From emotional arousal to executive action. Role of the prefrontal cortex.

Emotional arousal is caused by the activity of two parallel ascending systems targeting mostly the subcortical limbic regions and the prefrontal cortex. The aversive, negative arousal system is initiated by the activity of the mesolimbic cholinergic system and the hedonic, appetitive, arousal is initiated by the activity of the mesolimbic dopaminergic system. Both ascending projections have a diffused nature and arise from the rostral, tegmental part of the brain reticular activating system. The mesolimbic cholinergic system originates in the laterodorsal tegmental nucleus and the mesolimbic dopaminergic system in the ventral tegmental area. Cholinergic and dopaminergic arousal systems have converging input to the medial prefrontal cortex. The arousal system can modulate cortical EEG with alpha rhythms, which enhance synaptic strength as shown by an increase in long-term potentiation (LTP), whereas delta frequencies are associated with decreased arousal and a decrease in synaptic strength as shown by an increase in long-term depotentiation (LTD). It is postulated that the medial prefrontal cortex is an adaptable node with decision making capability and may control the switch between positive and negative affect and is responsible for modifying or changing emotional state and its expression.

Water Drinking Behavior Associated with Aversive Arousal in Rats: An Integrative Approach.

Cholinergic muscarinic stimulation of vast areas of the limbic brain induced a well-documented polydipsia in laboratory rats. This excessive water-drinking behavior has not received any convincing biological and physiological interpretation for the last 50 years. This review offers such an interpretation and suggests that cholinergically induced drinking response, mostly by carbachol, is associated with activation of the ascending mesolimbic cholinergic system that serves for initiation of emotional aversive arousal of the organism. The ascending cholinergic system originates from the laterodorsal tegmental nucleus, has a diffuse nature, and affects numerous subcortical limbic structures. It is proposed that the carbachol-induced drinking response is related to the state of anxiety and does not serve the regulation of thirst. Instead, the response is anxiety-induced polydipsia that might occur as a soothing procedure that decreases the aversiveness of the negative emotional state induced by carbachol. It is concluded that carbachol-induced water-drinking behavior is a rewarding process that contributes to alleviating the feeling of anxiety by bringing some relief from the cholinergically induced aversive state, and it is a homologue to anxiety-driven polydipsia in humans.

Biological Functions of Rat Ultrasonic Vocalizations, Arousal Mechanisms, and Call Initiation.

This review summarizes all reported and suspected functions of ultrasonic vocalizations in infant and adult rats. The review leads to the conclusion that all types of ultrasonic vocalizations subserving all functions are vocal expressions of emotional arousal initiated by the activity of the reticular core of the brainstem. The emotional arousal is dichotomic in nature and is initiated by two opposite-in-function ascending reticular systems that are separate from the cognitive reticular activating system. The mesolimbic cholinergic system initiates the aversive state of anxiety with concomitant emission of 22 kHz calls, while the mesolimbic dopaminergic system initiates the appetitive state of hedonia with concomitant emission of 50 kHz vocalizations. These two mutually exclusive arousal systems prepare the animal for two different behavioral outcomes. The transition from broadband infant isolation calls to the well-structured adult types of vocalizations is explained, and the social importance of adult rat vocal communication is emphasized. The association of 22 kHz and 50 kHz vocalizations with aversive and appetitive states, respectively, was utilized in numerous quantitatively measured preclinical models of physiological, psychological, neurological, neuropsychiatric, and neurodevelopmental investigations. The present review should help in understanding and the interpretation of these models in biomedical research.

Recent Studies of Rat Ultrasonic Vocalizations-Editorial.

Since the realization that human emotional experiences and behavior evolved from mammalian ancestors and are evolutionary continuations of animal emotional behavior [...].

Dissimilar interaction between dopaminergic and cholinergic systems in the initiation of emission of 50-kHz and 22-kHz vocalizations.

Rats emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) to signal their emotional state to other conspecifics. The 22-kHz USVs signal a negative emotional state while 50-kHz USVs reflect a positive affective state. The initiation of 22-kHz USVs is dependent on the activity of cholinergic neurons within the laterodorsal tegmental nucleus that release acetylcholine along the medial cholinoceptive vocalization strip. Emission of 50-kHz USVs is dependent upon the activation of dopaminergic neurons located within the ventral tegmental area that release dopamine into the medial shell of the nucleus accumbens. There have been reports that showed an antagonistic interaction between acetylcholine and dopamine during the expression of emotional states, and dopamine agonists decreased carbachol-induced emission of 22-kHz USVs. The current study tests the hypothesis that initial antagonism of dopamine receptors by systemic haloperidol or intraacumbens raclopride should increase the subsequent emission of 22 kHz USVs induced by carbachol from the lateral septum. Our findings showed that antagonism of dopaminergic signaling either via systemic haloperidol or via intracerebral raclopride did not alter the number of emitted 22-kHz USVs. Thus, inhibition of the mesolimbic dopamine system did not increase the magnitude of a negative emotional state. It was found, however, that prolonged emission of 22-kHz USVs initiated by carbachol caused a delayed rebound emission (R) of 50-kHz USVs appearing after 300 s of emission of 22-kHz USVs, i.e., when the response was subsiding. The R-50-kHz USVs were predominantly frequency modulated (FM) USVs and their number was directly proportional to the number of recorded 22-kHz USVs. The emission of R-50-kHz USVs was reversed by systemic pretreatment with haloperidol or intraacumbens injection of raclopride. It is argued that the R-50-kHz USVs represent a rebound emotional state that is opposite in valence and arousal induced by carbachol. Importantly, prolonged emission of amphetamine-induced 50 kHz USVs failed to show any vocalization rebound effect.

Association of medial corticostriatal regions with amphetamine-induced emission of 50 kHz vocalizations as studied by Zif-268 expression in the rat brain.

Behavioural sensitization of locomotion and 50 kHz ultrasonic vocalizations (USVs) following repeated amphetamine (AMPH) injections in rats has been extensively demonstrated. These two behaviours appear dissociable in their sensitization patterns and are thought to be reflective of underlying emotional states of the organism. Although AMPH is often used to induce 50 kHz USVs there is little research to date on the extent of cortical and subcortical forebrain region involvement in 50 kHz call production associated with the drug. Nor has general ergometric activity (a measure that in addition to locomotor activity includes all major muscular activity of the body) been investigated in such a framework. The present study sought to address this by performing a minimal sensitization protocol, utilizing only two injections, to investigate expression of the inducible transcription factor Zif-268 (Zif) among brain regions thought to be associated with 50 kHz USV emission. It was found that animals that spent a longer time emitting 50 kHz calls after a second AMPH injection showed statistically significant correlative patterns of Zif expression in medial prefrontal and striatal regions. These associations were not significant in animals that spent a shorter period of time calling after AMPH. There was also no significant correlation between any ergometric activity and time spent calling. The results provide evidence that the medial prefrontal cortices (prelimbic and infralimbic regions) of the rat may be involved with 50 kHz USV emission induced by AMPH in association with medial portions of the ventral and dorsal striatum.

The effects of selective breeding for differential rates of 50-kHz ultrasonic vocalizations on emotional behavior in rats.

Fifty-kHz ultrasonic vocalizations have previously been shown to be positively correlated with reward and appetitive social behavior in rats, and to reflect a positive affective state. In this study, rats selectively bred for high and low rates of 50-kHz vocalizations as juveniles were tested as adults in a battery of behavioral tests for social/emotional behaviors. We found that animals selectively bred for high rates of 50-kHz vocalizations exhibited more crosses into the center area of the open field apparatus, were more likely to show a preference for a dilute sucrose solution (.8%) compared to tap water, and were less aggressive than randomly bred animals. Conversely, animals bred for low rates of 50-kHz calls produced more fecal boli during both open field testing and "tickling" stimulation, and made less contact with conspecifics in a social interaction test compared to randomly bred animals. We also observed that low line rats have elevated brain levels of cholecystokinin (CCK) in the cortex, which is consistent with literature showing that CCK content in the cortex is positively correlated with rates of aversive 22-kHz USVs. Conversely, high line animals had elevated levels of met-enkephalin in several brain regions, which is consistent with the role of endogenous-opioids in the generation 50-kHz USVs and positive affect. These results suggest that animals bred for high rates of 50-kHz may show a stress resilient phenotype, whereas low line rats may show a stress prone phenotype. As such these animals could provide novel insights into the neurobiology of emotion.

Emission of 22 kHz vocalizations in rats as an evolutionary equivalent of human crying: Relationship to depression.

There is no clear relationship between crying and depression based on human neuropsychiatric observations. This situation originates from lack of suitable animal models of human crying. In the present article, an attempt will be made to answer the question whether emission of rat aversive vocalizations (22 kHz calls) may be regarded as an evolutionary equivalent of adult human crying. Using this comparison, the symptom of crying in depressed human patients will be reanalyzed. Numerous features and characteristics of rat 22 kHz aversive vocalizations and human crying vocalizations are equivalent. Comparing evolutionary, biological, physiological, neurophysiological, social, pharmacological, and pathological aspects have shown vast majority of common features. It is concluded that emission of rat 22 kHz vocalizations may be treated as an evolutionary vocal homolog of human crying, although emission of 22 kHz calls is not exactly the same phenomenon because of significant differences in cognitive processes between these species. It is further concluded that rat 22 kHz vocalizations and human crying vocalizations are both expressing anxiety and not depression. Analysis of the relationship between anxiety and depression reported in clinical studies supports this conclusion regardless of the nature and extent of comorbidity between these pathological states.

Intracerebral injection of R-(-)-Apomorphine into the nucleus accumbens decreased carbachol-induced 22-kHz ultrasonic vocalizations in rats.

Rats can produce ultrasonic vocalizations (USVs) in a variety of different contexts that signal their emotional state to conspecifics. Under distress, rats can emit 22-kHz USVs, while during positive pro-social interactions rats can emit frequency-modulated (FM) 50-kHz USVs. It has been previously reported that rats with increasing emission of FM 50-kHz USVs in anticipation of rewarding electrical stimulation or positive pro-social interaction decrease the number of emitted 22-kHz USVs. The purpose of the present investigation was to determine, in a pharmacological-behavioural experiment, if the positive emotional arousal of the rat indexed by the number of emitted FM 50-kHz USVs can decrease the magnitude of a subsequent negative emotional state indexed by the emission of 22-kHz USVs. To induce a positive emotional state, an intracerebral injection of a known D1/D2 agonist R-(-)-apomorphine (3.0 µg/0.3 µl) into the medial nucleus accumbens shell was used, while a negative emotional state was induced by intracerebral injection of carbachol (1.0 µg/0.3 µl), a known broad-spectrum muscarinic agonist, into the anterior hypothalamic-medial preoptic area. Our results demonstrated that initiation of a positive emotional state was able to significantly decrease the magnitude of subsequently expressed negative emotional state measured by the number of emitted 22-kHz USVs. The results suggest the neurobiological substrates that initiate positive emotional state indirectly antagonize the brain regions that initiate negative emotional states.

The antagonistic relationship between aversive and appetitive emotional states in rats as studied by pharmacologically-induced ultrasonic vocalization from the nucleus accumbens and lateral septum.

Rats can emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) in negative, as well as positive contexts which index their emotional state. 22-kHz USVs are emitted during aversive contexts and can be initiated by activation of the ascending cholinergic pathways originating from the laterodorsal tegmental nucleus or initiated pharmacologically by injection of cholinergic agonists into target areas of these pathways (medial cholinoceptive vocalization strip). Conversely, 50-kHz USVs are emitted during positive pro-social contexts and can be initiated by stimulation of ascending dopaminergic pathways originating from the ventral tegmental area or by injection of dopamine agonists into target areas of these pathways (nucleus accumbens shell). Recently, we have shown an inhibitory effect a positive emotional state has on the emission of carbachol-induced 22-kHz USVs from the anterior hypothalamic/medial preoptic area. However, this structure is a fragment of that cholinoceptive vocalization strip. We wanted to examine if we could observe similar effect when the aversive state is induced from the lateral septum, the most rostral division of the cholinoceptive vocalization strip. The results supported previous findings. First, microinjection of the dopamine agonist R-(-)-apomorphine into the nucleus accumbens shell resulted in increased emission of frequency modulated (FM) 50-kHz USVs that are regarded as signals expressing a positive emotional state in rats. Second, FM 50-kHz USVs and not flat (F) 50-kHz USVs were able to decrease 22-kHz USVs induced by microinjections of carbachol into the lateral septum. This research provides further support to the hypothesis that the initiation of a positive emotional state functionally antagonizes initiation of a negative emotional state in rats.

Effect of microinjections of dopamine into the nucleus accumbens shell on emission of 50 kHz USV: Comparison with effects of d-amphetamine.

Systemic pharmacological manipulation of dopamine (DA) signaling has been central to many investigations of 50 kHz ultrasonic vocalizations (USVs) in the rat. In particular, the indirect DA releaser d-amphetamine (AMPH) has been used extensively in many such investigations. The possible unique character of the native transmitter relative to DA-stimulating drugs such as AMPH in inducing and modulating emission of 50 kHz USVs has not been investigated. Adult male Long Evans rats were tested with intracerebral application of DA into the nucleus accumbens shell at several doses (3.75 µg-120 µg) to determine its capacity to induce 50 kHz USV emission. Additionally, the call profile characteristics of intracerebral DA injections were compared with those of intracerebral application of AMPH. Results indicated that local increases in DA signaling within the nucleus accumbens shell are sufficient to increase 50 kHz call rate, reduce latency to call, and increase the degree of frequency modulation of emitted USVs. However, our results found that microinjections of DA were not as efficacious in either inducing 50 kHz USVs or increasing frequency modulation without antagonism of the dopamine reuptake transporter when compared with AMPH. In summary, these results support the notion that the native transmitter DA is driving the increase in frequency modulation seen after administration of DA stimulating drugs. These results also suggest that drugs affecting dopamine may be altering the 50 kHz call profile in a distinct manner from the native transmitter and thus caution should be used in interpreting their effects.

Ultrasonic vocalizations of rats (Rattus norvegicus) during mating, play, and aggression: Behavioral concomitants, relationship to reward, and self-administration of playback.

Rats (Rattus norvegicus) emit a variety of ultrasonic vocalizations throughout their lifespan that reflect different forms of emotional arousal and accompanying affective states. In this study, high frequency recordings of ultrasonic vocalizations were made during mating, aggression, and both conspecific and heterospecific (dubbed "tickling") rough-and-tumble play behavior. We found that frequency modulated 50-kHz calls (trills and step calls) were positively correlated with positively valenced appetitive behavior during mating, play, and aggression. These calls were also positively correlated with the reward value of these social encounters. However, constant frequency (i.e., flat) 50-kHz calls were not related to appetitive behaviors or reward. In contrast, 22-kHz calls were positively related to aversive/withdrawal behaviors during mating, play, and aggression. Finally, we found that rats self-administered playback of frequency modulated 50-kHz trill calls and avoided playback of 22-kHz calls. Playback of flat 50-kHz calls or tape hiss was neutral. These results suggest that frequency modulated 50-kHz calls index a positively valenced, appetitive, social-emotional state in rats.

Individual behavioural predictors of amphetamine-induced emission of 50 kHz vocalization in rats.

Measurement of ultrasonic vocalizations (USVs) produced by adult rats represents a highly useful index of emotional arousal. The associations found between 50 kHz USV production and a variety of behavioural and pharmacological protocols increasingly suggests they serve as a marker of positive motivational states. This study used a powerful within-subjects design to investigate the relationships among individual differences in approach to a sweet-food reward, predisposition to emit 50 kHz USVs spontaneously, and 50 kHz USVs emission following acute systemic administration of amphetamine. Both approach motivation and predisposition to call were found to not correlate with each other but did predict 50 kHz USV response to acute amphetamine. These two behavioural phenotypes appear to represent dissociable predictors of acute amphetamine-induced emission of 50 kHz USVs in a non-sensitization paradigm. In contrast to that, a measure of sucrose preference was not found to predict 50 kHz USV emission following amphetamine. Acute amphetamine was also found to increase average sound frequency of emitted USVs and selectively increase the proportion of Trill subtype 50 kHz USVs. Together, these data demonstrate that acute amphetamine-induced 50 kHz USVs in the adult rat represent more than just a univariate motivational state and may represent the product of dissociable subsystems of emotional behavior.

Non-pharmacological induction of rat 50 kHz ultrasonic vocalization: Social and non-social contexts differentially induce 50 kHz call subtypes.

The emission of 50 kHz ultrasonic vocalizations (USVs) by rats is thought to represent a measurable expression of the individuals underlying emotional state. These calls are also posited as fulfilling important communicative functions among conspecifics. In addition to social situations, 50 kHz USVs are recorded in a variety of reward-related contexts including sugary foods and drink, consumable ethanol, and drugs of abuse. The current study sought to directly compare several of these behavioural contexts in their capability to induce and modulate 50 kHz USV emission in adult male rats. Using two social conditions (exposure of naïve rat to a naturally cycling female and reuniting with a same-sex cage partner) and two non-social conditions (access to consumables as Fruit Loops or 2% ethanol v/v), we analyzed USVs recorded in 6 stimulus-presentation sessions. Only the female-exposure condition was found to increase 50 kHz call rate significantly over baseline, and this induction sensitized across 4 standard recording sessions. The use of a same-sex cage-mate and the two consumable food rewards did not elicit higher than baseline 50 kHz calling. None of the behavioural contexts altered the acoustic parameters (peak frequency, duration, and bandwidth) of emitted 50 kHz calls. In counter-balanced recording sessions, calling across all groups was significantly reduced by pre-treatment with the dopamine antagonist haloperidol compared with vehicle. Non-social conditions appeared to induce a greater proportion of flat calls at the expense of non-trill FM calls, while the reverse was seen for social conditions. However, the type of food reward and the type of social context mattered for proportion of flat and trill calls respectively. When compared with a control, access to sugary food but not ethanol induced a greater proportion of flat calls, and the female but not the cage-mate stimulus induced a greater proportion of trill calls.

Rat 50-kHz ultrasonic vocalizations as a tool in studying neurochemical mechanisms that regulate positive emotional states.

BACKGROUND: Adolescent and adult rats emit 50-kHz ultrasonic vocalizations (USVs) to communicate the appetitive arousal and the presence of positive emotional states to conspecifics. NEW METHOD: Based on its communicative function, emission of 50-kHz USVs is increasingly being evaluated in preclinical studies of affective behavior, motivation and social behavior. RESULTS: Emission of 50-kHz USVs is initiated by the activation of dopamine receptors in the shell subregion of the nucleus accumbens. However, several lines of evidence show that non-dopaminergic receptors may influence the numbers of 50-kHz USVs that are emitted, as well as the acoustic parameters of calls. COMPARISON WITH EXISTING METHODS: Emission of 50-kHz USVs is a non-invasive method that may be used to study reward and motivation without the need for extensive training and complex animal manipulations. Moreover, emission of 50-kHz USVs can be used alone or combined with other well-standardized behavioral paradigms (e.g., conditioned place preference, self-administration). CONCLUSIONS: This review summarizes the current evidence concerning molecular mechanisms that regulate the emission of 50-kHz USVs. Moreover, the review discusses the usefulness of 50-kHz USVs as an experimental tool to investigate how different neurotransmitter systems regulate the manifestations of positive emotional states, and also use of this tool in preclinical modeling of psychiatric diseases.

Ultrasonic calls of rats as indicator variables of negative or positive states: acetylcholine-dopamine interaction and acoustic coding.

Adult rats produce two distinct types of ultrasonic vocalizations referred to as 22- and 50-kHz calls, respectively. Emission of the respective calls represents signaling a negative or positive state of the rat organism. The signaling has an adaptive value for survival and/or well-being of rats and their social groups. Literature is reviewed from studies on cats and rats, which indicates that the positive or negative states constitute a complex and integrated set of somatic, autonomic, endocrine, affective, and cognitive correlates. The basic states and their correlates are initiated, integrated, and maintained by activity of the subsets of the ascending cholinergic and dopaminergic systems originating from the reticular brainstem core. The cholinergic and dopaminergic systems interact mutually to form a dynamic balance, which is involved in a decision-making process of initiating and maintaining one or the other of these states. Activation of the relevant portion of the ascending cholinergic system invariably induces the negative state and releases 22-kHz calls while activation of the ascending dopaminergic system induces the positive state with 50-kHz calls. The 22- and 50-kHz calls have distinct and mostly non-overlapping acoustic parameters, which ensure unambiguous recognition of the calls and thus, the state of the emitter. The animal may only signal one of the states at any given time and emission of 22- or 50-kHz calls is mutually exclusive. It is postulated, therefore, that these two main types of ultrasonic calls are reliable indicator variables of two opposing states of the adult rat organism: negative or positive.

Amphetamine-induced 50 kHz calls from rat nucleus accumbens: a quantitative mapping study and acoustic analysis.

Emission of 50 kHz ultrasonic calls in rats is known to be associated with appetitive behavioural situations and positive social interactions. The purpose of the study was to pharmacologically characterize amphetamine-induced 50 kHz calls and to perform quantitative mapping of this response in the nucleus accumbens. Injections of amphetamine into the nucleus accumbens induced species-typical 50 kHz calls in adult rats. The acoustic parameters of the calls were not affected by different amphetamine doses or combination of agents. The increase in the number of calls occurred predominantly from the accumbens shell and to a lesser degree from the core region. This effect was dose-dependent within the range of 1-20 microg of amphetamine and was reversed by pretreatment with D1 or D2 dopamine antagonists (SKF-83566 or raclopride) administered to the same brain site. However, another D2 dopamine receptor antagonist, haloperidol, which is known to increase the accumbens dopamine level, was ineffective in reversing the increase in call number at the dose studied. On the contrary, intraacumbens haloperidol, when injected alone, caused an increase in 50 kHz calls. It is concluded that the release of dopamine, predominantly in the accumbens shell region, is responsible for production of 50 kHz calls and the calls may indicate an appetitive state compatible with anticipation of reward and positive affect. Both D1 and D2 subtypes of dopamine receptors may be necessary to induce 50 kHz calls and signal the appetitive state.

Acoustic characteristics of air puff-induced 22-kHz alarm calls in direct recordings.

Alarm calls were induced in adult Wistar rats by an air puff. Emitted calls were digitized and directly recorded on a computer hard drive. The long-duration 22-kHz calls were emitted almost exclusively in series. Initial calls in the series tended to have the longest durations, higher frequency range, and the highest degree of frequency modulation, as compared to other calls. The frequency modulation always appeared as a downward sweep and seemed to represent a tuning of individual calls to a 3 kHz communicatory band. Regardless of the maximum frequency, rats always reached approximately the same minimum frequency, common to all calls. Thus, the broader was the frequency range of a given call, the longer the call duration. It is postulated, therefore, that rats emit 22-kHz calls at the minimum possible ultrasonic frequency they are able to produce, which is synonymous with peak frequency. It is further postulated that production of alarm calls in series, with long call duration and the invariably low ultrasonic frequency, maximizes successful communication in dangerous situations. Exceptions to this rule were observed immediately following air puffs, suggesting that acoustic parameters of the initial calls may differ from the alarming properties of the remaining 22-kHz calls.

Pharmacology of Ultrasonic Vocalizations in adult Rats: Significance, Call Classification and Neural Substrate.

Pharmacological studies of emotional arousal and initiation of emotional states in rats measured by their ultrasonic vocalizations are reviewed. It is postulated that emission of vocalizations is an inseparable feature of emotional states and it evolved from mother-infant interaction. Positive emotional states are associated with emission of 50 kHz vocalizations that could be induced by rewarding situations and dopaminergic activation of the nucleus accumbens and are mediated by D1, D2, and partially D3 dopamine receptors. Three biologically significant subtypes of 50 kHz vocalizations have been identified, all expressing positive emotional states: (1) flat calls without frequency modulation that serve as contact calls during social interactions; (2) frequencymodulated calls without trills that signal rewarding and significantly motivated situation; and (3) frequency-modulated calls with trills or trills themselves that are emitted in highly emotional situations associated with intensive affective state. Negative emotional states are associated with emission of 22 kHz vocalizations that could be induced by aversive situations, muscarinic cholinergic activation of limbic areas of medial diencephalon and forebrain, and are mediated by M2 muscarinic receptors. Two biologically significant subtypes of 22 kHz vocalizations have been identified, both expressing negative emotional sates: (1) long calls that serve as alarm calls and signal external danger; and (2) short calls that express a state of discomfort without external danger. The positive and negative states with emission of vocalizations are initiated by two ascending reticular activating subsystems: the mesolimbic dopaminergic subsystem as a specific positive arousal system, and the mesolimbic cholinergic subsystem as a specific negative arousal system.

Role of the mesolimbic cholinergic projection to the septum in the production of 22 kHz alarm calls in rats.

The role of the ascending cholinergic projection from the laterodorsal tegmental nucleus (LDT) to septum in the production of 22 kHz ultrasonic vocalization was studied in adult rats, using behavioral-pharmacological and anatomical tracing methods. Direct application of carbachol, a muscarinic agonist, into the lateral septal region induced species-typical 22 kHz alarm calls. The septum receives cholinergic input from LDT, thus, activation with glutamate of predominantly cholinergic neurons of the LDT induced comparable 22 kHz alarm calls in the same animals. This glutamate-induced response from LDT was significantly reduced when the lateral septum was pretreated with scopolamine, a cholinergic antagonist. To investigate the localization of the cell groups projecting to septum, the fluorescent retrograde tracer, fluorogold, was pressure injected into the lateral septum and sections from these brains were also immunostained against choline acetyltransferase (ChAT) to visualize cholinergic cell bodies. Several ChAT-fluorogold double-labeled cells within the boundaries of the LDT were found, while other fluorogold-labeled regions did not contain double-labeled cells. These results provide both direct and indirect evidence that at least a part of the mesolimbic ascending cholinergic projection from LDT to septum is involved in the initiation of the 22 kHz vocalization. It is concluded that the septum is an integral part of the medial cholinoceptive vocalization strip and the 22 kHz alarm vocalization is triggered from septum by the cholinergic input from the LDT.