The release of H3-dopamine from cat brain following electrical stimulation of the substantia nigra and caudate nucleus

Neurophnrmncology.

1971,

10,733-741

Pergamon Press.Printed inGt.Britain.

THE RELEASE OF HS-DOPAMINE FROM CAT BRAIN FOLLOWING ELECTRICAL STIMULATION OF THE SUBSTANTIA NIGRA AND CAUDATE NUCLEUS* Department

P. F. VON VOrGTLANDERtand K. E. MOORE of Pharmacology, Michigan State University, East Lansing, Michigan 48823 (Accepted 23 March 1971)

Summary-After the intraventricular injection of Hs-dopamine, the cerebroventricular system of cats with spinal sections was perfused with artificial cerebrospinal fluid. Electrical stimulation of the caudate nucleus increased the perfusate concentration of Ha-dopamine, but not of Ha-3-methoxytyramine. After the intraventricular injection of C%trea, similar stimulation did not increase the efflux of this substance. When the substantia nigra pars compacta was stimulated the efflux of both H*-dopamine and Ha-3-methoxytyramine was increased ; the amount of Hs-dopamine released was dependent upon the intensity and frequency of the stimuli. IT HAS been

suggested that dopamine may function as an inhibitory neurotransmitter at the terminals of the nigro-striatal pathway. BLOOMet al. (1965) demonstrated a primarily inhibitory action of dopamine when it was applied microiontophoretically to caudate neurons. CONNOR(1968) observed that stimulation of the substantia nigra pars compacta caused a depression of firing in most responding caudate units. That the dopamine in the striatum is intimately associated with the nigro-striatal pathway has been demonstrated by several groups (POIRIERand SOURKES,1965 ; FAULL and LAVERTY,1969 ; GOLDSTEINet al., 1970). These workers have shown that lesions to the substantia nigra or the ascending nigro-striatal fibers caused a marked depletion of dopamine and a depression of the dopamine synthesizing enzymes in the caudate nucleus. These lesions may be related to those seen in parkinsonism where neuronal degeneration and depletion of dopamine have been observed in the striatum and the substantia nigra (HORNYKIEWICZ,1966). If dopamine does serve as the neurotransmitter of the nigro-striatal neurons, stimulation of these neurons should elicit the release of this putative transmitter. So far this has not been demonstrated, but in the present experiments, involving a cerebroventricular perfusion technique, electrical stimulation of the caudate nucleus and substantia nigra pars compacta released dopamine into the ventricular system. METHODS

Cats (2-3 kg) of either sex were anesthetized with methoxyflurane by the open drop method. After the animal was placed in a stereotaxic instrument, a mid-dorsal incision was made from the level of the supraorbital processes to the axis. The dorsal cervical muscles *Supported by USPHS Grant MH 13174. tPostdoctora1 fellow supported by USPHS Training Grant GM 1761. This study constitutes part of a thesis submitted to the Graduate School, Michigan State University, in partial fulfillment of the requirements for the M.S. degree. 733

734

P. F. VONVOIGTLANDER and K. E. MOORE

were reflected and cut away to expose the cisterna magna and the supraoccipital region. Anesthesia was withdrawn and artificial respiration commenced as the spinal cord was sectioned at the level of the atlas. All incisions and pressure points were treated with a local anesthetic (hexylcaine). Twenty-two gauge stainless steel screw-type cannulas (David Kopf Instruments) 14 mm in length were inserted stereotaxically into the lateral ventricles at 16.5 anterior, 3.5 lateral (left and right) and ~8.0 deep (SNIDERand NIEMER,1961). Five microcuries of H3-dopamine (New England Nuclear, 10.6 Ci/mmol) in an effective volume of 10 ~1. or 2.5 &i CY4-urea (New England Nuclear, 0.27 mCi/mmol) in 20 ~1. were injected through one of the cannulas. The purity of the H3-dopamine was checked routinely with thin layer chromatography as described by CARR and MOORE (1969). During the I-hr period allowed for isotope absorption, the supraoccipital region of the skull was removed, the cerebellum carefully lifted, and a cannula (5 cm x 2 mm outside diameter polyethlene, with a 5mmsilasticcuff)wasinsertedin the cerebroaqueduct. Perfusion of the ventricularsystemwith artificial cerebrospinal fluid (PAPPENHEIMER et al., 1962) was then commenced at a rate of 0.1 ml/min using the lateral ventricular cannula as the inflow and the cannula in the cerebroaqueduct as the outflow. During the 2-hr washout period, a femoral artery was cannulated for blood pressure recording and the stimulating electrodes were positioned. The caudate nucleus was stimulated by 2 bipolar electrodes (0.5 mm separation and O-5 mm exposed tip, David Kopf Instruments) spaced 5 mm apart at 18.0 and 13.0 anterior, 4-O lateral and $5.0 deep (SNIDERand NIEMER,1961) with the anterior electrode cathodal to the posterior. In experiments involving substantia nigra pars compacta stimulation, a single bipolar electrode (0.5 mm separation, 0.5 mm exposed tip, David Kopf Instruments) was placed at 4.1 anterior, 2.8 lateral and -4-S deep (BERMAN,1968). Ten minutes before the end of the 2-hr washout, the perfusion rate was increased to 0.5 ml/min. One millilitre perfusates were then collected every 2 min, and during one or more of the perfusion periods square wave constant current stimulation of various frequencies and intensities and 1 msec duration was applied to the electrodes. At the end of the perfusion the entire procedure (isotope injection, washout, electrode placement, perfusate collection and stimulation) was repeated on the opposite side. Throughout the experiments the rectal temperature was monitored with an electronic thermometer and maintained at 37.5f0.5”C with an electric heating pad. During all experiments the systemic blood pressure was monitored. Stimulation of the caudate nucleus did not alter the blood pressure but in preliminary experiments involving substantia nigra stimulation some increases in blood pressure were noted. Bilateral vagotomy blocked this pressor effect; accordingly, all substantia nigra stimulation experiments reported here were performed in vagotomized animals. At the end of the second perfusion, the brain was removed and fixed in 10 % formalin. In some experiments, the total radioactivity of the perfusates was determined by adding 0.1 ml of each perfusate to glass scintillation vials containing 0.5 % 2,5-diphenyloxazole (New England Nuclear) in 7 parts toluene and 3 parts 95 % ethanol. The sample was counted in a Beckman LS-100 liquid scintillation counter. The counts were corrected for counting efficiency and the data presented in units of absolute radioactivity. Factors for recovery of H3-dopamine and H3-3-methoxytyramine were applied to correct for loss of these compounds during separation procedures. The separation of the perfusate compounds into a catechol and non-catechol fraction, and the subsequent separation of the latter fraction into the 0-methylated and deaminated 0-methylated fractions were effected by alumina adsorption and ion-exchange chromatography respectively (CARRand MOORE,1970). The

Releaseof dopaminefrom brain

735

catechols were divided into deaminated catechols, norepinephrine, and dopamine by the following method. Six x 40 mm columns of Dowex 50 (200-400 mesh, H+) were prepared with flow rates of 5-7 drops per min and changed to Na+ form with 25 ml of 0.1 M NaH,PO,, pH 65 buffer. The samples were prepared by the addition of 100 pg of Na ascorbate, 100 pg of norepinephrine and 100 pg of dopamine each in a volume of 10 ~1. Before being added to the columns, the samples were adjusted to pH 6 with 1 N and 0.1 N KOH. After the samplehad run through the column, 5 ml H,O, 8 ml 1 N HCI, 10 ml of 1 N HCI and 4 ml 1 :l 6 N HCI-95 % ethanol solution were applied to the column in succession. The effluent and the H,O fraction contained Ha-deaminated catechols, the 10 ml 1 N HCI eluate contained H3-norepinephrine and the 4 ml HCI : ethanol eluate contained H3-dopamine. Two millilitres of each of these fractions were transferred to empty scintillation vials, dried, dissolved in 10 ml toluene-ethanol-2,5-diphenyloxazole scintillator and the radioactivity was determined. After at least 48 hr of formalin fixation, the brains were dissected to verify the cannula and electrode placement. In experiments involving direct caudate stimulation, gross horizontal sections were made of the brain and the positions of the cannulas and caudate electrodes verified directly. In the experiments involving substantia nigra stimulation, the electrode tract was dissected out, frozen and sectioned. The 20-pm section containing the electrode tract was stained with cresyl violet and examined microscopically to verify the electrode position. All data reported are the mean of at least 4 experiments. Statistical significance of the evoked release was calculated by comparing the perfusate concentration of H3-labelled compounds during the period just before stimulation to the concentration during the period of stimulation in a single-tailed paired Student’s t-test. P values of less than 0.05 were considered statistically significant. RESULTS

After 2 hr of washout, the radioactivity in the perfusates consisted of 43 % catechols and 57 % non-catechols. The latter fraction contained 70 % 3-methoxytyramine and 30 % 3-methoxy-deaminated metabolites. The catechol fraction consisted of 85 % dopamine, 14 % deaminated catechols and less than 1% norepinephrine; in the tables and figures the radioactivity in the catechol fraction is reported as H3-dopamine. The initial experiments involving caudate nucleus stimulation were designed to determine if depolarization of this region could increase the release of radioactivity into the ventricular perfusates (Fig. 1). A 2-min period of stimulation of the caudate nucleus increased the efflux of H3-dopamine but had no effect on the perfusate concentrations of H3-3methoxytyramine, H3-deaminated catechols or H3-3-methoxy-deaminated metabolites. Although the efflux of H3-dopamine was statistically greater during the period of stimulation, the greatest efflux of this amine occurred during the 2-min period immediately after the stimulation. To test the specificity of the stimulation-induced release of H3-dopamine, the efflux of urea, a substance not considered to be a transmitter, was examined. Electrical stimulation of the caudate nucleus failed to increase the efflux of C14-urea into the cerebroventricular perfusates (Fig. 2). Histofluorescent studies have demonstrated that many of the cell bodies giving rise to dopaminergic terminals in the neostriatum are localized in the substantia nigra pars compacta (AND~Net al., 1964; H~KFELTand UNGERSTEDT, 1969). A brief period of electrical

736

P. F.

VON VOIGTLANDER

and K. E.

MOORE

43-

-,

7 2* 2--

T d p? I ‘--

Stim.

.

T

T _

0

_I

4

6 Time,

8

IO

12

min

of 2 min of electrical stimulation (100 Hz, 350 !LA, I msec) of the caudate nucleus on the cerebroventricular effluent concentrations of H3-dopamine (WD) and H3-3-methoxytyramine (H33-MT). The height of each bar represents the mean concentration (vertical lines denote 1 SE.) of HSD or HS3-MT in the cerebroventricular effluent collected over 2-min periods from 4 cats. *The effluent concentration of H3D during the 2-min period of stimulation was significantly greater than during the 2-min prestimulation period (PcO.05). FIG. 1. Effects

i

1

Time, FIG. 2. Effect

min

of electrical stimulation of the caudate nucleus (100 Hz, 350 PA, 1 msec) upon the C14-urea concentration in ventricular perfusates. The height of the bars represents the concentration of C14-urea. The vertical lines denote standard errors determined from 4 experiments.

Release of

137

dopaminefrom brain

stimulation to the region of these cell bodies increased the perfusate concentration of H3-dopamine and H3-3-methoxytyramine (Table 1). The concentrations of H3-deaminated catechols and of deaminated-O-methyl metabolites were not altered during or after electrical stimulation. TABLE 1. RELEASE OF ~~~~~~~~~~~ AND H3-3-~~-r~ox~r~~~~I~~ DURING ELECTRICAL STIMULATION OF THE SUBSTANTIA NIGRA PARS COMPACTA Experiment number H3-dopamine

I

1.06

2 3 4

1.85

n+S.E. Ha-3-methoxytyramine

Control (nCi/ml)

Stimulation (nCi/ml)

0.98 0.85

I.18 1.99 I.18 1.00

1.19&0.23

1.34&0.22

Change

0.12 0.14 0.20 0.15

0.15+0.01*

I

I .60

2 3 4

1.29 1.08 0.92

I .70 1.50 1.25 0.98

0.10 0.21 0.17 0.06

1.22&-0.15

1.36hO.16

0.14+0.03*

before stimulation. was applied.

During

X&S.E.

The control period represents the 2-min perfusion period immediately stimulation period 30 Hz, 200 pA, 1 msec duration electrical stimulation *Increase in release statistically significant (PC 0.05).

the

The intensity-response relationships for substantia nigra stimulation were determined by randomly applying different strengths of stimulation in the same cat in order to avoid cat to cat variability in electrode placement. In one set of experiments, 100,200 and 400 ,uA intensities caused a progressive increase in the release of H3-dopamine (Fig. 3). In another experiment using lower intensities of stimulation (12.5-50 PA), there was also a progressive increase in the efflux of H3-dopamine, but because of variability in the small amounts released the responses were not statistically significant. When higher intensities were used (600-800 PA) the responses could not be repeated consistently. Accordingly, frequency responses were determined using 100 PA stimulation. Substantia nigra stimulation-induced release of H3-dopamine appeared to be frequency-dependent (Fig. 4). While 10 and 30 Hz stimulation caused a significantly increased efflux of H3-doapmine, 3 and 100 Hz failed to do so. To test the specificity of the substantia nigra stimulation-induced release of H3-dopamine, a series of double label experiments were performed with H3-dopamine and C14-urea. The results of one of these experiments is illustrated in Fig. 5. Stimulation of substantia nigra increased the perfusate concentration of H3-dopamine but failed to alter the efflux of P4-urea. In 4 such experiments the mean (i 1 S.E.) increase in dopamine was 0.33 f0.03 nCi/ml while the C14-urea concentrations decreased by 0.03 fO-02 nCi/ml. DISCUSSION

Within the

years, evidence has With histofluorescent techniques, dopaminehave been identified have cell bodies in substantia and

738

P. F. VON VOIGTLANDER and K. E. MOORE

I

I

IO0

i

4c

200

Intensity,

pA

FIG. 3. The relationship of substantia nigra pars compacta stimulation intensity and the increased cerebroventricular perfusate concentration of H*-dopamine (H3D). Each point represents the mean increase in HSD upon stimulation (30 Hz, 1 msec) at the indicated intensity as compared to the prestimulation H3D perfusate concentrations in a total of 8 experiments. The vertical lines denote 1 S.E. *Increased concentration of H3D is significant (P~0.05).

/

I-

T”

Frequency,

Hz

FIG. 4. The increase in H3-dopamine (HSD) released into the ventricular perfusate upon stimulation of the substantia nigra pars compacta at various frequencies (100 PA, 1 msec). The height of each bar represents the mean (vertical lines denote 1 S.E.) increase in H3D release upon stimulation for a total of 8 experiments. Increase in release is calculated as the difference between the perfusate concentration of H3D during the 2-min prestimulation period and the 2-min period of stimulation. *Increase is statistically significant (PCO.05).

Release of dopamine from brain

0

2

4 Time,

6

739

8

min

FIG.5. The effect of stimulation (30 Hz, 200 PA, 1 msec) of the substantia nigra pars compacta upon ventricular perfusateconcentrations of Ha-dopamine (HSD) and CY-urea. The height of the open bars represents HSD concentrations; cross-hatched bars represent CWxea.

terminals in the caudate nucleus (AND~N et al., 1964). Lesions in substantia nigra, or along the proposed nigro-striatal pathway, cause a loss of dopamine and enzymes for the synthesis of this amine in the neostriatum (GOLDSTEINet al., 1970; H~~KFJZLT and UNGERSTEDT,1969; POIRIERand SOURKES,1965; FAULL and LAVERTY, 1969). Conversely, destruction of the neostriatum causes retrograde degeneration of neurons in the substantia nigra (BEDARD ef al., 1969). In the present study it was demonstrated that electrical stimulation of the caudate nucleus and substantia nigra pars compacta increased the release of dopamine into the cerebroventricular system. These results lend further support to the existence of a functional dopaminergic nigro-striatal neuronal system. Other workers have attempted to detect electrically evoked release of endogenous dopamine from brain in situ. MCLENNAN (1964, 1965), using a push-pull catmula in the caudate nucleus and putamen, was able to detect the release of dopamine after electrical stimulation of the nucleus centromedianus and substantia nigra. The amounts of endogenous dopamine analyzed were near the limits of assay, and the push-pull cannula probably damaged the region of tissue that was perfused (MCKENZIE and SZERB, 1968; VOGT, 1969; CHASE and KOPIN, 1968). Using a cerebroventricular perfusing technique which causes little damage to the perfused tissues, PORTIGand VOGT (1969) measured the release of dopamine and its metabolite, homovanillic acid, from the brain. Stimulation of the sciatic nerve and the substantia nigra resulted in inconsistent changes in the perfusate concentrations of dopamine, but in some of the experiments both modes of stimulation increased the efflux of homovanillic acid. In the present experiments, dopamine stores in the caudate nucleus were labelled with H3-dopamine (CARR and MOORE,1969) thus allowing the detection of the release of 5 pg of dopamine. Direct stimulation of the caudate nucleus increased the efflux of H8-dopamine, but not of its metabolites. It was assumed that the Hs-dopamine was taken up by nerve terminals in the caudate nucleus and that it was from these nerve endings that the amine

P. F. VONVOIGTLANDER and K. E. MCXXE

740

was released when depolarizing electrical stimulation was applied. Direct electrical stimulation, however, through possible gross ionic shifts, metabolic alterations, etc. may release the amine through of an increased

some nonselective

efflux of Cr4-urea

is some selectivity exogenous

also accumulates

is taken up exclusively

in the terminals

factor

administration

of C14-tyrosine.

Catecholamines

Nevertheless,

by labelling

brain

In this way the labelled

released

from

peripheral

evoked release of amines, labelled

in the present

it is doubtful

neurons;

stores

amine

neurons).

of dopamine

would

upon

This

by the

be localized

is specifically

neurons

that

it probably

to

located in these

electrical

stimulation

from a newly synthesized pool (KOPIN et al., 1968). Accordingly,

be even greater than that demonstrated described

stores. The lack suggests that there

by dopamine-containing

neurons since tyrosine hydroxylase

appear to come preferentially the electrically

stimulation

of other neurons as well (e.g. serotonergic

may be circumvented

catecholamine-containing neurons.

or from non-neuronal

in the evoked release of H3-dopamine.

dopamine

complicating

mechanism

during direct electrical

in vivo from radioactive precursors,

in the present experiments.

study, but using C14-tyrosine,

may

Studies similar to those

are currently

in progress.

The direct electrical stimulation of the caudate nucleus reported in the present study is similar to the in vitro field stimulation of brain slices (BALDESSARINI and KOPIN, 1966) in that the nerve terminals will be depolarized.

are depolarized

Since H3-dopamine

released H3-dopamine

may not originate

other hand, the release of H3-dopamine the substantia substantia

directly.

Thus,

all nerve endings in the region

may be taken up by various nerve terminals, exclusively

from dopaminergic

after stimulation

of dopaminergic

nigra must reflect a release from dopaminergic

nigra is some distance from the ventricular

neurons.

the

On the

cell bodies

nerve terminals

in

since the

system and the site of H3-dopamine

accumulation. Since deaminated

catechols,

0-methylated

metabolites,

and deaminated

metabolites

are all present in the brain of cats after intraventricular

cholamines

(CARR and MOORE, 1969),

and MOORE, 1970,

and the present

and are also in the ventricular

study),

it might be expected

mediated increase in the etlIux of radioactivity was not the case.

Direct

caudate

nucleus

0-methylated

injection

of H3-cate-

perfusates

(CARR

that a non-neuronally

would include all of these compounds. stimulation

released

This

only H3-dopamine

and

H3-norepinephrine (VON VOIGTLANDER and MOORE, 1971). AXELROD (1968) has suggested that the post-synaptic receptor in adrenergic synapses may be adjacent to catechol-Omethyltransferase.

If this is true, some of the H3-dopamine

should appear in the ventricular of the substantia synapses. caudate

The nucleus

perfusates

nigra this did occur suggesting lack

of release

suggests

the amines originated

of H3-3-methoxytyramine

that this release

released at adrenergic

as H3-3-methoxytyramine.

occurs

after

predominately

direct

During

synapses

stimulation

from dopaminergic stimulation

of the

from non-dopaminergic

nerve terminals. Acknowledgements-The

technical assistance of Mrs. MIRDZAGRAMATINS is gratefully acknowledged. REFERENCES

ANDEN, N. E., CARLSSON,A., DAHLSTROM, A., FIJXE, K., HILLARP,N. A. and LARWN, K. (1964). Demonstration and mapping out of nigral neostriatal dopamine neurons. Life Sci. 3: 523-530. AXELROD, J. (1968). The fate of norepinephrine and the effect of drugs. The Physiologist 11: 63-73.

BALDESSAWNI, R. J. and KOPIN,1. J. (1966). Tritiated norepinephrine release from brain stores by electrical stimulation. Science, N. Y. 152: 1630-1631. BEDARD,P., LAROCHELLE, L., PARENT,A. and POIRIER,L. J. (1969). The nigro-striatal pathway: a correlative study based on neuroanatomical and neurochemical criteria in the cat and the monkey. Expl Neural. 25: 365-377.

Release of dopamine from brain

741

BERMAN,A. L. (1968). The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates. The University of Wisconsin Press, Madison. BLOOM,F. E., COSTA,E. and SALMOIRAGHI, G. C. (1965). Anesthesia and the responsiveness of individual neurons in the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine administered by microelectrophoresis. J. Pharmac. exp. Ther. 150: 244252. CARR, L. A. and MOORE,K. E. (1969). Distribution and metabolism of norepinephrine after its administration into the cerebroventricular system of the cat. Biochem. Pharmac. 18: 1907-1918. CARR, L. A. and MOORE, K. E. (1970). The effects of amphetamine on the contents of norepinephrine and its metabolites in the effluent of perfused cerebral ventricles of the cat. Biochem. Pharmac. 19: 2361-2374. CHASE, T. N. and KOPIN, I. J. (1968). Stimulus induced release of substances from olfactory bulb using the push-pull cannula. Nature, Lond. 217: 464469. CONNOR, J. D. (1968). Caudate unit responses to nigral stimuli: Evidence for a possible nigro-striatal pathway. Science, N. Y. 160:899-900. FAULL, R. L. M. and LAVERTY,R. (1969). Changes in dopamine levels in the corpus striatum following lesions in the substantia nigra. Expl Neurol. 23: 332-340. GOLDSTEIN,M., ANAGNOSTE,B., BATTISTA,A. F., OWEN, W. S. and NAKATANI,S. (1970). Studies of amines in striatum in monkeys with nigral lesions. The disposition, biosynthesis and metabolites of H3-dopamine and C14-serotonin in the striatum. J. Neurochem. 16: 645-654. HGKFELT,T. and UNGERSTEDT,V. (1969). Electron and fluorescence microscopical studies on the nucleus caudatus putamen of the rat after unilateral lesions of ascending nigro-neostriatal dopamine neurons. Actaphysiol.

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HORNYKIEWICZ,0. (1966). Dopamine (3-hydroxytyrarnine) and brain function. Pharmac. Rev. 18: 925-964. KOPIN, I. J., BREESE,G. R., KRAUSS,K. R. and WEISE,V. K. (1968). Selective release of newly synthesized norepinephrine from cat spleen during sympathetic nerve stimulation. J. Pharmac. exp. Ther. 161: 271-278.

MCKENZIE, G. M. and SZERB, J. C. (1968). The effect of dihydroxyphenylalanine, pheniprazine and d-amphetamine on the in vivo release of dopamine from the caudate nucleus. J. Pharmac. exp. Ther. 162: 302-308.

MCLENNAN, H. (1964). The release of acetylcholine and 3-hydroxytyramine from the caudate nucleus. J. Physiol., Lond. 174: 152-161. MCLENNAN,H. (1965). The release of dopamine from the putamen. Experientia 21: 725-726. PAPPENHEIMER, J. R., HEISEY, S. R., JORDAN, E. F. and DOWNER, J. (1962). Perfusion of the cerebroventricular system in unanesthetized goats. Am. J. Physiol. 203: 763-774. POIRIER,L. J. and SOURKES,T. L. (1965). Influence of the substantia nigra on catecholamine content of striatum. Brain 88: 181-192. PORTIG,P. J. and VOGT, M. (1969). Release into the cerebral ventricles of substances with possible transmitter function in the caudate nucleus. J. Physiol., Lond. 204: 687-715. SNIDER,R. S. and NIEMER,W. T. (1961). A Stereotaxic Atlas ofthe Cat Brain. University of Chicago Press, Chicago. VOGT, M. (1969). Release from brain tissue of compounds with possible neurotransmitter function: Interaction of drugs with these substances. Br. J. Pharmac. Chemother. 37: 325-337. VON VOIGTLANDER, P. F. and MOORE,K. E. (1971). In vivo electrically evoked release of H%oradrenaline from cat brain. J. Pharm. Pharmac. 23: 381-382.