Intraventricular administration of MSH induces hyperalgesia in rats.

In a completely crossed, double blind designed study, six rats received intraventricular injections of 0.1, 1.0 and 10 micrograms of alpha-MSH and a placebo. The rats were tested for response to painful thermal stimuli with the tail-flick test. All of the doses of alpha-MSH produced hyperalgesia during the first 20 min of testing. Only the 1.0 microgram dose of alpha-MSH produced hyperalgesia throughout the 80 min course of the experiment. This study, coupled with previous reports that MSH/ACTH fragments may attenuate morphine-induced analgesia, suggest that MSH can have opposite actions from those of the endorphins. It is possible that alpha-MSH and related peptides may be endogenous anti-opiates.


MSH
Hyperalgesia Tail-flick test TItERE are striking anatomical and neurophysiologieal relationships between melanoeyte stimulating hormone (MSH) and fl-endorphin. Both of these peptides have been localized in the same cells of the arcuate nucleus [17,23], both are stored and released by the intermediate lobe of the pituitary [4], and both peptides are contained in a larger precursor molecule [14]. It may be argued that these shared relationships suggest that the peptides subserve redundant functions and indeed there is scattered support for such a speculation [8,12,16,18]. However, there is also support for the possibility that these two peptides (including the 4-10 fragment shared by MSH and ACTH) have reciprocal functions. For instance, these molecules can have opposing actions on cyclic AMP; MSH/ACTH and their fragments increase cyclic AMP in the brain [6,9], whereas 3-endorphin decreases it in vitro in neuroblastoma cells [15]. Differences in learning [22], social behavior I2, 22], and electrical activity of the brain [22] further suggest a reciprocal relationship between these substances. Among the most direct evidence of a modulating relationship between these peptides is the attenuation of opioid-induced analgesia by MSH/ACTH compounds [14]. Recently, Szekely et al. [23] reported that ~-MSH administered concomitantly with morphine also reduces tolerance and dependence on morphine.
There are many reports that /3-endorphin produces analgesia when injected centrally [6,25]. Two recent reports [1, 3,4] suggested that hyperalgesia resulted from central administration of ACTH. Bertolini et aL 13] proposed that ACTH may be an endogenous anti-endorphin. Since major behavioral differences have been related to small changes in thestructure of MSH/ACTH fragments [21], the current study was designed to investigate the hyperalgesic influence of a-MSH.

Snbjects
Nine Sprague-Dawley, male, ninety day old rats served as subjects. Histological confirmation of placement of the cannulae indicated that six rats reliably received intraventricular administration of the peptide,

Surgery
At least a week before testing, ventricular cannulae were implanted into the left ventricle of the rats. Stereotaxic coordinates were 1 mm posterior to bregma, 1.5 mm lateral of the midline, and 4.1 mm below the skull surface. The cannulae were secured with dental acrylic and stainless steel hooks. The cannulae were constructed from 22 ga stainless steel hypodermic tubing beveled at the tips. A 26 ga needle of ~Address reprint requests to Curt A. Sandman, Ph.D., Fairview ttospital, 2501 Harbor Boulevard, Costa Mesa, CA 92626.

SANDMAN AND KASTIN
a microsyringe was extended 1 mm below the cannulae for injections of the a-MSH. Histological verification of the cannulae placement was done by observation of marker dyes injected into the ventricle before sacrifice.

Apparatus
Response to thermal stimulation was measured with the tail-flick test of D'Amour and Smith [8]. The apparatus consisted of an adjustable heat source directed to the rat's tail. A solid state timer was. initiated automatically when the heat was applied. The timer and the heat were terminated when the tail was withdrawn from the source of heat. The latency of response, to tenths of a second, provided a measure of pain sensitivity.

Procedure
Immediately before testing, each rat was administered an intraventricular injection of 0.1, 1.0 or I0 p.g ofa-MSH or the vehicle solution. The vials containing the peplides were coded so that the experimenter was unaware of the solutions administered. The completely crossed design ensured that every animal received every treatment. At least one week elapsed between treatments to control for the possible lingering effects of the peptidcs. The heat source was calibrated so that the mean latencies were between 3.5 and 4.5 see during the pretreatment phase. Tail-flick latencies were measured for each animal in each session before treatment, and the p.roper calibrations were performed. The measures confirmed that there was no long-term influence of MSH on the tail-flick test. The criterion of 6 consecutive latencies within 1 sec was established during the pretreatment period in order to obtain reliable measures. The mean of the 6 consecutive trials was established as the zero point and all results after treatment with the solutions were calculated as a percentage of change from baseline measurement. Latencies were measured every 2-3 rain throughout the entire session. The data were summarized into 10 min intervals by averaging all of the latencies within each 10 min epoch. dependent manner. Although all doses of a-MSH produced hyperalgesia during the first 20 min, only the 1.0 #g dose produced long-lasting effects. The reason for this dose response is unknown, although quadratic functions often describe drug/peptide-behavior relationships [21]. In related studies using much higher 9 of ACTH 1-24 (20--50 #g/rat), linear relations with hyperalgesia were reported with the highest dose producing the greatest effect [3]. Direct comparisons are tenuous since the ACTH 1-24 molecule produces different behavioral effects than a-MSH [21]. Indeed, there is evidence that an analog of ACTH 4-9 (ORG 2766) injected directly into the midbrain central gray produced significant analgesia [24]. This effect was only apparant for the largest dose (30/zg) injected and was not observed when injected into the lateral ventricles. Further, the current study provides dose characteristics only at the lower end of the dosage curve. It is conceivable that with larger pharmacological doses, linear relationships between hyperalgesia and dose of peptide may exist.
Perhaps the potent influence of ACTH 1-24 in the study of Bertolini et al. [3] was a function of its steroidogenic properties. For instance, Holaday et al. [11] have reported that morphine-induced analgesia can be attenuated in adrenalectomized rats by treatment with dexamethasone. Thus, ACTH 1-24 may have a dual or additive influence; one effect may be due solely to the peptide and a second effect may be related to the stimulation of corticosterone by ACTH 1-24. In the current study, hyperalgesia was produced by a-MSH, the actions of which are not mediated by secretion of corticosterone. Our results suggest that a-MSH, like ACTH [4] and perhaps MIF-I [12], may represent endogenous antiopiates.