Transition from random to phasic firing induced in neurons cultured from the hypothalamic supraoptic area

Brain Research, 193 (1980) 415-425 © Elsevier/North-Holland Biomedical Press

415

T R A N S I T I O N F R O M R A N D O M TO PHASIC F I R I N G I N D U C E D IN NEURONS C U L T U R E D F R O M T H E H Y P O T H A L A M I C SUPRAOPTIC A R E A

B. H. G/~HWILER and J. J. DREIFUSS Pharmaceutical Division, Preclinical Research, Sandoz Ltd., Basle and Department of Physiology, University of Geneva Medical School, Geneva (Switzerland)

(Accepted January 10th, 1980) Key words: neuropeptides - - nerve tissue cultures - - intracellular recording - - hypothalamus - -

firing pattern - - drug effects

SUMMARY Intracellular recordings of the spontaneous activity were obtained from neurons in long-term cultures from the area of the supraoptic nucleus of rats. The effects of various substances known from in situ studies to cause vasopressin release were analyzed. Application of nicotine or acetylcholine induced a transition from a random to a phasic discharge pattern. Similar alterations in firing patterns were observed with enkephalin analogues, an effect which was blocked by the opiate antagonist, naloxone. Glutamate excited hypothalamic neurons in a dose-dependent manner, but did not induce phasic firing. Angiotensin II increased the firing rate in randomly firing cells and the duration of bursts in phasic cells. In an attempt to identify the transmitter(s) involved in the generation of phasic activity, several antagonists to excitatory transmitters were applied. Of the agents tested, only saralasin reduced the duration of bursts, but it is questionable whether this effect is due to its angiotensin-antagonistic property.

INTRODUCTION While many nerves cells in invertebrate ganglia have been shown to fire in intermittent burstsaa,39, 44, this type of neural activity has been less intensively studied in the mammalian nervous system. Phasic discharges are, however, observed in endocrine neurons in the hypothalamus11,26,40, 51. In the rat, phasic activity is believed to be a characteristic of vasopressin-secreting neuronsg,la,aa,aa, 49. Short interspike intervals in these cells have been shown to be particularly effective in causing vasopressin release 16. In order to study the mechanisms involved in determining the phasic spiking activity we have organ-cultured the area of the hypothalamic supraoptic nucleus of

416 young rats in which a proportion of neurons display this pattern of activityee, "':~. Amongst these, a few appear to be true pacemaker neurons 2t. This in vitro preparation allows stable intracellular recordings and bath application of drugs. In the present study, various compounds were chosen from those known, from in situ studies, to induce vasopressin release: several of these substances caused randomly firing neurons in culture to adopt a bursting pattern of activity or, otherwise, modulated the characteristics of pre-existing bursts. METHODS AND MATERIALS Cultures of hypothalamic tissue including the supraoptic nucleus were prepared from 1-7-day-old rats as previously described 23. Standard electrophysiological techniques were used to record intracellularly the spontaneous activity of large nerve cells. Electrodes were filled with 1.6 M potassium citrate or 3 M KC1 and had a resistance of 40-80 M ~). During electrophysiological recordings, the cultures were continuously perfused at a constant rate of 1 ml/min with Hanks' balanced salt solution. The composition of the medium could be repeatedly altered without interruption of the perfusion flow. Substances directly added to Hanks' balanced salt solution included: acetylcholine, Damino-adipate, atropine, curare, GABA, glutamate and mecamylamine (all from Sigma), angiotensin I1 (Calbiochem), dihydro-fl-erythroidine (Merck), muscimol (Ciba-Geigy), nicotine (Schuchardt), nuciferine (dimethoxyaporphine, Smith, Kline and French), saralasin (Sarl-AlaS-angiotensin II, Bachem) and THIP (Lundbeck). The enkephalin analogues FK 33-824 and FW 34-569 are Sandoz substances, and bicuculline methochloride was also synthesized at Sandoz from bicuculline (Fluka). RESULTS Spontaneous activity was recorded intracellularly from 48 large cells (25-35 #m in diameter) in cultures of the hypothalamic supraoptic nucleus area. Action potentials with amplitudes in excess of 40 mV were recorded for several hours. Bath application of 10-4 M glutamate depolarized the cells and rapidly and reversibly increased their average firing rate (Fig. 1A). At 10-3 M, the spike generating mechanism was partially inactivated. In contrast to glutamate, nicotine (17 cells tested) in a concentration of 10-5-10 -3 M changed the firing pattern by inducing bursts of activity in previously randomly firing cells (Fig. 1A). At 10-3 M, the firing was characterized by short rhythmic bursts followed by silent periods (Fig. 2). Excitatory effects similar to those produced by nicotine were noticed with 10-5 M acetylcholine in all 4 cells tested. Prior application of 10-5-10 -4 M dihydro-fl-erythroidine partially prevented the nicotine action, whereas neither hexamethonium nor mecamylamine (up to 10-3 M) affected the nicotinic response. Two actions of angiotensin II (10-6-10 -5 M) could be distinguished. First, it increased the average firing rate of randomly discharging cells (Fig. tB). Second, in 7 of 13 cells displaying a tendency for phasic activity, angiotensin increased the duration

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Fig, 1. Effects of drugs on spontaneous activity. Ratemeter records of spontaneous bioelectric activity recorded intracellularly from 3 cells of the supraoptic nucleus area. A: bath application of 10-4 M glutamate (GLU) increased the firing rate. The nicotine-induced excitation was accompanied by a transition from random to phasic activity. B: angiotensin 1I (All, 10-~ M) increased the average firing rate of a random ceil. C: GABA agonists abolished the firing of a phasic cell. THIP and muscimol (MUSC) were more potent than GABA. Simultaneous application of 10 -5 M bicuculline methochloride (BMC) antagonized the muscimol action. Drug concentrations are given as the log of the molarity. Age of the cultures: A, 50 days in vitro (DIV); B, 36 DIV; C, 46 DIV.

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Fig. 2. Contrasting effects of glutamate and nicotine. Excerpts of spike trains recorded from a neuron of the supraoptic nucleus area. At 10 .4 M glutamate (GLU) increased the firing rate, whereas 10.3 M nicotine elicited phasic activity. After each drug application, control solution was perfused until the firing returned to near-control values. Resting potential, --52 mY. Same cell as in Fig. 1A.

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5s~ Fig. 3. Angiotensin effects. Bath application of 10-~ M angiotensin II (AII) reversibly increased the duration of spontaneous bursts. Resting potential, --68 mV. Age of the culture, 35 D1V. but not the number of bursts (Fig. 3). Despite continuous perfusion, the angiotensin effect was usually short-lived. Randomly firing neurons became phasic when enkephalin analogues 41 were added to the bathing medium (Fig. 4). F W 34-569 ( M e T y r - D - A l a - G l y - M e P h e - M e t (O)-ol) induced bursting activity in 6 cells and had no effect in 2 cells whereas F K 33824 (Tyr-D-Ala-Gly-MePhe-Met(O)-ol) elicited bursts in 5 cells, inhibited 3 and had no effect on 4 cells. In general, the enkephalin action was rapid in onset and reversible, but relatively high concentrations (10-5-10 -4 M) of the peptides were needed in order

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Fig. 4. Effects of an enkephatin analogue. Reversible transition from random to phasic firing induced by 10-4 M FW 34-569 (ENK). Resting potential, --54 mV. Age of the culture, 21 DIV.

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5.~c Fig. 5. Effects of GABA antagonists. Spike trains recorded during application of 10 -4 M o-tubocurarine (dTc) and 10-5 M bicuculline methochloride (BMC). In contrast to dTc and BMC, 10-4 M hexamethonium (HEX) induced no bursting activity. Resting potential, - - 6 0 inV. Age of the culture, 20 DIV.

to produce this random-to-phasic transition of the firing pattern. In preliminary experiments, the opiate antagonist naloxone 0 0 -6 M) (4 cells) blocked the excitatory effect of the opioid peptides. A consistent feature of cultured hypothalamic neurons was their pronounced sensitivity to GABAergic drugs. GABA in a concentration of 10-4 M reduced the occurrence of phases and finally abolished the activity of phasic neurons. Similar results were obtained with the more potent GABA-agonists, THIP 31 (10-5 M) and muscimol (10 -6 M). The effects of all three were shown to be bicuculline-sensitive (Fig. 1C). This GABA antagonist invariably induced phasic activity with highly rhythmic short bursts (Fig. 5). It is likely that D-tubocurarine, which produced a very similar change in firing pattern (Fig. 5), exerted this effect by virtue of its GABA-antagonistic, rather than its anticholinergic properties 2a. Hexamethonium (Fig. 5) and mecamylamine, substances with known anti-nicotinic activity, never elicited phasic discharges.

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Fig. 6. 'Abnormal electrical discharges' induced in hypothalamic cultures. In 4 different cells, o-tubocurarine (dTc), 10-5 M bicuculline methochloride (BMC) and exposure to Cl--free solution (C1 replaced by isethionate) caused bursts with plateau-formation during which there was a partial spike inactivation. The concentrations of dTc were 10 -4 M in A and 10-5 M in B. Resting potentials, --58 mV (A), --54 mV (B), ~ 6 8 mV (C) and --70 mV (D). Age of the cultures, 21 DIV (A), 21 DIV (B), 24 DIV (C) and 38 DIV (D).

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Fig. 7. Saralasin effects. Saralasin (SAR) in a concentration of 10 -s M reversibly reduced the duration of spontaneous bursts to single spikes and doublets. Resting potential, --78 mV. Age of the culture, 53 DIV.

421 In addition to the generation of phasic activity, GABA antagonists elicited in a proportion of cells (Fig. 6) bursts which resembled the 'abnormal bursting discharges' 10 induced in molluscan and crustacean neurons by convulsants such as metrazol and strychnine. These discharges were characterized by a plateau formation accompanied by bursting discharges. Following the first few spikes of a burst, the spike-generating mechanism was usually (but not always) partially inactivated. These bursts were followed by an hyperpolarization of the membrane. Since C1--free solutions (C1replaced by isethionate) elicited similar bursting discharges (Fig. 6), it is assumed that they are caused by a blockade of inhibitory synaptic transmission. In an earlier study 21, the phasic activity of cultured hypothalamic neurons was shown in the majority of cases to be synaptic in origin. In an attempt to identify the transmitter involved, we analyzed the effects of various antagonists to putative excitatory transmitters on cells which spontaneously displayed phasic activity. Atropine, nuciferine 18, D-aminoadipate 6, hexamethonium, mecamylamine, dihydro-fl-erythroidine, naloxone and saralasin were applied in concentrations varying from 10-6 M to 10-3 M. These substances were without any detectable effect with the exception of saralasin which in a concentration of 10-~ M reversibly reduced the duration of spontaneous bursts (Fig. 7). Although in some cells the activity was completely randomized, the specificity of this effect is doubtful since saralasin also decreased the firing rate of randomly firing neurons and, furthermore, inhibited cultured hippocampal and cerebeUar neurons (B.H.G., unpublished observations). DISCUSSION In the rat 14, monkeyl,~7 and sheep ~9 hypothalamus in situ, a proportion of supraoptic neurons are characterized by a phasic pattern of firing in which bursts of spiking activity alternate with periods devoid of action potentials. This phasic activity has also been observed in rat brain slices ~4,25 and in cultured neurons although, in the latter, the phases tended to be shorter and action potentials occurred at higher rates ~1,2z than those in situ. Stimuli which cause neurohypophysial hormone release increase the rate of firing of supraoptic neurons11,26,40,51 and, in the rat, vasopressinergic stimuli increase the proportion of those cells firing phasicallyla,4L Moreover, during prolonged in situ recordings, neurons tentatively identified as being vasopressinergic (because their firing was unrelated to the suckling-induced pulses of oxytocin release) actually switched from a random to a phasic pattern of activity during osmotic stimulation and haemorrhageg,3s. In the present study, a number of substances known to cause the secretion of vasopressin have been investigated for their effects on the pattern of firing of neurons cultured from the hypothalamic supraoptic nucleus area. Of the drugs tested, nicotine and enkephalin analogues were shown to cause a transition from random to phasic firing. A similar effect was noted with GABA-receptor antagonists. Angiotensin II prolonged the duration of preexisting bursts. Our results show that neuropeptides (and neurotransmitters) can modify neuronal firing patterns, as has been earlier observed in molluscan neurons with vasopressin3,4, 32.

422 Previous studies have indicated that the neurosecretory cells of the supraoptic nucleus are cholinoceptive. Thus, acetylcholine and nicotine applied to the organcultured hypothalamic-neurohypophysial explant from rats caused the release of vasopressin, while muscarinic agents were without effect on hormone secretiona6,4L Moreover, acetylcholine and nicotine excited supraoptic neurons in situ T M and in vitro 4a, while activation of muscarinic receptors has been claimed to reduce their rate of firing2,43. Recent data showed that iontophoretic acetylcholine could initiate bursts of activity in rat supraoptic neurons 5, whereas glutamate produced inconsistent effects: according to one report, iontophoretically administered glutamate was unable to cause bursting firing 5. In slices, however, a minority of previously silent supraoptic neurons exhibited phasic spike trains upon bath application of 5 × l0 -:~ M glutamate 24. Our results indicate that nicotine but not glutamate can elicit phasic activity in cultured cells. The mechanism of the nicotine action is nuclear, though, since only dihydro-fl-erythroidine, but neither hexamethonium nor mecamylamine, antagonized the effect of nicotine. It is conceivable that a weak GABA-antagonistic effect of nicotine which has been described in the frog spinal cord in vitro a5 also may explain the similar actions of nicotine and bicuculline in our preparation. Angiotensin has been shown to increase the level of plasma vasopressin when administered to dogs, goats and rats 8,19,3°. In organ cultures of the rat hypothalamicneurohypophysial system, low concentrations of angiotensin stimulated vasopressin release, an effect which was blocked by the antagonist, saralasin 45. The sensitivity of this release to tetrodotoxin 4~ indicated that action potentials were involved. A direct activation of supraoptic nucleus neurons by angiotensin has been demonstrated by electrophysiological studies both in situ a6 and in vitro 42. Our data extend these investigations by showing that angiotensin II not only accelerates randomly fring cells, but also increases the duration of bursts in already phasic cells. Opiates and opioid peptides have been shown by some authors to evoke an antidiuresis (refs. 7 and 12, but see also ref. 48). In rabbits, intravenously administered fl-endorphin was found to stimulate the secretion of vasopressin 5°, an effect interpreted to be caused by an action on the brain. It is therefore of interest that enkephalin analogues induced a transition from random to phasic firing in cultured hypothalamic neurons. It is unclear, however, why higher concentrations of the peptides were needed to induce phasic activity in these hypothalamic neurons than were required to excite hippocampal pyramidal ceils in culture z°. An inhibitory action of GABA on neurons in the cat and rat supraoptic area has been well documented, and this effect can be blocked by GABA-receptor antagonists 15,~7. Since GABA antagonists can produce bursting activity in cultures derived from various brain regions (B.H.G., unpublished observation), it is not surprising that bicuculline caused phasic firing when applied to the solution bathing the hypothalamic cultures. The GABA-antagonistic property of curare 2a,85 offers a plausible explanation for the bursts induced by this drug. We are unaware of any study demonstrating neurohypophysial hormone release following the application of GABA antagonists to the hypothalamus. However, Feldberg and Rocha e Silva Iv showed that several GABA antagonists caused vasopressin secretion when applied to the ventral surface of the brain stem in cats.

423 T h e effects o f G A B A a n d g l u t a m a t e a r e likely to be due to a direct a c t i o n o n the m e m b r a n e o f the i m p a l e d n e u r o n , since they were a c c o m p a n i e d b y a m a r k e d increase in m e m b r a n e c o n d u c t a n c e . In c o n t r a s t , the actions o f the o t h e r drugs a p p l i e d d u r i n g this s t u d y are p r o b a b l y synaptically m e d i a t e d . T h e identity o f the e x c i t a t o r y transm i t t e r involved in phasic activity r e m a i n s u n k n o w n , since neither acetylcholine a n t a g o n i s t s n o r acidic a m i n o acid a n t a g o n i s t s a b o l i s h e d the phasic activity, T h e possibility r e m a i n s t h a t a n g i o t e n s i n m i g h t be involved. The evidence, however, is n o t conclusive, since saralasin m a y have o t h e r effects in a d d i t i o n to angiotensin a n t a g o nism. ACKNOWLEDGEMENTS W e t h a n k Dr. E. van D e u s e n for his help with the m a n u s c r i p t a n d Ms. E. Hoffm a n n a n d L. Rietschin for excellent technical assistance.

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