Fever alters osmosensitivity of hypothalamic-vasopressin system in the rat.

The osmosensitivity of peripheral vasopressin release was studied during healthy thermoregulation and endotoxin-induced fever. There was an increase in osmosensitivity following bolus injection of saline in febrile rats. These animals displayed a steeper slope in the linear relationship between plasma osmolality and plasma vasopressin levels compared with afebrile animals. The change in regression slope was due to a significantly lower plasma osmolality in febrile rats. The plasma osmolality of animals infused with hypertonic saline was similarly decreased, but data analysis failed to show a significant change in the regression slope. Osmotic thresholds were not altered in either group. There was an increased urine output in febrile rats, and consequently these animals excreted greater amounts of salts than afebrile rats. This could account for the lower plasma osmolality observed in the febrile rat.

A simple cannula assembly for chronic bilateral brain infusion in freely moving rats.

Osmotic minipumps provide a means for the chronic infusion of agents into the brain of the awake unrestrained animal. This paper describes the construction of a simple and inexpensive cannula assembly to be used with an osmotic minipump for the bilateral infusion of brain regions in the rat. The cannula/catheter assembly employs the use of a Y-joint, thereby allowing the investigator to use only one osmotic minipump per animal. This reduces the flow rate and decreases the cost of an experiment. Chronic infusion of the ventral septal area with saline using this system failed to alter the circadian rhythm of body temperature, except for a significant decrease in the amplitude of the rhythm.

A critical role for central vasopressin in regulation of fever during bacterial infection.

Previous investigations on the antipyretic properties of arginine vasopressin have used bacterial endotoxins or pyrogens to induce fever. Because these experimental models of fever fail to mimic all aspects of the responses to infection, we felt it was important to examine the role of endogenously released vasopressin as a neuromodulator in febrile thermoregulation during infection. Therefore the present study examines the effects of chronic infusion of a V1-receptor antagonist or saline (via osmotic minipumps into the ventral septal area of the brain) on a fever induced by injection of live bacteria. Telemetry was used for continuous measurement of body temperature in the awake unhandled rat. Animals infused with the V1-antagonist exhibited fevers that were greater in duration compared with those of saline-infused animals. These results support the hypothesis that vasopressin functions as an antipyretic agent or fever-reducing agent in brain. Importantly, they suggest that endogenously released vasopressin may play a role as a neuromodulator in natural fever.

Bombesin, neuromedin C and neuromedin B given intrathecally facilitate the tail flick reflex in the rat.

Evidence from an earlier study suggested that bombesin, neuromedin C and neuromedin B may play a role in spinal nociceptive transmission; iontophoretic administration of these peptides onto dorsal horn neurones in the cat was found to preferentially depress those neurones activated by noxious stimulation. Therefore, to further examine the possible function of these peptides in the spinal cord, the present study compares the effects of intrathecal administration of bombesin, neuromedin C and neuromedin B on reaction time in the tail flick test in the rat. Intrathecal injection of bombesin and neuromedin C to the lower lumbar vertebral level produced a dose-dependent decrease in reaction time which lasted up to 46 min. Similar administration of neuromedin B had a biphasic effect; there was a dose-dependent decrease in reaction time lasting about 6 min followed by a delayed increase in reaction time to above control values at 31-46 min. In addition, administration of these peptides induced behavioral responses such as spontaneous vocalization and vocalization in response to innocuous touch. These results provide physiological evidence for a role of neuromedin C and neuromedin B in sensory transmission at the spinal level. In this model, bombesin was a potent agonist which may selectively activate the neuromedin C receptor.

Characteristics of precipitated withdrawal from spinal morphine: changes in [Met5]enkephalin levels.

This investigation was carried out to study the development of physical dependence on spinally administered morphine, and it was determined if this phenomenon is associated with altered levels of [Met5]enkephalin. Morphine was infused continuously into the intrathecal space of rats for three or six days. In morphine-dependent animals, an intrathecal naloxone challenge produced increased reaction to nociceptive stimuli, hypertension, hyperthermia, decreased urinary output, and loss of body weight. Chronic spinal infusion of morphine alone failed to alter levels of [Met5]enkephalin in sacral and lumbar spinal cord. However, 24 h after the naloxone challenge, there was a significant increase in spinal enkephalin levels in morphine-dependent animals. It is concluded that spinal morphine treatment leads to the development of physical dependence. Certain characteristics of this phenomenon, as reflected in the naloxone-precipitated withdrawal signs, differ from those associated with dependence on systemic morphine.

Intrathecal administration of CGRP in the rat attenuates a facilitation of the tail flick reflex induced by either substance P or noxious cutaneous stimulation.

Intrathecal (i.t.) administration of substance P in the awake rat decreases reaction time (RT) in the tail flick test. In the anaesthetized rat, noxious thermal stimulation of the tail (by immersion of tip in water at 55 degrees C) also decreases RT; this effect is blocked by a substance P antagonist. The present study examines the effects of calcitonin gene-related peptide (CGRP) on the decrease in RT produced in these two paradigms. Pretreatment with CGRP (3.25 nmol, i.t.) attenuated and abolished the decreases in RT produced by substance P (3.25 nmol, i.t.) and by noxious cutaneous stimulation, respectively. These results indicate an interaction of CGRP and substance P in the regulation of the tail flick reflex.

Facilitation of the tail-flick reflex by noxious cutaneous stimulation in the rat: antagonism by a substance P analogue.

Effects of noxious cutaneous stimulation on the tail flick reflex were examined in the anaesthetized rat. Noxious stimulation was applied by immersing the distal 4 cm of the tail in water at 55 degrees C for 1.5 min. The tail flick reflex was tested at 3 min intervals by applying a noxious radiant heat stimulus to a region of the tail 10 cm proximal to the tip. Tail immersion reduced reaction time to tail flick by 30% and 20% at 0.5 and 3.5 min after immersion, respectively. Reaction time returned to control at 6.5 min and tended to increase above baseline values at 9.5 and 12.5 min. Naloxone (10 mg/kg, i.p.) potentiated the effects of tail immersion on reaction time and prevented the increase above baseline. When the surface temperature of the skin used to evoke the tail flick reflex was raised by 10 degrees C using innocuous radiant heat, reaction time was not significantly different from the control, suggesting that an increase in skin temperature per se is insufficient to account for the response to immersion. Intrathecal administration of a substance P antagonist (1 nmol) attenuated the response to tail immersion. These results indicate that noxious cutaneous stimulation may release an agent in the spinal cord which facilitates the tail flick reflex, and that this agent is antagonized by a substance P antagonist.

An adrenal-mediated, naloxone-reversible increase in reaction time in the tail-flick test following intrathecal administration of substance P at the lower thoracic spinal level in the rat.

We have shown previously that intrathecal administration of substance P to the lower thoracic vertebral level increases sympathetic output and increases the adrenal output of catecholamines. As opioid peptides are co-released with catecholamines from the adrenal medullae, experiments were done to determine whether the intrathecal administration of substance P to the eighth thoracic vertebral level would alter reaction time in the tail-flick test. Intrathecal administration of substance P (6.5 nmoles in artificial cerebrospinal fluid) in the awake restrained rat increased the reaction time at 1 and 6 min after injection to about 130-140% of the preadministration values; reaction time returned to preadministration values by 11 min. Similar administration of cerebrospinal fluid was without effect. Administration of 6.5 nmoles of thyrotropin-releasing hormone or oxytocin, peptides which also increase sympathetic output, failed to mimic the effects of substance P. The substance P-induced increase in reaction time was absent in rats which had been medullectomized and in rats pretreated with naloxone (10 mg/kg). Pretreatment with 10 mg/kg of either phentolamine or the quaternary opiate antagonists, nalorphine methochloride and naloxone methobromide, had no effect on the substance P-induced increase in reaction time. These results suggest that substance P given intrathecally to the eighth thoracic vertebral level may activate spinal sympathetic neurons to the adrenal medullae to cause the release of an opioid into the circulation. This circulating opioid may in turn play a role in the regulation of the tail-flick reflex by a centrally-mediated depression.

N- and C-terminal fragments of substance P: spinal effects in the rat tail flick test.

Evidence exists to suggest that within the CNS, substance P may be enzymatically cleaved into fragments which may mediate some of the effects of substance P. As we have previously reported on the spinal effects of substance P, the present study examines the effects of selected substance P fragments on reaction time in the tail flick test. Peptides were administered via a chronically implanted intrathecal catheter to the L5 vertebral level in the rat. Administration of 6.5 nmoles of SP(1-7) produced a transient decrease in reaction time at 1 min after injection with a return to above control values by 5 min. Similar administration of SP(7-11) produced a smaller decrease in reaction time at 6 min which lasted until 16 min. Administration of 6.5 nmoles of SP(1-9), SP(8-11) and of CSF were without effect. As the effects of SP(1-7) on reaction time resembled those of similar administration of substance P in the earlier experiments, these results suggest that this fragment may be the active component involved in facilitating the tail flick reflex. Substance P may be degraded to the active fragment prior to receptor activation or alternatively, substance P and SP(1-7) may act on the same receptor.

Intrathecal administration of substance P in the rat: spinal transection or morphine blocks the behavioural responses but not the facilitation of the tail flick reflex.

Substance P (10 micrograms) was administered intrathecally (i.t.) via a chronic catheter to the L5 vertebral level in rats with spinal cords transected at the T10-12 level (n = 11) and in intact rats pretreated 25 min earlier with morphine (5 micrograms i.t., n = 5). In both experimental paradigms, substance P decreased the reaction time in the tail flick test at 1 min after injection; the magnitude of this effect resembled that which occurs in intact rats given substance P alone. In both paradigms, substance P failed to produce the behavioural responses typically observed in untreated rats given substance P. These results indicate that substance P-induced facilitation of the tail flick reflex may occur via spinal mechanisms alone. The results also suggest that in the spinal cord morphine preferentially blocks the relay of substance P-induced information to supraspinal structures. Finally, as the behavioural response is blocked by the analgesic and by spinal transection, this is interpreted as further support for the proposal that this behavioural response indicates that the animal has perceived a noxious stimulus.

Effects of intrathecal administration of neuropeptides on a spinal nociceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensin II.

The present study examines the effects of intrathecal administration of selected peptides on nociceptive responses in the rat. Each peptide was delivered via a chronically implanted catheter to the L5 vertebral level. In the tail flick test, VIP (0.65-6.5 nmoles) produced a dose-dependent decrease in reaction time (RT) from 1 to 6-16 min after injection; 6.5 nmoles decreased RT to 37% of control value at 1 min after injection. Galanin (0.65-6.5 nmoles) produced a dose-dependent increase in reaction time at 1 and 6 min; at high doses, many of the rats failed to flick the tail. CGRP (6.5 nmoles) produced a small, transient decrease in RT to 73% of control values at 1 min; 3.25 nmoles were without effect. CSF and 6.5 nmoles of somatostatin, TRH and angiotensin II were without effect. At high doses of galanin and CGRP, rats vocalized to innocuous touch of the tail, as reported for substance P. Von Frey hairs were thus applied to the tail after 6.5 nmoles of VIP, galanin, CGRP or substance P. Vocalization in response to a previously innocuous pressure stimulus was observed at 30 s after injection in all rats given galanin and some rats given CGRP or substance P; the effect lasted 4-8 min. VIP and CSF had no effect. These results suggest that VIP, galanin, CGRP and substance P may act as excitatory agents in nociceptive pathways and that specific peptides may function in the different types of pain modalities; VIP in thermal, galanin in mechanical and substance P and CGRP in both.

Distribution of label after intrathecal administration of 125I-substance P in the rat.

Despite the widespread use of the intrathecal route for the administration of neuroactive agents, little is known about the penetration of these agents into the spinal cord. In the present study, 125I-substance P was injected via a spinal catheter to the thoracic or sacro-coccygeal spinal cord in the rat (350-400 g) anesthetized with urethane (2.5 g/kg). Spinal cords were removed rapidly at 1 or 10 min after injection and immediately frozen in CCl2F2. Frozen sections, 20 micron thick, were cut and mounted for autoradiography. Autoradiographs of transverse sections demonstrated that the label penetrated 700 to 1800 micron from the surface of the spinal cord at both levels. In longitudinal sections, this penetration extended about 0.5 cm rostrally and caudally from the site of injection. Serial autoradiographs of transverse sections showed a similar penetration rostro-caudally. In addition, venous blood samples were taken at 1, 6, 11 and 16 min after injection of the labelled peptide. Quantification of the radioactivity in the samples revealed that 0.8 to 3.5% of the total CPM injected had passed into the general circulation at these times. These data indicate that after intrathecal administration of radiolabelled substance P, the label penetrates into the grey matter of the spinal cord to presumed sites of action. They also suggest that the rostro-caudal extent of penetration is more localized than suggested from earlier studies which looked only at levels of radioactivity in pieces of whole spinal cord. Finally, our study has indicated that passage of label into the circulation is negligible at least for substance P.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of substance P, neurokinin A, physalaemin and eledoisin in facilitating a nociceptive reflex in the rat.

The postulated existence of different types of tachykinin receptor in the spinal cord provided the basis for the present study; substance P, neurokinin A, eledoisin and physalaemin were administered intrathecally in the awake, restrained rat to compare their effects on reaction time in the tail-flick test. Each peptide was delivered via a chronically implanted subdural catheter to the lower lumbar vertebral level of the spinal cord. Intrathecal administration of 10 micrograms of substance P (6.5 nmol), eledoisin (8.0 nmol) or physalaemin (7.9 nmol) decreased the reaction time, respectively, to 22.5, 24.3 and 20.8% of the mean preadministration control values at 1 min after injection; similar administration of 6.5 nmol of neurokinin A produced a smaller decrease in reaction time, to only 49.5% of preadministration values. These effects were transient, the reaction times returning to preadministration values within 5 min. Each peptide also produced an initial vocalization followed by increased restlessness. Analysis of the dose-response curves indicated that the rank order of potency of the fitted curves for these peptides was physalaemin greater than or equal to substance P greater than or equal to eledoisin greater than neurokinin A. The results suggest that the receptor involved in facilitation of the tail-flick reflex resembles a substance P receptor rather than a receptor for one of the other endogenous neurokinins and that this receptor may bear some resemblance to the SP-P type postulated to exist in peripheral tissues.