The norepinephrine system has been implicated in separation vocalizations in a number of species but has been examined in greatest detail in the rat pup. Clonidine, an alpha-2 adrenoreceptor agonist, along with other norepinephrine agonists, are among the few pharmacological agents that increase isolation-induced calling in pups (13,30). Clonidine (0.025-.5mg/kg) even stimulated ultrasonic vocalizing in rat pups that had been pretreated with monoamine depletors such as reserpine and alpha-methyl-tyrosine, suggesting a postsynaptic norepinephrine mechanism (13). Idazoxan and yohimbine, alpha-2 antagonists, blocked the effect of clonidine and, in fact, reduced vocalizing in control pups while prazosin, an alpha-1 antagonist, blocked the clonidine effect but was ineffective alone. These findings are age specific in that by day 17 and 18 clonidine was less effective, prazosin no longer antagonized clonidine, and idazoxan was a more potent antagonist. It is possible that the pharmacological control of these receptor systems changes over time from a postsynaptic to a presynaptic mechanism. These studies suggest that the norepinephrine system is importantly involved in the onset of isolation-induced vocalizations in the infant rat.

Additionally, the norepinephrine agonists, desipramine (1-20 mg/kg), mazindol (.5mg/kg), and nortriptyline (1.0mg/kg) significantly increased calling in rat pups at 10 days of age (43). However, these effects were accompanied by reductions in rectal temperature. In a direct comparison of related drugs, desipramine (6-12 mg/kg) again increased isolation-induced vocalizing, but imipramine (6-12 mg/kg) had no effect on the number of calls (28). These results may reflect different potencies in inhibiting norepinephrine reuptake. That is, both drugs are moderate anticholinergics, but desipramine strongly inhibits reuptake of norepinephrine and weakly inhibits reuptake of serotonin, whereas imipramine has stronger serotonergic, and weaker noradrenergic effects.

In contrast to the noradrenergic system which enhances vocalizing by rat pups, stimulation of the serotonergic system seems to have both an inhibitory and facilitatory effect on calling. Serotonin-reuptake blockers such as clomipramine (1-5 mg/kg), paroxetine (1.0 mg/kg) and citalopram (1.0 mg/kg) reduced or even eliminated calling in isolated rat pups (43). Following acute subcutaneous injections of the serotonergic neurotoxin MDMA (3,4-methylenedioxy-methamphetamine), pups were monitored for both short- and long-term effects (42). Sixty minutes after MDMA (0.5-10 mg/kg) drug administration, 10-day-old pups emitted fewer vocalizations than did controls, but by 10 hours vocalizing had risen above control levels. These effects point to the possible importance of serotonergic modulation of the infant ratís response during isolation. Using another approach, Winslow and Insel (44) assessed the effectiveness of selective serotonin agonists and antagonists in modulating the vocalizing of 9-11-day-old pups. 5HT1a receptor agonists (buspirone and 8-OH-DPAT) profoundly decreased ultrasounds. 5HT2/5HT1c agonists decreased calling at low doses. In contrast, the 5HT1b agonist GC12066B produced a dose-related increase in the rate of vocalizing. Perhaps 5HT1a agonists produce an anxiolytic-like effect, as measured by ultrasound production, and 5HT1b agonists have an anxiogenic effect, as indicated by the same measure. Thus, the serotonin system seems to modulate the isolation-induced calling behavior of the infant rat in a receptor-dependent fashion.

Similar to the serotonergic system, the endogenous opioid system has receptor-specific influences on the ultrasonic vocalizing of isolated rat pups. Morphine, primarily a mu agonist, given either intraperitoneally or intracerebrally, significantly reduced the ultrasonic vocalizing of 10-day-old rat pups, and the opiate receptor blocker naltrexone returned the rate to control levels (6,26, 27). The mu agonist DAMGO and the delta receptor agonist DPDPE also reduced the rate of ultrasonic calling (9). Naltrexone alone has been found to increase vocalizing in some circumstances (6, 26,40) but not in others (7,45); apparently a subtle change in the test environment can determine the outcome. In fact, in those conditions in which naltrexone increased vocalizing, it appeared that endogenous release of central beta-endorphin and enkephalin were likely to have occurred, particularly in the midbrain (40). The particular conditions under which naltrexone increased vocalization rate were when some familiar object was present. In the case of bedding, or anesthetized dam or littermates, naltrexone was able to reverse the reduction in calling that occurs with those stimuli.

In contrast to the mu and delta receptor agonists, the kappa opioid receptor agonist U50,488 significantly increased isolation-induced vocalizations in infant rats (9, 29). The kappa agonist, given either intraperitoneally or intracisternally, produced high levels of calling, enhanced activity, and analgesia. How opioids interact with other neurochemical systems to produce their behavioral effects remains to be tested, but effects of opioids on dopamine activity offer an intriguing possibility. Microdialysis has shown that central extracellular dopamine levels are increased with mu opioid agonists in doses that are considered positively reinforcing, and are inhibited with kappa agonists in doses that produce aversive states (11, 41).

Cocaine, a potent dopamine reuptake inhibitor, in doses from 2.5 to 20 mg/kg caused a marked reduction in isolation vocalizations in 10-day-old pups (31). Although the opiates, like the stimulant cocaine, can decrease pupsí calls, opiates generally reduce activity levels whereas cocaine produces hyperactivity in the neonate. To control for the influence of higher activity on vocalization rate, a separate group of pups received caffeine prior to the isolation. Caffeine administration had no effect on calling behavior, although activity levels were significantly greater than in saline-administered controls. In addition, intraperitoneal injection of the dopamine antagonist haloperidol (0.5mg/kg & 1.0 mg/kg) prior to cocaine (1.25 & 2.5 mg/kg) produced a significant elevation in vocalizing compared with saline pretreatment. Haloperidol in the absence of cocaine had no effect on calling. These results suggest that the endogenous dopamine system involved with reinforcement and reward may quell the stress of isolation in the infant rat. Moreover, in studying the effects of psychotropic drugs on mouse pup calls, it was found that haloperidol and chlorpromazine reduced body temperature without affecting ultrasonic vocalizations (3).

The GABA system has been strongly implicated in the control of ultrasonic vocalizations in isolated rat pups. The administration of non-sedating doses of chlordiazepoxide (CDP) and diazepam (DZ) decreased the number of vocalizations emitted by rat (6,12,25) as well as mouse pups (3). The antagonist RO 15-1788 blocked the effects of the agonists and returned the number of calls to the level of controls. When administered alone, however, the antagonist, RO 15-1788 significantly reduced the number of ultrasounds, perhaps implicating involvement of the BDZ receptor. The putative anxiogenic pentylenetetrazol increased the number of vocalizations (12). Finally, the quieting effects of CDP were not reversed by naltrexone, suggesting that the GABA mediation of reduced calling was not functioning by way of the opioid system. These results suggest that the benzodiazepine-GABA receptor-chloride channel complex may be an important entity in the mediation of infant rat isolation behavior.

The stress-related neuropeptide corticotropin-releasing factor (CRF) affects the vocalizing of infant rat pups. Throughout the preweaning period, intracerebroventricular CRF reduces the rate at which isolated pups vocalize (15,24). This effect can be blocked with the CRF-receptor antagonist, alpha-helical CRF 9-41, and central administration of the antagonist alone (1ug) was observed in one study to increase vocalizing (24), indicating that the effect of intracerebroventricular CRF was receptor-mediated and mimicked the action of an endogenous CRF mechanism. However, the effect of CRF on vocalizing does not always appear to be inhibitory. When a large (20ug) dose of alpha-helical CRF 9-41 was injected alone, it too suppressed the vocalizing of pups (15). These findings suggest that the effect of CRF on vocalizing may be curvilinear, with substantial levels of CRF suppressing vocalizing, but low endogenous levels contributing to the onset of the vocalization response (15,24). Nonetheless, exogenous CRF alone is not sufficient to initiate vocalizing of pups in a warm familiar environment (15). All of the above effects appear to be due to central CRF activity because peripheral injection of CRF and the antagonist were without effects on vocalizing in these studies. In sum, these results suggest that during isolation in a novel environment at ambient temperature, low endogenous levels of central CRF, in concert with other neurochemical systems (e.g., norepinephrine), lead to the onset of vocalizing; but as levels continue to rise, the effect of CRF becomes inhibitory, contributing to the suppression of vocalizing that pups exhibit after an extended period of isolation.

More is known about the neuropharmacology of infant isolation calls in the rat than in any other mammalian species. Yet, the close and intricate relation between thermogenesis and USV production in the immature rat pup (4) raises doubts about the generality of findings to other species, especially to those such as various primates, including ourselves, in which the isolation call of infants appears much more unambiguously to be an emotional response. The precocial guinea pig provides a useful comparison. Guinea pig pups are able to ambulate from shortly after birth, and mothers are passive, so that it is the pupís responsibility from early on to maintain contact with the mother. Perhaps as a result, the guinea pig pup exhibits clearer evidence of attachment to the mother throughout the preweaning period than does the rat (35,21,14). Moreover, the young guinea pig thermoregulates well and the primarily audible ìwhistleî vocalization that the pup emits during isolation does not appear to be related to thermogenic mechanisms. Some of the same neurochemical systems examined in the isolated rat pup have also been studied in the young guinea pig.

In an early study (22), low doses of subcutaneous morphine (<1mg/kg) were found to dose-dependently reduce the vocalizing of young isolated guinea pigs, whereas naloxone enhanced the vocalization rate. These data are consistent with effects in the rat pup reported above. Moreover, in the guinea pig pup as in the infant rat, central CRF appears to inhibit vocalizing. Intracerebroventricular administration of the CRF antagonist alpha-helical CRF9-41 enhanced initial vocalization rates during isolation in a novel test cage (20). However, CRF (.05 and 5 ug) administered in the same fashion had no significant effect on vocalizing.

Unlike the case in the rat, however, peripherally injected CRF does affect vocalizing, greatly reducing vocalization rate during isolation (16). This effect was not duplicated with injection of ACTH nor blocked with naloxone, suggesting that downstream hormones of the pituitary and adrenal are not involved. Further, the effect cannot be explained by reduction of motor capacity, excessive freezing, or hypotension (2,18). Peripheral administration of the antagonist alone enhanced vocalizing and, when administered with CRF, prevented CRF-induced suppression (18,19,34). Although the site of action has not been determined, evidence indicates that peripherally injected CRF does not cross the blood-brain barrier to exert its effects at central CRF receptors (17). The results suggest the possibility of an endogenous mechanism in which peripheral CRF binds to peripheral CRF receptors to initiate a message along an indirect pathway to brain regions controlling vocalizing. For instance, the adrenal medulla contains both CRF and CRF receptors, and it may increase secretion of CRF during times of stress (5,10). Afferent fibers from the adrenal medulla to the cord (36) could potentially relay the peripheral signal to appropriate central regions.

Following up on an early preliminary report suggesting a suppressive effect of imipramine on guinea pig vocalizations (39), two recent studies have tested young guinea pigs with a variety of compounds possessing anxiolytic and/or antidepressant properties. Interestingly, reductions in vocalizing during isolation were achieved with the majority of such drugs examined. These include benzodiazapine ligands (diazepam, alpraloam, alcohol), serotonin and norepinephrine reuptake inhibitors (clomipramine, imipramine, fluvoxamine, fluvoxetine, desipramine, maprotaline), serotonin 5HT1A angonists (8-OH-DPAT, flesinoxan, buspirone), a MAO inhibitor (clorgyline), and Substance P receptor antagonists (L-760,735; L733-060; 33,37,39). These intriguing initial results pave the way for more mechanistic studies. Clearly, the neuropharmacology of isolation calls in the guinea pig is still in its infancy. Nevertheless, these initial studies reveal both similarities with results seen in rat pups (e.g., quieting effects diazepam and 8-OH-DPAT) as well as some inconsistencies between species (effects of imipramine and desipramine). Further work with the precocial and gregarious guinea pig may help better establish the neurochemical systems that underlie social cohesion and the psychogenic stress of social disruption.

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