Frequency-modulated (FM) 50-kHz ultrasonic vocalizations (USVs) in rats reflect a positive emotional state. The aim of this study was to evaluate whether anticipatory and cocaine-induced FM 50-kHz USVs can predict individual vulnerability to cocaine self-administration behavior. Twenty singly housed rats underwent behavioral testing (elevated plus maze, novel object recognition, and tickling-induced vocalizations) and amphetamine sensitization. The animals then participated in 22 self-administration (SA) sessions (2 days on / 1 day off), during which active nose pokes and FM 50-kHz USVs were recorded during both the anticipatory (cocaine-free) and cocaine phases. Despite pronounced individual differences in SA levels, none of the analyzed behavioral markers predicted SA acquisition. The FM 50-kHz USV response to the first dose of amphetamine showed a bimodal distribution; rats with a weak response exhibited significantly higher levels of cocaine SA. Due to substantial behavioral differences, further analyses were conducted in low-SA and high-SA groups. Anticipatory FM 50-kHz USVs did not correlate with SA intensity and were similarly high in both groups. During the cocaine phase, high-SA rats displayed significantly stronger vocal responses than low-SA rats. FM 50-kHz USVs reflect both contextual and drug-induced positive affect but are highly sensitive to housing conditions and experimental procedures. The response to the first amphetamine dose may serve as a predictive marker of vulnerability to cocaine SA. However, anticipatory FM 50-kHz USVs do not provide a reliable indicator of SA intensity. Accurate interpretation of FM 50-kHz USVs requires integration with behavioral and neurobiological markers to effectively identify addiction risk phenotypes.

Rationale Chronic amphetamine (AMPH) use leading to addiction results in adaptive changes within the central nervous system that persist well beyond the drug’s elimination from the body and can precipitate relapse. Notably, alterations in glutamatergic neurotransmission play a crucial role in drug-associated behaviours. Objectives This study aimed to identify changes induced by amphetamine in glutamate levels and the neuromodulators of glutamatergic neurotransmission (taurine and kynurenic acid) observable after 14 and 28 days of abstinence in key brain regions implicated in addiction: the cortex (Cx), nucleus accumbens (Acb), and dorsolateral striatum (CPu-L). Methods The rats were administered 12 doses of amphetamine (AMPH) intraperitoneally (i.p.) at 1.5 mg/kg. The behavioural response was evaluated through ultrasonic vocalizations (USV). High-performance liquid chromatography (HPLC) was used to measure the levels of glutamate, taurine, and kynurenic acid in the Cx, Acb, and CPu-L after 14 and 28 days of abstinence. Results AMPH administration led to sensitisation towards AMPH’s rewarding effects, as evidenced by changes in USV. There was a noticeable decrease in kynurenic acid levels and an increase in both taurine and glutamate in the CPu-L, along with an increase in glutamate levels in the Cx, 28 days following the final AMPH injection. Conclusions The most significant changes in the tissue levels of glutamate, taurine, and kynurenic acid were seen in the CPu-L 28 days after the last dose of AMPH. The emergence of these changes exclusively after 28 days suggests that the processes initiated by AMPH use and subsequent abstinence take time to become apparent and may be enduring. This could contribute to the incubation of craving and the risk of relapse. Developing pharmacological strategies to counteract the reduction in kynurenic acid induced by psychostimulants may provide new avenues for therapy development.

Background A hallmark of psychostimulants is the persistence of neurobiological changes they produce. The difficulty in reversing long-time effects of psychostimulants use is why addiction therapy is so ineffective. This study aimed to look for such drug-induced changes that can be detected even after many weeks of abstinence. Methods Rats were given 12 doses of amphetamine (Amph) at 1.5 mg/kg. The rewarding effect of Amph was assessed using ultrasonic vocalization. After 14 and 28 days of abstinence, tissue levels of dopamine (DA), serotonin (5-HT), and their metabolites were measured in the prefrontal cortex (PFC), nucleus accumbens (Acb), dorsomedial (CPuM), and dorsolateral (CPuL) striatum. Results After 28 days of abstinence, DA levels were increased in the dorsal striatum while 5-HT levels were decreased in all brain regions studied. The opposite direction of changes in DA and 5-HT tissue levels observed in the dorsal striatum may be related to the changes in the emotional state during abstinence and may contribute to the incubation of craving and relapses. Tissue levels of 5-HT and DA showed intra- and inter-structural correlations, most pronounced after 14 days of abstinence. Most of them were absent in the control group (ctrl), which may indicate that their appearance was related to the changes induced by earlier Amph administration. We did not find any associations between reward sensitivity and the persistence of Amph-induced neurochemical disturbances. Conclusions Administration of 12 moderate doses of Amph causes prolonged changes in DA and 5-HT tissue levels. The direction and severity of the changes are dependent on the brain region and the neurotransmitter studied.

A hallmark of addictive substances is the persistence of the neurobiological changes they produce. The difficulty in reversing them is why addiction therapy is so ineffective. This study aimed to look for permanent drug-induced changes that are visible even after many weeks of abstinence. Rats were given 12 doses of amphetamine (Amph) at 1.5 mg/kg. The behavioural effect was monitored using ultrasonic vocalization, that showed a rewarding effect of Amph and its sensitization. After 14, 28 and 42 days of abstinence, tissue levels of dopamine (DA), serotonin (5-HT) and their metabolites were measured in the brain regions key to the effects of addictive substances: the prefrontal cortex (PFC), nucleus accumbens (Acb), dorsomedial (CPuM) striatum and dorsolateral (CPuL) striatum. DA was decreased in the Acb after 42 days and increased in the dorsal striatum after 28 days of abstinence. 5-HT was decreased after 28 days of abstinence in all brain regions studied. The opposite direction of changes in tissue levels of DA and 5-HT in the dorsal striatum may be related to changes in the emotional state during abstinence and may contribute to the incubation of craving and relapses. Tissue levels of 5-HT and DA showed intra- and interstructural correlations, most pronounced after 14 days of abstinence. Most of those correlations were absent in the control group (ctrl), which may indicate that their appearance is related to changes induced by earlier Amph administration.In summary, administering only 12 moderate doses of amphetamine causes multiweek changes in tissue levels of DA and 5-HT. Their direction and severity are dependent on the brain structure and neurotransmitter studied.

Purpose: This article discusses the modulatory effects of the serotonergic system on the behavioral and neurochemical effects exerted by psychostimulants, mainly cocaine. Views: The mesocorticolimbic dopaminergic system plays an important role in the rewarding effects of psychostimulants and the long-lasting neuroadaptive changes underlying the development of addiction. Dopaminergic brain regions such as the ventral tegmental area (VTA) and substantia nigra (SN) and their projection fields (prefrontal cortex - PFC, nucleus accumbens - Acb, dorsal striatum) are innervated by serotonergic neurons that can modulate this system. Pharmacological manipulation of the ac-tivity of the serotonergic system in rats has shown that lowering or elevating its activity increases and decreases, respectively, most behavioral responses to cocaine. Studies on the role of serotonin receptors have shown that the serotonin 5-HT1B receptor agonists administered to the Acb during self-administration increase the reinforcing effects of cocaine, whereas when administered during abstinence they decrease cocaine seeking. Distinct populations of 5-HT2AR and 5-HT2CR in the PFC, Acb, and VTA differentially affect the output of the mesocorticolimbic dopaminergic pathway. 5-HT2B receptors exert independent control over the activity of the three ascending dopamine (DA) pathways through specific tonic excitatory and inhibitory control of DA efflux from the Acb and PFC and do not affect striatal activity. Conclusions: The serotonergic system exerts modulatory effects on the behavioral and neurochemical effects of psychostimulants. The pharmacological manipulation of serotonergic system activity makes it possible to attenuate the effects of psychostimulants, which gives hope for the development of effective pharmacotherapy. Currently, the main obstacle to this is the excessive side effects shown by potential drugs.

One characteristic feature of addictive drugs is their ability to induce, after a single exposure, a lasting sensitization to the next doses; the underlying neuroplastic changes supposedly involve the brain dopamine system. We aimed at identifying putative relationships between alterations in extracellular dorsal striatal dopamine, HVA and DOPAC levels and in frequency-modulated 50-kHz ultrasonic vocalizations rate response during repeated intraperitoneal amphetamine treatment. Measurements were performed before and after amphetamine doses 1, 2, 7 and 8 (Amph1, Amph2, Amph7 and Amph8; treatment days 1, 7, 12 and 23, respectively). Amphetamine was confirmed to induce sensitization of the vocalization response, but an extended recording time (180 instead of 20 min) revealed that sensitization of this response requires more time to develop than hitherto believed. Baseline extracellular dopamine level increased initially, declined after a series of daily amphetamine doses and showed some tendency for recovery after drug withdrawal. Baseline extracellular DOPAC (but not HVA) showed a continuous decline during the treatment. There was no significant change in the integrated short-term (3-h) extracellular dopamine response, whereas the respective DOPAC collection lowered significantly after repeated drug treatment. Extracellular DOPAC is believed to originate mostly from newly synthesized dopamine, hence the declines in its baseline and post-amphetamine levels suggest falling dopamine synthesis. These results indicate that sensitization of the appetitive vocalization response to amphetamine continues despite reduced dorsal striatal dopamine synthesis and involves no changes in amphetamine-induced dopamine release.

Measuring ultrasonic vocalizations (USVs) allows studying psychoactive drug use-related affective states in laboratory rats and may help understand changes underlying the progress of addictions. We aimed at finding an effective scheme for amphetamine self-administration training in rats, identifying factors affecting their anticipatory and drug-evoked, frequency-modulated 50-kHz USV responses, and verifying whether the rewarding action of amphetamine promotes current drug intake during the training. Therefore, we monitored amphetamine intake and anticipatory and drug-evoked USVs in two rat cohorts trained using two different training schemes. Then we retrospectively divided these cohorts into low-amphetamine and high-amphetamine intake subsets and analyzed their frequency-modulated 50-kHz USV responses accordingly. Anticipatory (i.e., drug-context-related) USVs as well as USVs induced by self-administration training-related non-pharmacological manipulations (tested in an additional rat group) showed surprisingly high call rates but faded spontaneously relatively quickly. Only the scheme employing short cycles of training sessions (two instead of six) and intermittent instead of continuous intra-session drug availability yielded long-lasting escalation of amphetamine intake in a sizable subset. This subset showed high initial amphetamine-evoked USV call rate, which suggests that a strong rewarding action of the drug early in the SA training favors intake escalation. A major decrease in the drug-evoked USVs during advanced training indicated the emergence of tolerance to the rewarding action in these rats, a phenomenon that is characteristic of addiction. Frequency-modulated 50-kHz rat USVs are a good index of the rewarding action of amphetamine at the absence of USVs induced by drug context and other training-related factors.

In our study expression of phospho-(Ser-10)-histone H3 (pH3S10), a marker for the early stage of neurogenesis, and cellular early response genes were investigated using c-Fos protein as an example of a transcription factor in the neurogenic process in rats. Neurogenesis in the adult brain is regulated by endo- and exogenous factors, which influence the proliferation potential of progenitor cells and accelerate the dendritic development of newborn neurons. D-amphetamine, a psychoactive substance, is one of the exogenous factors able to influence the process of neurogenesis. The rats were injected with D-amphetamine at a dose of 1.5 mg/kg/body weight (b.w.) under one administration scheme. Analysis of the pH3S10 and c-Fos expression levels in the group of D-amphetamine administered rats provided evidence of enhanced expression of these proteins in the regions of neurogenesis occurrence in rats. However, conclusions concerning stimulant effects of amphetamine on neurogenesis should be formulated with great caution, taking into account amphetamine dosage and the administration scheme. It should also be remembered that doses of psychoactive substances used in animal models can be lethal to humans..

Rationale Our previous studies showed promise for using sensitization of the frequency-modulated 50-kHz vocalization response to amphetamine (AMPH) as an index of rat vulnerability to AMPH addiction. Objective This study aimed to test the utility of sensitizing frequency-modulated (FM) 50-kHz vocalization in the AMPH self-administration paradigm as well as the ability of N-acetylcysteine to prevent self-administration relapse. Methods Rats were subjected to the so-called two-injection protocol of sensitization (TIPS) using AMPH and were categorized as low-sensitized callers (LC TIPS ) or high-sensitized callers (HC TIPS ) based on the individual outcomes. Then, they were given 44 sessions of AMPH self-administration followed by a 17-session N-acetylcysteine-aided extinction course and a single session of AMPH-primed self-administration reinstatement. Results LC TIPS compared to HC TIPS rats showed no considerable difference in the FM 50-kHz vocalization rate during the self-administration training or extinction course, but they were considerably more likely to acquire AMPH self-administration and experience drug-induced reinstatement of this trait. Moreover, the LC TIPS rats were more likely than HC TIPS rats to have a markedly higher FM 50-kHz vocalization rate after AMPH reinstatement. N-acetylcysteine did not affect the course of self-administration extinction or the instrumental or FM 50-kHz vocalization responses to AMPH reinstatement. Conclusions There is no link between the FM 50-kHz vocalization and key characteristics of AMPH self-administration. Additionally, N-acetylcysteine does not help prevent AMPH self-administration relapse. However, there is a high predictive value for poor sensitization of the FM 50-kHz vocalization response to AMPH with respect to the acquisition and maintenance of self-administration of this psychostimulant.

This study utilised the two injection protocol of sensitisation (TIPS) and the conditioned place preference test to validate and extend previous findings on the effects of amphetamine on positive reinforcement-related 50 kHz ultrasonic vocalisation (USV) in rats. We also examined changes in the expression of c-Fos and the NMDA receptor 2B (GluN2B) subunit, markers of neuronal activity and plasticity, in brain regions of rats in response to TIPS. We used low anxiety-responsive (LR) and high anxiety-responsive (HR) rats, which are known to exhibit different fear-conditioned response strengths, different susceptibilities to amphetamine in the TIPS procedure and different amphetamine-dependent 50 kHz USV responses. The LR rats, compared to the HR rats, not only vocalised much more intensely but also spent significantly more time in the amphetamine-paired compartment. After the second dose of amphetamine, the LR rats exhibited more c-Fos and GluN2B activation in layers II and III of the M1/M2 motor cortex area and prefrontal cortex (PRE, PRL, IL) and also presented with more GluN2B activation in the basal amygdala. These data reveal that HR and LR rats exhibit different levels of reactivity in the cortical-limbic pathway, which controls reward-related motivational processes. These findings contribute to the general hypothesis that heterogeneity in emotional processes is one of the causes of sensitisation to amphetamine and drug addiction.

Individuals predisposed to addiction constitute a minority of drug users, in both humans and animal models of the disorder, but there are no established characteristics that would allow identifying them beforehand. Our studies demonstrate that sensitization of rat 50-kHz ultrasonic vocalization (USV) response to amphetamine shows marked inter-individual diversity but substantial intra-individual stability. Low sensitization of the response shows relevance to the acquisition of self-administration of this drug and hence might be of predictive value regarding the risk of addiction. We compared amphetamine-induced Fos expression in 16 brain regions considered important for the development of addiction between rats preselected for low and high sensitization of the response and next given nine daily amphetamine doses followed by a 2-week withdrawal and final amphetamine challenge. Ventral tegmental area and nucleus accumbens shell Fos-positive nuclei counts correlated positively with 50-kHz USV response to the challenge in high-sensitized rats. Compared to those in amphetamine-untreated controls, Fos-positive nuclei counts were significantly and markedly (2–6 times) higher in 12 regions in high-sensitized rats, whereas in low-sensitized rats they were significantly higher in the cingulate cortex and dorsomedial striatum only. The difference in the counts between the latter two subsets reached statistical significance in dorsomedial and dorsolateral striatum and three out of four cortical regions studied. The fact that the diversification was most distinct in dorsal striatum that plays a critical role in the transition from controlled to compulsive drug intake suggests that the USV-based categorization may be related to divergent vulnerability of rats to AMPH addiction.

Acute administration of high doses of morphine reduced 50-kHz ultrasonic vocalizations (USVs). Although morphine meets the classical criteria for inducing 50-kHz USVs (it causes place preference and induces dopamine release in nucleus accumbens), it also inhibits appetitive vocalizations.

Behavioral sensitization and tolerance to repetitive exposure to addictive drugs are commonly used for the assessment of the early stages of the drug dependence progress in animals. The orchestra of tools for studying the progress of drug dependence in laboratory rodents has been considerably enriched in the 1980s by the introduction of ultrasonic vocalization (USV) detection and characterization. However, the relationship between the results of this technology and those of traditional behavioral tests is not clear. We attempted to elucidate some of the respective ambiguities by comparing the effects of an intermittent amphetamine treatment, which was aimed both at the induction of sensitization and tolerance to this drug and at testing the persistence of these effects, on the locomotor activity and 50-kHz USV responses to both the drug and the context of drug exposure in adult male rats showing diverging susceptibility for sensitization to amphetamine. Categorization of the rats into low and high responders/callers based on sensitization of their frequency-modulated 50-kHz USV responsiveness showed some correspondence with conditioned place preference effects, but not with responses to amphetamine. The study showed distinct changes in the rate and latency of the frequency-modulated 50-kHz USV responses to repetitive amphetamine treatment, which were reminiscent of classical behavioral signs of sensitization and tolerance. These results show the utility of the appetitive USV for monitoring of early phases of complex processes leading to drug dependence. However, USV, locomotor activity and conditioned place preference seem to reflect different aspects of these phenomena.

A satisfactory pharmacological cure for addictions to psychostimulants has not yet been developed. Because of the well-known role of changes in the corticoaccumbal and corticostriatal glutamatergic system(s) in drug seeking and relapses in psychostimulant addiction, much hope is presently linked to the use of agents that restore glutamate homeostasis. In this regard, one of the most promising agents is N-acetyl cysteine, which has been shown to reverse some changes in neuroplasticity associated with psychostimulant addiction/dependence. In this study, we used the enhancement of locomotor activity and the induction of frequency-modulated 50-kHz ultrasonic vocalization (FM 50-kHz USV) to test the possible stimulant properties of N-acetyl cysteine itself in various experimental settings (acute and subchronic administration in amphetamine-naïve and amphetamine-pretreated rats) and the capacity of N-acetyl cysteine to attenuate both the rewarding effects of amphetamine and the behavioral sensitization to this stimulant in rats showing considerable differences in their susceptibility to the FM 50-kHz USV sensitization. Our data showed no stimulant properties of N-acetyl cysteine and no acute effect of the drug on the rewarding properties of amphetamine. Moreover, no effect of N-acetyl cysteine on the pre-existing sensitization of the FM 50-kHz USV and locomotor activity responses to amphetamine were observed, independent of the susceptibility of the rats to the FM 50-kHz USV sensitization. Hence, N-acetyl cysteine seems to be ineffective at reversing the neurobiological changes underlying the sensitization of these responses to amphetamine in rats.

This study assessed behaviour, as measured by 50kHz calls related to positive affect, in rats with different fear conditioned response strengths: low-anxiety rats (LR) and high-anxiety rats (HR), after amphetamine injection in a two-injection protocol (TIPS). The results showed that the first dose of amphetamine evoked similar behavioural effects in frequency-modulated (FM) 50kHz calls in the LR and HR groups. The second injection of amphetamine resulted in stronger FM 50kHz calls in LR compared with HR rats. The biochemical data ('ex vivo' analysis) showed that the LR rats had increased basal levels of dopamine in the amygdala, and increased homovanilic acid (HVA), dopamine's main metabolite, in the amygdala and prefrontal cortex compared with HR rats. The 'in vivo' analysis (microdialysis study) showed that the LR rats had increased HVA concentrations in the basolateral amygdala in response to an aversively conditioned context. Research has suggested that differences in dopaminergic system activity in the amygdala and prefrontal cortex may be one of the biological factors that underlie individual differences in response to fear stimuli, which may also affect the rewarding effects of amphetamine.

The development of tolerance to alcohol with chronic consumption is an important criterion for an animal model of alcoholism and may be an important component of the genetic predisposition to alcoholism. The aim of this study was to determine whether the selectively bred Warsaw High Preferring (WHP) line of alcohol-preferring rats would develop behavioral and metabolic tolerance during the free-choice drinking of ethanol. Chronic tolerance to ethanol-induced sedation was tested. The loss of righting reflex (LRR) paradigm was used to record sleep duration in WHP rats. Ethanol (EtOH)-naive WHP rats received a single intraperitoneal (i.p.) injection of 5.0 g ethanol/kg body weight (b.w.), and sleep duration was measured. Subsequently, rats had access to a 10% ethanol solution under a free-choice condition with water and food for 12 weeks. After 12 weeks of the free-choice intake of ethanol, the rats received another single i.p. injection of 5.0 g ethanol/kg b.w., and sleep duration was reassessed. The blood alcohol content (BAC) for each rat was determined after an i.p. injection of 5 g/kg of ethanol in naive rats and again after chronic alcohol drinking at the time of recovery of the righting reflex (RR). The results showed that the mean ethanol intake was 9.14 g/kg/24 h, and both sleep duration and BAC were decreased after chronic ethanol intake. In conclusion, WHP rats exposed to alcohol by free-choice drinking across 12 weeks exhibited increased alcohol elimination rates. Studies have demonstrated that WHP rats after chronic free-choice drinking (12 weeks) of alcohol develop metabolic tolerance. Behavioral tolerance to ethanol was demonstrated by reduced sleep duration, but this decrease in sleep duration was not significant.