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Stimulation-Induced Release of Serotonin
Stimulation-Induced Release of Serotonin THOMAS N. CHASE,GEORGER. BREESE, DAVID0. CARPENTER, SAULM. SCHANBERG, AND IRWIN J. KOPIN Laboratory of Clinical Science and Laboratory of Neurophysiology, National Institute of Mental Health, Bethesda, Maryland
Int rod uct io n Considerable evidence has accumulated during the past decade which suggests that 5-HT may serve as a transynaptic mediator in a wide variety of species. This contention is supported by the high concentration of this amine within certain neurons together with the enzymes for its synthesis and inactivation (Bogdanski et al., 1957; Fuxe, 1965; Garattini and Valzelli, 1965), its storage within synaptic vesicles (Michaelson and Whittaker, 1963; Maynert and Kuriyama, 1964), its high rate of turnover in nervous tissue (Brodie et al., 1958; Udenfriend and Weissbach, 1958), and the effects of its microelectrophoretic application upon spontaneous and stimulated neuronal activity (Krnjevi6 and Phillis, 1963; Gerschenfeld and Tauc, 1964; Engberg and Ryall, 1966; Roberts and Straughan, 1967). Furthermore, biochemical and fluorescent histochemical evidence suggesting the release of neuronal 5-HT by electrical stimulation has recently been reported ( A n d h et al., 1964; Dahlstrom et aE., 1965; S.-R6zsa and PerBnyi, 1966; Aghajanian et al., 1967). I n this paper, studies on the stimulus-induced release of exogenous 5-HT from molluscan tissues in vivo or in vitro and from mammalian brain in vitro are described. These data provide additional support for the hypothesis that 5-HT may act as a transynaptic mediator.
Studies in the Aplysia Serotonin has been identified in the nervous system and other tissues in many classes of mollusks (Welsh and Moorhead, 1960; Dahl et al., 1962; Kerkut and Cottrell, 1963), together with the enzymes for its synthesis and metabolism (Cardot, 1964; Blaschko and Hope, 1957; Blaschko and Milton, 1960). Furthermore, 5-HT exerts a strong depolarizing and excitatory action upon specific molluscan neurons (Kerkut and Walker, 1962; Gerschenfeld and Stefani, 1966), while such serotonin antagonists as lysergic acid diethylamide (LSD) and bromolysergic acid diethylamide (BOL) block the spontaneous excitatory synaptic potentials as well as the effect of iontophoretically applied 5-HT on these neurons (Gerschenfeld and Tauc, 1964; Gerschenfeld and Stefani, 1966). The suggestion by Bacq and co-workers (1952) 35L
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KOPIN
that 5-HT may function in the cardioregulatory system of mollusks has been supported by the observation that the application of 5-HT excites the heart (Erspamer and Ghiretti, 1951; Bacq et al., 1952; Welsh, 1953) and that BOL and LSD block both the effect of 5-HT application (Welsh, 1955; Welsh and McCoy, 1957; Greenberg, 1960; Wright et al., 1962) and stimulation of the excitatory nerves to the heart (S.-R6zsa and Graul, 1964). Furthermore, S.-R6zsa and Perenyi (1966) have recently identified 5-HT in the cardiac perfusate of Helix following nerve stimulation. The present study on the sea hare, Aplysia californica, was done to obtain further evidence of the neurohumoral role of 5-HT in mollusks. ENDOGENOUS SEROTONIN CONTENT Serotonin in heart and nervous tissue of the Aplysia (Table I) was assayed by a modification of the method of Snyder and co-workers (1965).Moderately TABLE I SEROTONIN CONTENT IN VARIOUS TISSUESOF Aplysiq
Heart
a b
Auricle Ventricle Crista aortae
3 4 2
1.61 f 0.31 0.80 f 0.20 Traceb
Ganglia Visceral Pleural and pedal Cerebral Buccal
6 4 3 2
3.37 f 0.28 1.73 f 0.18 2.61 =k 0.06 Trace*
Nerves
6 4
0.63 f 0.11 2.66 f 0.47
Connective Posterior parapodial
Number of determinations on two to ten pooled tissues. Fluorescence spectrum differs from authentic 6-HT.
high concentrations of this amine were fotind in the auricle, posterior parapodia1 nerve, and visceral ganglion. Approximately equal amounts of 5-HT were found in each half of 'the visceral ganglion, but no significant amount was detected in the bag regions which contain neurosecretory cells (Kupfermann et al., 1966) but few synapses. Extracts of the crista aortae, a noncontractile ventricular appendage, and from the buccal ganglion contained no 5-HT. Serotonin was also identified in extracts of Aplysia tissue by native fluorescence spectra and chromatography.
STIMULATION-INDUCEDRELEASE
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OF SEROTONIN
ACCUMULATION OF EXOGENOUS SEROTONIN The capacity for heart and nervous tissue to accumulate 5-HT (Table 11) was examined by incubation for 30 minutes a t room temperature in artificial seawater (Lambert Kay Co., Los Angeles) containing 500 mpcuries/ml of tritiated 5-HT (specific activity, 2.1 curies/mM). The auricle attained the TABLE I1 SEROTONM-3HACCUMULATZQN IN VARIOUS TISSWES OF
Tissue Heart
N0.b
Auricle Ventricle Crista eortae
6 7
3 6
Ganglia Visceral Periesophageald Nerves
Connective Posterior parapodia1
THE
ApEysiaP
Accumulation ratioc 8.85 f 0.66 2.90 f 0.42 1.26 f 0.16
7
2.38 f 0.30 1.68 f 0.08
6 6
3.03 f 0.10 3.08 f 0.11
Tissues incubated for 30 minutes in artificial seawater at 25%. Number of determinations on two to six pooled tissues. C Accumulation ratio is the tissue: medtnm ratio. b t & - s H comxmtwtim in the medium was 500 mpcdeslml. d Periesophageal includes pleural and pedd and cerebral genglia. b
highest accumulation ratio of any of the tissues tested. The crista aortae, which contained no detectable endogenous 5-HT, failed to accumulate a significant amount of the tritiated amine. On the other hand, both the connective and posterior parapodial nerves accumulated the same amount of tritiated 5-HT, despite the marked differences in their endogenous 5-HT content (Table I). SEROTONIN
RELEASE IN
VITRO
The ability of electrical field stimulation to release labeled 5-HT was tested in the heart, ganglia, and nerve bundles. After incubation with tritiated 5-HT, the tissues were transferred to individual perfusion chambers, placed between platinum coil electrodes and superfused with fresh artificial seawater a t room temperature. Electrical field stimulation such as employed in these experiments, is known to produce depolarization of cell membranes (Hillman et al., 1963) and has recently been shown to release radioactive catecholamines from brain slices and other tissues which have an intact
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CHASE, BREESE, CARPENTER, SCWNBERG, AND KOPIN
sympathetic innervation (Baldessarini and Kopin, 1967). The superfusate was collected during the sequential 2-minute intervals and total tritium (tritiated 5-HT plus its metabolites) determined. The application of a 6 - ~ 0 l t , 20-impulse per second rectangular stimulus for 1 minute to isolated atria resulted in a rapid and significant increase in the efflux of total tritium (Fig. 1). During stimulation, no significant change in temperature or pH of the medium occurred. Superfusion with calcium-free seawater significantly reduced the release incident to electrical stimulation (Fig. 1).
FIQ.1. Release of d o a c t i v i t y from the Aplyaia atrium during electrical field stimulation. %ma were incubated for 30 minutes in artificial seawater containing 300 mpcof ~mtonin-W/ml. Six-volt, 20-impulseper second pulsatile stimulation was applied for 1 minute. Mean values for tritium efflux during superfusion with artificial seawater a d calcium-free,artificial seawater are given.
Stimulus-induced release of radioactivity after incubation with tritiated 5-HT has also been obtained from Splysia ventricle and visceral ganglion, both richly endowed with nerve terminals, while none could be demonstrated from the nerve fibers comprising the connective nerves. $&ROTONIN
RELEASE BY NERVE STIMULATION
Since atria concentrated b b e b d 5-HT to the greatest extent, presumably within nerve endings present in this structure, release of the tritiated 5-HT by remote nerve stimulation was attempted. The Aplysia heart was perfused
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STIMULATION-INDUCED RELEASE O F SEROTONIN
in situ by means of a cannula inserted into the branchial vein. Collections were made from a second cannula introduced into the ventricle. Tritiated 5-HT (500 mpcuries/ml of seawater) was circulated through the system for 15 minutes prior to each experiment. The heart was then washed with fresh seawater until the radioactivity in the perfusate reached a low level (about 60 minutes). The connective nerves to the visceral ganglion were then sbimulated with a 30 ma, 4/msec, 40 impulse/second rectangular current for 1
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FIG.2. Release of radioactivity by nerve stimulation and by LSD from the Aplysia heart perfused in situ. The heart WES perfused with seawater containing 5-HT-3H (600 mpcuries/ml) prior to each experiment and then washed (W) with fresh seawater. A 30-milliampere, 40-impulse per second stimulus was applied to the connective nerves to the visceral ganglion for 1 minute at the times indicated ( S ) .
minute. Such stimulation usually resulted in a marked initial increase in heart rate and amplitude together with a 4-fold rise in tritium eflux (Fig. 2). The addition of BOL (lo-' M ) to the perfusate blocked the excitatory effect of nerve stimulation upon the heart, but did not significantly alter the release of tritiated 5-HT produced by nerve stimulation. This result is consistent with the observation that BOL may block the excitatory postsynaptic potentials of 5-HT-sensitive cells in the Aplysia (Gerschenfeld and Tauc, 1964). LSD a t a concentration of 2 x M , on the other hand, produced a
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CHASE, BREESE, CARPENTER, SCHANBERO, AND KOPIN
large, spontaneous efflux of radioactivity, together with an increase in the amplitude and rate of cardiac contraction (Fig. 2). These findings suggest that 5-HT may mediate the stimulatory action of LSD upon the Aplysia heart. The release of radioactivity following nerve stimulation was substantially reduced in the presence of this concentration of L S D , and was totally abolished by higher concentrations. After washing with fresh seawater for 30 minutes, radioactivity could again be released by nerve stimulation (Fig. 2).
Studies in Rat Brain Slices Mammalian brain slices, when incubated in a suitable medium, maintain their bioelectrical and biochemical activity for several hours (Quastel and Quastel, 1961; McIlwain, 1966; Yamamoto and McIlwain, 1966). Such slices have been shown to accumulate labeled 5-HT and its precursors (Schanberg, 1963; Smith, 1963). There is convincing radioautographic evidence that tritiated 5-HT, when injected into the cerebral ventricles, is concentrated within certain nerve endings rich in endogenous 5-HT (Aghajanian et al., 1966; Aghajanian and Bloom, 1967). Intraventricularly administered radioactive 5-BT appears to displace endogenous 5-HT from its subcellular binding sites (Palaid et ad.,1967) and to be highly localized to a synaptosomal fraction prepared by centrifugation in a sucrose density gradient (Eccleston and Axelrod, personal communication). Preliminary observations in our laboratory indicate that part of the labeled 5-HT taken up by brain slices is also in the synaptosomal fraction. Nerve ending pacticles and synaptic vesicles isolated from cerebral tissue have also been shown to accumulate exogenous 5-HT (Maynert and Kuriyama, 1964; Robinson et al., 1965; Marchbanks, 1966). These observations suggest that exogenous 5-HT, a t least in part, enters 5-HT stores in brain and provides the basis for the present studies on the uptake and stimulation-induced release of labeled 5-HT in slices prepared from various regions of the rat brain and other organs.
ACCUMULATION OF LABELED SEROTONIN Unanesthetized, adult Sprague-Dawley rats were used throughout these experiments. The animals were killed by cervical fracture and their brains quickly removed. Either midcoronal slices of whole brain (160-200 mg) or 3 mm diameter slices (16-20 mg) from several regions of brain were prepared. Incubation was carried out a t 37°C in a Krebs-Ringer bicarbonate medium supplemented with glutamate, pyruvate, and fumarate (Krebs, 1960) and saturated with 5% carbon dioxide in oxygen. I n some experiments, Trowell’s T-8 medium was employed (Trowell, 1955). Fresh coronal slices or small slices (3 mm diameter) from specific areas
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STIMULATION-INDUCED RELEASE OF SEROTONIN
were incubated €or 30 minutes in a medium containing 800 mpcuries/ml of tritiated 5-HT. Coronal slices attained a concentration of radioactivity 2.3 times that of the medium (Fig. 3). Little or no radioactivity was accumulated by coronal slices subjected to postmortem autolysis or freezing and thawing prior to incubation.
T
FRESH
6 HOURS POST MORTEM
FROZEN AND THAWED
FIQ. 3. Accumulation of radioactivity by rat brain in vitro. Midcoronal slices were incubated with 800 mpcuries of 5-HT-3H per milliliter for 30 minutes at 37°C. Results are the mean values for four slices f SEM.
Significant regional differences in accumulation of the exogenous amine were demonstrated (Table 111). While cerebellar folia failed to concentrate tritiated 5-HT, appreciable accumulations of labeled 5-HT were attained TABLE I11 AND RELEASE OF TRITIUM FROM ACCUMULATION SLICES O F VARIOUS REGIONS O F RATBRAININCUBATED WITH SEROTONIN-3H'
Tritium Accumulation ratio* Electrically induced release= Percent of residual releasedd
Cerebellar folia
Frontal cortex
2.9 i 0.2 1.1 7tO.06 0.046 i 0.009 0.45 f 0.1 22 f 3 7&1
Corpus striatum
HYPOthalamus
3 . 2 i 0.3
3.5rt0.2
0.45 C! 0.1 23 f 3
0.47 f 0.09
16 k 2
Slices incubated in serotonin-3H (300 mpcuries/ml for 30 minutes. Accumulation ratio is the tissue : bath ratio. Results are the mean values for four or more slices + SEM. c Stimulus ( 8 v, 60 cps) applied for 1 minute. Results are the mean values for four or more slices f SEM, expressed as mpcurieslmin. Results are the means f SEM. a
b
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CHASE, BREESE, CARPENTER, SCHANBERO, AND KOPIN
by slices of frontal cortex, striatum, and hypothalamus. The accumulation of exogenous 5-HT appeared to be related to the endogenous concentration (Garattini and Valzelli, 1965) of the amine in each of the regions tested. The proportion of accumulated tritium accounted for by 5-HT and its deaminated metabolites was estimated by extraction into a salt-saturated toluene-isoamyl alcohol mixture a t pH 10 and into ether a t p H 1, respectively. The ratio of 5-HT to deaminated products in striatal slices a t the end of 30 minutes' incubation was found to be about 3:2.
RELEASE OF EXOGENOUS SEROTONIN Following incubation, slices were transferred to individual perfusion chambers through which fresh, oxygenated medium was circulated a t 37 "C. The effluent was assayed for total tritium, tritiated 5-HT and its deaminated 20 16 t ._
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FIG.4. Efflux of radioactivity from rat brain slices after incubation with 5-HT-3H. Slices were incubated with 900 mpcuries of 5-HT-3H/ml.After perfusion for 16 minutes, a 6-v, 60 cps sinusoidal stimulus was applied for 1 minute. Results are the mean values for four midcoronal slices f SEM.
metabolites as previously described. Efflux of radioactivity followed a multiphasic course during 30 minutes of superfusion (Fig. 4). After 20 minutes of superfusion, approximately 23% of the initially accumulated tritium was retained by the slice. This is in contrast with the results obtained with tritiated NE, where more than 80% of the radioactivity was retained by slices under similar conditions (Baldessarini and Kopin, 1967). Presumably, washout of the less specifically bound and more rapidly metabolized 5-HT and of
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STIMULATION-INDUCED RELEASE O F SEROTONIN
its metabolites is favored during this period. Sixteen to 22 minutes after the initiation of superfusion, the application of a 6-v, 60 cps sinusoidal alternating stimulus for 1 minute resulted in a rapid, but brief, increase in tritium efflux. Approximately 20% of the remaining tritium was released during such stimulation. I n some experiments, brain slices were prepared from rats which had received an intracisternal injection of tritiated 5-HT while under light ether anesthesia. After 15 minutes, when fully recovered from the anesthesia, the rats were killed and brain slices prepared in the usual manner. The electrically induced release of radioactivity from slices under these conditions was similar to that obtained from slices incubated with tritiated 5-HT in vitro.
Radioactive Compounds in Efluent
A t the beginning of superfusion, tritiated 5-HT accounted for approximately 44% of the total efflux of 5-HT and its deaminated products (Table IV). After 20 minutes of superfusion, just prior to stimulation, the proportion of 5-HT in the tritium efflux decreased to about 35%. Electrical stimulation (8-v, 60 cps sinusoidal current for 1 minute) of striatal tissues resulted in a sevenfold rise in the efflux of tritiated 5-HT, while the efflux of deaminated metabolites increased only 2.4 times. A threefold rise in the release of tritiated 5-HT without a significant change in the deaminated metabolites attended the stimulation of cerebellar slices. TABLE IV EFFECT
OF
ELECTRICAL STIMULATION O N EFFLUXOF 5-HYDROXYTRYPTAMINE-3H AND 5-HYDROXYINDOLEACETIC ACID-~H FROM RATBRAINSLICES" Time (minutes)
Tissue and Metabolite
4-6
20-22
Striatum 5-HT-3H 5-HIAA-3H
0.45 + 0.05 0.56 f 0.05
0.09 0.01 0.16 f 0.02
0.65 f 0.2 0.39 f 0.07
Cerebellum 5-HT-3H 5-HIAA-3H
0.18 f 0.02
0.03 f 0.006 0.06 f 0.006
0.09 f 0.01 0.07 f 0.01
0.24
+ 0.01
*
22-24b
a Slices incubated with 5-HT-3H (500 mpcuries/ml) for 30 minutes prior to superfusion. Results are expressed as mpcurieslmin and are the mean values for eight slices + SEM. * Stimulus ( 8 v, 60 cps) applied between the twenty-secondand twenty-thirdminutes after beginning superfusion.
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CHASE, BREESE, CARPENTER, SCHANBERG, AND KOPIN
The electrically induced release (the rise in tritium efflux during the 2minute interval which includes stimulation, above the level of the preceding 2 minutes) was found to vary with (1) surface area and viability of the slice, (2) concentration of 5-HT in the incubation medium, (3) parameters of the electrical stimulus, (4) origin of tissue tested, and (5)ionic composition of the superfusing medium.
Effect of Tissue Viability Stimulus-induced release (6-17, 60 cps, sinusoidal current applied for 1 minute) from slices prepared from brain left in situ for 4 hours postmortem, while still significant, was less than one-half that found in fresh control slices (Fig. 5). No significant release could be obtained from slices left in situ
T
FRESH
BEFORE
STI M UL ATlON
4 hours POST MORTEM
FROZEN AND THAWED
FIG.5 . The effect of tissue viability on the efflux of radioactivity from brain slices after incubation with serotonin-3H. All slices were incubated with 900 mpcuries 5-HTsH/ml. One group was prepared from brain left in situ for 4 hours after death. Freezing and thawing was carried out after incubation in another group of slicea. A 6-v, 60 cpa sinusoidal current was applied for 1 minute. Results are the mean values of tritium efflux for three or more midcoronal slices f SEM before and during stimulation.
€or more than 6 hours postmortem. Similarly, no significant alteration in tritium efflux with electrical stimulation was observed in slices which had been subjected to freezing and thawing after incubation but prior to superfusion.
Regional Differences in Release I n view of the well-established regional differences in the endogenous 5-HT content of the brain (Fuxe, 1965; Garattini and Valzelli, 1965) and in the
361
STIMULATION-INDUCED RELEASE OF SEROTONIN
corresponding variations in the accumulation of exogenous 5-HT found in the present study (Table 111),regional differences in the electrically induced release might also be expected. Spontaneous and stimulationinduced (8-v, 60 cps sinusoidal current applied for 1 minute) tritium efflux from cerebellar slices was only one-third to one-half that obtained from slices of striatum or hypothalamus (Fig. 6). Similarly, the electrically induced release from cerebellar slices was a smaller fraction of the spontaneous efflux and only one-fourth to one-sixth in absolute amount of that obtained from the other regions studied. There were also significant differences between the frontal cortex and hypothalamus in the spontaneous efflux of tritium as well as in its electrically induced release. The striatum and hypothalamus had
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FIG.6. Effect of electrical stimulation on the efflux of radioactivity from slices. Various regions of rat brain were incubated with 5-HT-3H (300 mpcuries 5-HT-3H/ml) and stimulated (8-v, 60 cps sinusoidal current) as previously described. The results are the mean for four or more 20-mg slices f SEM.
the largest electrically induced release of 5-HT of any of the areas tested. This finding is consistent with the high density of serotonergic nerve terminals known to be present in these regions (Fuxe, 1965; Anden et al., 1966). Both accumulation and release of 5-HT from brain slices appeared related to the regional concentration of endogenous 5-HT. However, the amount of electrically induced release did not appear to be a simple funcbion of the amount of 5-HT taken up. While the accumulation ratio for the cerebellar folia was about one-third that of the other regions tested, the stimulationinduced release was only one-tenth that of the other areas (Table 111). Furthermore, the ratio of electrically released radioactivity to residual radioactivity a t the time of stimulation was only one-half to one-third that of the other areas studied. Nonspecific uptake may account for these differences.
362
CHASE, BREESE, CARPENTER, SCIEANBERO, AND KOPIN
Drug and Ion Effects Raising the concentration of potassium to 40 mM resulted in a rapid increase in the eMux of radioactivity from both striatal and cerebellar slices (Fig. 7). This increase was quantitatively similar to that produced by electrical stimulation, and is consistent with the well-known effect of high potassium concentrations to depolarize neuronal membranes (Hillman and McIlwain, 1961). I
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FIG.7. Effect of increased potrtssium concentration in the superfusate on the efflux of radioactivity from brain slices previously incubated with 5-HT-3H.Slices were incubated with 320 mpcuries 5-HT-3H/ml. After 24 minutes of superfusion with Krebs-Ringer solution, the medium was changed to one containing 40 mM of K+. The results are the means for three slices f SEM.
M ) to the superfusing medium reduced the The addition of LSD (2 x stimulation-induced (8-v, 60 cps sinusoidal current for 1 minute) release of radioactivity from striatal slices to 27% f 3% (p< .001) that of control slices. The rise in tritiated 5-HT produced by stimulation was sevenfold in control slices and only 2.5-fold in the LSD-treated tissues. There was no significant difference in the rise in deaminated metabolites during stimulation between the control and the LSD-treated slices. Effect of Electrical Stimulation on Inert Substances As a test of the specificity of 5-HT release obtained in the present experiments, the release of tritiated water and urea-I4C wry attempted under the same conditions. None could be detected. These findings suggest that the stimulus-induced release obtained under the conditions of these experiments is not merely a nonspecific reflection of the ionic migrations and metabolic changes induced by field stimulation.
STIMULATION-INDUCED RELEASE OF SEROTONIN
363
Studies in Extracerebral Tissues of the Rat Accumulation and release of exogenous 5-HT from several extracerebral organs were studied in the same manner as brain slices. Both the liver and salivary gland concentrated only small amounts of tritiated 5-HT. The electrically induced release from these organs was only one-tenth that obtained from brain tissue other than cerebellum. The substantially greater accumulation and release of 5-HT from cerebral tissues is consistent with the view that exogenous 5-HT is concentrated in and mixes with endogenous stores within nerve terminals in the brain and can be liberated from these sites by depolarizing stimuli. The relatively small uptake and release of 5-HT from the cerebellum, liver, and salivary gland suggests a paucity of serotonergic terminals in these structures.
Conc Iusions Serotonin satisfies several important criteria for a neurotransmitter in the cardio-acceleratory system of the Aplysia. It is present in nervous tissue, it is releasable by nerve stimulation, and its local administration is accompanied by the same cardiotonic effect as nerve stimulation. Furthermore, pharmacological antagonists, in concentrations which block the effect of 5-HT, also block the effect of nerve stimulation. Slices of mammalian brain accumulate exogenous 5-HT which is releasable by procedures which depolarize neuronal membranes. Release is not directly related to the amount accumulated, but is considerably greater in tissues where the 5-HT is presumably stored within nerve endings rather than in extraneuronal sites. Release by electrical field stimulation is dependent upon the viability of the tissue studied. These findings, which are similar to the results obtained with NE, support the contention that 5-HT may serve as a synaptic mediator in the mammalian central nervous system. REFERENCES Aghajanian, G. K., and Bloom, F. E. (1967) J . Phamnacol. Exptl. Therap. 156, 23. Aghajanian, G. K., Bloom, F. E., Lovell, R. A., Sheard, M. H., and Freedman, D. X. (1966). Biochem. PharmacoE. 15, 1401. Aghajanian, G. K., Roscrans, J. A., and Sheard, M. H. (1967).Science, 156, 402. AndBn, N.-E., Carlsson, A., Hillarp, N.-A., and Magnusson, T. (1964). Life Sci. 3, 473. And&, N.-E., Dahlstrom, A., Fuxe, K., Larsson, K., Olson, L., and Ungerstedt, U. (1966). Acta Phyeiol. S c a d . 67, 313. Bacq, Z., Fischer, P., and Ghiretti, F. (1952). Arch. Intern. Physiol. 60, 165. Baldessarini, R. J., and Kopin, I. J. (1967).J . Phamzacol. Exptl. Therap. 156, 31. Blaschko, H., and Hope, D. B. (1957). Arch. Biochem. Biophys. 69, 10. Blaschko, H., rmd Milton, A. S. (1960). Brit. J . Phamacol. 15, 42.
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Bogdanski, D. F., Weissbach, H., and Udenfriend, S. (1957).J. Neurochem. 1, 272. Brodie, B. B., Spector, S., Kuntzman, R. G., and Shore, P. A., (1958).Naturwissenschajten 45, 243. Cardot, J. (1964).Contpt. Rend. 258, 1103. Dahl, E., Falck, B., Lundquist, M., and Mecklenburg, I. (1962).Kgl. Fysiograj. SaZlskap. Lund, Forh. 32, 89. Dahhtrom, A., Fuxe, K., Kernell, D., and SedvalI, G. (1965).Life Sci. 4, 1207. Engberg, I., and Ryall, R. W. (1966).J. Physiol. (London) 185, 298. Erspamer, V., and Ghiretti, F. (1951).J. Physiol. (London) 115,470. Fuxe, K. (1965). Acta Physiol. Scand. Suppl. 274, 39. Garattini, S., and Valzelli, L. (1965).“Serotonin,” Elsevier, Amsterdam. Gerschenfeld, H. M., and Stefani, E. (1966).J. Physiol. (London), 185, 684. Gerschenfeld, H. M., and Tsuc, L. (1964).J. Physiol. (Paris)56, 360. Greenberg, H. M. (1960).Brit. J. Pharmacol. 15,365. Hillman, H. H.,and McIlwain, H. (1961).J. Physiol. (London) 157, 263. Hillman, H. H., Campbell, W. J., and McIlwain, H. (1963).J. Neurochem. 10, 325. Kerkut, G. A., and Cottrell, G. A. (1963).Comp. Biochem. Physiol. 8, 63. Kerkut, G. A., and Walker, R. J. (1962).Contp. Biochem. Physiol. 7 , 277. Krebs, H.A. (1950).Biochim. Biophys. Acta 4, 249. Krnjevid, K., and Phillis, J. W. (1963).Brit. J . P h a m c o l . 20, 471. Kupfermann, I., Kandel, E., and Coggeshall, R. (1966). Physiologist 9, 223. McIlwain, H. (1966).“Biochemistry and the Central Nervous System.” Little, Brown, Boston, Massachusetts. Marchbanks, R. M. (1966).J. Neurochem. 13, 1481. Maynert, E. W., and Kuriyama, K. (1964).Life Sci. a, 1067. Michaelson, I. A., and Whittaker, V. P. (1963).Biochem. Pharmacol. 12, 203. Palaid, D., Page, I. H., and Khairallah, P. (1967).J . Neurochem. 14, 63. Quastel, J. H.,and Quastel, D. M. J. (1961). “The Chemistry of Brain Metabolism in Health and Disease.” Thomas, Springfield, Illinois. Roberts, M. H. T., and Straughan, D. W. (1967).J. Physiol. (London) 188, 27P. Robinson, J. D., Anderson, J. H., and Green, J. P. (1965).J . Pharmacol. Exptl. Therap. 147, 236. Schanberg, S. M. (1963).J. Pharmacol. Ezptl. Therap. 139, 191. Smith, S. E. (1963).Brit. J. Pharmacol. 20, 178. Snyder, S. H., Axelrod, J., and Zweig, M. (1965).Biochem. Pharmacol. 14, 331. S.-R6zsa, K.,and Graul, C. (1964).Ann. B i d . Tihany 31, 85. S.-R6zsa, K., and Perbnyi, L. (1966).Comp. Biochem. Physiol. 19, 105. Trowell, D. A. (1955).Exptl. CelE Res. 9, 258. Udenfriend, S., and Weissbach, H. (1958).Proc. SOC.Ezptl. Biol. Med. 97, 748. Welsh, J. H.(1953).Arch. Exptl. Pathol. Pharmakol. Nauynn-Schmiedebergs 219, 23. Welsh, J. H.(1955).I n “The Hormones” (G. Pincus and K. V. Thimann, eds.), Vol. 3, p. 97. Academic Press, New York. Welsh, J. H., and McCoy, A. C. (1957).Science 125, 348. Welsh, J. H.,and Moorhead, M. (1960).J . Neurochem. 6,146. Wright, A. McC., Moorhead, M., and Welsh, J. H. (1962).Brit. J. Pharmacol. 18, 440. Yamamoto, C., and McIlwain, H. (1966).3. Neurochem. 13, 1333.
Int rod uct io n Considerable evidence has accumulated during the past decade which suggests that 5-HT may serve as a transynaptic mediator in a wide variety of species. This contention is supported by the high concentration of this amine within certain neurons together with the enzymes for its synthesis and inactivation (Bogdanski et al., 1957; Fuxe, 1965; Garattini and Valzelli, 1965), its storage within synaptic vesicles (Michaelson and Whittaker, 1963; Maynert and Kuriyama, 1964), its high rate of turnover in nervous tissue (Brodie et al., 1958; Udenfriend and Weissbach, 1958), and the effects of its microelectrophoretic application upon spontaneous and stimulated neuronal activity (Krnjevi6 and Phillis, 1963; Gerschenfeld and Tauc, 1964; Engberg and Ryall, 1966; Roberts and Straughan, 1967). Furthermore, biochemical and fluorescent histochemical evidence suggesting the release of neuronal 5-HT by electrical stimulation has recently been reported ( A n d h et al., 1964; Dahlstrom et aE., 1965; S.-R6zsa and PerBnyi, 1966; Aghajanian et al., 1967). I n this paper, studies on the stimulus-induced release of exogenous 5-HT from molluscan tissues in vivo or in vitro and from mammalian brain in vitro are described. These data provide additional support for the hypothesis that 5-HT may act as a transynaptic mediator.
Studies in the Aplysia Serotonin has been identified in the nervous system and other tissues in many classes of mollusks (Welsh and Moorhead, 1960; Dahl et al., 1962; Kerkut and Cottrell, 1963), together with the enzymes for its synthesis and metabolism (Cardot, 1964; Blaschko and Hope, 1957; Blaschko and Milton, 1960). Furthermore, 5-HT exerts a strong depolarizing and excitatory action upon specific molluscan neurons (Kerkut and Walker, 1962; Gerschenfeld and Stefani, 1966), while such serotonin antagonists as lysergic acid diethylamide (LSD) and bromolysergic acid diethylamide (BOL) block the spontaneous excitatory synaptic potentials as well as the effect of iontophoretically applied 5-HT on these neurons (Gerschenfeld and Tauc, 1964; Gerschenfeld and Stefani, 1966). The suggestion by Bacq and co-workers (1952) 35L
352
CHASE,
BREESE,
CARPENTER,
SCHANBERG, AND
KOPIN
that 5-HT may function in the cardioregulatory system of mollusks has been supported by the observation that the application of 5-HT excites the heart (Erspamer and Ghiretti, 1951; Bacq et al., 1952; Welsh, 1953) and that BOL and LSD block both the effect of 5-HT application (Welsh, 1955; Welsh and McCoy, 1957; Greenberg, 1960; Wright et al., 1962) and stimulation of the excitatory nerves to the heart (S.-R6zsa and Graul, 1964). Furthermore, S.-R6zsa and Perenyi (1966) have recently identified 5-HT in the cardiac perfusate of Helix following nerve stimulation. The present study on the sea hare, Aplysia californica, was done to obtain further evidence of the neurohumoral role of 5-HT in mollusks. ENDOGENOUS SEROTONIN CONTENT Serotonin in heart and nervous tissue of the Aplysia (Table I) was assayed by a modification of the method of Snyder and co-workers (1965).Moderately TABLE I SEROTONIN CONTENT IN VARIOUS TISSUESOF Aplysiq
Heart
a b
Auricle Ventricle Crista aortae
3 4 2
1.61 f 0.31 0.80 f 0.20 Traceb
Ganglia Visceral Pleural and pedal Cerebral Buccal
6 4 3 2
3.37 f 0.28 1.73 f 0.18 2.61 =k 0.06 Trace*
Nerves
6 4
0.63 f 0.11 2.66 f 0.47
Connective Posterior parapodial
Number of determinations on two to ten pooled tissues. Fluorescence spectrum differs from authentic 6-HT.
high concentrations of this amine were fotind in the auricle, posterior parapodia1 nerve, and visceral ganglion. Approximately equal amounts of 5-HT were found in each half of 'the visceral ganglion, but no significant amount was detected in the bag regions which contain neurosecretory cells (Kupfermann et al., 1966) but few synapses. Extracts of the crista aortae, a noncontractile ventricular appendage, and from the buccal ganglion contained no 5-HT. Serotonin was also identified in extracts of Aplysia tissue by native fluorescence spectra and chromatography.
STIMULATION-INDUCEDRELEASE
353
OF SEROTONIN
ACCUMULATION OF EXOGENOUS SEROTONIN The capacity for heart and nervous tissue to accumulate 5-HT (Table 11) was examined by incubation for 30 minutes a t room temperature in artificial seawater (Lambert Kay Co., Los Angeles) containing 500 mpcuries/ml of tritiated 5-HT (specific activity, 2.1 curies/mM). The auricle attained the TABLE I1 SEROTONM-3HACCUMULATZQN IN VARIOUS TISSWES OF
Tissue Heart
N0.b
Auricle Ventricle Crista eortae
6 7
3 6
Ganglia Visceral Periesophageald Nerves
Connective Posterior parapodia1
THE
ApEysiaP
Accumulation ratioc 8.85 f 0.66 2.90 f 0.42 1.26 f 0.16
7
2.38 f 0.30 1.68 f 0.08
6 6
3.03 f 0.10 3.08 f 0.11
Tissues incubated for 30 minutes in artificial seawater at 25%. Number of determinations on two to six pooled tissues. C Accumulation ratio is the tissue: medtnm ratio. b t & - s H comxmtwtim in the medium was 500 mpcdeslml. d Periesophageal includes pleural and pedd and cerebral genglia. b
highest accumulation ratio of any of the tissues tested. The crista aortae, which contained no detectable endogenous 5-HT, failed to accumulate a significant amount of the tritiated amine. On the other hand, both the connective and posterior parapodial nerves accumulated the same amount of tritiated 5-HT, despite the marked differences in their endogenous 5-HT content (Table I). SEROTONIN
RELEASE IN
VITRO
The ability of electrical field stimulation to release labeled 5-HT was tested in the heart, ganglia, and nerve bundles. After incubation with tritiated 5-HT, the tissues were transferred to individual perfusion chambers, placed between platinum coil electrodes and superfused with fresh artificial seawater a t room temperature. Electrical field stimulation such as employed in these experiments, is known to produce depolarization of cell membranes (Hillman et al., 1963) and has recently been shown to release radioactive catecholamines from brain slices and other tissues which have an intact
354
CHASE, BREESE, CARPENTER, SCWNBERG, AND KOPIN
sympathetic innervation (Baldessarini and Kopin, 1967). The superfusate was collected during the sequential 2-minute intervals and total tritium (tritiated 5-HT plus its metabolites) determined. The application of a 6 - ~ 0 l t , 20-impulse per second rectangular stimulus for 1 minute to isolated atria resulted in a rapid and significant increase in the efflux of total tritium (Fig. 1). During stimulation, no significant change in temperature or pH of the medium occurred. Superfusion with calcium-free seawater significantly reduced the release incident to electrical stimulation (Fig. 1).
FIQ.1. Release of d o a c t i v i t y from the Aplyaia atrium during electrical field stimulation. %ma were incubated for 30 minutes in artificial seawater containing 300 mpcof ~mtonin-W/ml. Six-volt, 20-impulseper second pulsatile stimulation was applied for 1 minute. Mean values for tritium efflux during superfusion with artificial seawater a d calcium-free,artificial seawater are given.
Stimulus-induced release of radioactivity after incubation with tritiated 5-HT has also been obtained from Splysia ventricle and visceral ganglion, both richly endowed with nerve terminals, while none could be demonstrated from the nerve fibers comprising the connective nerves. $&ROTONIN
RELEASE BY NERVE STIMULATION
Since atria concentrated b b e b d 5-HT to the greatest extent, presumably within nerve endings present in this structure, release of the tritiated 5-HT by remote nerve stimulation was attempted. The Aplysia heart was perfused
355
STIMULATION-INDUCED RELEASE O F SEROTONIN
in situ by means of a cannula inserted into the branchial vein. Collections were made from a second cannula introduced into the ventricle. Tritiated 5-HT (500 mpcuries/ml of seawater) was circulated through the system for 15 minutes prior to each experiment. The heart was then washed with fresh seawater until the radioactivity in the perfusate reached a low level (about 60 minutes). The connective nerves to the visceral ganglion were then sbimulated with a 30 ma, 4/msec, 40 impulse/second rectangular current for 1
150-
w 100-
I3
3
f 5
\ v)
I-
z
3
50-
D H
lo-'
S
WLSO
s
2x10"
w
s
3omrutas
5 minutes
FIG.2. Release of radioactivity by nerve stimulation and by LSD from the Aplysia heart perfused in situ. The heart WES perfused with seawater containing 5-HT-3H (600 mpcuries/ml) prior to each experiment and then washed (W) with fresh seawater. A 30-milliampere, 40-impulse per second stimulus was applied to the connective nerves to the visceral ganglion for 1 minute at the times indicated ( S ) .
minute. Such stimulation usually resulted in a marked initial increase in heart rate and amplitude together with a 4-fold rise in tritium eflux (Fig. 2). The addition of BOL (lo-' M ) to the perfusate blocked the excitatory effect of nerve stimulation upon the heart, but did not significantly alter the release of tritiated 5-HT produced by nerve stimulation. This result is consistent with the observation that BOL may block the excitatory postsynaptic potentials of 5-HT-sensitive cells in the Aplysia (Gerschenfeld and Tauc, 1964). LSD a t a concentration of 2 x M , on the other hand, produced a
356
CHASE, BREESE, CARPENTER, SCHANBERO, AND KOPIN
large, spontaneous efflux of radioactivity, together with an increase in the amplitude and rate of cardiac contraction (Fig. 2). These findings suggest that 5-HT may mediate the stimulatory action of LSD upon the Aplysia heart. The release of radioactivity following nerve stimulation was substantially reduced in the presence of this concentration of L S D , and was totally abolished by higher concentrations. After washing with fresh seawater for 30 minutes, radioactivity could again be released by nerve stimulation (Fig. 2).
Studies in Rat Brain Slices Mammalian brain slices, when incubated in a suitable medium, maintain their bioelectrical and biochemical activity for several hours (Quastel and Quastel, 1961; McIlwain, 1966; Yamamoto and McIlwain, 1966). Such slices have been shown to accumulate labeled 5-HT and its precursors (Schanberg, 1963; Smith, 1963). There is convincing radioautographic evidence that tritiated 5-HT, when injected into the cerebral ventricles, is concentrated within certain nerve endings rich in endogenous 5-HT (Aghajanian et al., 1966; Aghajanian and Bloom, 1967). Intraventricularly administered radioactive 5-BT appears to displace endogenous 5-HT from its subcellular binding sites (Palaid et ad.,1967) and to be highly localized to a synaptosomal fraction prepared by centrifugation in a sucrose density gradient (Eccleston and Axelrod, personal communication). Preliminary observations in our laboratory indicate that part of the labeled 5-HT taken up by brain slices is also in the synaptosomal fraction. Nerve ending pacticles and synaptic vesicles isolated from cerebral tissue have also been shown to accumulate exogenous 5-HT (Maynert and Kuriyama, 1964; Robinson et al., 1965; Marchbanks, 1966). These observations suggest that exogenous 5-HT, a t least in part, enters 5-HT stores in brain and provides the basis for the present studies on the uptake and stimulation-induced release of labeled 5-HT in slices prepared from various regions of the rat brain and other organs.
ACCUMULATION OF LABELED SEROTONIN Unanesthetized, adult Sprague-Dawley rats were used throughout these experiments. The animals were killed by cervical fracture and their brains quickly removed. Either midcoronal slices of whole brain (160-200 mg) or 3 mm diameter slices (16-20 mg) from several regions of brain were prepared. Incubation was carried out a t 37°C in a Krebs-Ringer bicarbonate medium supplemented with glutamate, pyruvate, and fumarate (Krebs, 1960) and saturated with 5% carbon dioxide in oxygen. I n some experiments, Trowell’s T-8 medium was employed (Trowell, 1955). Fresh coronal slices or small slices (3 mm diameter) from specific areas
357
STIMULATION-INDUCED RELEASE OF SEROTONIN
were incubated €or 30 minutes in a medium containing 800 mpcuries/ml of tritiated 5-HT. Coronal slices attained a concentration of radioactivity 2.3 times that of the medium (Fig. 3). Little or no radioactivity was accumulated by coronal slices subjected to postmortem autolysis or freezing and thawing prior to incubation.
T
FRESH
6 HOURS POST MORTEM
FROZEN AND THAWED
FIQ. 3. Accumulation of radioactivity by rat brain in vitro. Midcoronal slices were incubated with 800 mpcuries of 5-HT-3H per milliliter for 30 minutes at 37°C. Results are the mean values for four slices f SEM.
Significant regional differences in accumulation of the exogenous amine were demonstrated (Table 111). While cerebellar folia failed to concentrate tritiated 5-HT, appreciable accumulations of labeled 5-HT were attained TABLE I11 AND RELEASE OF TRITIUM FROM ACCUMULATION SLICES O F VARIOUS REGIONS O F RATBRAININCUBATED WITH SEROTONIN-3H'
Tritium Accumulation ratio* Electrically induced release= Percent of residual releasedd
Cerebellar folia
Frontal cortex
2.9 i 0.2 1.1 7tO.06 0.046 i 0.009 0.45 f 0.1 22 f 3 7&1
Corpus striatum
HYPOthalamus
3 . 2 i 0.3
3.5rt0.2
0.45 C! 0.1 23 f 3
0.47 f 0.09
16 k 2
Slices incubated in serotonin-3H (300 mpcuries/ml for 30 minutes. Accumulation ratio is the tissue : bath ratio. Results are the mean values for four or more slices + SEM. c Stimulus ( 8 v, 60 cps) applied for 1 minute. Results are the mean values for four or more slices f SEM, expressed as mpcurieslmin. Results are the means f SEM. a
b
358
CHASE, BREESE, CARPENTER, SCHANBERO, AND KOPIN
by slices of frontal cortex, striatum, and hypothalamus. The accumulation of exogenous 5-HT appeared to be related to the endogenous concentration (Garattini and Valzelli, 1965) of the amine in each of the regions tested. The proportion of accumulated tritium accounted for by 5-HT and its deaminated metabolites was estimated by extraction into a salt-saturated toluene-isoamyl alcohol mixture a t pH 10 and into ether a t p H 1, respectively. The ratio of 5-HT to deaminated products in striatal slices a t the end of 30 minutes' incubation was found to be about 3:2.
RELEASE OF EXOGENOUS SEROTONIN Following incubation, slices were transferred to individual perfusion chambers through which fresh, oxygenated medium was circulated a t 37 "C. The effluent was assayed for total tritium, tritiated 5-HT and its deaminated 20 16 t ._
,E 12
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rn
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xE @ 3
4
6-VOLT
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4
I
I
8
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16 MINUTES
20
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24
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28
FIG.4. Efflux of radioactivity from rat brain slices after incubation with 5-HT-3H. Slices were incubated with 900 mpcuries of 5-HT-3H/ml.After perfusion for 16 minutes, a 6-v, 60 cps sinusoidal stimulus was applied for 1 minute. Results are the mean values for four midcoronal slices f SEM.
metabolites as previously described. Efflux of radioactivity followed a multiphasic course during 30 minutes of superfusion (Fig. 4). After 20 minutes of superfusion, approximately 23% of the initially accumulated tritium was retained by the slice. This is in contrast with the results obtained with tritiated NE, where more than 80% of the radioactivity was retained by slices under similar conditions (Baldessarini and Kopin, 1967). Presumably, washout of the less specifically bound and more rapidly metabolized 5-HT and of
359
STIMULATION-INDUCED RELEASE O F SEROTONIN
its metabolites is favored during this period. Sixteen to 22 minutes after the initiation of superfusion, the application of a 6-v, 60 cps sinusoidal alternating stimulus for 1 minute resulted in a rapid, but brief, increase in tritium efflux. Approximately 20% of the remaining tritium was released during such stimulation. I n some experiments, brain slices were prepared from rats which had received an intracisternal injection of tritiated 5-HT while under light ether anesthesia. After 15 minutes, when fully recovered from the anesthesia, the rats were killed and brain slices prepared in the usual manner. The electrically induced release of radioactivity from slices under these conditions was similar to that obtained from slices incubated with tritiated 5-HT in vitro.
Radioactive Compounds in Efluent
A t the beginning of superfusion, tritiated 5-HT accounted for approximately 44% of the total efflux of 5-HT and its deaminated products (Table IV). After 20 minutes of superfusion, just prior to stimulation, the proportion of 5-HT in the tritium efflux decreased to about 35%. Electrical stimulation (8-v, 60 cps sinusoidal current for 1 minute) of striatal tissues resulted in a sevenfold rise in the efflux of tritiated 5-HT, while the efflux of deaminated metabolites increased only 2.4 times. A threefold rise in the release of tritiated 5-HT without a significant change in the deaminated metabolites attended the stimulation of cerebellar slices. TABLE IV EFFECT
OF
ELECTRICAL STIMULATION O N EFFLUXOF 5-HYDROXYTRYPTAMINE-3H AND 5-HYDROXYINDOLEACETIC ACID-~H FROM RATBRAINSLICES" Time (minutes)
Tissue and Metabolite
4-6
20-22
Striatum 5-HT-3H 5-HIAA-3H
0.45 + 0.05 0.56 f 0.05
0.09 0.01 0.16 f 0.02
0.65 f 0.2 0.39 f 0.07
Cerebellum 5-HT-3H 5-HIAA-3H
0.18 f 0.02
0.03 f 0.006 0.06 f 0.006
0.09 f 0.01 0.07 f 0.01
0.24
+ 0.01
*
22-24b
a Slices incubated with 5-HT-3H (500 mpcuries/ml) for 30 minutes prior to superfusion. Results are expressed as mpcurieslmin and are the mean values for eight slices + SEM. * Stimulus ( 8 v, 60 cps) applied between the twenty-secondand twenty-thirdminutes after beginning superfusion.
360
CHASE, BREESE, CARPENTER, SCHANBERG, AND KOPIN
The electrically induced release (the rise in tritium efflux during the 2minute interval which includes stimulation, above the level of the preceding 2 minutes) was found to vary with (1) surface area and viability of the slice, (2) concentration of 5-HT in the incubation medium, (3) parameters of the electrical stimulus, (4) origin of tissue tested, and (5)ionic composition of the superfusing medium.
Effect of Tissue Viability Stimulus-induced release (6-17, 60 cps, sinusoidal current applied for 1 minute) from slices prepared from brain left in situ for 4 hours postmortem, while still significant, was less than one-half that found in fresh control slices (Fig. 5). No significant release could be obtained from slices left in situ
T
FRESH
BEFORE
STI M UL ATlON
4 hours POST MORTEM
FROZEN AND THAWED
FIG.5 . The effect of tissue viability on the efflux of radioactivity from brain slices after incubation with serotonin-3H. All slices were incubated with 900 mpcuries 5-HTsH/ml. One group was prepared from brain left in situ for 4 hours after death. Freezing and thawing was carried out after incubation in another group of slicea. A 6-v, 60 cpa sinusoidal current was applied for 1 minute. Results are the mean values of tritium efflux for three or more midcoronal slices f SEM before and during stimulation.
€or more than 6 hours postmortem. Similarly, no significant alteration in tritium efflux with electrical stimulation was observed in slices which had been subjected to freezing and thawing after incubation but prior to superfusion.
Regional Differences in Release I n view of the well-established regional differences in the endogenous 5-HT content of the brain (Fuxe, 1965; Garattini and Valzelli, 1965) and in the
361
STIMULATION-INDUCED RELEASE OF SEROTONIN
corresponding variations in the accumulation of exogenous 5-HT found in the present study (Table 111),regional differences in the electrically induced release might also be expected. Spontaneous and stimulationinduced (8-v, 60 cps sinusoidal current applied for 1 minute) tritium efflux from cerebellar slices was only one-third to one-half that obtained from slices of striatum or hypothalamus (Fig. 6). Similarly, the electrically induced release from cerebellar slices was a smaller fraction of the spontaneous efflux and only one-fourth to one-sixth in absolute amount of that obtained from the other regions studied. There were also significant differences between the frontal cortex and hypothalamus in the spontaneous efflux of tritium as well as in its electrically induced release. The striatum and hypothalamus had
-1
0BEFORE STIMULATION DURING STIMULATION ._
T
0.6
t I \
c)
M 04 ._ c
u 2
I0.2 0
CEREBELLUM
HYPOFRONTAL THALAMUS STRIATUM THALAMUS CORTEX
FIG.6. Effect of electrical stimulation on the efflux of radioactivity from slices. Various regions of rat brain were incubated with 5-HT-3H (300 mpcuries 5-HT-3H/ml) and stimulated (8-v, 60 cps sinusoidal current) as previously described. The results are the mean for four or more 20-mg slices f SEM.
the largest electrically induced release of 5-HT of any of the areas tested. This finding is consistent with the high density of serotonergic nerve terminals known to be present in these regions (Fuxe, 1965; Anden et al., 1966). Both accumulation and release of 5-HT from brain slices appeared related to the regional concentration of endogenous 5-HT. However, the amount of electrically induced release did not appear to be a simple funcbion of the amount of 5-HT taken up. While the accumulation ratio for the cerebellar folia was about one-third that of the other regions tested, the stimulationinduced release was only one-tenth that of the other areas (Table 111). Furthermore, the ratio of electrically released radioactivity to residual radioactivity a t the time of stimulation was only one-half to one-third that of the other areas studied. Nonspecific uptake may account for these differences.
362
CHASE, BREESE, CARPENTER, SCIEANBERO, AND KOPIN
Drug and Ion Effects Raising the concentration of potassium to 40 mM resulted in a rapid increase in the eMux of radioactivity from both striatal and cerebellar slices (Fig. 7). This increase was quantitatively similar to that produced by electrical stimulation, and is consistent with the well-known effect of high potassium concentrations to depolarize neuronal membranes (Hillman and McIlwain, 1961). I
I
I
0.6 -
< 0.4 c .-
I
10 UI
.-L
22
26 MINUTES
30
FIG.7. Effect of increased potrtssium concentration in the superfusate on the efflux of radioactivity from brain slices previously incubated with 5-HT-3H.Slices were incubated with 320 mpcuries 5-HT-3H/ml. After 24 minutes of superfusion with Krebs-Ringer solution, the medium was changed to one containing 40 mM of K+. The results are the means for three slices f SEM.
M ) to the superfusing medium reduced the The addition of LSD (2 x stimulation-induced (8-v, 60 cps sinusoidal current for 1 minute) release of radioactivity from striatal slices to 27% f 3% (p< .001) that of control slices. The rise in tritiated 5-HT produced by stimulation was sevenfold in control slices and only 2.5-fold in the LSD-treated tissues. There was no significant difference in the rise in deaminated metabolites during stimulation between the control and the LSD-treated slices. Effect of Electrical Stimulation on Inert Substances As a test of the specificity of 5-HT release obtained in the present experiments, the release of tritiated water and urea-I4C wry attempted under the same conditions. None could be detected. These findings suggest that the stimulus-induced release obtained under the conditions of these experiments is not merely a nonspecific reflection of the ionic migrations and metabolic changes induced by field stimulation.
STIMULATION-INDUCED RELEASE OF SEROTONIN
363
Studies in Extracerebral Tissues of the Rat Accumulation and release of exogenous 5-HT from several extracerebral organs were studied in the same manner as brain slices. Both the liver and salivary gland concentrated only small amounts of tritiated 5-HT. The electrically induced release from these organs was only one-tenth that obtained from brain tissue other than cerebellum. The substantially greater accumulation and release of 5-HT from cerebral tissues is consistent with the view that exogenous 5-HT is concentrated in and mixes with endogenous stores within nerve terminals in the brain and can be liberated from these sites by depolarizing stimuli. The relatively small uptake and release of 5-HT from the cerebellum, liver, and salivary gland suggests a paucity of serotonergic terminals in these structures.
Conc Iusions Serotonin satisfies several important criteria for a neurotransmitter in the cardio-acceleratory system of the Aplysia. It is present in nervous tissue, it is releasable by nerve stimulation, and its local administration is accompanied by the same cardiotonic effect as nerve stimulation. Furthermore, pharmacological antagonists, in concentrations which block the effect of 5-HT, also block the effect of nerve stimulation. Slices of mammalian brain accumulate exogenous 5-HT which is releasable by procedures which depolarize neuronal membranes. Release is not directly related to the amount accumulated, but is considerably greater in tissues where the 5-HT is presumably stored within nerve endings rather than in extraneuronal sites. Release by electrical field stimulation is dependent upon the viability of the tissue studied. These findings, which are similar to the results obtained with NE, support the contention that 5-HT may serve as a synaptic mediator in the mammalian central nervous system. REFERENCES Aghajanian, G. K., and Bloom, F. E. (1967) J . Phamnacol. Exptl. Therap. 156, 23. Aghajanian, G. K., Bloom, F. E., Lovell, R. A., Sheard, M. H., and Freedman, D. X. (1966). Biochem. PharmacoE. 15, 1401. Aghajanian, G. K., Roscrans, J. A., and Sheard, M. H. (1967).Science, 156, 402. AndBn, N.-E., Carlsson, A., Hillarp, N.-A., and Magnusson, T. (1964). Life Sci. 3, 473. And&, N.-E., Dahlstrom, A., Fuxe, K., Larsson, K., Olson, L., and Ungerstedt, U. (1966). Acta Phyeiol. S c a d . 67, 313. Bacq, Z., Fischer, P., and Ghiretti, F. (1952). Arch. Intern. Physiol. 60, 165. Baldessarini, R. J., and Kopin, I. J. (1967).J . Phamzacol. Exptl. Therap. 156, 31. Blaschko, H., and Hope, D. B. (1957). Arch. Biochem. Biophys. 69, 10. Blaschko, H., rmd Milton, A. S. (1960). Brit. J . Phamacol. 15, 42.
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