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Noradrenaline inhibits depolarization-induced 3H-serotonin release from slices of rat hippocampus
European Journal o f Pharmacology, 63 (.1980) 179--182
179
© Elsevier/North-Holland Biomedical Press
Short communication NORADRENALINE INHIBITS DEPOLARIZATION-INDUCED aH-SEROTONIN RELEASE FROM SLICES OF RAT HIPPOCAMPUS ABRAHAM L. F R A N K H U Y Z E N and ARIE H. MULDER
Department o f Pharmacology, Free University, Medical Faculty, Van der Boechorststraat 7, 1081 B T Amsterdam, The Netherlands Received 19 February 1980, accepted 25 February 1980
A.L. F R A N K H U Y Z E N and A.H. MULDER, Noradrenaline inhibits depolarization-induced 3H-serotonin release from slices o f rat hippocampus, European J. Pharmacol. 63 (1980) 179--182. The depolarization (26 mM K+)-induced release of 3H-serotonin and 3H-noradrenaline from slices of rat hippocampus was studied with a superfusion method. Exogenous NA (in the presence of 10 pM desipramine) inhibited 3H-5-HT release (ECs0 3 X 10 -7 M) as well as 3H-NA release {ECs0 10 -7 M) by more than 70%. Both of these effects were competitively antagonized by phentolamine, but not by propranolol. It is tentatively suggested that the inhibitory effect of NA on 3H-5-HT release from hippocampus slices reflects the activation of postsynaptic t~-receptors which are localized on serotonergic nerve terminals. Neuronal interactions Transmitter release
Noradrenaline
Hippocampus
1. Introduction
Noradrenaline (NA) and serotonin (5-HT) are the major monoaminergic neurotransmitters in the hippocampus. The noradrenergic fibres innervating this limbic brain structure arise from the nucleus locus coeruleus, while the serotonergic fibres originate from the raphe nuclei (Storm-Mathisen and Guldberg, 1974; Moore, 1975). Neuroanatomical and neurochemical studies have indicated the possibility of mutual interactions between both monoaminergic systems. Thus, the activity of serotonergic neurons might be influenced by noradrenergic neurons terminating in the raphe nuclei (Palkovits and Jocabowitz, 1974; Saavedra et al., 1976). Conversely, the activity of noradrenergic neurons might be modified through a serotonergic input in the locus coeruleus (Palkovits et al., 1974; Pickel et al., 1977). In the present study we have considered another possibility for interactions between
a-Receptors
Serotonin
noradrenergic and serotonergic systems in the hippocampus. In recent years evidence has been obtained for the existence of various types of receptors on or near the nerve terminals of catecholaminergic neurons (for reviews see Langer, 1977; Starke, 1978). These presynaptic receptors might be involved in local regulatory mechanisms to modulate transmitter biosynthesis and/or release from the nerve terminals on which they are located. In the hippocampus the distributions of noradrenergic and serotonergic fibres appear to overlap considerably (Moore, 1975). It is conceivable, therefore, that transmitter release from serotonergic nerve endings may be modulated by NA released from neighbouring noradrenergic nerve endings and/or vice versa. This possibility is supported by the data reported here showing that NA inhibits the depolarization-induced release of 3H-5-HT from hippocampus slices via activation of -receptors.
180
2. Materials and Methods
2.1. Experimental procedure Male Wistar rats of 140-160 g b o d y weight were killed by decapitation. After rapid dissection of the brain, transversal slices of 0.3 mm thickness from the dorsal part of the hippocampus were prepared. The slices were incubated with radiolabelled neurotransmitters and superfused essentially as described elsewhere (Wemer et al., 1979). In short, 5060 slices (total fresh tissue weight 25-30 mg) were incubated at 37°C for 15 min in 2.5 ml Krebs-Ringer-bicarbonate medium, containing 5 pCi 3H-5-HT or 5 pCi 3H-NA (final concentration in the medium about 0.1 pM in both cases). After labelling, the slices were transferred to the chambers (0.25 ml volume) of a superfusion apparatus (6 slices per chamber) and superfused at a rate of 0.25 ml/min. After a 30 min washout period 12 successive 5-min fractions were collected (i.e. from t = 30 to t = 90 min). Depolarization-induced release was effected twice by superfusion with medium containing 26 mM K ÷ for 5 min, at t = 45 (S~) and t = 7 0 m i n ($2). Drugs were present in the medium from t = 50 min (i.e. the antagonists phentolamine or propranolol) or from t = 60 min (i.e. the agonists NA or isoprenaline). In order to prevent uptake of exogenously added NA in noradrenergic nerve endings 10 -s M desipramine was present in the superfusion medium from t = 30 min. At the end of each experiment the radioactivity remaining in the slices was extracted with 2 ml 0.1 N HC1. The radioactivity in fractions and extracts was determined by liquid scintillation counting.
2.2. Evaluation of results The release of radioactivity in excess of basal efflux resulting from the stimulation with 26 mM K ÷ was calculated as the percentage of total radioactivity present at the onset of stimulation. The ratio of the percentages
A.L. FRANKHUYZEN, A.H. MULDER
of radioactivity released during the first and second stimulation was calculated for both control (S2/Sl)c and drug-treated slices (S2/S1)D. (S2/Sl)D was then expressed as percentage of ($2/$1)c. Statistical analysis of the differences was carried out in paired experiments, using Student's t-test.
2.3. Radiochemicals and drugs 3H-Serotonin creatinine sulfate, 27.4 Ci/ mmole (New England Nuclear), 3H-noradrenaline HC1, 19.0 Ci/mmole (Radiochemical Centre). L-noradrenaline hydrogen° tartrate (Fluka), phentolamine (Ciba-Geigy), isoprenaline sulfate (Sigma), propranolol HC1 (Sigma). Desipramine was a generous gift from Ciba-Geigy.
3. Results The percentages of radioactivity released by the first and second stimulation with 26 mM K ÷ from hippocampus slices previously labelled with 3H-5-HT were 3.7 + 0.3% and 2.7 + 0.3%, respectively, resulting in a $2/$1 ratio of 0.73 + 0.08. Much higher percentages of radioactivity were released by 26 mM K ÷ from slices previously incubated with 3H-NA, viz. 12.0 + 0.5% for the first and 6.0 +- 0.4% for the second stimulation, resulting in a S~/S1 ratio of 0.50 + 0.02. The radioactivity released by K÷-stimulation from slices labelled with either 3H-5-HT or 3H-NA was strongly calcium-dependent and consisted for the major part of the unmetabolized amines as revealed by chromatographic analysis. NA dose~lependently inhibited K+-induced aH-5-HT release with an ECs0 of about 3 × 10-7M (fig. l A ) . A slight but statistically significant (P < 0.05) inhibition could already be detected at a NA concentration of 3 × 10 -s M. The maximal inhibiton of 3H-5-HT release, 73 + 5%, was reached at 3 × 10 -6 M NA. Phentolamine (10 -7 M) by itself did not affect 3H-5-HT release, but caused a virtually parallel shift to the right of the log dose-
N O R A D R E N A L I N E INHIBITS SEROTONIN RELEASE 150-
[3.]_.A
I3HI-5 HT
A ,,=1
.°1
oI Z Iz O 100
100t
I.I.
o
-
i .......
so]
LIJ ILl ,J
i.u 50i
i 0" B
0- A lO-8
i- 5
~-8
'i) '-7
'-6
I-5
1()-7 lO 6 lO lO 1o 10 lO NORADRENALINE CONCENTRATION (M)
Fig. 1. Inhibition by NA of the K* (26 mM)-induced release of 3H-5-HT (A) and 3H-NA (B) from slices of the dorsal part of the hippocampus of the rat and antagonism by phentolamine (10-TM). © © log dose-response curve of NA; • e id. in the presence of phentolamine (10 -7 M). The vertical bars indicate the S.E.M. of at least 10 determinations.
response curve for NA (fig. 1A). The K*-induced release of 3H-NA was also dose-dependently inhibited by exogenous NA with an ECs0 of approximately 1 0 - 7 M (fig. 1B). The maximal inhibiton of 75 + 3% was reached at 10 -6 M NA. Phentolamine (10 -7 M} itself increased the release o f 3H-NA by 42 + 4% and caused a parallel shift to the right of the log dose-response curve for NA. The fl-receptor agonist isoprenaline (up to a concentration of 10 -s M) had no effect on the release of either 3H-5-HT or 3H-NA (data not shown). The fl-receptor antagonist propranolol (10-TM) did n o t influence the inhibitory effect of NA on 3H-5-HT or on 3H-NA release.
4. Discussion The present data demonstrate that in the hippocampus NA m a y inhibit depolarizationinduced 5-HT release,,which suggests the pos-
181
sibility of a local adjustment of transmitter release from serotonergic neurons b y noradrenergic neurons. The inhibitory effect of NA on the release of 3H-5-HT from hippocampus slices was antagonized by phentolamine, b u t not b y propranolol, indicating that a-receptors are involved. Moreover, the flreceptor agonist isoprenaline had no effect on 3H-5-HT release. It is not possible to draw definite conclusions about the localization of these inhibitory a-receptors on the basis of the results obtained in this study. With brain slices the possibility cannot be excluded that the inhibition of 5-HT release by NA is an indirect effect, e.g. mediated b y short interneurons. However, it is not unlikely that the a-receptors involved are localized on the serotonergic nerve terminals themselves. Preliminary experiments in our laboratory with synaptosomes from the hippocampus, showing that synaptosomal 3H-5-HT release was inhibited b y NA and adrenaline, support this contention. As found before with cortex slices and synaptosomes (Mulder et al., 1979; Wemer et al., 1979) 3H-NA release from hippocampus slices was inhibited by exogenous NA. This inhibiting effect was antagonized by phentolamine, which indicates that in this brain region also a-receptors are present on noradrenergic nerve endings. We would like to emphasize here that although the a-receptors mediating the inhibitory effect of NA on 5-HT release presumably have a presynaptic localization, i.e. on serotonergic nerve terminals, they are postsynaptic a-receptors in relation to the noradrenergic neurons. We tentatively suggest, therefore, that the inhibitory effect of NA on 3H-5-HT release from hippocampus slices reflects the activation of postsynaptic areceptors. Further studies, which are in progress in this laboratory to examine the effects of various a-receptor agonists and antagonists, m a y reveal possible pharmacological differences between these receptors and the autoreceptors on noradrenergic nerve terminals.
182
A final interesting point is our finding that phentolamine by itself increased the K+induced release of 3H-NA, but did not affect 3H-5-HT release. The increasing effect of phentolamine and other a-receptor antagonists on 3H-NA release from brain slices has recently been discussed extensively (Wemer et al., 1979). In all probability phentolamine antagonizes the effect of released endogenous NA, which in brain slices reaches a concentration high enough to partially activate presynaptic a-receptors. Along this line of reasoning the finding that phentolamine did not affect 3H-5-HT release would indicate that released endogenous NA does not attain a concentration high enough to cause a significant activation of the (po~tsynaptic) a-receptors mediating the inhibition of 5-HT release. This might be explained by assuming that released endogenous NA has to traverse a fairly large distance between the nerve terminals and the postsynaptic receptors. This assumption is supported by morphological evidence, since Descarries et al. (1977) have shown that the major proportion of noradrenergic varicosities lack classical synaptic contacts, suggesting that NA may act as a neuromodulator rather than as a synaptic transmitter. Conceivably, in such a situation the concentration of released endogenous NA which is relatively high near the varicosities (and, therefore, also in the vicinity of the presynaptic autoreceptors) may be lowered considerably by diffusion into extracellular spaces and by metabolism before reaching the postsynaptic receptors.
Acknowledgements We thank Victoria de Regt and George Wardeh for their skilful technical assistance.
A.L. FRANKHUYZEN, A.H. MULDER
References Descarries, L., K.C. Watkins and Y. Lapierre, 1977, Noradrenergic axon terminals in the cerebral cortex of rat. III. Topometric ultrastructural analysis, Brain Res. 133, 197. Langer, S.Z., 1977, Presynaptic receptors and their role in the regulation of transmitter release, Br. J. Pharmacol. 60,481. Moore, R.Y., 1975, Monoamine neurons innervating the hippocampal formation and septum: organization and response to injury, in: The Hippocampus, Vol. I, eds. R.L. Isaacson and K.H. Pribram (Plenum, New York) p. 215. Mulder, A.H., J. Wemer and C.D.J. de Langen, 1979, Presynaptic receptor-mediated inhibition of noradrenaline release from brain slices and synaptosomes by noradrenaline and adrenaline, in: Presynaptic Receptors, eds. S.Z. Langer, K. Starke and M.L. Dubocovich (Pergamon Press, Oxford) p. 219. Palkovits, M. and D.M. Jacobowitz, 1974, Topographic atlas of catecholamine and acetylcholinesterasecontaining neurons in the brain. II. Hindbrain (mesencephalon, rhombencephalon), J. Comp. Neurol. 157, 29. Palkovits, M., M. Brow~.lstein and J.M. Saavedra, 1974, Serotonin content of the brain stem nuclei in the rat, Brain Res. 80, 237. Pickel, V.M.,T.H. Joh and D.J. Reis, 1977, A serotonergic innervation of noradrenergic neurons in nucleus locus coeruleus: demonstration by immunocytochemical localization of the transmitter specific enzymes tyrosine and tryptophan hydroxylase, Brain Res. 131,197. Saavedra, J.M., H. Grobecker and J. Zivin, 1976, Catecholamines in the raphe nuclei of the rat, Brain Res. 114,339. Starke, K., 1978, Presynaptic regulation of release in the central nervous system, in: The Release of Catecholamines from Adrenergic Neurons, ed. D.M. Paton (Pergamon Press, Oxford), p. 143. Storm-Mathisen, J. and H.C. Guldberg, 1974, 5-Hydroxytryptamine and noradrenaline in the hippocampal region: effect of transection of afferent pathways on endogenous levels, high affinity uptake and some transmitter-related enzymes, J. Neurochem. 22,793. Wemer, J., J.C. van der Lugt, C.D.J. de Langen and A.H. Mulder, 1979, On the capacity of presynaptic alpha receptors to modulate norepinephrine release from slices of rat neocortex and the affinity of some agonists and antagonists for these receptors, J. Pharmacol. Exp. Ther. 211,445.
179
© Elsevier/North-Holland Biomedical Press
Short communication NORADRENALINE INHIBITS DEPOLARIZATION-INDUCED aH-SEROTONIN RELEASE FROM SLICES OF RAT HIPPOCAMPUS ABRAHAM L. F R A N K H U Y Z E N and ARIE H. MULDER
Department o f Pharmacology, Free University, Medical Faculty, Van der Boechorststraat 7, 1081 B T Amsterdam, The Netherlands Received 19 February 1980, accepted 25 February 1980
A.L. F R A N K H U Y Z E N and A.H. MULDER, Noradrenaline inhibits depolarization-induced 3H-serotonin release from slices o f rat hippocampus, European J. Pharmacol. 63 (1980) 179--182. The depolarization (26 mM K+)-induced release of 3H-serotonin and 3H-noradrenaline from slices of rat hippocampus was studied with a superfusion method. Exogenous NA (in the presence of 10 pM desipramine) inhibited 3H-5-HT release (ECs0 3 X 10 -7 M) as well as 3H-NA release {ECs0 10 -7 M) by more than 70%. Both of these effects were competitively antagonized by phentolamine, but not by propranolol. It is tentatively suggested that the inhibitory effect of NA on 3H-5-HT release from hippocampus slices reflects the activation of postsynaptic t~-receptors which are localized on serotonergic nerve terminals. Neuronal interactions Transmitter release
Noradrenaline
Hippocampus
1. Introduction
Noradrenaline (NA) and serotonin (5-HT) are the major monoaminergic neurotransmitters in the hippocampus. The noradrenergic fibres innervating this limbic brain structure arise from the nucleus locus coeruleus, while the serotonergic fibres originate from the raphe nuclei (Storm-Mathisen and Guldberg, 1974; Moore, 1975). Neuroanatomical and neurochemical studies have indicated the possibility of mutual interactions between both monoaminergic systems. Thus, the activity of serotonergic neurons might be influenced by noradrenergic neurons terminating in the raphe nuclei (Palkovits and Jocabowitz, 1974; Saavedra et al., 1976). Conversely, the activity of noradrenergic neurons might be modified through a serotonergic input in the locus coeruleus (Palkovits et al., 1974; Pickel et al., 1977). In the present study we have considered another possibility for interactions between
a-Receptors
Serotonin
noradrenergic and serotonergic systems in the hippocampus. In recent years evidence has been obtained for the existence of various types of receptors on or near the nerve terminals of catecholaminergic neurons (for reviews see Langer, 1977; Starke, 1978). These presynaptic receptors might be involved in local regulatory mechanisms to modulate transmitter biosynthesis and/or release from the nerve terminals on which they are located. In the hippocampus the distributions of noradrenergic and serotonergic fibres appear to overlap considerably (Moore, 1975). It is conceivable, therefore, that transmitter release from serotonergic nerve endings may be modulated by NA released from neighbouring noradrenergic nerve endings and/or vice versa. This possibility is supported by the data reported here showing that NA inhibits the depolarization-induced release of 3H-5-HT from hippocampus slices via activation of -receptors.
180
2. Materials and Methods
2.1. Experimental procedure Male Wistar rats of 140-160 g b o d y weight were killed by decapitation. After rapid dissection of the brain, transversal slices of 0.3 mm thickness from the dorsal part of the hippocampus were prepared. The slices were incubated with radiolabelled neurotransmitters and superfused essentially as described elsewhere (Wemer et al., 1979). In short, 5060 slices (total fresh tissue weight 25-30 mg) were incubated at 37°C for 15 min in 2.5 ml Krebs-Ringer-bicarbonate medium, containing 5 pCi 3H-5-HT or 5 pCi 3H-NA (final concentration in the medium about 0.1 pM in both cases). After labelling, the slices were transferred to the chambers (0.25 ml volume) of a superfusion apparatus (6 slices per chamber) and superfused at a rate of 0.25 ml/min. After a 30 min washout period 12 successive 5-min fractions were collected (i.e. from t = 30 to t = 90 min). Depolarization-induced release was effected twice by superfusion with medium containing 26 mM K ÷ for 5 min, at t = 45 (S~) and t = 7 0 m i n ($2). Drugs were present in the medium from t = 50 min (i.e. the antagonists phentolamine or propranolol) or from t = 60 min (i.e. the agonists NA or isoprenaline). In order to prevent uptake of exogenously added NA in noradrenergic nerve endings 10 -s M desipramine was present in the superfusion medium from t = 30 min. At the end of each experiment the radioactivity remaining in the slices was extracted with 2 ml 0.1 N HC1. The radioactivity in fractions and extracts was determined by liquid scintillation counting.
2.2. Evaluation of results The release of radioactivity in excess of basal efflux resulting from the stimulation with 26 mM K ÷ was calculated as the percentage of total radioactivity present at the onset of stimulation. The ratio of the percentages
A.L. FRANKHUYZEN, A.H. MULDER
of radioactivity released during the first and second stimulation was calculated for both control (S2/Sl)c and drug-treated slices (S2/S1)D. (S2/Sl)D was then expressed as percentage of ($2/$1)c. Statistical analysis of the differences was carried out in paired experiments, using Student's t-test.
2.3. Radiochemicals and drugs 3H-Serotonin creatinine sulfate, 27.4 Ci/ mmole (New England Nuclear), 3H-noradrenaline HC1, 19.0 Ci/mmole (Radiochemical Centre). L-noradrenaline hydrogen° tartrate (Fluka), phentolamine (Ciba-Geigy), isoprenaline sulfate (Sigma), propranolol HC1 (Sigma). Desipramine was a generous gift from Ciba-Geigy.
3. Results The percentages of radioactivity released by the first and second stimulation with 26 mM K ÷ from hippocampus slices previously labelled with 3H-5-HT were 3.7 + 0.3% and 2.7 + 0.3%, respectively, resulting in a $2/$1 ratio of 0.73 + 0.08. Much higher percentages of radioactivity were released by 26 mM K ÷ from slices previously incubated with 3H-NA, viz. 12.0 + 0.5% for the first and 6.0 +- 0.4% for the second stimulation, resulting in a S~/S1 ratio of 0.50 + 0.02. The radioactivity released by K÷-stimulation from slices labelled with either 3H-5-HT or 3H-NA was strongly calcium-dependent and consisted for the major part of the unmetabolized amines as revealed by chromatographic analysis. NA dose~lependently inhibited K+-induced aH-5-HT release with an ECs0 of about 3 × 10-7M (fig. l A ) . A slight but statistically significant (P < 0.05) inhibition could already be detected at a NA concentration of 3 × 10 -s M. The maximal inhibiton of 3H-5-HT release, 73 + 5%, was reached at 3 × 10 -6 M NA. Phentolamine (10 -7 M) by itself did not affect 3H-5-HT release, but caused a virtually parallel shift to the right of the log dose-
N O R A D R E N A L I N E INHIBITS SEROTONIN RELEASE 150-
[3.]_.A
I3HI-5 HT
A ,,=1
.°1
oI Z Iz O 100
100t
I.I.
o
-
i .......
so]
LIJ ILl ,J
i.u 50i
i 0" B
0- A lO-8
i- 5
~-8
'i) '-7
'-6
I-5
1()-7 lO 6 lO lO 1o 10 lO NORADRENALINE CONCENTRATION (M)
Fig. 1. Inhibition by NA of the K* (26 mM)-induced release of 3H-5-HT (A) and 3H-NA (B) from slices of the dorsal part of the hippocampus of the rat and antagonism by phentolamine (10-TM). © © log dose-response curve of NA; • e id. in the presence of phentolamine (10 -7 M). The vertical bars indicate the S.E.M. of at least 10 determinations.
response curve for NA (fig. 1A). The K*-induced release of 3H-NA was also dose-dependently inhibited by exogenous NA with an ECs0 of approximately 1 0 - 7 M (fig. 1B). The maximal inhibiton of 75 + 3% was reached at 10 -6 M NA. Phentolamine (10 -7 M} itself increased the release o f 3H-NA by 42 + 4% and caused a parallel shift to the right of the log dose-response curve for NA. The fl-receptor agonist isoprenaline (up to a concentration of 10 -s M) had no effect on the release of either 3H-5-HT or 3H-NA (data not shown). The fl-receptor antagonist propranolol (10-TM) did n o t influence the inhibitory effect of NA on 3H-5-HT or on 3H-NA release.
4. Discussion The present data demonstrate that in the hippocampus NA m a y inhibit depolarizationinduced 5-HT release,,which suggests the pos-
181
sibility of a local adjustment of transmitter release from serotonergic neurons b y noradrenergic neurons. The inhibitory effect of NA on the release of 3H-5-HT from hippocampus slices was antagonized by phentolamine, b u t not b y propranolol, indicating that a-receptors are involved. Moreover, the flreceptor agonist isoprenaline had no effect on 3H-5-HT release. It is not possible to draw definite conclusions about the localization of these inhibitory a-receptors on the basis of the results obtained in this study. With brain slices the possibility cannot be excluded that the inhibition of 5-HT release by NA is an indirect effect, e.g. mediated b y short interneurons. However, it is not unlikely that the a-receptors involved are localized on the serotonergic nerve terminals themselves. Preliminary experiments in our laboratory with synaptosomes from the hippocampus, showing that synaptosomal 3H-5-HT release was inhibited b y NA and adrenaline, support this contention. As found before with cortex slices and synaptosomes (Mulder et al., 1979; Wemer et al., 1979) 3H-NA release from hippocampus slices was inhibited by exogenous NA. This inhibiting effect was antagonized by phentolamine, which indicates that in this brain region also a-receptors are present on noradrenergic nerve endings. We would like to emphasize here that although the a-receptors mediating the inhibitory effect of NA on 5-HT release presumably have a presynaptic localization, i.e. on serotonergic nerve terminals, they are postsynaptic a-receptors in relation to the noradrenergic neurons. We tentatively suggest, therefore, that the inhibitory effect of NA on 3H-5-HT release from hippocampus slices reflects the activation of postsynaptic areceptors. Further studies, which are in progress in this laboratory to examine the effects of various a-receptor agonists and antagonists, m a y reveal possible pharmacological differences between these receptors and the autoreceptors on noradrenergic nerve terminals.
182
A final interesting point is our finding that phentolamine by itself increased the K+induced release of 3H-NA, but did not affect 3H-5-HT release. The increasing effect of phentolamine and other a-receptor antagonists on 3H-NA release from brain slices has recently been discussed extensively (Wemer et al., 1979). In all probability phentolamine antagonizes the effect of released endogenous NA, which in brain slices reaches a concentration high enough to partially activate presynaptic a-receptors. Along this line of reasoning the finding that phentolamine did not affect 3H-5-HT release would indicate that released endogenous NA does not attain a concentration high enough to cause a significant activation of the (po~tsynaptic) a-receptors mediating the inhibition of 5-HT release. This might be explained by assuming that released endogenous NA has to traverse a fairly large distance between the nerve terminals and the postsynaptic receptors. This assumption is supported by morphological evidence, since Descarries et al. (1977) have shown that the major proportion of noradrenergic varicosities lack classical synaptic contacts, suggesting that NA may act as a neuromodulator rather than as a synaptic transmitter. Conceivably, in such a situation the concentration of released endogenous NA which is relatively high near the varicosities (and, therefore, also in the vicinity of the presynaptic autoreceptors) may be lowered considerably by diffusion into extracellular spaces and by metabolism before reaching the postsynaptic receptors.
Acknowledgements We thank Victoria de Regt and George Wardeh for their skilful technical assistance.
A.L. FRANKHUYZEN, A.H. MULDER
References Descarries, L., K.C. Watkins and Y. Lapierre, 1977, Noradrenergic axon terminals in the cerebral cortex of rat. III. Topometric ultrastructural analysis, Brain Res. 133, 197. Langer, S.Z., 1977, Presynaptic receptors and their role in the regulation of transmitter release, Br. J. Pharmacol. 60,481. Moore, R.Y., 1975, Monoamine neurons innervating the hippocampal formation and septum: organization and response to injury, in: The Hippocampus, Vol. I, eds. R.L. Isaacson and K.H. Pribram (Plenum, New York) p. 215. Mulder, A.H., J. Wemer and C.D.J. de Langen, 1979, Presynaptic receptor-mediated inhibition of noradrenaline release from brain slices and synaptosomes by noradrenaline and adrenaline, in: Presynaptic Receptors, eds. S.Z. Langer, K. Starke and M.L. Dubocovich (Pergamon Press, Oxford) p. 219. Palkovits, M. and D.M. Jacobowitz, 1974, Topographic atlas of catecholamine and acetylcholinesterasecontaining neurons in the brain. II. Hindbrain (mesencephalon, rhombencephalon), J. Comp. Neurol. 157, 29. Palkovits, M., M. Brow~.lstein and J.M. Saavedra, 1974, Serotonin content of the brain stem nuclei in the rat, Brain Res. 80, 237. Pickel, V.M.,T.H. Joh and D.J. Reis, 1977, A serotonergic innervation of noradrenergic neurons in nucleus locus coeruleus: demonstration by immunocytochemical localization of the transmitter specific enzymes tyrosine and tryptophan hydroxylase, Brain Res. 131,197. Saavedra, J.M., H. Grobecker and J. Zivin, 1976, Catecholamines in the raphe nuclei of the rat, Brain Res. 114,339. Starke, K., 1978, Presynaptic regulation of release in the central nervous system, in: The Release of Catecholamines from Adrenergic Neurons, ed. D.M. Paton (Pergamon Press, Oxford), p. 143. Storm-Mathisen, J. and H.C. Guldberg, 1974, 5-Hydroxytryptamine and noradrenaline in the hippocampal region: effect of transection of afferent pathways on endogenous levels, high affinity uptake and some transmitter-related enzymes, J. Neurochem. 22,793. Wemer, J., J.C. van der Lugt, C.D.J. de Langen and A.H. Mulder, 1979, On the capacity of presynaptic alpha receptors to modulate norepinephrine release from slices of rat neocortex and the affinity of some agonists and antagonists for these receptors, J. Pharmacol. Exp. Ther. 211,445.