Jeffrey A. Vivian

Aggression, anxiety and vocalizations in animals: GABAA and 5-HT anxiolytics

A continuing challenge for preclinical research on anxiolytic drugs is to capture the affective dimension that characterizes anxiety and aggression, either in their adaptive forms or when they become of clinical concern. Experimental protocols for the preclinical study of anxiolytic drugs typically involve thesuppression of conditioned or unconditioned social and exploratory behavior (e.g., punished drinking or social interactions) and demonstrate the reversal of this behavioral suppression by drugs acting on the benzodiazepine-GABAA complex. Less frequently, aversive events engenderincreases in conditioned or unconditioned behavior that are reversed by anxiolytic drugs (e.g., fear-potentiated startle). More recently, putative anxiolytics which target 5-HT receptor subtypes produced effects in these traditional protocols that often are not systematic and robust. We propose ethological studies of vocal expressions in rodents and primates during social confrontations, separation from social companions, or exposure to aversive environmental events as promising sources of information on the affective features of behavior. This approach focusses on vocal and other display behavior with clear functional validity and homology. Drugs with anxiolytic effects that act on the benzodiazepine-GABAA receptor complex and on 5-HT1A receptors systematically and potently alter specific vocalizations in rodents and primates in a pharmacologically reversible manner; the specificity of these effects on vocalizations is evident due to the effectiveness of low doses that do not compromise other physiological and behavioral processes. Antagonists at the benzodiazepine receptor reverse the effects of full agonists on vocalizations, particularly when these occur in threatening, startling and distressing contexts. With the development of antagonists at 5-HT receptor subtypes, it can be anticipated that similar receptor-specificity can be established for the effects of 5-HT anxiolytics.

Ethology and Neuropharmacology: Rodent Ultrasounds

One of the most fascinating and instructive illustrations of how neural networks mediate complex communication comes from the neuroethological studies with zebra finches and canaries that sing to court mates and defend their territories (e.g., Nottebohm, 1985, 1981). The results from oscine songbirds inform importantly on the plasticity of the neural network, its seasonal androgen-dependent expression, and the behavioural significance in reproductive and agonistic contexts. However, less is known about the neurochemical characteristics of these communication-specific networks and how potential neural receptors in these networks may be targeted by substances with known effects of affective expression in mammals. Nonetheless these investigations point to multisynaptic networks involving aminergic and steroid-sensitive cells that mediate the initiation and production of important communicative signals.

Ultrasounds during morphine withdrawal in rats

Ultrasounds (US) in rats may communicate affective states, as they occur only in highly significant situations such as maternal care, sex and aggression. Withdrawal from morphine is a manipulation which dramatically alters autonomic, somatic and motor functions; the present experiment demonstrated the production of US in this context and the influence of previous social experience in their production. Sixty male Long-Evans rats with distinct social experiences (social inexperience, defeat or copulation) underwent 72 h of continuous morphine exposure (4 × 75 mg morphine or placebo pellets) and subsequent withdrawal. The rats were observed for 10 min in equally treated pairs and while solitary at 6, 24 and 96 h after pellet removal. US were emitted by all groups and consisted primarily of two distributions of pure tone whistles with little frequency modulation: 1–2 s 21–25 kHz (“low”) signals and the more prevalent 0.02–0.1 s 44–52 kHz (“high”) signals. Morphine withdrawn rats lost weight, displayed wet dog shakes, were hypoactive and emitted threefold more US vocalizations with a fourfold greater duration than placebo controls. Defeat-experienced morphine withdrawn rats were more hypoactive than either socially inexperienced or copulatory experienced rats while increasing vocalization rates and total duration. This increased duration of ultrasounds included a shift in the distribution of individual US durations from less than 0.3 s to greater than 1.0 s. US are readily emitted at high rates in morphine withdrawn laboratory rats, which may implicate an opioid involvement in their generation. Furthermore, relevant social experiences such as copulation and defeat facilitate the emission of US during morphine withdrawal and may serve as an index of the affective components of withdrawal.

Diazepam and gepirone selectively attenuate either 20–32 or 32–64 kHz ultrasonic vocalizations during aggressive encounters

Ultrasonic vocalizations (USV) in rats may communicate “affective” states, as they occur only in highly significant behavioral contexts such as during sex, aggression, exposure to painful or startling events. This proposal was evaluated in an experiment with adult male Long-Evans rats during agonistic encounters; specifically, the effects of diazepam, flumazenil and gepirone were studied on different types of USV emitted by intruder rats exposed to resident attacks and to “threat of attacks” (i.e., intruder protected within the home cage of the resident by a wire mesh cage). USV were readily emitted during agonistic encounters and consisted primarily of two distributions of pure tone whistles: 0.3- to 3-s, 20- to 32-kHz (“low”) signals and 0.02- to 0.3-s, 32- to 64-kHz (“high”) signals. A considerable repertoire of frequency modulated signals was observed and proved to be sensitive to the anxiolytic treatments. Diazepam (1–6 mg/kg) dose-dependently decreased high frequency USV during the threat of attack and decreased the mean pitch of the most predominant vocalizations but did not affect low frequency USV or the audible squeals (AS) in response to bites. Gepirone (0.3–6 mg/kg) dose-dependently decreased low frequency USV and did not affect high frequency USV or AS. Responses to thermal pain stimuli remained unaltered by all drugs, while walking duration was decreased and crouch postures were increased after diazepam but not after gepirone administration. Gepirone in the present dose range had minimal effects on submissive, exploratory and locomotor behaviors. The pattern of results is consistent with the proposal that low frequency USV reflect a heightened affective state which is ameliorated with 5HT1A but not benzodiazepine anxiolytics, and suggests that the suppression of high frequency USV in reaction to attacks or threats coincides with the sedative or muscle relaxant properties of these compounds.

Diazepam withdrawal: effects of diazepam and gepirone on acoustic startle-induced 22 kHz ultrasonic vocalizations.

It has proven difficult to demonstrate and study the “anxiogenic” quality of drug withdrawal states in animals. Ultrasonic vocalizations (USV) in response to acoustic startle stimuli have shown promise as a measure of affect and may represent “distress” responses during diazepam withdrawal. Three experiments evaluated the association between USV and “distress” by comparing the effects of diazepam as a prototypic benzodiazepine agonist and the putative anxiolytic gepirone with affinity for 5-hydroxytryptamine (5-HT1A) receptors in naive and diazepam-withdrawn subjects. Adult male Long-Evans rats were exposed to acoustic startle sessions consisting of nine 105 dB and nine 115 dB stimuli. USV at 20–30 kHz were readily emitted during startle and often commenced after the third or fourth stimulus presentation. Acutely, intraperitoneal (IP) administration of diazepam (0.1–3 mg/kg) and gepirone (0.1–1 mg/kg) decreased USV dose-dependently without affecting the startle reflex; gepirone also decreased tail flick latency. Startle-induced USV were also sensitive to the “anxiogenic” effects of withdrawal from diazepam exposure (0, 2.5, 5, 10 mg/kg b.i.d. IP×5 days). Twenty-four hours after the last diazepam injection, rats were hyperreactive to startle stimuli and doubled their rate of USV over vehicle-treated controls. Gepirone (0.1–1 mg/kg IP), but not diazepam (3–20 mg/kg IP) antagonized the increased rate of USV in rats withdrawn from 10 mg/kg b.i.d. diazepam. Diazepam (2.5–10 mg/kg IP) antagonized the increased rate of USV in rats withdrawn from 2.5 mg/kg b.i.d. diazepam. USV induced by acoustic startle stimuli are sensitive to the anxiolytic effects of benzodiazepine and 5-HT1A receptor agonists and permit the assessment of the “anxiogenic” properties of diazepam withdrawal. The potent effect of gepirone on USV suggests a serotonergic amelioration of the “anxiogenic” aspects of diazepam withdrawal.

Morphine attenuates ultrasonic vocalization during agonistic encounters in adult male rats

Ultrasonic vocalizations (USV) in rats may communicate “affective” states during pain, sex and aggression. This proposal was evaluated in an experiment with adult male Long-Evans rats during agonistic encounters; specifically, morphine and naltrexone effects were studied on different types of USV by intruder rats exposed to resident attacks and to “threat of attacks” (i.e., intruder residing within the home cage of the resident but prevented from physical contact by a wire mesh cage). Intruders readily emitted USV during agonistic encounters. These calls consisted primarily of two distinct distributions of pure tone whistles: 0.3–3 s, 19–32 kHz (“low”) calls and 0.02–0.3 s, 32–64 kHz (“high”) calls. Sonographic analysis revealed a considerable repertoire of frequency modulated calls. Different types of vocalizations proved to be differentially sensitive to the opiate treatments: morphine (1–10 mg/kg SC) dose-dependently decreased the rate, duration and pitch of both low and high frequency USV during the threat of attack; this decrease in rate and duration measures was naltrexone-reversible (0.1 mg/kg IP). Interestingly, audible vocalizations were also emitted but were unaffected by morphine in this dose range. Concomitant with the decrease in USV after morphine was a dose-dependent decrease in rearing, walking and nasal contact behavior with increases in submissive crouch behavior and tail flick analgesia. The decreases in rate and duration of both low and high USV and the pitch of specific frequency modulated calls after morphine administration may reflect an attenuation of affective aspects of pain, and the many characteristics of US (rate, duration, pitch, frequency modulation, pre-and suffix attributes and temporal structure) point to potentially diverse functions. Morphines pervasive effects on ultrasonic but not audible vocalizations, in addition to reflexive and submissive responses, provides evidence for opioid influences on “affective” as well as somatomotor responses to socially aversive situations.

Automatic quantification of withdrawal from 5-day diazepam in rats: ultrasonic distress vocalizations and hyperreflexia to acoustic startle stimuli

The purpose of the present work was to develop an objective and precise method to quantify withdrawal responses from anxiolytics relying on ethologically valid responses. Behavioral effects of diazepam withdrawal in rats are automatically measured that appear to correspond to clinically relevant disturbances in affective and sensory-motor functions. Ultrasonic vocalizations and startle reflexes in response to acoustic stimuli were measured as indices of withdrawal 24 h after 5 days of 2.5, 5 or 10 mg/kg diazepam, b.i.d., IP in male Long-Evans rats. About 60% of male rats emit 22–26 kHz ultrasonic calls when exposed to acoustic startle stimuli (18 presentations, 9 at 105 dB and 9 at 115 dB, each 30 s apart on average). Diazepam-withdrawn rats exhibited startle responses with larger maximal and average amplitude and emitted more frequent 22–26 kHz ultrasonic vocalizations than vehicle-treated control animals. The magnitude of the withdrawal changes in ultrasonic calls and in startle reflex amplitude increased significantly already at the low 2.5 mg/kg diazepam dose in spite of considerable individual variability. The increased ultrasound rates during diazepam withdrawal contrast with the suppressive effects of acutely administered diazepam in drug-naive rats. The current methodology offers the opportunity to more adequately characterize withdrawal from anxiolytic substances in a quantitative, objective and automated manner.

The muscarinic M1 receptor positive allosteric modulator PQCA improves cognitive measures in rat, cynomolgus macaque, and rhesus macaque

RationaleThe current standards of care for Alzheimer’s disease, acetylcholinesterase inhibitors, have limited efficacy due to a host of mechanism-related side effects arising from indiscriminate activation of muscarinic and nicotinic receptors. The M1 muscarinic receptor is predominantly expressed in the brain in regions involved in cognition, and therefore selective activation of the M1 receptor would be expected to boost cognitive performance with reduced risk of peripheral side effects.ObjectivesHere we investigated whether the selective M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance and cerebral blood flow.ResultsPQCA attenuated a scopolamine-induced deficit in novel object recognition in rat, self-ordered spatial search in cynomolgus macaque, and the object retrieval detour task in rhesus macaque. Beneficial effects in each of these assays and species were observed at similar plasma drug concentrations. Furthermore, at similar drug concentrations that were effective in the behavioral studies, PQCA increased blood flow in the frontal cortex of mice, providing a translational biomarker that could be used to guide dose selection for clinical studies.ConclusionsThese findings provide a framework for appropriately testing an M1 selective compound in patients with Alzheimer’s disease.

Effects of μ and δ opioid agonists and antagonists on affective vocal and reflexive pain responses during social stress in rats

Abstract The present experiments evaluated the influence of intraventricular μ and δ opioid receptors on affective vocal and reflexive responses to aversive stimuli in socially inexperienced, as well as defensive and submissive responses in defeated, adult male Long-Evans rats. Defeat stress consisted of: (1) an aggressive confrontation in which the experimental intruder rat exhibited escape, defensive and submissive behaviors [i.e., upright, supine postures and ultrasonic vocalizations (USV)], and subsequently, (2) protection from the resident stimulus rat with a wire mesh screen for 10–20 min. Defeat stress was immediately followed by an experimental session with tactile startle (20 psi). The μ opioid receptor agonists morphine (0.1–0.6 μg ICV) and [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO; 0.01–0.3 μg ICV), and the δ opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE; 10–100 μg ICV) dose-dependently decreased startle-induced USV and increased tail-flick latencies in socially inexperienced and defeated rats. Of greater interest, morphine, DAMGO and DPDPE increased the occurrence of the submissive crouch posture, and defeated rats were more sensitive than socially inexperienced rats to the startle-induced USV-suppressive and antinociceptive effects of morphine and DPDPE. The antinociceptive effects of DAMGO were likewise obtained at lower doses in defeated rats. Finally, the USV-suppressive effects of morphine and DAMGO were reversed with the μ receptor antagonist naltrexone (0.1 mg/kg IP), but the USV-suppressive effects produced by DPDPE were not reversed with the δ receptor antagonist naltrindole (1 mg/kg IP). These results confirm μ, but not δ opioid receptor activation as significant in affective vocal, passive-submissive behavior, as well as reflexive antinociception. Furthermore, similar to previous studies with restraint and electric shock stress, the facilitation of μ opioid effects on vocal responses and antinociception is consistent with the proposal that defeat stress activated endogenous opioid mechanisms.

Interactions between social stress and morphine in the periaqueductal gray : effects on affective vocal and reflexive pain responses in rats

Abstract Rationale: Endogenous opioid systems within the mesencephalic periaqueductal gray matter (PAG) appear to be intricately involved in many affective, defensive, submissive, and reflexive responses, and these systems are activated by aversive stimuli. Objectives: The present experiments evaluated the influence of opioid receptors within the PAG on affective vocal and reflexive responses to aversive stimuli in socially inexperienced, as well as defensive and submissive responses in defeated, adult male Long-Evans rats. Methods: Defeat stress consisted of: (1) an aggressive confrontation with a ”resident” stimulus rat in which the experimental ”intruder” rat exhibited escape, defensive and submissive behaviors [i.e. upright, supine postures and ultrasonic vocalizations (USV)], and subsequently, (2) protection from the resident rat with a wire mesh screen for ca. 25 min. Defeat stress was immediately followed by an experimental session with thermal antinociceptive and tactile startle stimuli (20 psi airpuffs). Results: The µ opioid receptor agonist morphine (0.3, 1, 3 µg IC) attenuated startle-induced USV and the tail-flick reflex in socially inexperienced and defeated rats, with both groups of rats demonstrating equal sensitivity to morphine. Morphine decreased defeat-induced USV and increased the display of the crouch posture in defeated rats; these morphine effects in socially inexperienced and defeated rats were re- versed with the opioid receptor antagonist naltrexone (0.1 mg/kg IP). Conclusions: These results reveal that the ventrolateral PAG is an important site in which µ opioid receptor agonists such as morphine mediate affective vocal and submissive responses, yet this structure is not critical in the display of defeat stress-augmented effects of morphine. Endogenous opioid mechanisms appear to participate in the organization of defensive behavior, namely, to facilitate a shift from active to passive forms of coping.