Hiroshi Kannan

CCK-8 excites oxytocin-secreting neurons in the paraventricular nucleus in rats-possible involvement of noradrenergic pathway

Systemic administration of CCK-8 increased plasma oxytocin (OXT) level in rats anesthetized with a mixture of urethane and alpha-chloralose. Extracellular recordings were made from magnocellular neurosecretory neurons in the paraventricular nucleus (PVN) of the hypothalamus in anesthetized rats to examine the effects of CCK-8 on the firing of PVN neurons. Thirteen out of 16 nonphasic neurons (putative OXT-secreting neurons) were excited by intravenous and/or intraperitoneal administration of CCK-8. By contrast, 8 out of 10 phasic cells, vasopressin(AVP)-secreting cells, were inhibited by systemic administration of CCK-8. Four out of five nonneurosecretory neurons in the PVN were excited by the administration of CCK-8. Moreover, microiontophoretically applied phentolamine blocked the excitatory responses induced by CCK-8 in nonphasic neurons. We measured extracellular noradrenaline (NA) level in the PVN, using in vivo microdialysis. Intravenous administration of CCK-8 induced NA release in the PVN. These results suggest that CCK-8 activates the excitatory afferent pathway to putative OXT-secreting neurons in the PVN which may, at least in part, be involved in the central noradrenergic projection.

Interleukin-1β directly excites hypothalamic supraoptic neurons in rats in vitro

Responses of 42 neurosecretory neurons in the supraoptic nucleus (SON) to human recombinant interleukin-1 beta (IL-1 beta) were examined during intracellular recordings in rat brain slices. IL-1 beta (10(-9)-10(-8) M) depolarized the membrane and caused increased firing in 25 neurons (59.5%). In 11 other neurons (26.6%), depolarization was also seen, followed by membrane hyperpolarization. The IL-induced depolarizing effect remained in the presence of tetrodotoxin (TTX) but was abolished by sodium salicylate. The results suggest that IL-1 beta mainly exerts a direct excitatory effect on SON neurons and further, that prostaglandins may be involved in such an effect.

Inward sodium current involvement in regenerative bursting activity of rat magnocellular supraoptic neurones in vitro.

1. The rat hypothalamic slice preparation was used to investigate the involvement of inward Na+ currents as well as inward Ca2+ currents in the generation of bursting activity by supraoptic (SON) neurones. Intracellular records were made from thirty‐two SON neurones which showed regenerative bursting activity. The bursting activity consisted of spontaneous, intermittent bursts of action potentials with subsequent silent periods. During the bursts, plateau potentials on which action potentials were superimposed were frequently observed. 2. Perfusion of a low‐Na+ medium, a tetrodotoxin (TTX)‐containing medium or a Ca(2+)‐free medium suppressed the regenerative bursting activity. 3. Addition of 3‐10 microM veratridine to Ca(2+)‐free medium elicited regenerative bursting activity and spontaneous plateau potentials. The veratridine‐induced regenerative bursting activity and plateau potentials were blocked by 1 microM TTX. Addition of 5 mM TEA allowed regenerative bursting activity to persist in Ca(2+)‐free medium. 4. These results suggest that TTX‐sensitive Na+ inward currents as well as Ca2+ inward currents contribute to the generation of bursting activity in rat SON cells.

Pathways between the nucleus tractus solitarius and neurosecretory neurons of the supraoptic nucleus: Electrophysiological studies

In anesthetized cats recordings were made from hypothalamo-neurohypophysial neurons in a supraoptic nucleus (SON) of the hypothalamus. The region of the nucleus tractus solitarius in the medulla, identified electrophysiologically as the site of termination of the first relay neurons of the sinus and aortic nerves, was stimulated with single or short trains of pulses (2-3 at 200 Hz). Out of 133 SON neurons 67 were affected by such stimuli. In 14 cells (21% of responsive neurons) the stimulus produced profound inhibition of SON neuron activity after a latency of 10-30 msec. In another 8 neurons (12%) the inhibitory effect was observed after a longer latency of over 100 msec. An increase in intensity of stimulus merely prolonged or increased the inhibitory effect without changing the response qualitatively. The other 45 (67%) SON neurons were excited by stimulation of the nucleus tractus solitarius. In a small proportion of these neurons (5 cells, 7%) the stimulus evoked discharges, even in spontaneously silent neurosecretory cells, after a latency of 10-20 msec with little fluctuation. In the remaining 40 neurons, i.e. 60% of the responsive neurons, the excitatory effect was observed after a latency of 40-120 msec. Again, changes in intensity of stimulation did not alter the nature of this response. The results indicate that both fast as well as slow pathways between the nucleus tractus solitarius and SON neurons exist and impulses travelling through the latter pathway from the carotid sinus or aortic nerve affect the larger proportion of SON neurons.

The Actions of Endothelin on Single Cells in the Anteroventral Third Ventricular Region and Supraoptic Nucleus in Rat Hypothalamic Slices

Endothelin (ET), a peptide consisting of 21 amino‐acid residues was recently isolated from the culture supernatant of porcine aortic endothelial cells. ET has been reported to be a more potent vasoconstrictor than angiotensin II. Other studies suggest that ET is involved in central control of the autonomie nervous system and body water regulation. Extracellular recordings were made from neurons in the anteroventral third ventricle (AV3V) and supraoptic nucleus (SON) in rat hypothalamic slice preparations. ET‐3 was applied at concentrations of 10−10 M to 3x 10−7 M. Of 226 AV3V neurons tested, 48 (21%) were excited, 8 (4%) were inhibited, and 170 (75%) were unaffected by ET‐3 at 10−7 M. The threshold concentration to evoke the responses was approximately 10−9 M. Of 144 SON neurons tested, 64 had a phasic firing pattern and 80 had a non‐phasic firing pattern. Of 64 phasic neurons tested, 39 (61%) were inhibited by ET‐3 at 10−7 M, 25 (39%) were non‐responsive and none was excited. Of 80 non‐phasic neurons tested, 14 (17.5%) were inhibited by ET‐3 at 10−7 M, 66 (82.5%) were non‐responsive and none was excited. The effects of ET‐1 were compared with those of ET‐3. The number of neurons responding to ET‐1 and their responsiveness were almost the same as for ET‐3. To investigate whether the ET responses are dependent on Ca2+ influx, a Ca2+ free medium and the Ca2+ antagonist, nicardipine, were used. The excitatory responses of AV3V neurons to ET were maintained in the Ca2+ free medium. Nicardipine at 10−5 M suppressed neither the excitatory responses of AV3V neurons nor the inhibitory responses of SON neurons to ET‐3. After 8 to 12 h preincubation of slices with islet activating peptide (IAP: pertussis toxin) at 10−7 M, none of the 48 AV3V neurons tested was excited by either ET‐3 or ET‐1. On the other hand, inhibitory responses were still observed in 12 (52%) out of 23 phasic SON neurons tested after IAP treatment. These results suggest that ET has a direct action as a neuropeptide on hypothalamic neurons through the ETB receptor and that the mechanisms underlying the responses may be different in the AV3V and the SON.

Effects of centrally administered endothelin-3 on renal sympathetic nerve activity and renal blood flow in conscious rats

Effects of intracerebroventricular (i.c.v.) administration of endothelin-3 (ET-3) on renal sympathetic nerve activity (RSNA) and renal blood flow (RBF), arterial blood pressure and heart rate were examined in conscious rats. Administration of ET-3 (1-50 pmol) through a chronically implanted cannula evoked an increase in arterial blood pressure and decreases in heart rate and RSNA, whereas RBF measured by Doppler flow probes did not change. Maximum changes in these responses occurred 10-15 min after i.c.v. administration of ET-3 and the responses returned to the control level after approximately 60 min. In sinoaortic denervated (SAD) rats, the decrease in RSNA induced by i.c.v. ET-3 was attenuated but still significantly persistent. During the experiments, we found that the injection of ET-3 (50-100 pmol) induced a barrel rotation, with an onset latency of 10-15 min. In those cases, prominent increases in arterial blood pressure and RSNA were observed, and these lasted for more than 60 min. The result shows that ET-3 can have centrally mediated effects on autonomic nerve activity as well as on cardiovascular function.

Intraperitoneal administration of recombinant human interleukin-1β inhibits osmotic thirst in the rat

Decrease in water intake after intraperitoneal injection of interleukin-1 beta (IL-1 beta) was studied in the rat. Administration of IL-1 beta at a dose of 20 micrograms/kg attenuated osmotic thirst induced by intraperitoneal injection of hypertonic saline, but did not affect hypovolemic thirst induced by subcutaneous injection of either polyethylene glycol or angiotensin II. Interleukin-1 beta also decreased spontaneous intake of water but not that of 1.8% saline. The results suggest that the decrease in water intake by IL-1 beta is caused, at least in part, by suppression of osmotic thirst but not by general suppression of behavior. The effects of IL-1 beta were not secondary responses accompanied by feeding behavior, since food supply was removed during the experiments. Pretreatment with indomethacin blocked the decrease in water intake by IL-1 beta, suggesting the involvement of production of prostaglandins.

Influences of the limbic system on hypothalamo-neurohypophysial system

(1) Effects of stimulations of various limbic structures (the olfactory bulb, olfactory tubercle, prepyriform cortex, endopyriform nucleus and various parts of amygdaloid nuclei) on the neurosecretory neurons in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus were studied. All regions stimulated received strong inputs from the olfactory bulb. (2) Out of 195 identified neurosecretory neurons tested one-half or more (49-74%, depending on the structures stimulated) were inhibited by stimuli consisting of 1-3 short pulses. The inhibition occurred immediately after the stimulus in approximately one-fifty of all inhibited neurons, in the remaining four-fifths inhibition occurred after more than 20 ms latency. Inhibition of neurosecretory neuron activity lasted for several hundred milliseconds, often followed by clear post-inhibitory excitation or rebound. (3) In 23 neurons, a distinct evoked response of brief duration occurred with a 30 ms latency following stimulation of the lateral and medical amygdala, olfactory tubercle and prepyriform cortex. In another 17 neurons, a general increase in background activity with a longer latency (50-100 ms) occurred following stimulation of nearly all amygdaloid nuclei, olfactory tubercle and the pyriform cortex: lateral amygdala stimulation caused an excitation of the largest proportion of neurosecretory cells (30%) while none was excited by stimulation of the olfactory bulb and endopyriform cortex, except those occurring as post-inhibitory excitation. (4) There was a convergence of afferent impulses on single neurosecretory cells. A large proportion (42%) of the neurons received inputs from 2 to 4 limbic regions. (5) Neurosecretory cells which were influenced by limbic stimuli were also inhibited by baroreceptor activation and excited by osmotic stimulation. Unidentified neurons within SON and PVN and atypical neurosecretory cells (those responding to pituitary stalk stimulation with varying latencies) were also affected by the forebrain stimulation; some of these were also affected by an osmotic stimulus. A part of this group may send their axons to the median eminence.

Lateral preoptic neurons inhibit thirst in the rat

Kainic acid (KA) and muscimol were injected into the lateral preoptic area (LPO) of the rat to study their effects on drinking behavior. A low dose (5 ng) of KA, which stimulates neurons, decreased the amount of water intake induced by hypertonic saline (IP) and angiotensin II (SC). Injection of 2 ng muscimol, a potent GABAA receptor agonist that suppresses neurons, facilitated drinking responses induced by hypertonic saline, but did not affect angiotensin II-induced drinking. Rats injected with a high dose (150 ng) of KA, which destroys neurons, showed marked polydipsia accompanied by increased urination. One week after the KA lesion, drinking and urine output recovered to normal. During the polydipsia, a small volume of concentrated urine could be excreted if water intake was restricted. After recovery, excessive drinking responses followed water deprivation and hypertonic saline load. The rats normally drank water in response to angiotensin II and to polyethylene glycol solution. The results show that activation of LPO neurons inhibits water intake, and that suppression of LPO neurons facilitates osmotically induced water intake. Therefore, LPO neurons are probably involved in the inhibition of thirst.

Synaptic inputs from the stomach to tuberoinfundibular neurons in the paraventricular nucleus of the hypothalamus in rats

In male rats anesthetized with urethane (600 mg/kg) and alpha-chloralose (60 mg/kg), extracellular recordings were made from tuberoinfundibular (TI) neurons in the paraventricular nucleus (PVN) identified by antidromic stimulation of the median eminence. Electrical stimulation of the gastric branches of the vagus nerve excited 26 (72%) of 36 TI neurons tested. I.v. (0.5-1 microgram/rat) or i.p. (5 micrograms/rat) administration of cholecystokinin (CCK-8) excited 10 (63%) of 16 TI neurons tested. The excitatory responses induced by CCK-8 were abolished by bilateral cervical vagotomy. These results suggest that TI neurons in the PVN receive excitatory inputs from gastric afferents via vagus nerves.