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The pharmacology of recurrent inhibition in the supraoptic neurosecretory system
BRAIN RESEARCH
501
T H E P H A R M A C O L O G Y OF R E C U R R E N T I N H I B I T I O N IN T H E SUPRAOPTIC N E U R O S E C R E T O R Y SYSTEM
ROGER A. NICOLL AND JEFFERY L. BARKER* Laboratory of Neuropharmaeology, Division of Speeial Mental Health Research, National Institute of Mental Health, Saint Elizabeth's Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted June 24th, 1971)
INTRODUCTION Axons of supraoptic neurosecretory neurons terminate on fine capillaries and release their hormonal products into the circulationlS, a2. However, these neurosecretory cells also possess basic neuronal properties 4 including synaptic functions 6,17,aa-aT. We6, 2~ and otherslV,lS,a6, 37 have demonstrated an inhibitory pathway to neurosecretory cells which appears to be mediated by neurosecretory axons. Since our neurophysiologic investigation has failed to reveal a suitable interneuron mediating this inhibition, the possibility of a direct inhibitory interaction between neurosecretory cells should be considered. If neurosecretory cells participate in synaptic events, they must either utilize their secretory product as the synaptic transmitter or elaborate another substance in addition to the hormone. We have investigated the pharmacology of this inhibitory pathway in the supraoptic neurosecretory system of the cat by applying various pharmacologic agonists and antagonists both intravenously and microelectrophoretically in an attempt to identify the synaptic transmitter mediating the inhibition. METHODS Eighteen cats were anesthetized with intraperitoneal sodium pentobarbital (35 mg/kg), tracheotomized and placed supine in a stereotaxic apparatus. Small doses of sodium pentobarbital were given intravenously during the experiment to maintain a light level of anesthesia. Prior to the intravenous administration of convulsant drugs, the animal was paralyzed with intravenous gallamine triethiodide (5 mg/kg) and artificially ventilated with room air. A buccal approach was used to expose the optic chiasm and pituitary gland. * Present address: Electroencephalography Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Md. 20014, U.S.A. Brain Research, 35 (1971) 501-511
502
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N I C O L L A N D J. L. B A R K E R
(Further details on methodology may be found in a recent paper~.) Extracellular unit recordings were made through the 5 M NaCl-filled center barrels of 5-barrel micropipettes, prepared according to the methods previously described30. In several experiments fine glass microelectrodes were glued to 5-barrel micropipettes with lucite chips so that the single, recording barrel protruded 5-10/zm from the micropipette assembly27. This allowed excellent recording of cellular activity as well as easy passage of large currents from the drug barrels. The unit activity was displayed on an oscilloscope, gated with an adjustable window and then relayed either to a 1-sec integrator and recorded on a Beckman pen-writer or to a computer of average transients (CAT, Mnemotron). Supraoptic unit activity in response to neurohypophyseal stimuli was summated on the CAT for a period of 80 msec before and 420 msec after the onset of each stimulus. Generally, 100 stimuli summated in this fashion proved adequate to demonstrate the inhibition of neurosecretory cells following such stimuli. The pharmacologic agents employed were dissolved in distilled water as follows: 0.5 M DL-homocysteate (pH 7.5; NaOH) (DLH), 0.1 M atropine sulfate, 1 M glycine (pH 3.5; HC1), 1 M gamma-aminobutyric acid (GABA) (pH 3.5; HC1), and 0.1 M strychnine phosphate. Bicuculline was prepared as a 0.01 M solution of 0.165 M NaC1 (pH adjusted to 3.5 with 0.1 N HC1) and ejected with cationic current. Picrotoxin was made up as a 0.01 M solution of 0.165 M NaC1 (pH 4.5) and ejected with cationic current. 0.025 M antidiuretic hormone (lysine vasopressin) (Calbiochem) was dissolved in distilled water (pH 4-5) and ejected with cationic current. (Lysine vasopressin rather than the naturally occurring arginine vasopressin was used because of its ready availability. Although the ADH is reported to be chromatographically pure, we cannot entirely exclude the possibility that some breakdown of the peptide may have occurred.) Previously described electrical methods prevented polarization of the electrode tip during drug ejection, as well as diffusion of drugs from the micropipette 30. Most supraoptic neurons encountered were silent and therefore a continuous application of DLH was required to produce a suitable background level of activity against which drug effects could be assessed. The drug responses observed in DLHexcited cells did not appear to differ from responses obtained in spontaneously active cells. Antidiuretic hormone (ADH) usually required relatively large ejection currents to obtain drug responses. Since it was often difficult to exclude the electrical effects of large, direct currents on cells, each cell requiring large current passage for the presumed drug effect was thoroughly tested with currents of equal strength passed through the 3 M NaC1 (balancing) barrel. Any cell responding to these latter currents was removed from further study. At the conclusion of the experiments poststimulus histograms of neurosecretory inhibition were obtained before and during the intravenous administration of strychnine, bicuculline and atropine. Two cats were injected intraventricularly with 6-hydroxydopamine (6-HODA), a substance which is known to selectively destroy norepinephrine terminals7. A total of 2 mg/kg was given in two doses 3 days apart. Acute experiments on these cats Brain Research, 35 (1971) 501-511
PHARMACOLOGY OF RECURRENT INHIBITION
503
were performed 3 days after the second dose. Following the experiments, areas of the brain including the supraoptic region were frozen in liquid nitrogen and prepared for histofluorescence. RESULTS
Acetylcholine. Since microelectrophoretically applied acetylcholine (ACh) causes inhibition of supraoptic neurosecretory cells by acting at muscarinic sites 5 and since ACh is present in both the supraoptic nucleus and the neurohypophysisz2, ACh may be acting at muscarinic sites to mediate the observed inhibition. Therefore, the muscarinic antagonist atropine13,16, which blocks ACh-induced depressions of supraoptic neurons 5, was used to test this possibility. Atropine was microelectrophoretically applied to 6 supraoptic neurons in which control postimulus histograms had demonstrated recurrent inhibition (RI). Atropine delivered in this way did not affect the RI. Furthermore, intravenous injection of atropine in doses up to 1.3 mg/kg also had no effect on the RI (Fig. 3A). Norepinephrine. In a previous study we have shown that norepinephrine (NE) inhibits the activity of supraoptic neurosecretory cells 5 which receive numerous NE terminals 8. Thus we considered NE as a candidate for the inhibitory transmitter.
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Fig. 1. Action of glycine and strychnine on supraoptic neurosecretory cells. A, Continuous polygraph record of 1-sec integrated unit activity showing dose-response depression of activity to microelectrophoretic application of glycine (solid lines with current in nanoamperes above trace). Calibration bracket to left of record: 0-10 spikes/sec. Antidromically activated neurosecretory cell spike upper right. Calibration: 5 msec/100 pV. B, Strychnine ejected with 20 nA for the duration of center record readily antagonizes the glycine-induced depressions (solid lines) of another neurosecretory cell (antidromic spike above trace at right with same calibrations as in A).
Brain Research, 35 (1971) 501-511
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Fig. 2. Action of GABA. bicuculline and pircotoxin on neurosecreto~ cells. A, same cell as in Fig. 1A,
Dose-response depression of activity by microelectrophoretic application of GABA (solid lines with current in nanoamperes above record). Calibration bracket at left of trace: 0-20 spikes/sec. B, Continuous record showing bicuculline antagonism (at 125 hA) of GABA depressions (solid lines, 5 nA) in a neurosecretory cell displaying recurrent inhibition (poststimulus histogram beneath trace showing inhibition following neurohypophysial stimuli delivered at time indicated by upward arrow; calibrations: vertical bar: 0-5 counts/address; horizontal bar: 100 msec). C, Picrotoxin antagonism (at 200 nA)of GABA depression (solid lines, 20 nA). Calibration bracket at left: 0--5 spikes/sec. Identifying antidromic spike above trace at left. Calibration: 5 msec; 100/~V. Cats pretreated with 6 - H O D A (which selectively destroys NE endings) were examined for RI. RI was present in these cats despite the fact that nearly all the NE-fluorescent terminals in the supraoptic nucleus were gone. Glycine. It has been proposed that glycine is an important inhibitory transmitter in the spinal cord za. The convulsant strychnine blocks both glycine-induced depressions of spinal neurons and spinal postsynaptic inhibition 10. Glycine applied microelectrophoretically to supraoptic neurons inhibited all 79 tested. Relatively small doses were sufficient to cause complete inhibition of activity (Fig. 1A). The average current required to inhibit these neurons was 20 nA. Glycine depressions were readily antagonized by concomitant microelectrophoretic application of strychnine in 14 of 15 cells tested (Fig. 1B). However, neither microelectrophoretic administration of strychnine at a time when glycine depression was blocked nor intravenous injections up to 1.0 mg/kg affected the RI (Fig. 3C). GABA. Recent studies have shown that the convulsants picrotoxin 26 and bicuculline 11 specifically antagonize the depressant action of G A B A on neuronal activity and also block a number of supraspinal inhibitory pathways11,12,z3, 25, suggesting
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t Fig. 3. Action of intravenous atropine, bicuculline and strychnine on the recurrent inhibitory pathway in the supraoptic neurosecretory system. Control poststimulus histograms of neurosecretory cell inhibition in response to neurohypophysial stimuli (arrows beneath bottom histograms) are across top row. Calibration bracket (0-5 counts/address) and time scale same for all records. A, 1.3 mg/kg atropine i.v. does not effect inhibition profile. B, Middle record shows slight reduction in inhibition following0.67 mg/kg bicuculline i.v., the threshold dose for seizure activity. A similar profile is evident in the bottom trace, after a total of 1.0 mg/kg bicuculline. C, Strychnine (1.0 mg/kg i.v.) does not alter the recurrent inhibition. an important role for G A B A in supraspinal postsynaptic inhibition. In the supraoptic nucleus G A B A inhibited all 74 neurons studied. Small ejecting currents were able to produce profound depressions of activity (Fig. 2A). The average current necessary to completely inhibit unit activity was 10 nA. Microelectrophoresis of either picrotoxin (8 of 10 cells tested) or bicuculline (9 of 10 cells tested) antagonized the GABAinduced depressions of unit activity (Fig. 2B and C). However, microelectrophoretic application of both picrotoxin and bicuculline failed to block RI. Similarly, intravenous injections of bicuculline did not affect the synaptic inhibition when given in subconvulsant doses. Yet, at doses which produced generalized seizures and bursting of neurosecretory cells some reduction in inhibition was observed (Fig. 3B). The obvious changes in neuronal excitability during convulsions make it difficult to relate this reduction in inhibition to a specific antagonism of the pathway. A n t i d i u r e t i c h o r m o n e . The possibility that antidiuretic hormone (ADH) might in fact be the inhibitory transmitter was investigated by applying A D H microelectrophoretically to cells in the supraoptic nucleus. In general, A D H appeared to have a weak pharmacologic action on the excitability of neurons as evidenced by the need to use large ejecting currents in order to obtain an effect. In those neurons which were either antidromically invaded and/or exhibited RI, 80 ~ of the responsive cells were inhibited by A D H (Table I; Fig. 4A and B). Occasionally neurosecretory cells TABLE I NEURONAL
RESPONSES TO
ADH
Neuron type
Supraoptic neurosecretory neurons Unidentified supraoptic neurons Cortical neurons
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22 63 35 Brain Research, 35 (1971) 501-511
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Fig. 4. Responses of supraoptic neurosecretory cells to microelectrophoresis of ADH. A, Doseresponse depression of activity in a cell exhibiting recurrent inhibition (histogram beneath record; calibration: 5 counts/address; 100 msec) by ADH (solid lines above trace, currents in nA). Calibration bracket at/eft: 0-20 spikes/sec. B, ADH depression of neurosecretory cell activity. Polygraph calibration same as in A. Antidromic spike below trace. Calibration: 5 msec/100 /~V. C, ADH excitation of a neurosecretory cell. Calibration bracket: 0-5 spikes/sec. Antidromic spike centered above trace. Calibration: 2 msec/4 inV.
were excited by A D H (Fig. 4C), a l t h o u g h larger ejecting currents were needed to p r o d u c e this effect. A D H was also tested on cortical n e u r o n s in o r d e r to serve as a c o n t r o l for the responses seen in the s u p r a o p t i c n e u r o s e c r e t o r y system. Microelect r o p h o r e s i s o f A D H in this a r e a excited nearly 9 0 ~ o f r e s p o n d i n g cells (Table I; Fig. 5A), a l t h o u g h several n e u r o n s were inhibited using large ejecting currents (Fig. 5B). DISCUSSION T h e present p h a r m a c o l o g i c investigation was p r o m p t e d by the recent finding o f s u p r a o p t i c n e u r o s e c r e t o r y cell i n h i b i t i o n following electrical s t i m u l a t i o n o f the post e r i o r pituitary18,24, ~7. A l t h o u g h the a n a t o m y o f this i n h i b i t o r y p a t h w a y is n o t en-
Brain ResearCh, 35 (1971) 501-511
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Fig. 5. Responses of neurons in the sensorimotor cortex to microelectrophoresis of ADH. A, Examples of excitation in response to ADH (solid lines above trace, nA current). B, ADH depression of a cortical neuron. Calibration bracket: 40 spikes/sec (A, left); 20 spikes/sec (A, right and B). tirely clear, recurrent collaterals have been describedL F r o m our neurophysiological observations we have been unable to identify an interneuron participating in this pathway6, is, which suggests that neurosecretory cells and/or collaterals synapse directly with other neurosecretory cells. Since supraoptic neurosecretory cells possess synaptic ability, they must either release their hormonal product (ADH) at synapses or elaborate a separate transmitter substance. If Dale's Principle (that the same substance is released from all branches of a particular neuron) 14 holds for neurosecretory cells, then one might expect A D H to be released at synapses as well as into fine capillaries. Axo-axonic synapses between neurosecretory and non-neurosecretory neurons have been described in several species2,19, 2s and 'neurosecretomotor junctions' with epithelial cells of the pars intermedia have recently been reported in the cat 3, but synapses between two neurosecretory elements have not yet been found. The second possibility - - that neurosecretory cells possess a dual secretory role - - is based primarily on electron microscopic evidence of two morphologically different populations of storage site in neurosecretory cells: large, elementary neurosecretory granules 20 and small, clear 'synaptoid' vesicles 21 which stain selectively with ZnI-OsO4 (ref. 29). Although this stain has been correlated with synaptic vesicles elsewhere in the nervous system 1, the functional significance of these 'vesicles' is not clear and it has been suggested that they may represent a different stage in the neurosecretory process~L Our investigation was designed to explore these possibilities by considering 5 suspected transmitter substances: ACh, NE, glycine, G A B A and A D H . Brain Research, 35 (1971) 501-511
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Acetylcholine. The depressant action of ACh on neurosecretory cells is mediated through muscarinic sites and is blocked by atropine 5. However, the fact that atropine administered either microelectrophorerically or intravenously failed to reduce the RI, makes it unlikely that ACh is the inhibitory transmitter. Norepinephrine. Although NE inhibits the activity of supraoptic neurosecretory cells 4 and is present in the terminals ending on these cellss, there is no anatomic evidence that neurosecretory axons contain NE. Furthermore, NE-fluorescent cell bodies have not been described in the supraoptic region. Thus, it is unlikely that NE is involved in RI. Our demonstration of the inhibition in cats which had their NE terminals destroyed by 6-HODA further reduces the possibility that NE is the inhibitory transmitter. Glycine and GABA. In agreement with results obtained elsewhere in the central nervous systemX0,11,25,34, we have found that both glycine and GABA are potent depressants of neurosecretory cell activity, as evidenced by their rapid onset of action and quick reversibility of effect at relatively low ejecting currents. GABA appeared to be slightly more potent in this regard (based on the average amount of current required to inhibit neurosecretory cells). Strychnine, a convulsant which blocks both spinal postsynaptic inhibition34 and glycine-induced depression of cellular activity in many areas of the nervous system10,2z,25,26 was quite effective in blocking glycine inhibition of neurosecretory cells. However, neither microelectrophoretic application, nor intravenous injection of convulsant doses of strychnine (up to 1.0 mg/kg) were able to antagonize the RI in the supraoptic neurosecretory system. Both picrotoxin and bicuculline have been reported to block several supraspinal inhibitory pathways and to antagonize the depressant actions of GABA when administered microelectrophoretically11, 9,6. GABA-induced depression of neurosecretory cells was also effectively blocked by microelectrophoresis of either picrotoxin or bicuculline. However, neither of these convulsants were effective in antagonizing the RI when applied in this manner. The slight reduction in inhibition observed following intravenous convulsant doses of bicuculline was difficult to interpret owing to the generalized seizure activity and therefore could not be related to a specific antagonism of the pathway. This, however, does not rule out GABA as the inhibitory transmitter in this pathway. Antidiuretic hormone. When ADH was applied to neurons exhibiting RI, a high proportion of responding cells (80~) was depressed. On the contrary, application of ADH to cortical neurons caused excitation in a great majority (nearly 90 ~) of responsive cells. These results suggests that the excitatory effect of ADH is nonspecific, and the inhibitory effect related to supraoptic neurosecretory cells. Although we have been able to block the depressant actions of the suspected transmitters considered in this study (ACh, glycine, GABA) with microelectrophoresis of their antagonists (atropine, strychnine, picrotoxin and bicuculline), we have not been able to block the inhibitory pathway by either microelectrophoretic or intravenous administration of these antagonists. We feel it unlikely that either ACh or glycine is the suspected transmitter mediating RI. The present findings related to GABA do not permit excluding it from a possible role in this pathway. Our results Brain Research, 35 (1971) 501-511
PHARMACOLOGYOF RECURRENTINHIBITION
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also suggest that NE is not involved in this pathway. On the other hand, wefinda good correlation between cells exhibiting RI and those depressed by microelectrophoretic application of ADH. Complementary to this is the report that A D H is released (in response to hemorrhage) into the cerebrospinal fluid independent of plasma concentrations 3a. This suggests an intracranial release of A D H coincident with the increase in supraoptic neurosecretory cell activity. These lines of evidence support the notion that A D H may be the transmitter mediating RI in the supraoptic neurosecretory system; however, more anatomic evidence is needed to further strengthen this hypothesis. SUMMARY A pharmacologic study of recurrent inhibition in the supraoptic neurosecretory system of the cat has been conducted in an attempt to identify the inhibitory transmitter in this pathway. Acetylcholine (muscarinic component), glycine and GABA were considered as possible transmitters mediating this inhibitory pathway. Antagonists to the depressant actions of these substances on neurosecretory cells were tested on both unit responses and the inhibitory pathway. Although atropine, strychnine, picrotoxin and bicuculline were able to block the pharmacologic depressions, they were allIineffective in antagonizing the recurrent inhibition when applied either microelectrophoretically or intravenously. It is thus unlikely that either acetylcholine, glycine or GABA participate in this inhibitory pathway. Norepinephrine was also considered as a candidate for inhibitory transmitter. However, since recurrent inhibition was present in cats whose norepinephrine-containing terminals were destroyed by pretreatment with 6-hydroxydopamine, we do not think norepinephrine is the transmitter. The possibility that the neurosecretory product A D H might also serve a synaptic function and mediate the recurrent inhibition was investigated by microelectrophoretic administration of A D H to neurosecretory cells. The fact that a good correlation was found between those cells exhibiting recurrent inhibition and those depressed by A D H supports the idea that A D H may serve as a transmitter in this recurrent inhibitory pathway. ACKNOWLEDGEMENTS We thank A. P. Oliver and B. Hamstrom for fine technical assistance. We are grateful for a sample of vasopressin received from Dr. R. Walter. We are indebted to Mrs. Jean Alwine for her work on the histochemistry and to Dr. F. Bloom for interpreting the fluorescent findings and reviewing the manuscript. REFERENCES 1 AKERT,K., AND SANDRI,C., An electron-microscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions, Brain Research, 7 (1968) 286-295.
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2 BARGMANN,W., Neurosecretion, Int. Rev. Cytol., 19 (1966) 183-201. 3 BARGMANN,W., LINDER, E., UND ANDRES, K. H., t0ber Synapsen an endokrinen Epithelzellen und die Definition sekretorischer Neurone, Z. Zellforsch., 77 (1967) 282-298. 4 BARKER,J. L., CRAVTON,J. W., AND NICOLL, R. A., Supraoptie neurosecretory cells: Autonomic modulation, Science, 171 (1971) 206-207. 5 BARKER,J. L., CRAYTON,J. W., AND NICOLL, R. A., Supraoptic neurosecretory cells: Adrenergic and cholinergic sensitivity, Science, 171 (1971) 208-210. 6 BARKER,J. L., CRAYTON,J. W., AND NICOLL, R. A., Antidromic and orthodromic responses of paraventricular and supraoptic neurosecretory cells, Brain Research, 33 (1971) 353-366. 7 BLOOM,F., ALGERLS., GROPPEa~rI,A., REVUELTA,A., AND COSTA, E., Lesions of central norepinephrine terminals with 6-OH-dopamine: Biochemistry and fine structure, Science, 166 (1969) 1284-1286. 8 CARLSSON,A,, FLACK, B., AND HILLARP, N. A., Cellular localization ofbrainmonoamines, Acta physiol, scand., 56, Suppl. 196 (1962) 1-28. 9 CHRIST, J. F., Nerve supply, blood supply and cytology of the neurohypophysis. In G. W. HARRIS AND B. T. DONOVAN(Eds.), The Pituitary Gland, Vol. 3, Univ. California Press, Berkeley, Calif., 1966, pp. 62-130. 10 CURTIS, D. R., H6SLI, L., JOHNSTON, G. A. R., AND JOHNSTON,I. H., The hyperpolarization of spinal motoneurons by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 11 CURTIS, D. R., DUGGAN, A. W., FELIX, D., AND JOHNSTON, G. A. R., GABA, bicuculline and central inhibition, Nature (Lond.), 226 (1970) 1222-1224. 12 CURTIS, D. R., FELIX, D., AND MCLENNAN, H., GABA and hippocampal inhibition, Brit. J. Pharmacol., 40 (1970) 881-883. 13 DALE, H. A., The action of certain esters and ethers on choline, and their relation to muscarine, J. Pharmacol., 6 (19t4) 147-190. 14 DALE, H. A., Pharmacology and nerve endings, Proc. roy. Soc. Med., 28 (1935) 319-332. 15 GINSBURG, M., Production, release, transportation and elimination of the neurohypophysial hormones. In B. BEROE (Ed.), Handbook of Experimental Pharmacology, Vol. 23, Springer, Berlin, 1968, pp. 286-371. 16 HENDERSON,V. E., AND ROEPKE, M. H., Drugs affecting parasympathetic nerves, Physiol. Rev., 17 (1937) 373-407. 17 KANDEL, E. R., Electrical properties of hypothalamic neuroendocrine cells, J. gen. Physiol., 47 (1964) 691-717. 18 KELLY, J. J., AND DREIFtJSS, J. J., Antidromic inhibition of identified rat supraoptic neurones, Brain Research, 22 (1970) 406-409. 19 KOBAYASHI,H., HIRANO, T., AND OOTA, Y., Electron microscopic and pharmacological studies on the median eminence and pars nervosa, Arch. Anat. ruler., 54 (1965) 277-294. 20 KUROSUr~LK., MATSUZAWA,T., AND SHIBASAKLS., Electron microscope studies on the fine structure of the pars nervosa and pars intermedia, and their morphological interrelation in the normal rat hypophysis, Gen. comp. Endocr., 1 (1961) 433-452. 21 LE•ERIs• K.• A pre•iminary rep•rt •n the u•trastructure •f the human neur•hyp•physis• J. End••r.• 27 (1963) 133-135. 22 LEDERIS,K., AND LIVINGSTON,A., Acetylcholine and related enzymes in the neural lobe and anterior hypothalamus of the rabbit, J. Physiol. (Lond.), 201 (1969) 695-709. 23 NICOLL, R. A., GABA and dendrodendritic inhibition in the olfactory bulb, Pharmacologist, 12 (1970) 236. 24 NICOLL, R. A., CRAYTON,J. W., AND BARKER, J. L., Antidromic and orthodromic responses in supraoptic neurosecretory cells, Physiologist, 13 (1970) 272. 25 OBATA,K., TAKEDA,K., AND SHINOZAKI,H., Further study on pharmacological properties of the cerebellar induced inhibition of Deiters' neurons, Exp. Brain Res., 11 (1970) 327-342. 26 OBATA,K., ANDHIGEISTEIN,S. M., Blocking by picrotoxin of both vestibular inhibition and GABA action on rabbit oculomotor neurons, Brain Research, 18 (1970) 538-541. 27 OLIVER,A. P., A simple, rapid method for preparing 'parallel' micropipette electrodes, Electroenceph, clin. Neurophysiol., 31 (1971) 284-286. 28 OOTA, Y., AND KOBAYASHI,H., Synapses between neurosecretory axons and the processes of nonneurosecretory neurons, Dobutsugaku Zasshi, 72 (1963) 35-39. 29 RUFENER, C., AND DREIFUSS, J. J., Selective zinc iodide-osmium tetroxide impregnation of synaptoid vesicles in the rat neurohypophysis, Brain Research, 22 (1970) 402-405.
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30 SALMOIRAGHI,G. C., AND WEIGHT,F. F., Micromethods in neuropharmacology: an approach to the study of anaesthetics, Anaesthesiology, 28 (1967) 54-64. 31 SCHARRER,B., Neurohumors and neurohormones: definitions and terminology, J. Neuro- Viscerol. Relat., 9 (1969) 1-20. 32 SCHARRER,E., AND SCHARRER,B., Hormones produced by neurosecretory cells, Recent Progr. Hormone Res., 10 (1954) 183-240. 33 VORHERR,H., BRADBURG,M. W. B., HOGHARGHI,M., ANDKEEEMAN,C. R., Antidiuretic hormone in cerebrospinal fluid during endogenous and exogenous changes in its blood level, Endocrinology, 83 0968) 246-250. 34 WERMAN, R., DAVIDOFF, R. A., AND APRISON, M. H., Inhibitory action of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. 35 YAGI, K., AZUMA, T., AND MATSUDA,K., Neurosecretory cell: capable of conducting impulse in rats, Science, 154 (1966) 778-779. 36 YAMASHITA,H., AND KOIZUMI, K., Excitation and inhibition of mammalian neurosecretory cells, Physiologist, 13 (1970) 351. 37 YAMASHITA,H., KOIZUMI, K., AND BROOKS, C. McC., Electrophysiological studies of neurosecretory cells in the cat hypothalamus, Brain Research, 20 (1970) 462-466.
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T H E P H A R M A C O L O G Y OF R E C U R R E N T I N H I B I T I O N IN T H E SUPRAOPTIC N E U R O S E C R E T O R Y SYSTEM
ROGER A. NICOLL AND JEFFERY L. BARKER* Laboratory of Neuropharmaeology, Division of Speeial Mental Health Research, National Institute of Mental Health, Saint Elizabeth's Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted June 24th, 1971)
INTRODUCTION Axons of supraoptic neurosecretory neurons terminate on fine capillaries and release their hormonal products into the circulationlS, a2. However, these neurosecretory cells also possess basic neuronal properties 4 including synaptic functions 6,17,aa-aT. We6, 2~ and otherslV,lS,a6, 37 have demonstrated an inhibitory pathway to neurosecretory cells which appears to be mediated by neurosecretory axons. Since our neurophysiologic investigation has failed to reveal a suitable interneuron mediating this inhibition, the possibility of a direct inhibitory interaction between neurosecretory cells should be considered. If neurosecretory cells participate in synaptic events, they must either utilize their secretory product as the synaptic transmitter or elaborate another substance in addition to the hormone. We have investigated the pharmacology of this inhibitory pathway in the supraoptic neurosecretory system of the cat by applying various pharmacologic agonists and antagonists both intravenously and microelectrophoretically in an attempt to identify the synaptic transmitter mediating the inhibition. METHODS Eighteen cats were anesthetized with intraperitoneal sodium pentobarbital (35 mg/kg), tracheotomized and placed supine in a stereotaxic apparatus. Small doses of sodium pentobarbital were given intravenously during the experiment to maintain a light level of anesthesia. Prior to the intravenous administration of convulsant drugs, the animal was paralyzed with intravenous gallamine triethiodide (5 mg/kg) and artificially ventilated with room air. A buccal approach was used to expose the optic chiasm and pituitary gland. * Present address: Electroencephalography Branch, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Md. 20014, U.S.A. Brain Research, 35 (1971) 501-511
502
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N I C O L L A N D J. L. B A R K E R
(Further details on methodology may be found in a recent paper~.) Extracellular unit recordings were made through the 5 M NaCl-filled center barrels of 5-barrel micropipettes, prepared according to the methods previously described30. In several experiments fine glass microelectrodes were glued to 5-barrel micropipettes with lucite chips so that the single, recording barrel protruded 5-10/zm from the micropipette assembly27. This allowed excellent recording of cellular activity as well as easy passage of large currents from the drug barrels. The unit activity was displayed on an oscilloscope, gated with an adjustable window and then relayed either to a 1-sec integrator and recorded on a Beckman pen-writer or to a computer of average transients (CAT, Mnemotron). Supraoptic unit activity in response to neurohypophyseal stimuli was summated on the CAT for a period of 80 msec before and 420 msec after the onset of each stimulus. Generally, 100 stimuli summated in this fashion proved adequate to demonstrate the inhibition of neurosecretory cells following such stimuli. The pharmacologic agents employed were dissolved in distilled water as follows: 0.5 M DL-homocysteate (pH 7.5; NaOH) (DLH), 0.1 M atropine sulfate, 1 M glycine (pH 3.5; HC1), 1 M gamma-aminobutyric acid (GABA) (pH 3.5; HC1), and 0.1 M strychnine phosphate. Bicuculline was prepared as a 0.01 M solution of 0.165 M NaC1 (pH adjusted to 3.5 with 0.1 N HC1) and ejected with cationic current. Picrotoxin was made up as a 0.01 M solution of 0.165 M NaC1 (pH 4.5) and ejected with cationic current. 0.025 M antidiuretic hormone (lysine vasopressin) (Calbiochem) was dissolved in distilled water (pH 4-5) and ejected with cationic current. (Lysine vasopressin rather than the naturally occurring arginine vasopressin was used because of its ready availability. Although the ADH is reported to be chromatographically pure, we cannot entirely exclude the possibility that some breakdown of the peptide may have occurred.) Previously described electrical methods prevented polarization of the electrode tip during drug ejection, as well as diffusion of drugs from the micropipette 30. Most supraoptic neurons encountered were silent and therefore a continuous application of DLH was required to produce a suitable background level of activity against which drug effects could be assessed. The drug responses observed in DLHexcited cells did not appear to differ from responses obtained in spontaneously active cells. Antidiuretic hormone (ADH) usually required relatively large ejection currents to obtain drug responses. Since it was often difficult to exclude the electrical effects of large, direct currents on cells, each cell requiring large current passage for the presumed drug effect was thoroughly tested with currents of equal strength passed through the 3 M NaC1 (balancing) barrel. Any cell responding to these latter currents was removed from further study. At the conclusion of the experiments poststimulus histograms of neurosecretory inhibition were obtained before and during the intravenous administration of strychnine, bicuculline and atropine. Two cats were injected intraventricularly with 6-hydroxydopamine (6-HODA), a substance which is known to selectively destroy norepinephrine terminals7. A total of 2 mg/kg was given in two doses 3 days apart. Acute experiments on these cats Brain Research, 35 (1971) 501-511
PHARMACOLOGY OF RECURRENT INHIBITION
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were performed 3 days after the second dose. Following the experiments, areas of the brain including the supraoptic region were frozen in liquid nitrogen and prepared for histofluorescence. RESULTS
Acetylcholine. Since microelectrophoretically applied acetylcholine (ACh) causes inhibition of supraoptic neurosecretory cells by acting at muscarinic sites 5 and since ACh is present in both the supraoptic nucleus and the neurohypophysisz2, ACh may be acting at muscarinic sites to mediate the observed inhibition. Therefore, the muscarinic antagonist atropine13,16, which blocks ACh-induced depressions of supraoptic neurons 5, was used to test this possibility. Atropine was microelectrophoretically applied to 6 supraoptic neurons in which control postimulus histograms had demonstrated recurrent inhibition (RI). Atropine delivered in this way did not affect the RI. Furthermore, intravenous injection of atropine in doses up to 1.3 mg/kg also had no effect on the RI (Fig. 3A). Norepinephrine. In a previous study we have shown that norepinephrine (NE) inhibits the activity of supraoptic neurosecretory cells 5 which receive numerous NE terminals 8. Thus we considered NE as a candidate for the inhibitory transmitter.
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Fig. 1. Action of glycine and strychnine on supraoptic neurosecretory cells. A, Continuous polygraph record of 1-sec integrated unit activity showing dose-response depression of activity to microelectrophoretic application of glycine (solid lines with current in nanoamperes above trace). Calibration bracket to left of record: 0-10 spikes/sec. Antidromically activated neurosecretory cell spike upper right. Calibration: 5 msec/100 pV. B, Strychnine ejected with 20 nA for the duration of center record readily antagonizes the glycine-induced depressions (solid lines) of another neurosecretory cell (antidromic spike above trace at right with same calibrations as in A).
Brain Research, 35 (1971) 501-511
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Dose-response depression of activity by microelectrophoretic application of GABA (solid lines with current in nanoamperes above record). Calibration bracket at left of trace: 0-20 spikes/sec. B, Continuous record showing bicuculline antagonism (at 125 hA) of GABA depressions (solid lines, 5 nA) in a neurosecretory cell displaying recurrent inhibition (poststimulus histogram beneath trace showing inhibition following neurohypophysial stimuli delivered at time indicated by upward arrow; calibrations: vertical bar: 0-5 counts/address; horizontal bar: 100 msec). C, Picrotoxin antagonism (at 200 nA)of GABA depression (solid lines, 20 nA). Calibration bracket at left: 0--5 spikes/sec. Identifying antidromic spike above trace at left. Calibration: 5 msec; 100/~V. Cats pretreated with 6 - H O D A (which selectively destroys NE endings) were examined for RI. RI was present in these cats despite the fact that nearly all the NE-fluorescent terminals in the supraoptic nucleus were gone. Glycine. It has been proposed that glycine is an important inhibitory transmitter in the spinal cord za. The convulsant strychnine blocks both glycine-induced depressions of spinal neurons and spinal postsynaptic inhibition 10. Glycine applied microelectrophoretically to supraoptic neurons inhibited all 79 tested. Relatively small doses were sufficient to cause complete inhibition of activity (Fig. 1A). The average current required to inhibit these neurons was 20 nA. Glycine depressions were readily antagonized by concomitant microelectrophoretic application of strychnine in 14 of 15 cells tested (Fig. 1B). However, neither microelectrophoretic administration of strychnine at a time when glycine depression was blocked nor intravenous injections up to 1.0 mg/kg affected the RI (Fig. 3C). GABA. Recent studies have shown that the convulsants picrotoxin 26 and bicuculline 11 specifically antagonize the depressant action of G A B A on neuronal activity and also block a number of supraspinal inhibitory pathways11,12,z3, 25, suggesting
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t Fig. 3. Action of intravenous atropine, bicuculline and strychnine on the recurrent inhibitory pathway in the supraoptic neurosecretory system. Control poststimulus histograms of neurosecretory cell inhibition in response to neurohypophysial stimuli (arrows beneath bottom histograms) are across top row. Calibration bracket (0-5 counts/address) and time scale same for all records. A, 1.3 mg/kg atropine i.v. does not effect inhibition profile. B, Middle record shows slight reduction in inhibition following0.67 mg/kg bicuculline i.v., the threshold dose for seizure activity. A similar profile is evident in the bottom trace, after a total of 1.0 mg/kg bicuculline. C, Strychnine (1.0 mg/kg i.v.) does not alter the recurrent inhibition. an important role for G A B A in supraspinal postsynaptic inhibition. In the supraoptic nucleus G A B A inhibited all 74 neurons studied. Small ejecting currents were able to produce profound depressions of activity (Fig. 2A). The average current necessary to completely inhibit unit activity was 10 nA. Microelectrophoresis of either picrotoxin (8 of 10 cells tested) or bicuculline (9 of 10 cells tested) antagonized the GABAinduced depressions of unit activity (Fig. 2B and C). However, microelectrophoretic application of both picrotoxin and bicuculline failed to block RI. Similarly, intravenous injections of bicuculline did not affect the synaptic inhibition when given in subconvulsant doses. Yet, at doses which produced generalized seizures and bursting of neurosecretory cells some reduction in inhibition was observed (Fig. 3B). The obvious changes in neuronal excitability during convulsions make it difficult to relate this reduction in inhibition to a specific antagonism of the pathway. A n t i d i u r e t i c h o r m o n e . The possibility that antidiuretic hormone (ADH) might in fact be the inhibitory transmitter was investigated by applying A D H microelectrophoretically to cells in the supraoptic nucleus. In general, A D H appeared to have a weak pharmacologic action on the excitability of neurons as evidenced by the need to use large ejecting currents in order to obtain an effect. In those neurons which were either antidromically invaded and/or exhibited RI, 80 ~ of the responsive cells were inhibited by A D H (Table I; Fig. 4A and B). Occasionally neurosecretory cells TABLE I NEURONAL
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22 63 35 Brain Research, 35 (1971) 501-511
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Fig. 4. Responses of supraoptic neurosecretory cells to microelectrophoresis of ADH. A, Doseresponse depression of activity in a cell exhibiting recurrent inhibition (histogram beneath record; calibration: 5 counts/address; 100 msec) by ADH (solid lines above trace, currents in nA). Calibration bracket at/eft: 0-20 spikes/sec. B, ADH depression of neurosecretory cell activity. Polygraph calibration same as in A. Antidromic spike below trace. Calibration: 5 msec/100 /~V. C, ADH excitation of a neurosecretory cell. Calibration bracket: 0-5 spikes/sec. Antidromic spike centered above trace. Calibration: 2 msec/4 inV.
were excited by A D H (Fig. 4C), a l t h o u g h larger ejecting currents were needed to p r o d u c e this effect. A D H was also tested on cortical n e u r o n s in o r d e r to serve as a c o n t r o l for the responses seen in the s u p r a o p t i c n e u r o s e c r e t o r y system. Microelect r o p h o r e s i s o f A D H in this a r e a excited nearly 9 0 ~ o f r e s p o n d i n g cells (Table I; Fig. 5A), a l t h o u g h several n e u r o n s were inhibited using large ejecting currents (Fig. 5B). DISCUSSION T h e present p h a r m a c o l o g i c investigation was p r o m p t e d by the recent finding o f s u p r a o p t i c n e u r o s e c r e t o r y cell i n h i b i t i o n following electrical s t i m u l a t i o n o f the post e r i o r pituitary18,24, ~7. A l t h o u g h the a n a t o m y o f this i n h i b i t o r y p a t h w a y is n o t en-
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Fig. 5. Responses of neurons in the sensorimotor cortex to microelectrophoresis of ADH. A, Examples of excitation in response to ADH (solid lines above trace, nA current). B, ADH depression of a cortical neuron. Calibration bracket: 40 spikes/sec (A, left); 20 spikes/sec (A, right and B). tirely clear, recurrent collaterals have been describedL F r o m our neurophysiological observations we have been unable to identify an interneuron participating in this pathway6, is, which suggests that neurosecretory cells and/or collaterals synapse directly with other neurosecretory cells. Since supraoptic neurosecretory cells possess synaptic ability, they must either release their hormonal product (ADH) at synapses or elaborate a separate transmitter substance. If Dale's Principle (that the same substance is released from all branches of a particular neuron) 14 holds for neurosecretory cells, then one might expect A D H to be released at synapses as well as into fine capillaries. Axo-axonic synapses between neurosecretory and non-neurosecretory neurons have been described in several species2,19, 2s and 'neurosecretomotor junctions' with epithelial cells of the pars intermedia have recently been reported in the cat 3, but synapses between two neurosecretory elements have not yet been found. The second possibility - - that neurosecretory cells possess a dual secretory role - - is based primarily on electron microscopic evidence of two morphologically different populations of storage site in neurosecretory cells: large, elementary neurosecretory granules 20 and small, clear 'synaptoid' vesicles 21 which stain selectively with ZnI-OsO4 (ref. 29). Although this stain has been correlated with synaptic vesicles elsewhere in the nervous system 1, the functional significance of these 'vesicles' is not clear and it has been suggested that they may represent a different stage in the neurosecretory process~L Our investigation was designed to explore these possibilities by considering 5 suspected transmitter substances: ACh, NE, glycine, G A B A and A D H . Brain Research, 35 (1971) 501-511
508
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Acetylcholine. The depressant action of ACh on neurosecretory cells is mediated through muscarinic sites and is blocked by atropine 5. However, the fact that atropine administered either microelectrophorerically or intravenously failed to reduce the RI, makes it unlikely that ACh is the inhibitory transmitter. Norepinephrine. Although NE inhibits the activity of supraoptic neurosecretory cells 4 and is present in the terminals ending on these cellss, there is no anatomic evidence that neurosecretory axons contain NE. Furthermore, NE-fluorescent cell bodies have not been described in the supraoptic region. Thus, it is unlikely that NE is involved in RI. Our demonstration of the inhibition in cats which had their NE terminals destroyed by 6-HODA further reduces the possibility that NE is the inhibitory transmitter. Glycine and GABA. In agreement with results obtained elsewhere in the central nervous systemX0,11,25,34, we have found that both glycine and GABA are potent depressants of neurosecretory cell activity, as evidenced by their rapid onset of action and quick reversibility of effect at relatively low ejecting currents. GABA appeared to be slightly more potent in this regard (based on the average amount of current required to inhibit neurosecretory cells). Strychnine, a convulsant which blocks both spinal postsynaptic inhibition34 and glycine-induced depression of cellular activity in many areas of the nervous system10,2z,25,26 was quite effective in blocking glycine inhibition of neurosecretory cells. However, neither microelectrophoretic application, nor intravenous injection of convulsant doses of strychnine (up to 1.0 mg/kg) were able to antagonize the RI in the supraoptic neurosecretory system. Both picrotoxin and bicuculline have been reported to block several supraspinal inhibitory pathways and to antagonize the depressant actions of GABA when administered microelectrophoretically11, 9,6. GABA-induced depression of neurosecretory cells was also effectively blocked by microelectrophoresis of either picrotoxin or bicuculline. However, neither of these convulsants were effective in antagonizing the RI when applied in this manner. The slight reduction in inhibition observed following intravenous convulsant doses of bicuculline was difficult to interpret owing to the generalized seizure activity and therefore could not be related to a specific antagonism of the pathway. This, however, does not rule out GABA as the inhibitory transmitter in this pathway. Antidiuretic hormone. When ADH was applied to neurons exhibiting RI, a high proportion of responding cells (80~) was depressed. On the contrary, application of ADH to cortical neurons caused excitation in a great majority (nearly 90 ~) of responsive cells. These results suggests that the excitatory effect of ADH is nonspecific, and the inhibitory effect related to supraoptic neurosecretory cells. Although we have been able to block the depressant actions of the suspected transmitters considered in this study (ACh, glycine, GABA) with microelectrophoresis of their antagonists (atropine, strychnine, picrotoxin and bicuculline), we have not been able to block the inhibitory pathway by either microelectrophoretic or intravenous administration of these antagonists. We feel it unlikely that either ACh or glycine is the suspected transmitter mediating RI. The present findings related to GABA do not permit excluding it from a possible role in this pathway. Our results Brain Research, 35 (1971) 501-511
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also suggest that NE is not involved in this pathway. On the other hand, wefinda good correlation between cells exhibiting RI and those depressed by microelectrophoretic application of ADH. Complementary to this is the report that A D H is released (in response to hemorrhage) into the cerebrospinal fluid independent of plasma concentrations 3a. This suggests an intracranial release of A D H coincident with the increase in supraoptic neurosecretory cell activity. These lines of evidence support the notion that A D H may be the transmitter mediating RI in the supraoptic neurosecretory system; however, more anatomic evidence is needed to further strengthen this hypothesis. SUMMARY A pharmacologic study of recurrent inhibition in the supraoptic neurosecretory system of the cat has been conducted in an attempt to identify the inhibitory transmitter in this pathway. Acetylcholine (muscarinic component), glycine and GABA were considered as possible transmitters mediating this inhibitory pathway. Antagonists to the depressant actions of these substances on neurosecretory cells were tested on both unit responses and the inhibitory pathway. Although atropine, strychnine, picrotoxin and bicuculline were able to block the pharmacologic depressions, they were allIineffective in antagonizing the recurrent inhibition when applied either microelectrophoretically or intravenously. It is thus unlikely that either acetylcholine, glycine or GABA participate in this inhibitory pathway. Norepinephrine was also considered as a candidate for inhibitory transmitter. However, since recurrent inhibition was present in cats whose norepinephrine-containing terminals were destroyed by pretreatment with 6-hydroxydopamine, we do not think norepinephrine is the transmitter. The possibility that the neurosecretory product A D H might also serve a synaptic function and mediate the recurrent inhibition was investigated by microelectrophoretic administration of A D H to neurosecretory cells. The fact that a good correlation was found between those cells exhibiting recurrent inhibition and those depressed by A D H supports the idea that A D H may serve as a transmitter in this recurrent inhibitory pathway. ACKNOWLEDGEMENTS We thank A. P. Oliver and B. Hamstrom for fine technical assistance. We are grateful for a sample of vasopressin received from Dr. R. Walter. We are indebted to Mrs. Jean Alwine for her work on the histochemistry and to Dr. F. Bloom for interpreting the fluorescent findings and reviewing the manuscript. REFERENCES 1 AKERT,K., AND SANDRI,C., An electron-microscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions, Brain Research, 7 (1968) 286-295.
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2 BARGMANN,W., Neurosecretion, Int. Rev. Cytol., 19 (1966) 183-201. 3 BARGMANN,W., LINDER, E., UND ANDRES, K. H., t0ber Synapsen an endokrinen Epithelzellen und die Definition sekretorischer Neurone, Z. Zellforsch., 77 (1967) 282-298. 4 BARKER,J. L., CRAVTON,J. W., AND NICOLL, R. A., Supraoptie neurosecretory cells: Autonomic modulation, Science, 171 (1971) 206-207. 5 BARKER,J. L., CRAYTON,J. W., AND NICOLL, R. A., Supraoptic neurosecretory cells: Adrenergic and cholinergic sensitivity, Science, 171 (1971) 208-210. 6 BARKER,J. L., CRAYTON,J. W., AND NICOLL, R. A., Antidromic and orthodromic responses of paraventricular and supraoptic neurosecretory cells, Brain Research, 33 (1971) 353-366. 7 BLOOM,F., ALGERLS., GROPPEa~rI,A., REVUELTA,A., AND COSTA, E., Lesions of central norepinephrine terminals with 6-OH-dopamine: Biochemistry and fine structure, Science, 166 (1969) 1284-1286. 8 CARLSSON,A,, FLACK, B., AND HILLARP, N. A., Cellular localization ofbrainmonoamines, Acta physiol, scand., 56, Suppl. 196 (1962) 1-28. 9 CHRIST, J. F., Nerve supply, blood supply and cytology of the neurohypophysis. In G. W. HARRIS AND B. T. DONOVAN(Eds.), The Pituitary Gland, Vol. 3, Univ. California Press, Berkeley, Calif., 1966, pp. 62-130. 10 CURTIS, D. R., H6SLI, L., JOHNSTON, G. A. R., AND JOHNSTON,I. H., The hyperpolarization of spinal motoneurons by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 11 CURTIS, D. R., DUGGAN, A. W., FELIX, D., AND JOHNSTON, G. A. R., GABA, bicuculline and central inhibition, Nature (Lond.), 226 (1970) 1222-1224. 12 CURTIS, D. R., FELIX, D., AND MCLENNAN, H., GABA and hippocampal inhibition, Brit. J. Pharmacol., 40 (1970) 881-883. 13 DALE, H. A., The action of certain esters and ethers on choline, and their relation to muscarine, J. Pharmacol., 6 (19t4) 147-190. 14 DALE, H. A., Pharmacology and nerve endings, Proc. roy. Soc. Med., 28 (1935) 319-332. 15 GINSBURG, M., Production, release, transportation and elimination of the neurohypophysial hormones. In B. BEROE (Ed.), Handbook of Experimental Pharmacology, Vol. 23, Springer, Berlin, 1968, pp. 286-371. 16 HENDERSON,V. E., AND ROEPKE, M. H., Drugs affecting parasympathetic nerves, Physiol. Rev., 17 (1937) 373-407. 17 KANDEL, E. R., Electrical properties of hypothalamic neuroendocrine cells, J. gen. Physiol., 47 (1964) 691-717. 18 KELLY, J. J., AND DREIFtJSS, J. J., Antidromic inhibition of identified rat supraoptic neurones, Brain Research, 22 (1970) 406-409. 19 KOBAYASHI,H., HIRANO, T., AND OOTA, Y., Electron microscopic and pharmacological studies on the median eminence and pars nervosa, Arch. Anat. ruler., 54 (1965) 277-294. 20 KUROSUr~LK., MATSUZAWA,T., AND SHIBASAKLS., Electron microscope studies on the fine structure of the pars nervosa and pars intermedia, and their morphological interrelation in the normal rat hypophysis, Gen. comp. Endocr., 1 (1961) 433-452. 21 LE•ERIs• K.• A pre•iminary rep•rt •n the u•trastructure •f the human neur•hyp•physis• J. End••r.• 27 (1963) 133-135. 22 LEDERIS,K., AND LIVINGSTON,A., Acetylcholine and related enzymes in the neural lobe and anterior hypothalamus of the rabbit, J. Physiol. (Lond.), 201 (1969) 695-709. 23 NICOLL, R. A., GABA and dendrodendritic inhibition in the olfactory bulb, Pharmacologist, 12 (1970) 236. 24 NICOLL, R. A., CRAYTON,J. W., AND BARKER, J. L., Antidromic and orthodromic responses in supraoptic neurosecretory cells, Physiologist, 13 (1970) 272. 25 OBATA,K., TAKEDA,K., AND SHINOZAKI,H., Further study on pharmacological properties of the cerebellar induced inhibition of Deiters' neurons, Exp. Brain Res., 11 (1970) 327-342. 26 OBATA,K., ANDHIGEISTEIN,S. M., Blocking by picrotoxin of both vestibular inhibition and GABA action on rabbit oculomotor neurons, Brain Research, 18 (1970) 538-541. 27 OLIVER,A. P., A simple, rapid method for preparing 'parallel' micropipette electrodes, Electroenceph, clin. Neurophysiol., 31 (1971) 284-286. 28 OOTA, Y., AND KOBAYASHI,H., Synapses between neurosecretory axons and the processes of nonneurosecretory neurons, Dobutsugaku Zasshi, 72 (1963) 35-39. 29 RUFENER, C., AND DREIFUSS, J. J., Selective zinc iodide-osmium tetroxide impregnation of synaptoid vesicles in the rat neurohypophysis, Brain Research, 22 (1970) 402-405.
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30 SALMOIRAGHI,G. C., AND WEIGHT,F. F., Micromethods in neuropharmacology: an approach to the study of anaesthetics, Anaesthesiology, 28 (1967) 54-64. 31 SCHARRER,B., Neurohumors and neurohormones: definitions and terminology, J. Neuro- Viscerol. Relat., 9 (1969) 1-20. 32 SCHARRER,E., AND SCHARRER,B., Hormones produced by neurosecretory cells, Recent Progr. Hormone Res., 10 (1954) 183-240. 33 VORHERR,H., BRADBURG,M. W. B., HOGHARGHI,M., ANDKEEEMAN,C. R., Antidiuretic hormone in cerebrospinal fluid during endogenous and exogenous changes in its blood level, Endocrinology, 83 0968) 246-250. 34 WERMAN, R., DAVIDOFF, R. A., AND APRISON, M. H., Inhibitory action of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. 35 YAGI, K., AZUMA, T., AND MATSUDA,K., Neurosecretory cell: capable of conducting impulse in rats, Science, 154 (1966) 778-779. 36 YAMASHITA,H., AND KOIZUMI, K., Excitation and inhibition of mammalian neurosecretory cells, Physiologist, 13 (1970) 351. 37 YAMASHITA,H., KOIZUMI, K., AND BROOKS, C. McC., Electrophysiological studies of neurosecretory cells in the cat hypothalamus, Brain Research, 20 (1970) 462-466.
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