release of N E f r o m visual cortex was 14.53 -4- 5.78 pg/min/sq.cm as compared to 25.80 -q- 8.91 pg/min/sq.cm f r o m somatosensory cortex. F o r D A the average basal release of
100
40-
~CONTROL ~ STIM.
30
•~
20-
O.
10"
. Fig. 1. The effect of stimulating the visual system on the release of norepinephrine (NE) and dopamine (DA) from the visual cortex. All experiments were performed using a stimulating frequency of 15.0/ see; in 8 animals photic stimulation of the visual field was used and in 3 clerical stimulation of the optic nerve at 3 times the evoked potential threshold (see Material and Methods). Each bar represents the mean 4- S.E. of 11 animals.
visual cortex was 21.50 ~ 5.58 as c o m p a r e d to 55.52 i 12.28 pg/min/sq,cm for somatosensory cortex. We were unable to establish any consistent correlation between the E E G pattern and the differences in release o f N E or D A in the resting state.
Endogenous cortical tissue concentration of CA The endogenous C A were measured in the contralateral non-perfused cortical areas. The values showed an average o f 0.202 -4- 0.027 # g / g (n = 13) for N E and o f 0.199 ~: 0.024 #g/g (n = 13) for D A . I n 6 animals basal release values o f C A were c o m p a r e d with the endogenous content in the tissues but no correlation was f o u n d either for D A or for NE. These values are similar to those obtained by Gessa et al. 17, using a fluorometric m e t h o d o f measurement.
Effects of sensory stimulation During stimulation o f the visual system at a frequency o f 15.0/sec (Fig. 1) a
101
6o-
1CONTROL ~O.N. STIM.
._c
lid_ NOREPINEPHRINE
DOPAMINE
Fig. 2. Recovery following reduction in release of CA from visual cortex by optic nerve stimulation (O.N. STIM.) at 15.0/secat intensity 3 times threshold for visual evoked potentials. Each bar represents the mean -4- S.E. of 7 animals for NE and 6 animals for DA.
decrease in the release of both N E and DA was observed in 11 successful experiments. In these animals the average basal release of NE was of 24.13 ± 5.56 pg/min/sq.cm (n = I 1) and during stimulation the release dropped to 11.75 -4- 2.43 pg/min/sq.cm (n = 11). The release of DA was also decreased during the periods of stimulation from 37.86 4- 11.49 pg/min/sq.cm (n = 11) to 12.61 4- 4.17 pg/min/sq.cm (n = 11). This decrease in the release of CA from the visual cortex was obtained either with photic stimulation or with electrical stimulation of the optic nerve. This effect of visual stimulation on CA release was reversible. Fig. 2 shows the recovery periods in 6 animals after stimulating the optic nerve (electrical stimulation). The control (basal) levels of CA returned to their previous levels and in the case of D A there was a slight increase as compared to the first control period. In some animals the reduction in CA release persisted for 30 min to 1 h after the period of stimulation, while in others there seemed to be a rebound increase above the basal level following stimulation. In order to study the specificity of the decrease in CA release induced by visual stimulation in 6 animals, two chambers were placed: one over the visual cortex and the other over the postcruciate cortex (somatosensory) (see Materials and Methods). Either somatic or visual stimulation decreased the release of DA and N E in both the somatosensory and visual cortical areas (Fig. 3). Somatic stimulation seemed to be somewhat more effective than visual stimulation, in general, for either visual or somatosensory areas and for both N E and DA. From these results it would appear that the effect of sensory stimulation is non-specific and not clearly related to local projecting sensory pathways. These results may be compared with the non-specific increase in the release of ACh with sensory stimulation under similar physiological conditions. This raises the question of some form of interaction between ACh and catecholamine release mechanisms which could in some way explain the reduction in CA release under conditions which cause an increase in release of ACh ~,9,aa,aS-aT.
102 30-
VISUAL CORTEX
SOMATOSENSORY CORTEX
VISUAL CORTEX 80-
m
SOMATOSENSORY CORTEX
CONTROL
]VISUAL STIMULATION [-~'1 SOMATICSTIMULATION 20 -
60-
lo 2O
0
0
NOREPINEPHRINE
DOPAMINE
Fig. 3. The effects of sensory stimulation on the release of CA from the visual and somatosensory cortical areas. Observe that either visual stimulation or somatic stimulation reduced the release of CA from both the visual and the somatosensory cortical areas. These experiments were performed with two chambers simultaneously perfused in the same animal. Both optic nerve and paw were stimulated at 15.0/sec with intensities 3 times threshold for cortical evoked potentials. Each bar represents the mean S: S.E. of 6 animals.
Effects of nicotine and atropine Perfusion with nicotine in Elliott's Solution at a concentration of 5 × 10-3 M caused a large increase in the release of both NE and D A as shown in Fig. 4. In this experiment evoked potentials in response to photic stimulation were sampled at 2-sec intervals throughout the entire experiment. Nicotine at the concentration of 10-8 M caused only a 1 0 ~ increase in CA release but at a concentration of 5 × 10-a M, CA release was increased 4-5 times the previous basal value. The increase was maximum during the first 30-min collection period and tended to fall to a lower value during the second 30-min period while still remaining well above the control levels before nicotine, which levels remained relatively constant for about 5 h previous to perfusion with the higher concentration of nicotine. In another experiment (Fig. 5) a moderate increase in CA release was obtained by perfusion with 10-3M nicotine solution and more importantly stimulation of the optic nerve after nicotine caused an increase rather than a decrease in DA. Although there was a slight reduction in NE during optic nerve stimulation after nicotine, these values remained well above the control value before nicotine. Atropine perfused in a concentration of 10-4M also caused an increase in basal release of both N E and D A as shown in Fig. 5, Stimulation of the optic nerve following atropine was found to cause an increase rather than a decrease in DA, while NE returned to its basal control level in this experiment during optic nerve stimulation. The stimulating effect of nicotine upon CA release is similar to that which has
103
~CONTROL 260
I
IO0t~NICOTINE' 220
180t
30 20
Ii]1
! 6O
,:.~
J 20
120 240 360 rain. NOREPINEPHRINE
120 240 360 rain. DOPAMINE
Fig. 4. The effects of nicotine on the release of CA from the visual cortex. In this experiment all collections were performed while stimulating photically the visual field at a frequency of 0.5/sec. Nicotine (free base) was employed at a concentration of 10-aM for the first period and in the two last collection periods at a concentration of 5 x 10-3M. Observe the relative stability of the basal release values (control periods) throughout the length of the experiment. The higher dose of nicotine (5 x 10-aM) induced a very important release of both CA, but in a second period of collection this release declined although it still remained well above the control values.
been observed in the periphery and in slices of brain tissue in vitro 1s,g2,44,46,4s. This suggests the presence of nicotinic ACh receptors capable of increasing CA release and of modifying the effects of sensory stimulation. The effect of atropine may be due to blocking muscarinic inhibitory ACh receptors thus also modifying the effects of sensory stimulation. The site of action of both of these effects are presumed to be upon presynaptic CA terminals. DISCUSSION
In the present studies we have shown that the visual cortex of the cat contains
104 ATROPINE 20
NICOTINE
m CONTROL ]
20- NE
ON.STtM
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Fig. 5. The effects of optic nerve stimulation, nicotine and atropine on the release of CA from the visual cortex. The concentrations of the agents employed were 10-4M for atropine sulphate and lO-ZM for nicotine. Observe that optic nerve stimulation (O.N.STIM.) reduced the release of both DA and NE. The release of CA was increased by both atropine and nicotine. During O.Ni stimulation while continuing nicotine perfusion the release of NE was reduced but remained above the control level. O.N. stimulation produced an increase in DA release in both atropine and nicotine treated cortex. about equal concentrations of endogenous N E and D A (0.2 #g/g). Preliminary studies using the histofluorescent method of Falck-Hillarp 18 have shown that the visual cortex of the cat is supplied with characteristic CA axonal terminals which disappear following perfusion of the cortical surface with 6-hydroxydopamine (unpublished results). Such terminals are relatively few in number and this is consistent with the relative sparsity of such terminals found in the cortex of the rat by the quantitative electron microscopic autoradiographic studies of Descarries and Lapierre 13 and Lapierre et al. 3°. It has been pointed out recently by Descarries et al. 12, that CA and 5-HT terminals in the cerebral cortex are rarely found to be associated with postsynaptic membrane specializations as seen in the electron microscope. This pattern of innervation is compatible with the suggestion that these biogenic amine terminals may not have specific punctate or local synaptic action as has been postulated for classical synapses. These findings and interpretations raise important questions regarding the functional significance of CA and 5-HT terminals in the cerebral cortex, and the mechanisms governing their release. By means of a recently developed highly sensitive enzymatic assay method 1°, 11,47 it has been possible to measure N E and D A concentrations in samples of extracellular fluid obtained by superfusion of a limited area of cortical surface, a method previously found useful in studies on the liberation of other putative neurotransmitters
105
in the intact animal under a variety of physiological conditions (including specific sensory stimulation, sleep and waking, and stimulation of the brain stem) s,9,za-2o,aa, 85,86. The quantities of NE and DA measured in the superfusates in our experiments are smaller by 2 and 3 orders of magnitude than those reported for ACh s,26,3a,35 and for glutamic acid2~ respectively and using the same superfusion techniques. However, these low values of CA are not surprising since DA and NE fibers seem to be rather few in number and on the other hand re-uptake and degradative mechanisms may account for a great reduction in the overflow. It is worth pointing out here that no pharmacological agents known to inhibit the activities of monoamine oxydase or catechol-O-methyltransferase or to block the re-uptake mechanisms were used in the experiments here reported in order to keep the animals as near to the physiological state as possible. Therefore the actual 'real' or 'true' amount of CA released by the nerve terminals and acting in the central nervous system at a specific site may be several times greater than what is measured with the superfusion method. This fact does not invalidate the method but shows that it is only an indirect measurement of the total release of free CA which have escaped metabolic degradation or re-uptake. Such a sampling may be of particular importance if biogenic amines act as local neurohormones affecting a large population of neurones by diffusion into extracellular spaces distant from their site of liberation. With regard to the specificity of the changes in CA release induced by sensory stimulation it was apparent from our experiments (in single or double chamber experiments) that suitable somatic stimulation was capable of altering the release of CA in visual cortex and stimulation of the visual system was able to reduce CA liberation in the somatosensory cortex and vice versa. This suggests a non-specific effect of sensory stimulation and opposite in direction to that which has been observed for the release of ACh s,9,26,35-3:. Recently the hypothesis has been proposed that ACh released from cholinergic fibres might cause an inhibition of NE fibres and a decrease in the release of NE by an action upon presynaptic receptors. Such an effect of ACh on CA release has been documented in the periphery. A muscarinic inhibitory effect of ACh on the release of NE for the cat heart 19, the rabbit heart al, the perfused rat mesenteric arterya4 and the central ear artery of the rabbit 89 has been reported. That such an inhibitory effect may be also present in the cerebral cortex is suggested by the increase in the release of both DA and NE when muscarinic ACh receptors are blocked with atropine (Fig. 5). Furthermore, visual stimulation in atropinized visual cortex caused an increase rather than a decrease in the release of DA, and reduced the usual effect of stimulation on NE release. This reversal of the effect of stimulation by atropine would suggest the possibility that there may be also facilitatory actions of ACh on CA release unmasked when the inhibitory mechanism is blocked by atropine. This facilitatory action would be magnified by the fact that atropine has been shown to increase extracellular ACh concentrations in the cerebral cortex s,35,42. Nicotine has been shown to cause a direct release of CA from peripheral adrenergic neurones81, 45, to lower the CA content of the rat diencephalon and the
106 mouse brain 4°, to show amphetamine-like effects releasing NE from central neurones 7,22,48 and to release [14C]NE and [14C]DA from rat brain striatum and hypothalamus slices 18. In a recent article Westfal144 has reported that nicotine produces a significant increase in the release of [aH]NE from incubated slices of rat cortex, hypothalamus and cerebellum. He presents further pharmacological evidence suggesting that ACh may control the release of CA from central neurones by means of nicotinic presynaptic receptors on the terminals of CA fibres, similar to the mechanism proposed for N E in peripheral adrenergic neuroneslL The marked increase in DA and NE release observed in the present studies when superfusing with nicotine (Figs. 4 and 5) is consistent with its effect on peripheral CA terminals and brain slices. These results suggest that the release of CA in cerebral cortex may be increased by similar nicotinic presynaptic facilitatory mechanisms which become apparent when muscarinic receptors are blocked with atropine. Further speculation regarding ACh-CA interaction is not warranted at the present time since non-specific effects of atropine and nicotine on cerebral cortex cannot be entirely ruled out 29,a8. We do not mean to imply that the observed effects of sensory stimulation upon CA release in cerebral cortex are all mediated by interactions with ACh. There are very probably direct effects of sensory stimulation upon CA neuronal systems of the brain stem which are known to project diffusely to the cerebral cortex. The relation of cortical CA release and metabolism to states of sleep, waking, and arousal has yet to be determined since it was not possible to make such observations in the above experiments. Such studies are presently being undertaken, as well as studies of the action of CA upon cortical synaptic function at the unitary level in order to gain further insight into the functional significance of CA terminals in the cerebral cortex. ACKNOWLEDGEMENTS This work was supported by the Medical Research Council Group in Neurological Sciences at the Universit6 de Montr6al (Drs. H. H. Jasper and J. de Champlain) and by a Post-doctoral Fellowship from the Medical Research Council (Canada) to Dr. T. A. Reader. The authors also acknowledge the skilful technical assistance of Miss Lise Farley.
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