Separation-induced Vocalizations Research Articles

Deficiency of the paternally inherited gene Magel2 alters the development of separation-induced vocalization and maternal behavior in mice.

The behavior of offspring results from the combined expression of maternal and paternal genes. Genomic imprinting silences some genes in a parent-of-origin specific manner, a process that, among all animals, occurs only in mammals. How genomic imprinting affects the behavior of mammalian offspring, however, remains poorly understood. Here, we studied how the loss of the paternally inherited gene Magel2 in mouse pups affects the emission of separation-induced ultrasonic vocalizations (USV). Using quantitative analysis of more than 1000 USVs, we characterized the rate of vocalizations as well as their spectral features from postnatal days 6-12 (P6-P12), a critical phase of mouse development that covers the peak of vocal behavior in pups. Our analyses show that Magel2 deficient offspring emit separation-induced vocalizations at lower rates and with altered spectral features mainly at P8. We also show that dams display altered behavior towards their own Magel2 deficient offspring at this age. In a test to compare the retrieval of two pups, dams retrieve wildtype control pups first and faster than Magel2 deficient offspring. These results suggest that the loss of Magel2 impairs the expression of separation-induced vocalization in pups as well as maternal behavior at a specific age of postnatal development, both of which support the pups' growth and development.

CRF1 but not glucocorticoid receptor antagonists reduce separation-induced distress vocalizations in guinea pig pups and CRF overexpressing mouse pups. A combination study with paroxetine

RationaleGiven the large number of patients that does not respond sufficiently to currently available treatment for anxiety disorders, there is a need for improved treatment. ObjectivesWe evaluated the anxiolytic effects of corticotropin releasing factor (CRF)1 receptor antagonists and glucocorticoid receptor (GR) antagonists in the separation-induced vocalization test in guinea pigs and transgenic mice with central CRF overexpression. Furthermore, we explored effects of these drugs when given in combination with a suboptimal dose of a selective serotonin re-uptake inhibitor (SSRI). MethodsIn guinea pig pups, the CRF1 receptor antagonists CP-154,526 and DMP695, and the GR antagonists mifepristone and Org34517 (all at 2.5, 10 and 40mg/kg intraperitoneally (IP)) were tested alone or in combination with 0.63mg/kg paroxetine IP. In CRF overexpressing mouse pups and wild type littermates, effects of CP-154,526 (10, 20 and 40mg/kg subcutaneously (SC)) and mifepristone (5, 15, 45mg/kg SC) were studied alone or in combination with 0.03mg/kg paroxetine SC. ResultsCRF1 but not GR antagonists reduced the number of calls relative to vehicle in guinea pigs and mice, independent of genotype. Treatment of CRF1 receptor or GR antagonists with paroxetine had no combined effect in guinea pigs, wild type or CRF overexpressing mice. ConclusionsCurrent results indicate robust anxiolytic properties of CRF1 receptor antagonists in guinea pigs and mice overexpressing CRF, and lack thereof of GR antagonists. Although no combined treatment effects were observed, it would be interesting to study combined treatment of CRF1 receptor antagonists with SSRIs following chronic drug administration.

Pharmacological and methodological aspects of the separation-induced vocalization test in guinea pig pups; a systematic review and meta-analysis

The separation-induced vocalization test in guinea pig pups is one of many that has been used to screen for anxiolytic-like properties of drugs. The test is based on the cross-species phenomenon that infants emit distress calls when placed in social isolation.Here we report a systematic review and meta-analysis of pharmacological intervention in the separation-induced vocalization test in guinea pig pups. Electronic databases were searched for original research articles, yielding 32 studies that met inclusion criteria. We extracted data on pharmacological intervention, animal and methodological characteristics, and study quality indicators.Meta-analysis showed that the different drug classes in clinical use for the treatment of anxiety disorders, have comparable effects on vocalization behaviour, irrespective of their mechanism of action. Of the experimental drugs, nociception (NOP) receptor agonists proved very effective in this test. Analysis further indicated that the commonly used read-outs total number and total duration of vocalizations are equally valid. With regard to methodological characteristics, repeated testing of pups as well as selecting pups with moderate or high levels of vocalization were associated with larger treatment effects. Finally, reporting of study methodology, randomization and blinding was poor and Egger’s test for small study effects showed that publication bias likely occurred.This review illustrates the value of systematic reviews and meta-analyses in improving translational value and methodological aspects of animal models. It further shows the urgent need to implement existing publication guidelines to maximize the output and impact of experimental animal studies.

Characterization of a novel vasopressin V1b receptor antagonist, V1B-30N, in animal models of anxiety-like and depression-like behavior

Overactivity of the hypothalamic–pituitary–adrenal (HPA) axis has been linked to affective disorders such as anxiety and depression. Dampening HPA activity has, therefore, been considered as a possible means of treating affective disorders. Given the important role of vasopressin in modulating the HPA axis, one strategy has focused on inhibiting activity of the vasopressin 1b (V1b) receptor. In animals, V1b receptor antagonists reduce plasma stress hormone levels and have been shown to have an anxiolytic-like effect. Recently, V1B-30N was identified as a highly potent V1b receptor antagonist with selectivity over other vasopressin receptors, which is evaluated here in rodent models of anxiety-like and depression-like behaviors. V1B-30N (1–30mg/kg, IP) dose-dependently reduced separation-induced vocalizations in rat pups without producing any sedative effects in the animals. Similarly, V1B-30N (3–30mg/kg, IP) dose-dependently reduced separation-induced vocalizations in guinea pig pups. In a conflict assay, conditioned lick suppression, V1B-30N (3–30mg/kg, IP) increased punished licking. To assess antidepressive-like properties, V1B-30N (1–30mg/kg) was tested in the mouse and rat forced-swim tests but was found to be inactive. These results are consistent with previous findings with other V1b antagonists, which suggest that acute pharmacological antagonism of the V1b receptor has anxiolytic-like but not antidepressant-like properties.

Preclinical Pharmacology and Pharmacokinetics of AZD3783, a Selective 5-Hydroxytryptamine 1B Receptor Antagonist

The preclinical pharmacology and pharmacokinetic properties of (2R)-6-methoxy-8-(4-methylpiperazin-1-yl)-N-(4-morpholin-4-ylphenyl)chromane-2-carboxamide (AZD3783), a potent 5-hydroxytryptamine 1B (5-HT(1B)) receptor antagonist, were characterized as part of translational pharmacokinetic/pharmacodynamic hypothesis testing in human clinical trials. The affinity of AZD3783 to the 5-HT(1B) receptor was measured in vitro by using membrane preparations containing recombinant human or guinea pig 5-HT(1B) receptors and in native guinea pig brain tissue. In vivo antagonist potency of AZD3783 for the 5HT(1B) receptor was investigated by measuring the blockade of 5-HT(1B) agonist-induced guinea pig hypothermia. The anxiolytic-like potency was assessed using the suppression of separation-induced vocalization in guinea pig pups. The affinity of AZD3783 for human and guinea pig 5-HT(1B) receptor (K(i), 12.5 and 11.1 nM, respectively) was similar to unbound plasma EC(50) values for guinea pig receptor occupancy (11 nM) and reduction of agonist-induced hypothermia (18 nM) in guinea pig. Active doses of AZD3783 in the hypothermia assay were similar to doses that reduced separation-induced vocalization in guinea pig pups. AZD3783 demonstrated favorable pharmacokinetic properties. The predicted pharmacokinetic parameters (total plasma clearance, 6.5 ml/min/kg; steady-state volume of distribution, 6.4 l/kg) were within 2-fold of the values observed in healthy male volunteers after a single 20-mg oral dose. This investigation presents a direct link between AZD3783 in vitro affinity and in vivo receptor occupancy to preclinical disease model efficacy. Together with predicted human pharmacokinetic properties, we have provided a model for the quantitative translational pharmacology of AZD3783 that increases confidence in the optimal human receptor occupancy required for antidepressant and anxiolytic effects in patients.

Moderate recurrent hypoglycemia during early development leads to persistent changes in affective behavior in the rat

Recurrent hypoglycemia is a common problem among infants and children that is associated with several metabolic disorders and insulin-dependent diabetes mellitus. Although studies have reported a relationship between a history of juvenile hypoglycemia and psychological health problems, the direct effects of recurrent moderate hypoglycemia have not been fully determined. Thus, in this study, we used an animal model to examine the effects of recurrent hypoglycemia during the juvenile period on affective, social, and motor function (assessed under euglycemic conditions) across development. To model recurrent hypoglycemia, rats were administered 5 U/kg of insulin or saline twice per day from postnatal day (P)10 to P19. Body weight gain was retarded in insulin-treated rats during the treatment period, but recovered by the end of treatment. However, insulin-treated rats displayed increases in affective reactivity that emerged early during treatment and persisted after treatment into early adulthood. Specifically, insulin-treated pups showed increased maternal separation-induced vocalizations as infants, and an exaggerated acoustic startle reflex as juveniles and young adults. Moreover, young adult rats with a history of recurrent juvenile hypoglycemia exhibited increased fear-potentiated startle and increases in behavioral and hormonal responses to restraint stress. Some of these effects were sex-dependent. The changes in affective behavior in insulin-exposed pups were accompanied by decreases in adolescent social play behavior. These results provide evidence that recurrent, transient hypoglycemia during juvenile development can lead to increases in fear-related behavior and stress reactivity. Importantly, these phenotypes are not reversed with normalization of blood glucose and may persist into adulthood.

The Anxiolytic-Like Effects of the Novel, Orally Active Nociceptin Opioid Receptor Agonist 8-[bis(2-Methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)

Orphanin FQ/nociceptin (OFQ/N) is the endogenously occurring peptide ligand for the nociceptin opioid receptor (NOP) that produces anxiolytic-like effects in mice and rats. The present study assessed the anxiolytic-like activity of 8-[bis(2-methylphenyl)-methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510), a novel potent piperidine NOP agonist (EC(50) = 12 nM) that binds with high affinity (K(i) = 0.3 nM) and functional selectivity (>50-fold over the mu-, kappa-, and delta-opioid receptors). The anxiolytic-like activity and side-effect profile of SCH 221510 were assessed in a variety of models and the benzodiazepine, chlordiazepoxide (CDP), was included for comparison. The effects of chronic dosing of SCH 221510 were also assessed. Furthermore, the specificity of the anxiolytic-like effect of SCH 221510 was investigated with the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and the opioid receptor antagonist naltrexone. Like CDP (1-30 mg/kg i.p.), SCH 221510 (1-30 mg/kg p.o.) produced anxiolytic-like effects in the elevated plus-maze (rat and gerbil), Vogel conflict (rat), conditioned lick suppression (rat), fear-potentiated startle (rat), and pup separation-induced vocalization (guinea pig) assays. In the Vogel conflict, the anxiolytic-like effect of SCH 221510 (10 mg/kg) was attenuated by J-113397 (3-10 mg/kg p.o.), but not naltrexone (3-30 mg/kg i.p.). Additionally, the anxiolytic-like effects of SCH 221510 did not change appreciably following 14-day b.i.d. dosing in rats (10 mg/kg). Furthermore, unlike CDP, SCH 221510 (3-30 mg/kg) produced anxiolytic-like activity at doses that did not disrupt overt behavior. Collectively, these data suggest that NOP agonists such as SCH 221510 may have an anxiolytic-like profile similar to benzodiazepines, with a reduced side-effect liability.

ATC0175: An Orally Active Melanin-Concentrating Hormone Receptor 1 Antagonist for the Potential Treatment of Depression and Anxiety

Melanin-concentrating hormone (MCH) has been implicated in a variety of physiological events. Recent studies clearly suggest that MCH plays an important role in the regulation of stress and emotion. To date, two receptor subtypes of MCH (MCH1R and MCH2R) have been identified. MCH1R has been suggested to mediate most of the physiological functions of MCH. Recently, we synthesized an orally active, nonpeptidic antagonist of MCH1R, N-(cis-4-{[4-(dimethylamino)quinazolin-2-yl]amino}cyclohexyl)-3,4-difluorobenzamide hydrochloride (ATC0175). This compound is a potent antagonist with a high affinity for MCH1R and additional affinities for 5-HT1A and 5-HT2B receptors. The receptor binding and the functional assay (MCH-induced increase in [Ca2+]i) indicated that ATC0175 is a noncompetitive antagonist at MCH1Rs. ATC0175 exhibited anxiolytic effects in numerous animal models of anxiety including the elevated plus-maze test, social interaction test, stress-induced hyperthermia and maternal separation-induced vocalization. Like with other stress-related peptide receptor antagonists, such as antagonists of corticotropin-releasing factor or vasopressin V1b receptor antagonists, anxiolytic effects of ATC0175 were more pronounced in models containing a stress component. ATC0175 also exhibited antidepressant effects in the forced swimming test. ATC0175 increased swimming performance without altering climbing behavior, as observed with selective serotonin reuptake inhibitors. ATC0175 has adequate ADME profile (reasonable oral bioavailability and brain penetration) and potent oral activity in animal models. In contrast, ATC0175 did not affect spontaneous locomotor activity, hexobarbital-induced sleeping time and did not impair rotarod performance. Thus, ATC0175 may be devoid of unwanted central nervous system side effects, which are sometimes observed with current medications. In addition, ATC0175 was well tolerated in rat repeated toxicity study, and had no genotoxic liability. Therefore, ATC0175 has the potential to be effective in the treatment of patients with depression and/or anxiety disorders.

Anxiolytic- and Antidepressant-Like Profile of ATC0065 and ATC0175: Nonpeptidic and Orally Active Melanin-Concentrating Hormone Receptor 1 Antagonists

Melanin-concentrating hormone (MCH) is a cyclic peptide produced in the lateral hypothalamus. It has been implicated in a number of physiological processes including feeding behavior, energy balance, and the regulation of emotional states. Here, we report in vitro and in vivo profiles of ATC0065 [N(2)-[cis-4-({2-[4-bromo-2-(trifluoromethoxy)phenyl]ethyl}amino)cyclohexyl]-N(4), N(4)-dimethylquinazoline-2,4-diamine dihydrochloride] and ATC0175 [N-(cis-4-{[4-(dimethylamino)quinazolin-2-yl]amino}cyclohexyl)-3,4-difluorobenzamide hydrochloride], newly synthesized MCH receptor 1 (MCHR1) antagonists. Both ATC0065 and ATC0175 had high affinities for human MCHR1 with IC(50) values of 15.7 +/- 1.95 and 7.23 +/- 0.59 nM, respectively. Both ATC0065 (IC(50) = 21.4 +/- 1.57 nM) and ATC0175 (IC(50) = 13.5 +/- 0.78 nM) showed potent antagonist activities at MCHR1, as assessed by MCH-increased guanosine 5'-O-(3-[(35)S]thio)phosphate ([(35)S]GTPgammaS) binding to human MCHR1. Oral administration of ATC0065 (3-30 mg/kg) or ATC0175 (1-10 mg/kg) significantly reduced immobility time in the forced swimming test in rats, indicating antidepressant-like effects. Both ATC0065 and ATC0175 significantly reversed swim stress-induced anxiety in the elevated plus-maze test in rats and stress-induced hyperthermia in mice. ATC0175 significantly increased social interaction between unfamiliar rats and reduced separation-induced vocalizations in guinea pig pups, indicating anxiolytic potential. In contrast, ATC0065 and ATC0175 did not affect spontaneous locomotor activity or rotarod performance in rats. These findings indicate that ATC0065 and ATC0175 are potent and orally active MCHR1 antagonists with anxiolytic and antidepressant activity in rodents.

Behavioral pharmacology of AR-A000002, a novel, selective 5-hydroxytryptamine(1B) antagonist.

The present review summarizes the behavioral pharmacology conducted to profile the anxiolytic and antidepressant potential of the selective 5-hydroxytryptamine (HT)(1B) antagonist (R)-N-[5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4-tetrahydro-2-naphthyl]-4-morpholinobenzamide (AR-A000002). AR-A000002 functions as a 5-HT(1B) antagonist in vivo, which was shown by the antagonism of the discriminative stimulus effects in the guinea pig of the 5-HT(1B) agonist 3-(N-methylpyrrolidin-2R-ylmethyl)-5-(3-nitropyrid-2-ylamino)-lH-indole (CP135,807). Anxiolytic activity of AR-A000002 was demonstrated in the separation-induced vocalization paradigm in guinea pig pups, and in a suppressed responding procedure in pigeons and guinea pigs, but only a weak trend was noted in a suppressed responding procedure in squirrel monkeys. Antidepressant efficacy was shown in a number of paradigms. In pigeons and guinea pigs responding under a differential reinforcement of low rates schedule of reinforcement (DRL), AR-A000002 increased the number of reinforcers earned without altering the number of responses made. In guinea pigs trained under a response duration differentiation paradigm, AR-A000002 increased mean lever-press duration. Finally, AR-A000002 was shown to block escape failures in guinea pigs submitted to a learned helplessness paradigm. Taken together, these data suggest utility for 5-HT(1B) antagonists in the treatment of both anxiety and affective disorders.

4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1, 3-thiazol-2-amine hydrochloride (SSR125543A), a potent and selective corticotrophin-releasing factor(1) receptor antagonist. II. Characterization in rodent models of stress-related disorders.

The present study investigated the effects of the novel corticotrophin-releasing factor (CRF)(1) receptor antagonist 4-(2-chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thiazol-2-amine hydrochloride (SSR125543A) in a variety of rodent models of anxiety, including conflict procedures (punished drinking and four-plate), exploration models (elevated plus-maze and light/dark), a fear/anxiety defense test battery, and several procedures based on stress-induced changes in physiological (isolation-induced hyperthermia and tail pinch-induced cortical norepinephrine release) or behavioral (social defeat-induced anxiety, maternal separation-induced vocalization) parameters. Moreover, the effects of SSR125543A were investigated in acute (forced swimming) and chronic (chronic mild stress; CMS) models of depression. SSR125543A and the CRF(1) receptor antagonist antalarmin displayed limited efficacy in exploration-based anxiety models. In contrast, both compounds produced clear-cut anxiolytic-like activity in models involving inescapable stress, including the conflict procedures, the social defeat-induced anxiety paradigm and the defense test battery (3-30 mg/kg i.p. or p.o.). These effects paralleled those of the anxiolytic diazepam. In addition, SSR125543A and antalarmin antagonized stress-induced hyperthermia, distress vocalization, and cortical norepinephrine release. In the forced swimming test, 30 mg/kg p.o. SSR125543A and 3 to 30 mg/kg p.o. antalarmin produced clear antidepressant-like effects. These latter results were strengthened by the findings from the CMS, which showed that repeated administration of 10 mg/kg i.p. SSR125543A for 30 days improved the degradation of the physical state, the reduction of body weight gain, and anxiety produced by stress. Together, these data indicate that SSR125543A shows good activity in acute and chronic tests of unavoidable stress exposure, suggesting that it may have a potential in the treatment of depression and some forms of anxiety disorders.

Behavioral and magnetic resonance spectroscopic studies in the rat hyperserotonemic model of autism

Autism is classified as a pervasive developmental disorder, with several cardinal features including sensory disturbances, obsessive–compulsive-like behavior, lack of bonding to caregivers and motor disturbances. To date, there is a lack of an animal model of the disease. The current work is aimed at producing such a model by treating developing rat pups with a serotonergic agonist, 5-methoxytryptamine (5-MT; 1 mg/kg) during development (from gestational age 12 days to postnatal day 20), thus mimicking one of the hallmark neurochemical features of the illness—increases in the neurotransmitter, serotonin. Animals were then tested in behavioral paradigms that may resemble the human illness. Treated rat pups were found to be overreactive to auditory or tactile sensory stimuli, to display changes in the negative geotaxic test of motor development, to show lack of separation-induced vocalizations when their dam was removed and to show decreased alternation in the spontaneous alternation task. As well, the animals showed metabolic abnormalities in the brain using in vivo proton magnetic resonance spectroscopy, which are consistent with those observed in autistic children. In summary, the model we are proposing shows some of the behavioral and metabolic features of autism, as well as being produced through alteration of a neurochemical system known to be altered in autism.

Effects of the CRF 1 receptor antagonist, CP 154,526, in the separation-induced vocalization anxiolytic test in rat pups

CRF 1 receptor antagonists have been proposed as novel pharmacological treatments for depression, anxiety and stress disorders. The primary goal of the present study was to evaluate the effects of the CRF 1 receptor antagonist, CP 154,526, in the separation-induced vocalization (SIV) model of anxiety. Nine- to 11-day-old rat pups were separated from their litter and the effects of intraperitoneally administered test compounds on the elicited ultrasonic vocalizations were measured. Side-effect potential was assessed using a modifed inclined plane test (‘time on an inclined plane’, or TIP), and using negative geotaxis. SIV was reduced by CP 154,526 at doses that did not affect TIP or negative geotaxis, a profile like that of the 5-HT 1A partial agonist buspirone. The benzodiazepine anxiolytic, diazepam, decreased SIV but also produced significant side effects at one to three-fold higher doses. Additional pharmacological characterization of SIV demonstrated anxiolytic-like effects of the atypical antipsychotic, clozapine, but not the typical antipsychotic, haloperidol, and of the serotonin reuptake inhibitor, zimelidine, but not the norepinephrine reuptake inhibitor, desipramine. In summary, the data support the conclusion that selective CRF 1 receptor antagonists may have utility in anxiety and stress disorders. The data further support the use of separation-induced vocalizations for identifying mechanistically diverse compounds with anxiolytic actions in man.

Benzodiazepine receptor function in the chick social separation-stress procedure.

The role of benzodiazepine (BZ) receptors in modulating social separation-induced distress vocalizations (DVocs) and stress-induced analgesia (SIA) were examined in 8-day-old cockerels (Gallus gallus). In Experiment 1, the BZ agonist chlordiazepoxide (CDP; 5.0 mg/kg) reversed both DVocs and SIA in isolated chicks. Coadministration of the BZ antagonist flumazenil (0.01, 0.03, or 0.10 mg/kg) reversed CDP anxiolytic effects. In Experiment 2, the BZ agonists alprazolam (ALP; 0.065, 0.125, 0.25, or 1.50 mg/kg) and lorazepam (LOR; 0.125, 0.25, 0.50, or 1.0 mg/kg) dose dependently reversed social separation-induced DVocs and SIA. The ED50s for ALP and LOR in attenuating DVocs were 0.19 and 0.34 mg/kg, respectively. These data strongly support the theory that CDP anxiolytic effects are mediated by BZ receptor activity in the chick social separation procedure (Experiment 1) and that this model is sensitive to BZ agonists of different potencies (Experiment 2).

Pharmacological characterization of MDL 105,519, an NMDA receptor glycine site antagonist

MDL 105,519, ( E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid, is a potent and selective inhibitor of [ 3H]glycine binding to the NMDA receptor. MDL 105,519 inhibits NMDA ( N-methyl- d-aspartate)-dependent responses including elevations of [ 3H] N-[1,(2-thienyl)cyclohexyl]-piperidine ([ 3H]TCP) binding in brain membranes, cyclic GMP accumulation in brain slices, and alterations in cytosolic Ca 2+ and Na +-Ca 2+ currents in cultured neurons. Inhibition was non-competitive with respect to NMDA and could be nullified with d-serine. Intravenously administered MDL 105,519 prevented harmaline-stimulated increases in cerebellar cyclic GMP content, providing biochemical evidence of NMDA receptor antagonism in vivo. This antagonism was associated with anticonvulsant activity in genetically based, chemically induced, and electrically mediated seizure models. Anxiolytic activity was observed in the rat separation-induced vocalization model, but muscle-relaxant activity was apparent at lower doses. Higher doses impair rotorod performance, but were without effect on mesolimbic dopamine turnover or prepulse inhibition of the startle reflex. This pattern of activities differentiates this compound from (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cyclohepten-5,10-imine (MK-801) and indicates a lower psychotomimetic risk. © 1997 Elsevier Science B.V. All rights reserved.

Monoamine activity in anterior hypothalamus of guinea pig pups separated from their mothers.

Brief isolation in a novel environment increased the ratios of 3-methoxy-4-hydroxyphenylethylene glycol to norepinephrine (MHPG:NE) and dihydroxyphenylacetic acid to dopamine (DOPAC:DA) in the anterior hypothalamus of guinea pig pups. Ratios were significantly elevated after 90 min of isolation and for MHPG:NE, after 30 min of isolation; changes were due to increases in MHPG and DOPAC. Home cage isolation produced no change in any measure of catecholamine activity. No changes in levels of serotonin or its metabolite were observed. In 1 experiment, resting levels of NE and DOPAC:DA were predictive of the rate of separation-induced vocalization. Maternal separation in the context of novelty increases hypothalamic NE and DA activity; however, both isolation and novelty are required because neither maternal separation in the home cage nor exposure to a novel cage together with the mother had any discernible effect.

Tolerance and withdrawal to chronic morphine treatment in the week-old rat pup

Chronic treatment of adult animals with morphine results in tolerance but there are fewer reports on the effects of chronic opiates during ontogeny. The present experiments assessed the development of morphine-induced tolerance and withdrawal in infant rats. Pups were injected with morphine twice daily from ages 1–7 days and then tested on day 7 for morphine-induced analgesia in a hot-water immersion test, and separation-induced ultrasonic vocalizations in response to isolation from the dam and littermates at 7 and 10 days of age. Tolerance occurred to the analgesic effects of morphine but not to its suppression of ultrasonic vocalizations. Separation-induced vocalizations were greatly increased in chronic morphine-treated pups following naltrexone-precipitated withdrawal at 7 days of age. The increase in ultrasonic vocalizations following naltrexone treatment in morphine exposed pups may be a developmentally unique sign of opiate withdrawal.

NMDA receptor complex antagonists have potential anxiolytic effects as measured with separation-induced ultrasonic vocalizations

Pre-weaning rat pups emit ultrasonic vocalizations when removed from the litter. These ‘separation-induced vocalizations’ (SIV) are suppressed by classical benzodiazepine anxiolytics and by non-benzodiazepine anxiolytics which lack muscle relaxant and sedative propertes. The present study used the SIV model to assess potential anxiolytic properties of compounds which target different sites associated with the NMDA receptor complex. Comparison was made to drugs which affect benzodiazepine or serotonin (5-HT) receptors. Muscle relaxant potential was assessed using ‘TIP’ (time on an inclined plane), the amount of time a pup was able to retain its position on a steeply inclined surface. Mephenesin, a centrally acting muscle relaxant, significantly suppressed TIP but not SIV. The benzodiazepine agonist diazepam suppressed both SIV and TIP, whereas the 5-HT 1A partial agonists, buspirone and MDL 73,005EF, suppressed SIV without affecting TIP. The 5-HT 2 antagonist MDL 11,939 suppressed TIP but not SIV, whereas neither measure was affected by the 5-HT 3 antagonist MDL 73,147EF. SIV was suppressed by NMDA a antagonist including those acting at the glutamate recognition site (D,L-amino-phosphonovaleric acid (AP5) and MDL 100,453) or at the ion channel (MK-801), or by the strychnine-insensitive glycine antagonist 5,7-dichlorokynurenic acid (5,7-DCKA). TIP was suppressed even more potently by AP5, MDL 100,453 and MK-801, whereas 5,7-DCKA was inactive on this measure. Thus, antagonists acting at different sites present on the glutamate recognition site exhibit potential anxiolytic activity, but the glycine antagonist was unusual in its lack of prominent muscle relaxant side effects.

Nimodipine alters acquisition of a visual discrimination task in chicks

Chicks 5 days old received intraperitoneal injections of nimodipine 30 min before training on either a visual discrimination task (0, 0.5, 1.0, or 5.0 mg/kg) or a test of separation-induced distress vocalizations (0, 0.5, or 2.5 mg/kg). Chicks receiving 1.0 mg/kg nimodipine made significantly fewer visual discrimination errors than vehicle controls by trials 41-60, but did not differ from controls 24 h later. Chicks in the 5 mg/kg group made significantly more errors when compared to controls both during acquisition of the task and during retention. Nimodipine did not alter separation-induced distress vocalizations at any of the doses tested, suggesting that nimodipine's effects on learning cannot be attributed to a reduction in separation distress. These data indicate that nimodipine's facilitation of learning in young subjects is dose dependent, but nimodipine failed to enhance retention.

Opiate modulation of separation-induced distress in non-human primates

Infant rhesus monkeys respond to separation from their mothers with a dramatic increase in vocalizations and activation of autonomic and pituitary-adrenal systems. Using the mother-infant separation paradigm in rhesus monkeys, we focused on the role of opiate systems in modulating the behavioral and neuroendocrine consequences of a brief, naturally occurring stressor. In the first experiment, morphine 0.1 mg/kg significantly decreased separation-induced vocalizations without affecting activity levels. In the second experiment, naloxone 1.0 mg/kg increased distress vocalizations but lower doses had no effect. In the third experiment we blocked the effect of morphine 0.1 mg/kg with naloxone 0.1 mg/kg, a dose of naloxone that had no intrinsic effects of its own. This suggests that the reduction of separation-induced vocalizations by morphine is mediated by opiate receptors. The last experiment demonstrated that separation-induced increases in pituitary-adrenal hormones can also be modulated by opiate agonists and antagonists. These findings are consistent with work in non-primate species and support the hypothesis that opiate receptors are specifically involved in mediating separation-induced vocalizations and pituitary-adrenal activation in primates.