Toshimasa Osaka

Noradrenergic inputs to sleep-related neurons in the preoptic area from the locus coeruleus and the ventrolateral medulla in the rat.

Responses of sleep-related neurons in the preoptic area (POA) to stimulation of the locus coeruleus (LC) and the ventrolateral medulla (VLM), components of the reticular activating system, were recorded in the unanesthetized, head-restrained rat. Single-pulse stimulation of the LC and the VLM, respectively, inhibited 50% and 54% of 30 sleep-active neurons and excited 47% and 67% of 34 waking-active neurons. The remaining neurons were mostly unaffected. Seventy-three neurons that were not related to a sleep-wake state were mostly (i.e., 73-80%) unresponsive to stimulation. The high incidence of responses by sleep-related neurons suggests that neural inputs from the LC and VLM regulate the hypnogenic mechanisms in the POA. Stimulation of the LC antidromically activated 15% of sleep-active neurons and 11% of waking-active neurons. Thus, some of the sleep-related neurons in the POA may regulate LC neurons. In a later stage of the experiment, we used isoflurane-anesthetized rats that had been used for recording sleep-related neurons. Antagonists for adrenoceptors at a concentration of 10 microM were applied to neurons through a multibarrel micropipette to examine the involvement of noradrenaline in the responses as a neurotransmitter. Application of the alpha 2-blocker, yohimbine, attenuated the inhibitory responses in all 7 neurons tested. The beta-blocker, timolol, and the alpha 1-blocker, prazosin, did not alter any of the inhibitory responses. On the other hand, timolol attenuated the excitatory responses in 4 of 7 neurons, and prazosin attenuated the excitatory responses in 5 of 12 neurons. Yohimbine did not affect the excitatory responses. Thus, the LC and the VLM probably inhibit sleep-active neurons through alpha 2-adrenoceptors and excite waking-active neurons through either beta- or alpha 1-adrenoceptors.

Paraventricular neurosecretory neurons: Synaptic inputs from the ventrolateral medulla in rats

Effects of electrical stimulation of the ventrolateral medulla on discharge activity of neurosecretory neurons in the paraventricular nucleus (PVN) were studied in male rats anesthetized with urethane-chloralose. Among 35 phasically firing neurosecretory neurons, stimulation of the lateral reticular nucleus and its vicinity produced excitation in 10 and inhibition in 2. The stimulation also enhanced the activity of 40% of the PVN neurosecretory neurons that fired continuously (n = 81); of these responsive neurons, half of the neurons tested (n = 12) were inhibited by i.v. administration of phenylephrine. The result suggests that both vasopressin- and oxytocin-secreting neurons in the PVN receive mainly excitatory synaptic inputs from the ventrolateral medulla.

Noradrenaline inhibits preoptic sleep-active neurons through α2-receptors in the rat

Effects of noradrenaline (NA) on the activity of sleep-related neurons in the preoptic area (POA) and the neighboring basal forebrain were examined in the rat. Of 36 sleep-active neurons tested, 19 were inhibited and the other 17 were unaffected by NA applied through a multibarrel pipette. The alpha 2-agonist clonidine inhibited 11 of 14 sleep-active neurons and did not affect the other 3 neurons, whereas the alpha 1-agonist methoxamine (n = 13) and the beta-agonist isoproterenol (n = 11) had no effect on any of the sleep-active neurons tested. Thus, alpha 2-receptors mediated the NA-induced inhibition. Of 22 waking-active neurons tested, NA excited 10, inhibited 1, and had no effect on the remaining 11. Methoxamine excited 4 of 13 waking-active neurons tested, whereas isoproterenol (n = 9) and clonidine (n = 4) were without effect on any of the waking-active neurons tested. Accordingly, alpha 1-receptors probably mediated the NA-induced excitation. Seventy-seven state-indifferent neurons, which lacked activity related to the sleep-waking state, and 20 paradoxical sleep-active neurons were mostly (65%-70%) insensitive to NA. These results suggest that NA promotes wakefulness by inhibiting sleep-active neurons and by exciting waking-active neurons.

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.

Excitation of hypothalamic paraventricular neurons by stimulation of the raphe nuclei

Extracellular recordings were made from 467 anti-dromically identified neurosecretory neurons and 148 non-neurosecretory neurons in the paraventricular nucleus of the hypothalamus of hemispherectomized cats under pentobarbital anesthesia. Stimulation of the dorsal, median, and pontine raphe nuclei excited 31%, 26%, and 12% of neurosecretory neurons tested, respectively, and inhibited 9%, 7%, and 8%. The excitatory responses in 13 of 14 neurons tested were blocked by either of two intravenously administered 5-HT2 antagonists, cyproheptadine or methysergide. The 5-HT1A antagonist, (-)pindolol, partially blocked the excitatory responses elicited by raphe stimulation in three of five neurons tested. The inhibitory responses to raphe stimulation were not affected by application of these antagonists. More non-neurosecretory neurons than neurosecretory neurons were excited in response to raphe stimulation and these excitatory responses were also blocked by these antagonists. We conclude that most electrically stimulated synaptic inputs from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus are excitatory and are mainly mediated by 5-HT2 receptors.

A novel apparatus that permits multiple routes for infusions and body-fluid collections in a freely-moving animal

A novel apparatus is described for simultaneous performance of multi-channel infusions/body-fluid collections and multichannel electrical recordings/stimulations in a freely behaving animals. This apparatus consists of a cylindrical cage and other devices described below. Electrical contacts are achieved via a slip-ring commutator. A rotation detector detects the turning of the animal in the cage, and a controller rotates the floor under the animal in the opposite direction by means of a stepping motor. Thus, excessive twisting of the fluid tubing between the animal and experimental equipment is released by rotation of the floor. Floor rotation starts when the animal turns in either direction and exceeds a present number of rotations. When the turning exceeds certain rates, the floor is rotated with higher angular velocities. This floor rotation little affected sleep-wake activities, brain temperature, food and water intake, and general behavior of the rat. Thus, multi-channel fluid routes have been realized; and by excluding the swivel apparatus from the fluid lines, advantages such as no fluid leakage at the swivel, no extra dead volume, and low torque to rotate the device have been gained.

γ-Aminobutyric acid antagonist blocks baroreceptor-activated inhibition of neurosecretory cells in the hypothalamic paraventricular nucleus of rats

The effects of gamma-aminobutyric acid (GABA) antagonists on baroreceptor-activated inhibition of neurosecretory cells in the paraventricular nucleus (PVN) were examined in urethane-chloralose anesthetized rats. In 11 neurosecretory cells which were inhibited by baroreceptor activation induced by intravenous application of phenylephrine, microiontophoretically applied bicuculline and/or picrotoxin blocked the inhibition (n = 9) completely or partially, whereas strychnine (n = 4) did not. The results suggest that GABA is, at least in part, involved in the baroreceptor-activated inhibition of PVN neurosecretory cells.

Inhibitory inputs to the subfornical organ from the AV3V: involvement of GABA.

Action potentials were recorded extracellularly from single neurons in the subfornical organ (SFO) of the pentobarbital-anesthetized cat following stimulation of the regions surrounding the anteroventral third ventricle (AV3V). Of 328 SFO neurons studied, 103 were antidromically activated, showing direct projections from the SFO to the AV3V. However, the major effects of stimulations of the AV3V on SFO neurons were orthodromic inhibition; almost 30% of SFO neurons were inhibited by various sites in the AV3V, while a smaller proportion of cells were excited. Local application of bicuculline, an antagonist for GABA, attenuated the inhibitory responses induced by stimulation of the AV3V in seven out of eight neurons tested. Application of GABA inhibited 16 out of 24 neurons, while that of bicuculline alone excited 11 out of 26 neurons, suggesting the tonic inhibitory action of GABA on some SFO neurons. On the other hand, application of kynurenic acid, a nonspecific antagonist for the excitatory amino acids, did not affect the excitatory responses induced by stimulation of the AV3V, but kynurenic acid itself inhibited 6 out of 18 neurons tested. Application of glutamate excited most SFO neurons. This suggests that the excitatory amino acids may be the transmitter(s) of interneurons in the SFO but may not mediate the excitation from the AV3V.

Osmotic responses of rat paraventricular neurons by pressure ejection method.

Extracellular recordings were made from 44 spontaneously active neurosecretory and 43 unidentified neurons in the paraventricular nucleus (PVN) of the hypothalamus in urethane-anesthetized rats. Physiologically hypertonic (+30 mOsm/kg, NaCl or mannitol) and hypotonic (-30 mOsm/kg) solutions were applied by pressure through multibarrel micropipettes to the immediate vicinity of neurons. Of 44 neurosecretory neurons tested, 31 (70%) were unaffected by locally applied osmotic stimulation, and the remaining 13 were excited. Of these 31 osmotically unresponsive neurosecretory neurons, 21 were further tested with very strong hypertonic (+260 mOsm/kg, NaCl) solution, and only 7 were affected. Of 43 unidentified neurons, 28 (65%) were unaffected by osmotic stimulation, and 12 were excited. Accordingly, only parts (30%) of both the neurosecretory and unidentified neurons in the PVN were found to be osmosensitive. We concluded that some neurons in the rat PVN are osmosensitive and osmosensitivity is not specific to neurosecretory neurons in the PVN.

Effects of iontophoretically applied cortisol on tuberoinfundibular neurons in hypothalamic paraventricular nucleus of anesthetized rats

Effects of cortisol on 22 tuberoinfundibular (TI) neurons in the paraventricular nucleus (PVN) of the hypothalamus were examined in urethane-anesthetized rats. Iontophoretically applied cortisol excited 10 (45%) of 22 TI-neurons tested, whereas the cortisol inhibited 4 (31%) of 13 neurons in the periventricular hypothalamic nucleus. Intravenously applied cortisol (0.5 mg in 0.5 ml saline) excited 4 (36%) of 11 TI neurons tested and inhibited 1 (9%). These results suggest that cortisol may have an excitatory effect on TI neurons in the PVN.