Using rat ultrasonic vocalization to study the neurobiology of emotion: from basic science to the development of novel therapeutics for affective disorders.

The use of ultrasonic vocalizations as an experimental tool for studying emotional states in rodents has led to an increased understanding of the basic science of affect as well as the development of novel diagnostics and therapeutics for the treatment of affective disorders. At the behavioral level, the rules that govern the generation of affective 'feeling' states are similar to those of the psychophysics of sensory perception. Emotions are elicited primarily in response to active social stimuli. A linear increase in affective response requires a logarithmic increase in stimulation and habituation of a given affective response allows for transition across the cycle of emotional/affective states (approach→consummatory phase→avoidance). At the neuronal level, the coordinated expression of affective responses in the medial prefrontal cortex is orchestrated by rhythmic activity, which is initiated and maintained by a variety of short-term and long-term synaptic plasticity processes. An objective measure of affective states may emerge from these psychophysical and neuronal properties of emotion. Enhancing synaptic plasticity with pharmacological agents that modulate NMDA receptor activity as well as IGFI receptor activity may have therapeutic potential for the treatment of affective disorders.

Positive emotional learning is regulated in the medial prefrontal cortex by GluN2B-containing NMDA receptors.

In rats, hedonic ultrasonic vocalizations (USVs) is a validated model of positive affect and is best elicited by rough-and-tumble play. Here we report that modulation of GluN2B-containing NMDA receptors (NMDAR) in the medial prefrontal cortex (MPFC) is involved in positive emotional learning. Rough and tumble play increased both GluN1 and GluN2B NMDAR subunit mRNA and protein levels in the frontal cortex. GLYX-13, a GluN2B-preferring, NMDAR glycine-site partial agonist (1 mg/kg, i.v.) significantly increased positive emotional learning whereas the GluN2B receptor-specific antagonist, ifenprodil (10 mg/kg, i.p.), inhibited positive emotional learning. Animals selectively bred for low rates of hedonic USVs were returned to wild-type levels of positive emotional learning following GLYX-13 treatment. MPFC microinjections of GLYX-13 (0.1-10 µg/side) significantly increased rates of positive emotional learning. Thus GluN2B-containing NMDARs may be involved in positive emotional learning in the MPFC by similar mechanisms as spatial/temporal learning in the hippocampus.

Heightened locomotor-activating effects of amphetamine administered into the nucleus accumbens in adolescent rats.

There is a shift in sensitivity to systemically administered psychostimulants in adolescence, as evidenced by less amphetamine-induced locomotor activity in adolescent compared to adult rodents. Locomotor activating effects of amphetamine are dependent on drug actions in the core of the nucleus accumbens (NAc), but the contribution of this region to age differences in amphetamine sensitivity has not been studied directly. In the present study, we investigated the development of the NAc using targeted injections of amphetamine (0, 3, or 6 µg/side) directly into the NAc core in early (postnatal day 30; P30) or late (P45) adolescence, or in adulthood (P75). Locomotor activity was recorded during two 1h sessions, 48 h apart. Amphetamine increased locomotor activity at all ages. P45 rats were more active than adults only at the 3 µg/side dose, but this difference was not significant when baseline activity was taken into account. In contrast, P30 rats were more active than adults at the 6 µg/side dose, indicating that the magnitude of the locomotor response is highest in early adolescence. Results of the present study are the first to directly show a developmental difference in the sensitivity of the NAc to amphetamine under conditions in which the influence of pharmacokinetic factors and regulatory brain regions is minimized.

Rats selectively bred for low levels of 50 kHz ultrasonic vocalizations exhibit alterations in early social motivation.

In rats, the rates of 50 kHz ultrasonic vocalizations (USVs) can be used as a selective breeding phenotype and variations in this phenotype can be an indicator of affective states. The 50 kHz USV is elicited by rewarding stimuli (e.g., food, sexual behavior) and therefore can express a positive affective state. Conversely, the 22 kHz USV is elicited by aversive stimuli (e.g., presence of a predator, social defeat) indicating a negative affective state. In the present study, we tested the effect of selectively breeding for 50 kHz USVs on a variety of maternal social/emotional behaviors in young rat pups (PND 10-12). These measures consisted of an assessment of isolation calls and conditioned odor preference paradigm. Results indicate that animals selected for low levels of 50 kHz USVs show the greatest alterations in social behaviors compared to the control animals. The low line animals had an increase in isolation calls tested during place preference conditioning and a decrease in 50 kHz ultrasonic calls in all conditions. These same low line animals failed to show a typical preference for a maternally-associated odor during the place preference test. The different social behaviors of the high line animals did not consistently vary from those of the control group. These results have important implications for the study of genetic and epigenetic mechanisms underlying emotional states, and possibly contribute to the research underlying the emotional changes in developmental disorders such as autistic spectrum disorder by providing a novel animal model that displays communication deficits that are interdependent with significant social behavioral impairments.

The related roles of dopamine and glutamate in the initiation of 50-kHz ultrasonic calls in adult rats.

Effects of amphetamine on the production of 50-kHz ultrasonic calls were studied. Calls were emitted spontaneously or were induced by an intrahypothalamic-preoptic injection of glutamate. Sonographic analysis of recorded calls revealed that they were within the 35-70-kHz sound frequency range reported for the 50-kHz call type. Systemic amphetamine (AMPH, 2 mg/kg) significantly increased the number of spontaneously emitted 50-kHz calls and the effect of AMPH was dose-dependent. Low dose of intracerebral glutamate (17 microg) had no additive effect on the number of AMPH-induced calls. Higher dose of intracerebral glutamate alone (34 microg) significantly increased the number of 50-kHz calls, which was completely reversed by systemic application of haloperidol (2 mg/kg), a dopamine antagonist. The results suggest that glutamate-induced or spontaneously occurring 50-kHz calls in adult rats are dependent upon dopaminergic transmission. It is postulated that this type of calls may be indicative of dopamine mediated affective state in adult rats.

The effect of cholinergic stimulation on rat pup ultrasonic vocalizations.

As cholinergic stimulation increases vocalizations in adult rats, the present study investigated the effects of systemic oxotremorine, a cholinergic agonist, on the production of separation calls in rat pups of different ages and whether these effects are in response to central versus peripheral stimulation. The first experiment examined the dose-response effects of oxotremorine on the number of vocalizations and acoustic parameters of 10-, 15-, and 17-day-old rat pups. In contrast to other studies on adult rats, pup vocalizations were decreased while marginally changing acoustic parameters. The second experiment, using muscarinic antagonists, showed that pretreatment with atropine sulfate, which can cross the blood-brain barrier (BBB), reversed the call-reducing effect of oxotremorine whereas pretreatment with atropine methyl nitrate, which does not cross BBB, did not. Suppression of vocalizations by oxotremorine may be explained by central activation and not the peripheral effects of the drug. Dissimilar effects of cholinergic stimulation of infant and adult rat brains may be attributed to a differential role of the cholinergic system during development and maturity.

Pharmacological and behavioral characteristics of 22 kHz alarm calls in rats.

The present review is focused on the neural mechanisms and acoustic features of 22kHz alarm calls emitted by adult rats as a defensive measure in numerous behavioral situations. The alarm calls are initiated by activity of the cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and a subsequent release of acetylcholine at the target areas, collectively termed as the medial cholinoceptive vocalization strip. Injection of carbachol, a predominantly muscarinic agent, into any portion of the cholinoceptive strip, or direct stimulation of the LDT, induced species-typical 22kHz calls comparable to those emitted in natural situations. The pharmacologically induced 22kHz calls contained their alarming properties for naïve rats. The 22kHz calls induced either by carbachol or by stimulation of the LDT could be antagonized by atropine, or scopolamine applied into the cholinoceptive strip. Our recent behavioral studies have shown that the combination of long call duration and constant sound frequency (20-30kHz) convey the alarming message. Anatomical and neurochemical organization of the vocalization strip and acoustic properties of the calls lead to the conclusion that 22kHz calls indicate a fundamental, negative affective state common for many behavioral situations.

Sonographic structure of isolation-induced ultrasonic calls of rat pups.

Sonographic analysis of isolation-induced calls of 10- to 17-day-old Sprague-Dawley rat pups showed that average acoustic parameters of calls changed with pups' age. Average call duration increased with age from 80 ms to over 140 ms in 15-day-old pups. The peak frequency increased from approximately 50 kHz to an average of 64 kHz in 17-day-old pups, and the average bandwidth increased from 34 to 45 kHz in 17-day-old pups. Analysis of the sonographic structure of pup calls additionally revealed a tendency to produce two or more alternating sweeps of sound frequency in each call. Development of sweeps is the most typical feature of pup calls with a dominant call type resembling "U" or inverted "U" shape in the sonogram. Number of "U" or inverted "U" call types significantly increased with pups' age. It is concluded that pups developed and strengthened those acoustic features of distress calls which play a role in intraspecific communication and maximize pup survival.

A role of subicular and hippocampal afterdischarges in initiation of locomotor activity in rats.

The possible role of a hippocampal afterdischarge (AD) episode in eliciting locomotor movements was evaluated in freely moving rats. Electrical stimulation of either the ventral subiculum (VSB) or the hippocampal CA1 region evoked an AD of 6-50 s in duration, which was followed by an increase in locomotor activity. Similar results were also observed after unilateral injection of N-methyl-d-aspartic acid (NMDA, 0.25 microg or 1 microg), a glutamate receptor agonist, into the VSB. Locomotor activity was not observed when either electrical or chemical stimulation of the VSB, or electrical stimulation of the CA1 region did not elicit an AD. In addition, the duration of the AD was positively correlated with the number of locomotor movements induced by stimulation of VSB or CA1 region. It is suggested that the hippocampal/subicular AD may be a necessary condition to induce locomotor activity by either chemical or electrical stimulation of the hippocampus in rats.

Mesolimbic component of the ascending cholinergic pathways: electrophysiological-pharmacological study.

The cholinergic input from the pontomesencephalic cholinergic neurons to the diencephalic and basal forebrain structures has been implicated in a number of limbically controlled overt behaviors. The cellular mechanism by which the cholinergic terminals initiate behavioral manifestations is not clear. The objective of this study was to investigate the effects of the ascending cholinergic projection from the laterodorsal tegmental nucleus (LDT) on neuronal firing in the anterior hypothalamic-medial preoptic region (AHMP), known to be involved in agonistic behavior. Experiments were performed on urethan-anesthetized rats. Iontophoretic application of carbachol (CCh) into the vicinity of single cells in the AHMP caused a dose-dependent decrease in the mean firing rate of 83% of units and an increase in 10% of units. The inhibitory effect of CCh, but not the excitatory effect, was reversed by iontophoretic pretreatment with scopolamine. The inhibition of the firing rate was repeatable for the same dose of CCh and dose dependent. Electrical stimulation of neurons in the LDT caused a comparable, current-dependent decrease in the mean firing rate of AHMP neurons that also was reversed by pretreatment of neurons in the AHMP with scopolamine. The antagonizing effects of scopolamine were reversible with time. The same units in the AHMP that inhibited their firing to stimulation of the LDT also responded with a similar inhibition to local iontophoretic CCh. Finally, the fluorescent carbocyanine dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide, (DiA), has been used as a retrograde axonal tracer and was injected into the recording sites immediately after the electrophysiological recordings. After 1 wk, DiA dye was found in numerous neurons in the LDT as shown by the fluorescence confocal microscopy. Results of the study suggest that LDT cholinergic neurons project and terminate in the AHMP and that their activation causes a decrease in the mean firing rate of the AHMP neurons. It is postulated that this inhibitory effect is implicated in the initiation of some of the behavioral patterns like defensive or alarm vocalization and behavioral inhibition.

Analysis of locomotor activity in the rat: parallelism index, a new measure of locomotor exploratory pattern.

Several measures of locomotor activity in rats, including the distance covered, movement time, speed of progression, and sinuosity showed significant changes in extreme situations after prolonged preadaptation or after stimulation with I.P. amphetamine. The same measures, however, either did not change or poorly reflected the changes in locomotor activity consistent with progressive habituation in successive daily or weekly recordings. Other movement parameters, such as the average angle of turns, did not change or changed marginally, even in extreme situations. A new locomotor parameter, the parallelism index, has been proposed. It reflects the overall tendency to turn and the angular magnitude of turns. The parallelism index is not directly dependent on the distance covered by the animal, and seems to significantly reflect subtle changes in the pattern of locomotor activity that is characteristic of the exploration of an unfamiliar environment compared to the locomotor movement in frequently visited areas. A significant decrease of the parallelism index with time or repeated exposure to the cage indicates that rats perform more turns, and/or more turns under larger angles, in a more familiar or a more explored environment. It is postulated that the parallelism index decreases with the familiarity of the area being explored.

Release of dopamine in the nucleus accumbens caused by stimulation of the subiculum in freely moving rats.

Stimulation of the ventral subiculum of the hippocampus activates the hippocampal-accumbens pathway and increases locomotor activity. Dopaminergic terminals in the nucleus accumbens have also been implicated in initiation of locomotor activity, and the release of dopamine in the nucleus accumbens is critical for locomotor responses initiated from the subiculum to occur. We have demonstrated release of dopamine in the nucleus accumbens using in vivo microdialysis after stimulation of the ventral subiculum with NMDA. Extracellular dopamine level in the nucleus accumbens was significantly increased by 40% over baseline as a result of NMDA stimulation of the ventral subiculum. This stimulation also caused more than a 40-fold increase in horizontal activity and total distance covered by the rats. Injection of saline into the subiculum caused neither a change in the dopamine level nor an increase in animal's activity. The dynamics of the measured changes in dopamine overflow correlated with the time course of locomotor changes. The results demonstrate that stimulation of the ventral subiculum causes release of dopamine in the nucleus accumbens which parallels the increase in locomotor activity.

C-Fos immunohistochemical localization of neurons in the mesencephalic locomotor region in the rat brain.

The projection from the limbic system via the subpallidal region to the mesencephalic locomotor region is implicated in limbic-motor integration. The goal of this study was to visualize neurons of the mesencephalic locomotor region which are active during locomotor activity induced by the disinhibition of the subpallidal region. The subpallidal region was disinhibited by picrotoxin, which antagonizes the effects of GABA. The unilateral injection of picrotoxin into the subpallidal region caused a significant increase in locomotor activity. Active tegmental neurons were subsequently visualized by immunocytochemical staining of c-Fos protein. There were significantly more immunostained neurons in the picrotoxin-injected animals than in the saline-treated rats. Heavily stained neuronal nuclei, prevailing on the brain side ipsilateral to the injection of picrotoxin, were localized within a narrow strip of tissue which stretched from the ventrolateral periaqueductal gray (including the dorsal raphe), the cuneiform nucleus, through the region of the dorsal tegmental bundle to the pedunculopontine nucleus. There were 3.5 times more immunostained neurons in the cuneiform/pedunculopontine region and 2.5 times more stained neurons in the periaqueductal region of the picrotoxin-injected rats, as compared to the saline group. This strip of immunostained cells represents neurons which are involved in the initiation and maintenance of locomotor activity due to subpallidal activation (predominantly pedunculopontine and cuneiform nuclei), as well as neurons possibly involved in the inhibition of locomotor activity (ventrolateral periaqueductal gray) and other feedback regulations. This study will help identify the neuronal pool involved in coupling the motivational commands with the locomotor system for execution of behaviour.

Subpallidal outputs to the nucleus accumbens and the ventral tegmental area: anatomical and electrophysiological studies.

The goal of this study was to investigate the functional organization of the subpallidal-->accumbens direct and indirect feedback loops by both anatomical and electrophysiological methods. The results of the dextran-conjugated rhodamine injections into the subpallidal area has shown three distinct projections: (1) a substantial pathway from the subpallidal area to the ventral tegmental area, (2) a more diffuse rostral projection from the subpallidal area to the core area of the nucleus accumbens, and (3) a sparse pathway projecting rostrodorsally from the subpallidal area toward the thalamic regions. Electrical or chemical stimulation of the subpallidal region, which was studied by the axonal tracer, evoked inhibitory responses in the majority (60 and 80%, respectively) of the accumbens and ventral tegmental area neurons in a standard extracellular recording study. Less than 1/3 of the accumbens or ventral tegmental area cells showed an increase in the mean firing rate. The majority (77.5%) of all responded neurons had a latency of less than 10 ms. Furthermore, injection of glutamate into the subpallidal area not only altered the firing pattern of the accumbens neurons, but also attenuated their excitatory responses elicited by the electrical stimulation of the ventral subiculum. Our results indicate that the subpallidal area plays a predominantly inhibitory role in the ventral tegmental area-accumbens-subpallidal circuitry, presumably by its GABAergic projections, and may also modulate subicular input into the nucleus accumbens.

Involvement of the nucleus accumbens-ventral pallidal pathway in postictal behavior induced by a hippocampal afterdischarge in rats.

The hypothesis that postictal motor behaviors induced by a hippocampal afterdischarge (AD) are mediated by a pathway through the nucleus accumbens (NAC) and ventral pallidum (VP) was evaluated in freely moving rats. Tetanic stimulation of the hippocampal CA1 evoked an AD of 15-30 s and an increase in number of wet-dog shakes, face washes, rearings and locomotor activity. Bilateral injection of haloperidol (5 micrograms/side) or the selective dopamine D2 receptor antagonist, (+/-)-sulpiride (200 ng/side) before the hippocampal AD, into the NAC selectively reduced rearings and locomotor activity, but not the number of wet-dog shakes and face washes. Injection of R(+)-SCH-23390 (1 microgram/side), a D1 receptor antagonist, or rimcazole (0.4 mg/side), a sigma opioid receptor antagonist, into the NAC did not significantly alter postictal behaviors. Bilateral injection of muscimol (1 ng/side), a gamma-aminobutyric acid (GABAA) receptor agonist, into the VP before the AD significantly blocked all postictal behaviors. It is concluded that postictal locomotor activity induced by a hippocampal AD is mediated by activation of dopamine D2 receptors in the NAC and a pathway through the VP.

Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat.

It has been well documented that cholinergic stimulation of the mediobasal forebrain structures induces 20-30 kHz ultrasonic vocalization in adult rats. If the cholinergic system plays a triggering role for ultrasonic vocalization, the question arises as to where the source of the cholinergic fibres, which innervate the mediobasal forebrain and induce vocalization, is located. In the present study, the role of the ascending cholinergic projection from the ponto-mesencephalic cholinergic nuclei to the mediobasal hypothalamic-preoptic region in production of 22 kHz calls was investigated. Cholinergic neurons were stimulated by local injection of L-glutamate and eventual vocalization was recorded by a S200 bat detector and analyzed sonographically. Intracerebral injection of L-glutamate into the laterodorsal tegmental nucleus induced short latency, 20-30 kHz ultrasonic calls. Sound frequency (pitch) and single call duration of the L-glutamate-induced vocalization did not differ from those obtained by cholinergic stimulation of the mediobasal hypothalamic-preoptic region with carbachol. However, L-glutamate stimulation of the laterodorsal tegmental nucleus was ineffective or less effective in 70% of responses, when the terminal fields in the mediobasal hypothalamic-preoptic region were pretreated with scopolamine, a muscarinic antagonist. The results demonstrate that the ascending cholinergic projection from the laterodorsal tegmental nucleus plays a triggering role for 20-20 kHz vocalization in adult rats.

Mesolimbic dopamine terminals and locomotor activity induced from the subiculum.

The role of the mesolimbic dopamine terminals in the nucleus accumbens in the initiation of locomotion in rats was studied. Locomotor activity was initiated by activation of the excitatory input from the ventral subiculum to the nucleus accumbens with NMDA. Measurements of locomotor activity, induced by unilateral administration of NMDA into the ventral subiculum, were compared before and after destruction of the mesolimbic dopamine terminals in the nucleus accumbens. The dopamine terminals were destroyed by injection of 6-OHDA into the ventral tegmental area which projects to the nucleus accumbens. Injection of NMDA into the ventral subiculum caused an almost four-fold increase in locomotor activity. However, this increase was abolished after the destruction of the mesolimbic dopamine terminals in the nucleus accumbens. The results suggest that the mesolimbic dopamine terminals are essential in transmitting subicular signals to the output neurones within the nucleus accumbens.

Behavioural responses of laboratory rats to playback of 22 kHz ultrasonic calls.

It has been demonstrated that cholinergic stimulation of the anterior hypothalamic-preoptic region induces 22 kHz ultrasonic vocalization in rats. Acoustic features of the cholinergically induced vocalization did not differ from those of 22 kHz calls emitted in natural situations and, therefore, could have a behavioural significance for other conspecifics. The 22 kHz calls induced by intracerebral injection of carbachol were played back to rats and their responses were compared with responses to playback of 22 kHz calls induced by tactile stimuli and to those with background noise. Animal responses were measured by an accelerometric sensor as an average ergometric activity. The average activity count was not changed during presentation of acoustic stimuli, however, striking differences were found in animal responses immediately after discontinuation of the sound. Activity of the rats consistently and significantly decreased after presentation of 22 kHz calls induced by tactile stimuli or by injection of carbachol. Animal responses to calls induced by carbachol were indistinguishable from responses to calls induced by tactile stimuli. No significant changes in the general activity of the animals were observed after presentation of the background noise or during the sessions without stimuli. The results demonstrate that carbachol-induced ultrasonic calls have behavioural significance for other conspecifics and could serve as an alarm call in a similar way to naturally produced 22 kHz vocalization.

Cholinergic mechanisms in generalized seizures: importance of the zona incerta.

OBJECTIVE: Stimulation of the central cholinergic system results in generalized epileptic seizures. The goal of this study was to map the epileptogenic effects of the cholinergic agonist, carbachol injected into different sites of the basal forebrain and diencephalon of the rat brain. METHODS: Carbachol was injected directly into the brain in a dose of 1 or 3 micrograms. Seizures were assessed behaviourally on a five-stage scale with electroencephalographic controls. Seizures at stage 1 were the least severe and those at stage 5 the most severe. RESULTS: Injections of high dose carbachol (3 micrograms) induced seizures from 40% of all injected brain sites. Injections of low dose carbachol (1 microgram) or isotonic saline into the same brain sites did not cause any behavioural or electrographic seizures. The majority of sites (84%) producing generalized seizures (stage 5) were concentrated in or around the zona incerta. CONCLUSIONS: Within the anatomical limits of the study, the zona incerta is the area most sensitive to carbachol-induced generalized seizures.

Regional specificity of the emotional-aversive response induced by carbachol in the cat brain: a quantitative mapping study.

In this paper, the emotional-aversive response induced by intracerebral injection of carbachol has been studied in cats by recording their vocalization as an index of emotional behavior. The carbachol-induced responses were quantitatively mapped in the basal forebrain and diencephalic regions using the cumulative time of the animal's vocalization as a measure of response. The areas inducing the emotional- aversive response extended along two axes: 1. longitudinally along the neuraxis, from the reticular formation through the hypothalamus to the rostro-basal forebrain; and 2. vertically along the fornix, from the mediobasal hypothalamus to the septal area. The highest magnitude of the response (vocalization time) was obtained from a strip of tissue extending from the septum and preoptic area to the dorsal perifornical area, the dorsomedial hypothalamic nucleus, the paraventricular nucleus and the periventricular stratum. The pattern of distribution of reactive and nonreactive sites showed anatomic specificity, with the highest sensitivity found within the close periventricular tissue of the third ventricle. It is suggested that carbachol mapping selectively delineates the muscarinic cholinoceptive portion of the aversive, emotional brain system.