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We studied the effects of water deprivation on the expression of c-fos protein (Fos) in the brain of inbred polydipsic mice, STR/N strain, that show extreme polydipsia without a lack of vasopressin in the body. Non-polydipsic mice, ICR strain, were used as controls. All male animals were deprived of water for 24 and 48 h. Fos-like immunoreactivity (Fos-LI) in the brain was studied by immunohistochemical techniques. In both groups of mice water deprivation induced a remarkable increase in Fos-LI in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, the median preoptic nucleus (MnPO), the organum vasculosum laminae terminalis (OVLT) and the subfornical organ (SFO). A far more increase, however, was seen in the MnPO, the SFO and the area postrema (AP) of the polydipsic mice compared to those of the non-polydipsic control mice. In the nucleus of the tractus solitarius (NTS) and in the anteroventral part of the PVN (avPVN), water deprivation caused a clear increase in Fos-LI in the polydipsic mice, while in the non-polydipsic mice the same treatment induced no Fos-LI in the NTS and no change in the avPVN. These results indicate that neurons in the circumventricular organs and the NTS are strongly activated by water deprivation in the polydipsic mice, suggesting that these brain structures play an important role in the polydipsia.

The inbred mice, STR/N, are known to possess extreme polydipsia with no known abnormality in vasopressin system and the kidney function. Our previous studies indicate that the opiate antagonists given intracerebroventricularly strongly attenuated spontaneous drinking. To determine the site(s) of action, the present study was undertaken. Microinjections of naltrexone methobromide and a selective kappa-receptor antagonist, nor-binaltorphimine (nor-BNI), into the paraventricular nucleus of the hypothalamus (PVN) greatly attenuated drinking of the STR/N for 0.5 to 16 h after injections, while in the two control groups, non-polydipsic STR/1N and Swiss/Webster strains, drinking was not affected by the injections. Food intake was not much altered in all groups. Studies of PVN neurons in vitro (n = > 160 for each group) showed that basal firing rates and patterns were similar in the STR/N and the control groups. Morphine added to the medium inhibited some but excited none in all strains tested. The threshold for the inhibitory action was higher in the polydipsic STR/N mice (10(-8) M), compared to that in the control, S/W mice (10(-9) M). Further, a proportion of neurons inhibited by morphine in the PVN was significantly smaller (P < 0.01) in the STR/N (41.7%), compared to that in the control (64.9%). Dynorphin had very similar effect to that of morphine, but the proportion of cells inhibited was 25.4% in the STR/N, and 70.4% in the S/W. Prior applications of naloxone to the medium prevented the action of both morphine and dynorphin. Under the synaptic blockade (in a low Ca2+ and high Mg2+ medium) the inhibitory effect of the opiates persisted. We concluded that the PVN is at least one of the possible sites where the opiates are acting to cause the polydipsia in the STR/N mice.

Effects of opioid antagonists on the genetic polydipsia of the STR/N strain of mice were investigated. Naltrexone (0.5-5.0 mg/kg) injected subcutaneously before dark period attenuated spontaneous drinking for the first 3 h after injection only in the inbred polydipsic mice (STR/N), whose water intake was 5 times that of controls (non-polydipsic mutant, STR/1N, and Swiss/Webster mice). The highest dose (5 mg/kg) of naltrexone administration reduced drinking also during the next 3-6 h period and overnight feeding. Cerebroventricular (i.c.v.) injection of naltrexone, 1.0 and 2.5 micrograms (per mouse), suppressed drinking only in the polydipsic mice, while the higher dose (5.0 micrograms) attenuated drinking and feeding of both the polydipsic mice and their controls. However, i.c.v. injection of specific kappa-receptor antagonist, nor-binaltorphimine (nor-BNI, 0.5-2.5 micrograms), suppressed drinking only in the polydipsic strain of mice at one-half dose of that needed for naltrexone. Furthermore, even a higher dose of nor-BNI administration was without effect on food intake in all strains. These findings suggest that the central opioid system plays an important role in causing the polydipsia in the STR/N mice, probably through the kappa-opioid receptor.

In previous studies we found that in the extremely polydipsic special strain of mice, STR/N, spontaneous drinking was greatly attenuated by injection of the opioid antagonists given intracerebroventricularly as well as subcutaneously. Therefore, we investigated, using hypothalamic slice preparations, responses of neurons in the anteroventral third ventricle region (AV3V) of the STR/N and its control, Swiss/Webster (S/W) mice to morphine and opiate peptides. An application of morphine at 10(-6) M to the circulating medium inhibited activities of 44% of AV3V neurons (45 of 102) in the STR/N, and 59% (76/129) in the S/W, demonstrating that morphine affected a smaller proportion of neurons of the polydipsic mice than that of controls. Opioid agonists for 3 receptor types, mu, delta and kappa, at 10(-6) to 10(-5) M inhibited AV3V neurons in both the STR/N and S/W mice, but to a different degree. No cell of either strain was excited by morphine or any of the opioids. The mu-receptor agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), was the most potent inhibitor of AV3V neurons; in the STR/N 53% (25/47), and in the S/W 77% (34/44) were inhibited. The kappa-agonist, dynorphin A-(1-13) (DYN), inhibited fewer cells in the STR/N (9%, 4/47), compared with the S/W (36%, 16/44). Only a few cells responded to the delta-agonist, [D-Pen2,5]enkephalin (DPDPE), in both strains. The inhibitory actions of the opiates were reversibly blocked by naloxone, and persisted under synaptic blockade. The threshold concentration of morphine or DAGO for inhibition of AV3V neurons was higher in the STR/N (approximately 10(-8) M for both morphine and DAGO) than in S/W mice (approximately 10(-9) M for morphine and less than 10(-9) M for DAGO). Although the AV3V also contains angiotensin II-sensitive neurons, they were not affected by morphine (10(-6) M). Similarly neurons inhibited by morphine were not excited by angiotensin II (10(-7) M); some neurons were unresponsive to both chemicals. We conclude that morphine and opiate peptides directly inhibit the AV3V neurons of both the STR/N and S/W strains of mice and the sensitivity of these neurons to the opiates is lower in the polydipsic inbred mice compared to their controls. The results, together with our behavioral studies, suggest involvement of the central opioid system in the polydipsia of the STR/N mice.

The polydipsic inbred mice, STR/N, are known to possess an extremely strong appetite for drinking but no abnormality in the vasopressin system and renal functions. In brain slice preparations the sensitivity of neurons in the anteroventral region of the third ventricle (AV3V) and the subfornical organ (SFO) to angiotensin II (ANG II) was investigated using extracellular recordings in the STR/N and its control, Swiss/Webster (S/W) mice. In the AV3V, less proportion of neurons (15 out of 168; 9%) of the STR/N than that in the S/W (49/206; 24%) was excited by ANG II added to the medium. In the SFO, a proportion of neurons excited by ANG II was again lower in the STR/N (27/104; 26%) than in the S/W mice (64/118; 54%). The threshold concentration of ANG II for excitation of the AV3V and SFO neurons was, however, similar for both strains, 10(-9) M or less. Only one neuron in the SFO of a S/W mouse was inhibited by ANG II application. The excitatory effect of ANG II on AV3V and SFO neurons of both strains of mice persisted under synaptic blockade and was reversibly antagonized by an ANG II antagonist, saralasin. Such differences in sensitivity to ANG II of neurons in the SFO and AV3V, the regions thought to be involved in drinking behavior, suggest the involvement, at least in part, of the central angiotensin system in the polydipsia of the STR/N mice.

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.

(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.

(1) In chloralose anesthetized dogs, effects of the left and the right atrial stretch were studied in the same animal. Stretch of the sino-atrial region of the right atrium produced acceleration of the heart rate during, and reversal of response at the termination of, the stretch. Stretch of the left pulmonary vein-atrial junctional region evoked an initial decrease followed by an increase in heart rate. The responses were similar in all animals, despite initial heart rates ranging from 120 to 200 beats per minute. (2) Activity in vagal and sympathetic nerve branches innervating the heart was recorded simultaneously. Care was taken to identify the vagal fibers innervating the heart, and record their activity without contamination of sympathetic impulses. The right atrial stretch evoked an augmentation of sympathetic activity which reached its peak at 20 sec after the beginning of stimulus. The stimulus slightly increased the vagal activity; this change occurred slowly and reached its peak in about 40-60 sec after stretch. At the release of stretch, sympathetic activity generally showed a reversal of response, i.e. activity was inhibited for 30 sec. (3) Stretch of the left atrium produced biphasic changes in cardiac sympathetic nerves; their activity was strongly inhibited for the first 15 sec, then augmented throughout the remainder of the stretch. This effect lasted 30 sec after the cessation of stimulus. Effects on vagal cardiac nerve fibers were smaller; mild augmentation in activity was produced. The onset of this effect was faster than that seen in case of the right atrium stretch. (4) Reciprocal action between vagal and sympathetic cardiac nerves was obvious only in the early phase of left atrium stretch. Effects on the heart were determined by balances in activity of these antagonistic nerves. In contrast with what occurred in cardiac reflexes, carotid sinus distension even in the same animal produced a large increase in vagal activity, and near complete inhibition of sympathetic nerve activity. Thus, good reciprocal action between the two sets of nerves was demonstrated. A difference in the two types of reflexes was thus revealed. (5) Stretch of the right atrium evoked during carotid sinus distension caused an increase in heart rate from the new low level which was produced by baroreceptor activation. Vagal activity which was greatly augmented by sinus distension was decreased by atrial stretch, while previously inhibited sympathetic activity due to sinus distension was augmented by stretch of the atrium. The effect of stretch on vagal activity seems to depend to a degree on the prestimulus level. It is of interest that the powerful baroreceptor reflexes do not mask the cardiac reflexes studied. (6) The relative importance of sympathetic and vagal efferents to atrial stretch reflexes was discussed.

The existence of monosynaptic connections between neurons in the paraventricular nucleus (PVN) of the hypothalamus and the intermediolateral cell column (ILC) of the spinal cord was studied by electrophysiological techniques in chloralose-anesthetized cats. Sympathetic preganglionic discharges (recorded from the 2nd or 3rd thoracic white ramus) were evoked by microstimulation of certain regions in or near the PVN with short train of pulses and below 50 microA current. By recording responses of 'identified' and 'non-identified' neurosecretory cells in the PVN and supraoptic nucleus (SON) to stimulation of the ILC of the thoracic cord, it was possible to identify antidromically evoked action potentials in 9 out of 297 neurons tested. Among them, 2 neurons were also antidromically excited by the pituitary stalk stimulation, 5 were orthodromically excited by the same stimulus and the remaining 2 were not excited by the stalk stimulation. Our results indicate that some PVN neurons, though small in number, send axons directly to the ILC of the cord, and that a very few neurons among these also send their axons to the pituitary gland.

(1) The effects of arterial baroreceptor stimulation on the activity of 'identified' neurosecretory cells in supraoptic nuclei (SON) of the hypothalamus were investigated in anesthetized cats. (2) Stimulation of baroreceptors by distension of an 'isolated' carotid sinus greatly inhibited SON activity. A linear relationship was found to exist between stimulus intensity (an increase in the sinus pressure) and the degree of inhibition of SON neuron activity. This inhibitory effect was abolished by section of the sinus nerve. (3) The inhibitory effect of baroreceptors on SON neuron activity was most pronounced in the first 5 sec during stimulation, and the effect became less when the stimulus lasted for a long period. At the end of stimulation there was a transient reversal of the response. (4) The excitation of aortic baroreceptors by occluding descending aorta strongly inhibited SON neuron activity. (5) Occlusion of carotid arteries augmented the SON neuron activity. Electrical stimulation of the sinus nerve caused an excitation or an inhibition of SON neurons, depending on the stimulus intensities. Apparently these stimuli produced different degrees of excitation in baro- and chemoreceptors. (6) Combination of chemo- and baroreceptor stimulations revealed that the excitatory effect exerted by chemoreceptor stimulation on SON neurons was reversed or blocked by baroreceptor activation, indicating the powerful inhibitory influence of baroreceptors.

(1) In anesthetized (chloralose-urethane) rats, strong mechanical stimuli which were applied to the abdominal skin always inhibited motility of the small intestine. This reflex is referred to as an 'inhibitory cutaneo-intestinal reflex'. Similar stimuli applied to the skin of the upper chest, neck, forepaws, or hindpaws, however, evoked the opposite effect, which is referred to as a 'facilitatory cutaneo-intestinal reflex'. (2) By recording the activity of efferent sympathetic nerves to the small intestine and by transecting intestinal sympathetic or parasympathetic nerves we found that the inhibitory cutaneo-intestinal reflex was largely due to an increase in intestinal sympathetic efferent activity, and that the facilitatory cutaneo-intestinal reflex was due to decrease in the intestinal sympathetic efferent nerve activity; both changes reflexly evoked. (3) The inhibitory cutaneo-intestinal reflex was shown to be a propriospinal reflex which was caused by excitation of group IV (unmyelinated) cutaneous afferent nerve fibers. On the other hand, the facilitatory cutaneo-intestinal reflex seemed to be mediated through supraspinal pathways, and was evoked by excitation of mainly group III (A-delta group) cutaneous afferent nerve fibers. (4) Interaction between the cutaneo-intestinal reflex and intestino-intestinal reflex was demonstrated. (5) The possibility of a dorsal root reflex contribution to cutaneo-intestinal reflex was eliminated. (6) Significance of the cutaneo-intestinal reflex in neural control of the gastro-intestinal tract was discussed.