FACULTY OF PHARMACY DEPARTMENT OF DRUG CHEMISTRY
EGLĖ MARIJA RUGYTĖ
Direct and conditioned effects of repeated
administration of amphetamine and morphine on
50-kHz ultrasonic vocalizations in rats
Master’s thesis
Supervisors: Assoc. prof. Ph.D. Raimondas Benetis Ph.D. Nicola Simola
LITHUANIAN UNIVERSITY OF HEALTH SCIENCES FACULTY OF PHARMACY
DEPARTMENT OF DRUG CHEMISTRY
APPROVED BY:
Dean of the Faculty of Pharmacy prof. Vitalis Briedis
Direct and conditioned effects of repeated
administration of amphetamine and morphine on
50-kHz ultrasonic vocalizations in rats
Master’s thesis
Supervisors:
Assoc. prof. Ph.D. Raimondas Benetis Ph.D. Nicola Simola
Reviewer:
Assoc. prof. Ph.D. Konradas Vitkevičius
The thesis performed by:
Master's student Eglė Marija Rugytė
TABLE OF CONTENTS
SUMMARY ... 5 SANTRAUKA ... 6 ABBREVIATIONS ... 7 DEFINITIONS ... 8 1. INTRODUCTION ... 92. AIM OF THE STUDY ... 11
3. OBJECTIVES OF THE STUDY ... 11
4. LITERATURE REVIEW ... 12
4.1 Characteristics of ultrasonic vocalizations emitted by adult rats ... 12
4.1.1 22-kHz ultrasonic vocalizations ... 12
4.1.2 50-kHz ultrasonic vocalizations ... 14
4.1.3 “Trill” subtype vocalizations ... 16
4.2 Registration and examination of ultrasonic vocalizations ... 16
4.3 Neural circuits involved in the emission of ultrasonic vocalizations ... 18
4.3.1 22-kHz ultrasonic vocalizations ... 18
4.3.2 50-kHz ultrasonic vocalizations ... 18
4.4 Ultrasonic vocalizations and pharmacological treatment ... 19
5. MATERIALS AND METHODS ... 21
5.1 Animals ... 21
5.2 Drugs ... 21
5.3 Experimental protocol ... 21
5.4 Recording of ultrasonic vocalizations ... 23
5.5 Data collection, analysis and statistics ... 24
6. RESULTS ... 25
6.1 Emission of ultrasonic vocalizations stimulated by the administration of vehicle, and emission of 22-kHz ultrasonic vocalizations ... 25
6.2 Emission of 50-kHz ultrasonic vocalizations ... 25
6.2.1 Amphetamine ... 25
6.2.2 Morphine ... 27
6.3.1 Amphetamine ... 29
6.3.2 Morphine ... 30
6.4 Emission of “trill” subtype of 50-kHz ultrasonic vocalizations during the repeated and intermittent administration of drugs and following test cage re-exposure ... 32
7. DISCUSSION ... 33
7.1 Direct effects of amphetamine and morphine on 50-kHz ultrasonic vocalizations ... 33
7.2 Conditioned effects of amphetamine and morphine on 50-kHz ultrasonic vocalizations ... 35
7.3 Direct and conditioned effects of amphetamine and morphine on “trill” 50-kHz ultrasonic vocalizations ... 35
7.4 Role of the administration protocol in the emission of 50-kHz ultrasonic vocalizations... 36
8. CONCLUSIONS ... 38
9. PRACTICAL RECOMMENDATIONS ... 39
10. REFERENCES ... 40
SUMMARY
Master thesis by Eglė Marija Rugytė. Supervisors – dr. R.Benetis and dr. N.Simola. Lithuanian University of Health Sciences, Faculty of Pharmacy, Department of Drug Chemistry, Kaunas. The thesis has been prepared in cooperation with the University of Cagliari (Italy), experiments were performed at the University of Cagliari, Department of Biomedical Sciences, Section of Neuropsychopharmacology.
Title of work. Direct and conditioned effects of repeated administration of amphetamine and morphine on 50-kHz ultrasonic vocalizations in rats.
The aim and objectives. The purpose of this study was to evaluate the effects of a repeated treatment with either amphetamine or morphine on the emission of 50-kHz ultrasonic vocalizations (USVs) by male rats. 50-kHz USVs were evaluated both after drug administration (direct effect) and after rats’ re-exposure to the environment where they previously received the drug (conditioned effects). This study also evaluated the extinction of conditioned USVs. In the present study, the administrations of amphetamine and morphine were performed on either consecutive or alternate days, in order to evaluate the role of the protocol of administration on the effects of these drugs. Furthermore, the present study evaluated the modifications in both the direct and conditioned “trill” USVs, that have been proposed to be specifically emitted in response to rewarding stimuli, to understand the importance of this USVs subtype in the study of the effects of substances of abuse.
Materials and methods. Male rats were used in this study. Distinct groups of rats were treated with either amphetamine or morphine, and each rat received only one substance. Rats treated with vehicle (purified water) were used as controls. Vocalizations were registered with microphones for ultrasounds. All the recorded vocalizations were collected with a computer and analyzed using a suited software for sonographic analysis. Statistical analysis was performed with the software Statistica for Windows.
Results. Direct effects: rats treated with amphetamine in consecutive and in intermittent manner have shown a significant emission of 50-kHz USVs while rats treated with morphine have not. Conditioned effects: re-exposure of rats to the test cage, which was associated with the administration of the substances of abuse, provoked a significant emission of 50-kHz USVs in rats treated with either amphetamine or morphine. The emission of conditioned 50-kHz USVs following the re-exposure to the test cage 7 days after repeated treatment discontinuation was stronger in rats treated on alternate days.
Practical recommendations. This study suggests that evaluating modifications in the emission of 50-kHz USVs can be a useful rat model to evaluate the expectation towards substances of abuse. The emission of 50-kHz USVs could be used to implement new experimental models to study the psychological component of drug dependence.
SANTRAUKA
Eglės Marijos Rugytės magistro baigiamasis darbas. Moksliniai vadovai dr. R.Benetis ir dr. N.Simola; Lietuvos sveikatos mokslų universiteto Farmacijos fakulteto Vaistų chemijos katedra; Kaunas. Baigiamasis darbas buvo parengtas bendradarbiaujant su University of Cagliari (Italija), eksperimentai buvo atlikti University of Cagliari, Biomedicinos mokslų katedroje, Neuropsichofarmakologijos skyriuje.
Darbo pavadinimas. Pakartotinio poveikio amfetaminu ir morfinu tiesioginiai ir sąlygoti efektai žiurkių 50-kHz ultragarso artikuliacijoms.
Tikslas ir uždaviniai. Šio tyrimo tikslas buvo įvertinti pakartotinio poveikio amfetaminu ir morfinu efektus, žiurkių patinų 50-kHz ultragarso artikuliacijų (USVs) emisijai. 50-kHz USVs buvo įvertintos po vaisto davimo (tiesioginis poveikis) ir po žiurkių pakartotinio patekimo į aplinką, kurioje jos anksčiau gavo vaistus (sąlygotas poveikis). Sąlygotų USVs išnykimas taip pat buvo tirtas žiurkes pakartotinai veikiant aplinka susieta su vaistu, po gydymo nutraukimo. Šiame tyrime amfetaminas ir morfinas buvo leidžiami arba kas antrą, arba kiekvieną dieną, tam, kad įvertinti eksperimento protokolo įtaką šių vaistų poveikiams. Taip pat, šis tyrimas įvertino „trill“ artikuliacijas (manoma, jog jos gali būti specifinis atsakas į malonumą suteikiančius dirgiklius) esant ir tiesioginiam, ir sąlygotam poveikiui, kad išsiaiškintume šio USVs tipo svarbą tiriant vaistų poveikį.
Tyrimo metodika. Šiame tyrime buvo naudojami žiurkių patinai. Atskiros žiurkių grupės buvo veikiamos arba amfetaminu, arba morfinu ir kiekviena žiurkė gavo tik vieną šių medžiagų. Žiurkės veikiamos tirpikliu (išgrynintuoju vandeniu) buvo naudojamos kaip kontrolinė grupė. Artikuliacijos buvo registruojamos mikrofonu skirtu ultragarsams. Artikuliacijų įrašai buvo sukaupti kompiuteryje ir analizuoti, naudojant programinę įrangą skirtą sonografinei analizei. Statistinė analizė buvo atlikta naudojant programą Statistica for Windows.
Rezultatai. Tiesioginis poveikis: žiurkės, gavusios amfetaminą kasdien ir kas antrą dieną, parodė reikšmingą 50-kHz USVs emisijos padidėjimą, tuo tarpu, žiurkės, gavusios morfiną – ne. Sąlygotas poveikis: žiurkių pakartotinis patekimas į aplinką, susietą su vaistu, sukelė reikšmingą 50-kHz USVs emisiją ir žiurkėse gavusiose amfetaminą, ir morfiną. Sąlygotų 50-kHz USVs emisija, 7 dienos po pakartotino gydymo nutraukimo, buvo stipresnė žiurkėse, veiktose vaistais kas antrą dieną.
Praktinės rekomendacijos. Šis tyrimas kelia prielaidą, jog 50-kHz USVs emisijos pokyčių tyrimas gali būti naudingas eksperimentinis modelis, tiriant priklausomybę sukeliančias medžiagas. 50-kHz USVs emisijos tyrimas gali būti panaudotas diegiant naujus eksperimentinius modelius tirti psichologinį faktorių priklausomybėje nuo vaistų.
ABBREVIATIONS
1. ANOVA – analysis of variance;2. i.p. – intraperitoneal; 3. kHz – kilohertz;
4. NAc – nucleus accumbens; 5. s.c. – subcutaneous;
6. S.E.M. – standard error of the mean; 7. USVs – ultrasonic vocalizations; 8. VTA – ventral tegmental area.
DEFINITIONS
1. Ultrasonic vocalizations – form of communication between rats, vocal sounds, having a frequency above the human ear's audibility limit of about 20 kHz.
2. Sonogram – the visual image produced by recorded sound waves.
3. Drug challenge – an acute drug administration performed after a period of withdrawal from a previous repeated drug administration.
1. INTRODUCTION
Communication is an important activity carried out by all living beings in order to exchange various information. Rats communicate among them not just through the emission of sounds within frequency audible to humans, but also by means of ultrasonic vocalizations (USVs) [1,2]. There are several types of USVs. In this study we focused on 50-kHz USVs, which are mainly emitted in response to rewarding stimuli [3,4,5].
Ultrasounds have an average frequency that is inaudible by experimenters. Therefore their examination requires the use of electronic computerized equipments. Signals are recorded with microphones for ultrasounds and collected with a computer. These registered signals are then analyzed through a specific sonographic software, which generates graphs that are called sonograms. These graphs can then be analyzed visually and by means of a suited software to calculate the number of vocalizations and their acoustic parameters.
The notable advantage in measuring USVs induced by pharmacological stimulation is related to the fact that the evaluation of this behavioral response does not require the animals to undergo training or stressful manipulations.
Regarding 50-kHz USVs, it is interesting to highlight that previous studies have demonstrated that the emission of these vocalizations can be stimulated by some substances with abuse potential, especially dopaminergic psychostimulants like amphetamine [6,7,8,9,10,11,12,13]. Therefore, 50-kHz USVs have recently attracted growing interest regarding their possible use as a tool to evaluate the effects of different psychoactive substances, as well as the influence these substances can have on the emotional state of rats.
The emission of 50-kHz USVs following pharmacological treatments appears to be related not just to direct, acute effects, but also to the conditioned effects of psychoactive substances. Previous studies have shown that the emission of conditioned 50-kHz USVs by rats previously treated with substances of abuse can indicate the animal’s expectation towards the substance [14,15,16]. Based on these considerations, it can be hypothesized that 50-kHz USVs could represent a useful tool for studying the factors that are responsible of compulsive search for substances of abuse.
In this regard, it is also noteworthy that the majority of studies performed so far on this issue have chiefly focused on the acute effects of amphetamine and, partly, of morphine. Conversely, very limited data are available on the long-term effects of the repeated treatment with substances of abuse on the emission of 50-kHz USVs.
Therefore, although the use of USVs could potentially be a useful tool for studying the properties of substances of abuse, it is necessary to characterize in detail the appropriate experimental conditions for the use of this method, and how 50-kHz USVs can be affected by the repeated administration of substances with abuse potential. In this context, it is particularly relevant to characterize the changes in vocalizations that are associated with the repeated administration of two major substances of abuse, amphetamine and morphine. Studying the effects of amphetamine is interesting, as it is one of the most used recreational drugs. Effects of morphine are important, because the use of this substance is associated with a very high risk of developing drug addiction. It is also interesting to consider that these two substances have different mechanisms of action, which may help to further clarify the relevance of 50-kHz USVs to the study of drug dependence.
The aim of the present study was to evaluate the effects of a repeated treatment with either amphetamine or morphine on the emission of 50-kHz USVs by male rats, in order to thoroughly characterize the effects of the drugs studied.
2. AIM OF THE STUDY
The aim of the present study was to evaluate the effects of a repeated treatment (5 administrations) with either amphetamine or morphine on the emission of 50-kHz USVs by male rats, in order to thoroughly characterize the effects of the drugs studied.
3. OBJECTIVES OF THE STUDY
1. To evaluate 50-kHz USVs emitted immediately after drug administration (direct effect). 2. To evaluate 50-kHz USVs emitted after rats’ re-exposure to the environment where they
previously received the drug (conditioned effects), and extinction of these vocalizations. 3. To evaluate the role of the protocol of administration (consecutive or alternate days) on
the effects of used drugs.
4. To evaluate the modifications in both the direct and conditioned “trill” USVs stimulated by amphetamine and morphine.
4. LITERATURE REVIEW
Rats produce all vocal sounds by means of the larynx which can function in two different ways: • The first, common to all mammals, is vibration of vocal cords and generation of sounds that are
audible to humans (squeaks) and have a frequency of 2-4 kHz [17].
• The second involves constriction of vocal cords and consequent emission of ultrasounds. Each vocalization is emitted when a tiny orifice is formed between vocal cords, which generates sort of a whistle [1,2].
USVs are emitted by rats to communicate their emotional states to conspecific individuals in response to either “positive” (e.g., presence of food, non aggressive social interactions) or “negative” stimuli (e.g., dangerous situations, presence of predators nearby, fear, anxiety). In this regard, it is worth mentioning that several previous studies have suggested that the different types of USVs have primarily developed as antipredator behavior, and that only later these vocalizations have been employed for social interactions between conspecifics [18,19,20,21]. In fact, numerous predators are not able to perceive ultrasounds. Moreover ultrasounds are difficult to localize, because they propagate in several directions and can overcome environmental obstacles, including bad meteorological conditions [5,22].
Rats are able to emit different types of USVs, the features of which differ depending on the animal’s age and emotional state, and on environmental factors [14,23,24].
USVs can be subdivided in three major distinct categories, based on their average frequency: • Vocalizations with an average frequency of 40 kHz, emitted by rats that are below three weeks of
age.
• Vocalizations with an average frequency of 22 kHz, emitted by young and adult rats in response to aversive stimuli (e.g., encounter with a predator, anxiety).
• Vocalizations with an average frequency of 50 kHz, emitted in response to rewarding stimuli [3,4,5], or for maintaining social coordination among animals belonging to the same group [25].
4.1 Characteristics of ultrasonic vocalizations emitted by adult rats
These USVs, often referred to as “alarm vocalizations”, are characterized by an average duration in the range between 100 and 300 ms, and a peak frequency in the range between 20 and 23 kHz, although this acoustic parameter can reach values as high as 30 kHz [5,26]. In general, rats emit 22-kHz USVs in short trains of 2-7 long vocalizations (100-300 ms). However, independent studies have consistently reported the existence of 22-kHz USVs of shorter duration (less than 100 ms), although the behavioral significance of these vocalizations is not yet clear [27,28,29]. Both female and male rats can emit 22-kHz USVs, with the only difference that female rats can emit vocalizations of shorter duration and slightly lower frequency than male rats [4,26]. Moreover, breeding conditions can influence the number and duration of 22-kHz USVs. In fact, it has been reported that the exposure to aversive stimuli induces a more marked emission of 22-kHz USVs in rats reared in pairs compared with rats reared individually. Finally, with regard to rats reared in pairs, it has been demonstrated that the dominant rats emit less 22-kHz USVs in response to aversive stimuli compared with subordinate rats [30]. Remarkably, rats possess an inborn mechanism of response to “alarm” vocalizations that allow them to quickly associate aversive situations with 22-kHz USVs, and to retain this association in their memory for a long period of time. The same phenomenon is not observed for events associated with other types of USVs, which suggests that the 22-kHz USVs are those with a stronger behavioral significance for rats [31].
It is now clearly established that rat emit 22-kHz USVs in response to various aversive stimuli, which may include:
• Potential dangerous situations, like the encounter with a predator (e.g., a cat or a fox), which represent an anxiogenic stimulus for the rat. If the danger is imminent (e.g., the predator is at short distance), the rat will remain silent. However, if the rat is in the ideal conditions to escape, it will remain silent until it reaches a safe place, and after that it will communicate with the other conspecifics by emitting 22-kHz USVs, to inform them about the presence of a danger [3,32]. • Situations that are unfamiliar for the rats, such as exposure to strong noises or the presence of
unknown humans in the housing environment [29,33,34,35,36,37,38,39].
• Fight with another rat [40,41,42]. In general, these vocalizations are emitted by the defeated animal in order to both signal a potentially dangerous situation to other conspecifics and appease the attacker.
• During mating, such vocalizations can be emitted by males either following the failure of the intromission [43], or if the female presents low level of lordosis behavior, which makes mating difficult [44]. In addition, male rats can emit 22-kHz USVs immediately after mating, to signal a change in their affective state and the onset of refractory period [40,45].
• Painful stimuli that have an anxiogenic component (e.g., in tests of conditioned avoidance and fear conditioning).
It has to be remarked that, although rats emit 22-kHz USVs mainly in aversive situations that may be associated with painful stimuli, these vocalizations cannot simply be considered a parameter indicative of physical discomfort and of the degree of pain perceived by the animal. On the other hand, 22-kHz USVs appear rather as an indicator of the anxiety elicited by stressful situations that can result in a potential danger for the animal [46]. In this regard, it has been demonstrated that application of an avoidable electric shock to the paw stimulates a more marked emission of 22-kHz USVs than application of an unavoidable shock. This finding appears in line with the fact that the avoidable shock possesses a strong anxiogenic component, while the unavoidable but expected shock does not [47].
4.1.2 50-kHz ultrasonic vocalizations
These USVs are characterized by an average frequency that differs from that featuring 22-kHz USVs. In fact, 50-kHz USVs have a peak of maximum frequency generally comprised in the range between 45 and 55 kHz, although it can be as high as 70 kHz. Moreover, these USVs are very short, having an average duration between 30 and 40 ms.
Previous studies have demonstrated the existence of two major and distinct categories of 50-kHz USVs [48,25], which are represented in Figure 1:
• Vocalizations with an almost constant frequency, called “flat”.
• Vocalizations which present a modulation of their frequency, called “frequency modulated”.
Further studies have demonstrated that rats emit “flat” or “frequency modulated” USVs in different situations. In fact, “flat” USVs seem to have almost exclusively the function of social coordination among animals of the same group [49,25], while “frequency modulated” USVs are emitted preferably in response to rewarding stimuli and/or to express a “positive” affective state [49]. Furthermore, the latter category of 50-kHz USVs was recently further subdivided in twelve subcategories, based on the form, duration, and acoustic peak of vocalizations [50]. However, it is not yet clear if vocalizations in these subcategories have their own biological and communicative significance [11]. Similarly to what reported for 22-kHz USVs, the emission of 50-kHz USVs is influenced by rearing conditions. In fact, the emission of these vocalizations decreases in case of repeated exposure to familiar conspecifics [5]. Moreover, rearing in group reduces the number of 50-kHz USVs which young rats emit in response to the experimental procedure called “tickling” [51], which is also known as “heterospecific hand play” and consists in the contact with the hand of a familiar human according to specific standardized procedures.
50-kHz USVs have a distinct communicative significance compared with 22-kHz USVs, and are involved in the following behavioral processes:
• Social coordination [3,4,5], communication between rats, and searching for, or expectation of interaction with, other conspecifics. In this regard, it has been demonstrated that young rats emit this type of vocalizations if they are placed in cages previously occupied by other rats [8].
• Manifestation of pleasure, satisfaction, and gratification [5,22,52,53]. Thus, the emission of 50-kHz USVs is stimulated by situations such as mating, presence of food, friendly non-aggressive social encounters with conspecifics, including social play or the presence of anesthetized, and therefore easily approachable, conspecifics [3,8,54,55,56]. Moreover, as mentioned above, young rats emit a considerable number of 50-kHz USVs during the “tickling” procedure [51,53,57].
Regarding the communicative significance of 50-kHz USVs, it is interesting to notice that these vocalizations can also be emitted after the administration of psychoactive substances with rewarding properties, such as amphetamine, cocaine, and methylphenidate, as well as following the electric stimulation of brain areas involved in reward processes [11,14,52,58]. Furthermore, the emission of 50-kHz USVs following pharmacological treatments appears to be related not just to the direct, acute, effects, but also to the conditioned effects of psychoactive substances. Regarding this, it is interesting to notice that rats which previously received the administration of either amphetamine or morphine in a novel environment emit 50-kHz USVs when later re-exposed to the same drug-paired environment [5,22,52,53]. Taken together, these considerations indicate that 50-kHz USVs can be emitted in response to hedonic
stimuli, which has suggested the possible use of these USVs as a tool to evaluate the rewarding properties of substances of abuse and the effects that they exert on the affective state.
4.1.3 “Trill” subtype
vocalizations
These vocalizations are described as rapid frequency oscillations (usually appearing as a sinusoid) with a period of approximately 15 ms [11,50], shown in Figure 2. It has been demonstrated how “trill” subtype vocalizations are emitted in response to administration of amphetamine. Moreover, some of the previous studies which have isolated and characterized various subtypes of 50-kHz USVs have suggested that the “trill” vocalizations might be those specifically correlated to euphoric states [49,50,59]. This has led to the hypothesis that “trill” vocalizations might represent a specific indicator of the changes in the affective states induced by drugs of abuse, and prompted the use of “trill” vocalizations in the investigation of these states.
Fig. 2. Example of “trill” 50-kHz USVs (adapted from Wright et al., 2010)
4.2 Registration and examination of ultrasonic vocalizations
Since ultrasounds have an average frequency that is higher than the human auditory threshold, and are therefore inaudible by experimenters, their examination requires the use of electronic computerized equipments. These equipments are designed to obtain acoustic signals and transform them into digitized signal, in turn generating vocalizations that are audible to the human ear. The registered signals are then subjected to acoustic analysis through a specific sonographic software, which elaborates the registrations and generates graphs called sonograms. Sonograms represent the temporal proceeding of
the frequency of the registered sound, reported on the ordinates axis, as a function of time, reported in the abscissa axis (Fig.1). In addition, the software used to analyze USVs can generate specific spectrograms called “spectrum of potency” which show all the individual frequencies of a sound (in kHz) and their relative potency (in dB), allowing the detailed analysis of the registered vocalizations by the experimenter. In order to render USVs audible to humans, the software modifies their original frequency through Fourier transformation. Therefore, the vocalizations obtained in this way present a different tone compared with the original USVs emitted by rats. However, transformed vocalizations maintain some fundamental acoustic parameters unchanged, including:
• Duration of the signal (in ms).
• Bandwidth (difference between the upper and the lower frequencies of the vocalization, in kHz). • Frequency modulation.
• Shape of the vocalization [5].
The different categories of USVs result in easily distinguishable signals in the sonogram. In particular, 22-kHz USVs appear as straight lines with a bandwidth in the range between 1 and 4 kHz, and with a near constant frequency. On the other hand, 50-kHz USVs display a higher bandwidth, ranging between 5 and 7 kHz [5,22]. As mentioned previously, these latter vocalizations can either have a near constant frequency or display a modulation of their frequency [25,60].
As described above, USVs have a fundamental communicative role for rats, and the clear and unambiguous understanding of their significance is crucial for animals’ survival [22]. In this regard, it has been demonstrated that rats are able to clearly distinguish 22-kHz USVs from 50-kHz USVs. Thus, these two categories of USVs possess acoustic parameters that are peculiar and clearly distinguishable by animals:
• The frequencies of the two categories of USVs are separated by a definite margin of about 15 kHz. Moreover, if 22-kHz and 50-kHz USVs are emitted concomitantly, they are always separated by a time interval sufficient to give the animal the ability to identify individual vocalizations with certainty.
• 50-kHz USVs have a much greater bandwidth than 22-kHz USVs.
• 22-kHz USVs have a quite longer duration (within seconds), than 50-kHz USVs.
Therefore, since the different categories of USVs are emitted in clearly distinguishable circumstances and possess peculiar acoustic parameters, they allow rats to communicate either “negative” or “positive” emotional states in a unique manner [5].
4.3 Neural circuits involved in the emission of ultrasonic vocalizations
The emission of 22-kHz and 50-kHz USVs is regulated by distinct cerebral mechanisms: 22-kHz vocalizations are dependent on the activation of the ascending cholinergic system, while 50-kHz vocalizations depend on the activation of the ascending dopaminergic system. Both systems belong to the reticular system of the brainstem, which regulates the state of excitation of the organism, attention, and emotions [61].
4.3.1 22-kHz ultrasonic vocalizations
22-kHz USVs are emitted following the activation of the ascending cholinergic system [62,63], which originates from the laterodorsal tegmental nucleus and projects to the basal proencephalon and limbic areas. This system prepares the individual to respond to stimuli that may represent threats or dangers [22] and that require increased vigilance. Importantly, the activation of the ascending cholinergic system not only triggers modifications involving the somatic, autonomic, and endocrine systems, but promotes the manifestation of dysphoric or “negative” affective states.
Besides ethological adverse stimuli (e.g., the presence of a predator), pharmacological treatments which are able to act, either directly or indirectly, on cholinergic transmission can as well stimulate the emission of 22-kHz USVs [6,7,8,9,12,13,62). Thus, activation of the ascending cholinergic system by means of the administration of cholinomimetic drugs, such as carbachol, induces an intense emission of 22-kHz USVs by rats [62]. Support to the crucial role of the ascending cholinergic system in the emission of 22-kHz USVs, comes also from the results of studies which have demonstrated how the administration of anticholinergic drugs attenuates the emission of these vocalizations in rats exposed to aversive stimuli [62]. Finally, the emission of 22-kHz ultrasonic vocalizations has been documented in rats subjected to the administration of naloxone or lithium, two substances that possess dysphoric properties [64], further substantiating the ethological significance of these vocalizations.
4.3.2 50-kHz ultrasonic vocalizations
The neural circuit mainly involved in the emission of 50-kHz USVs is the ascending dopaminergic system, which is activated by pleasant and rewarding stimuli and originates in the ventral
tegmental area (VTA). Stimulation of the ascending dopaminergic system induces modifications in somatic, autonomic, and endocrine systems, as well as the manifestation of euphoric, or “positive”, affective states [65]. The activation of the dopaminergic transmission can occur not only in response to natural stimuli, such as mating, social interactions, or presence of food, but also following treatment with substances of abuse. In fact, it is widely described how amphetamine significantly stimulates the emission of 50-kHz USVs after either systemic administration or intracranial infusion in the nucleus accumbens (NAc), a nucleus critically involved in reward and reinforcement processes [12,13].
4.4 Ultrasonic vocalizations and pharmacological treatment
As described in the introduction, the ability of certain substances of abuse to stimulate the emission of 50-kHz USVs suggests that the analysis of these vocalizations can represent a useful tool to study the rewarding and aversive effects of psychoactive substances, and to evaluate the influence of these substances on the emotional state of animals. The notable advantage in measuring USVs induced by pharmacological stimulation is related to the fact that the evaluation of this behavioral parameter does not require the animals to undergo training or stressful manipulations (e.g., food or water deprivation). Therefore this method can provide direct information on the properties of the administered substances that are devoid of the influence of stressful interfering factors.
However, it should be considered that pharmacological treatments could induce effects on the emission of USVs that may differ from those observed following the exposure to natural stimuli. For example, the release of neurotransmitters provoked by a drug can differ from that induced by natural stimuli, both in terms of intensity (e.g., strong vs. weak) and kinetic of effect (e.g., fast vs. slow). Moreover, previous studies which have examined the acute effects of amphetamine, cocaine and caffeine on the emission and acoustic parameters of 50-kHz USVs, in male adult rats have demonstrated striking differences in the effects of those drugs. In fact, it has been reported that both cocaine and amphetamine induce a robust emission of 50-kHz USVs [10,12,13,58], while caffeine is able to modify the acoustic parameters of these vocalizations, but not their number [10]. Subsequent studies have shown further differences in the effects of some substances with abuse potential on 50-kHz USVs, by demonstrating that while both amphetamine and methylphenidate stimulate these vocalizations following their acute administration, only methylphenidate, but not amphetamine, modifies the maximum peak frequency, bandwidth, and duration of 50-kHz USVs [11].
Therefore, although the use of 50-kHz USVs could potentially be a useful tool for studying the properties of substances of abuse, it is necessary to characterize in detail the appropriate experimental conditions for the use of this method, and how these vocalizations can be affected by the repeated administration of substances with abuse potential. In this context, it is particularly relevant to characterize the changes in vocalizations that are associated with the repeated administration of two major substances of abuse, such as amphetamine and morphine. Studying the effects of amphetamine is interesting, as this substance is the prototype of a large class of amphetamine-derived psychostimulants which are, together with cannabis, the most used recreational drugs [66]. Studying the effects of morphine is also interesting, because this substance is the parent compound of heroin and related opiates, the use of which is associated with a very high risk of developing drug addiction [67]. In this regard, it is also interesting to consider that these two substances have different mechanisms of action. Thus, amphetamine promotes the release of dopamine from cytoplasmic vesicles and reverses the direction of the dopamine transporter, which reuptakes this neurotransmitter; therefore this substance has a direct effect on dopaminergic transmission [68]. On the other hand, morphine induces dopamine release in an indirect way. In fact, morphine, by acting on the μ receptors located on GABAergic interneurons, reduces the inhibitory influence that these interneurons have on the dopaminergic neurons located in the VTA, hence promoting dopamine release in the ventral striatum [68]. Therefore, it is conceivable that these different mechanisms of action may be reflected by different long-term effects of amphetamine and morphine on the emission of 50-kHz USVs.
5. MATERIALS AND METHODS
5.1 Animals
Male Sprague-Dawley rats (Harlan, Italy), weighing 275–300 g were used in this study. Animals were housed in groups of 4-5 in standard polycarbonate cages, with the bottom covered with sawdust, and were kept under a 12 h light–dark cycle (lights on at 8:00 hours). Rats had free access to food (standard laboratory rodents chow) and water, except during the experiments, which were performed from 10:00 to 15:00 hours. All the experiments were performed in accordance with the Directive of the European Parliament and of the Council on the protection of animals used for scientific purposes (2010/63/EU; L. 276; 22/09/2010), and with the guidelines for animal experimentation approved by the Ethics Committee of the University of Cagliari.
5.2 Drugs
This study has evaluated the effects of the following drugs:
• Amphetamine (D-amphetamine sulfate) (purity ≥ 99.0 %), purchased from Sigma-Aldrich (Milan, Italy);
• Morphine (morphine hydrochloride) (purity ≥ 98.0 %) purchased from Franchini Prodotti Chimici srl (Mozzate, Como, Italy).
Both drugs were dissolved in distilled water. Amphetamine was administered at the dose of 2 mg/kg through intraperitoneal (i.p.) injection, in a volume of 3 ml/kg. Morphine was administered at the dose of 7.5 mg/kg by subcutaneous (s.c.) injection in a volume of 1 ml/kg. The doses of drugs used in this study were selected based on previous experiments which evaluated the acute effects of these substances on 50-kHz USVs [11] and on preliminary experiments which demonstrated how repeated administration of morphine at doses lower than that used in this study failed to stimulate the emission of 50-kHz USVs by rats (N.Simola, unpublished observations).
5.3 Experimental protocol
Rats were handled for 5 minutes a day, for 2 consecutive days before the beginning of the experiments, which included 6 phases:
1. Habituation to the test cage (2 daily sessions of 15 minutes each for two consecutive days). 2. Acute administration of vehicle in the test cage to quantify the basal emission of USVs. 3. Repeated administration of amphetamine, morphine or vehicle in the test cage on either consecutive or alternate days (5 administrations in total), and recording of USVs.
4. Drug withdrawal (7 days) in the home cage.
5. Evaluation of the emission of conditioned USVs following the re-exposure of rats to the test cage, immediately followed by the administration of either amphetamine or morphine and the recording of USVs.
6. Drug withdrawal (7 days) in the home cage, followed by repeated re-exposure of rats to the test cage (5 days) to evaluate the extinction of USVs.
A detailed description of the experimental protocol is provided in Figure 3. The intermittent drug administration regimen was used to prevent the onset of tolerance to the effects of the substances evaluated, as reported elsewhere [69], and to verify whether the protocol of administration could influence the effects of the substances evaluated on the emission of 50-kHz USVs. Distinct groups of rats were treated with either amphetamine (2 mg/kg, i.p.) or morphine (7.5 mg/kg, s.c.), and each rat received only one substance. Rats treated with vehicle were used as controls. The experimental groups of rats treated with amphetamine were composed of 6 animals treated continuously and 6 animals treated intermittently. The experimental groups of rats treated with morphine were composed of 8 rats treated continuously and 8 rats treated intermittently.
Fig. 3. Experimental plan. H = habituation to the test cage; V = vehicle administration; D = drug
administration in the test cage; R = re-exposure to the test cage; C = drug challenge; USVs = 50-kHz ultrasonic vocalizations; vUSV = USVs recorded after vehicle administration; dUSV = USVs recorded
after drug administration; cUSV = USVs recorded after re-exposure to the test cage in drug-free conditions
5.4 Recording of ultrasonic vocalizations
The recording of USVs was performed in a quiet room. Each rat was individually placed in a Plexiglas cylinder (diameter 25 cm, height 30 cm), surrounded by 4 cardboard walls (height 65 cm, distance from cylinder 15 cm). The cylinder had the bottom covered with sawdust taken from the home cage of the specific rat subjected to the experiment, in order to reduce the influence that the exposure to a completely new environment may have on the emission of USVs [10,70]. The cylinder was topped with a lid equipped with a microphone for ultrasounds recording having high directional properties (CM 16/CMPA, Avisoft, Berlin, Germany). 50-kHz USVs stimulated by vehicle, amphetamine, or morphine were recorded for 1 hour, while USVs emitted in response to the re-exposure of rats to the environment previously associated with drug administration were recorded for 10 minutes.
5.5 Data collection, analysis and statistics
All the recorded vocalizations were collected with a computer and analyzed using the software for sonographic analysis SAS Lab Pro 4.52 (Avisoft, Berlin, Germany). The following settings were used to obtain the spectrograms: Hamming window, FFT length of 512, and 75% overlapping of frame set-up [10,71]. After visual inspection and deletion of all the signals that could be not univocally classified as USVs, the spectrograms were analyzed with the software SAS Lab Pro 4.52, which automatically calculates the number of vocalizations and the acoustic parameters for each vocalization.
This study found that the emission of 50-kHz USVs recorded immediately after drug administration from rats that received either amphetamine or morphine on consecutive days was similar to that recorded from rats that received the same drugs on alternate days. Conversely, this study observed a significant emission of conditioned 50-kHz USVs only in those rats that underwent intermittent drug administration. Based on this latter finding, and in order to compare direct and conditioned effects of drugs, the analysis of “trill” vocalizations was narrowed to those rats that received the drugs intermittently.
Data are presented as mean ± S.E.M. (standard error of the mean) of the total number of 50-kHz USVs and “trill” USVs, and of the percentage of “trill” USVs. Data were analyzed by means of Student’s t-test or two-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test, when appropriate. Statistical analysis was performed with the software Statistica for Windows (StatSoft, USA).
6. RESULTS
6.1 Emission of ultrasonic vocalizations stimulated by the administration of vehicle,
and emission of 22-kHz ultrasonic vocalizations
The day before the beginning of the pharmacological treatments, rats received an acute administration of vehicle. Vehicle scarcely stimulated the emission of 50-kHz USVs with no significant difference between the various experimental groups (data not shown). Moreover, one rat emitted 22-kHz USVs during the treatment with amphetamine administered in consecutive days. This rat was excluded from further experiments, to avoid the possible influence of negative affective states on the results observed.
6.2 Emission of 50-kHz ultrasonic vocalizations
6.2.1 Amphetamine
Rats treated with amphetamine (2 mg/kg, i.p.) in either consecutive or intermittent administration exhibited a marked emission of 50-kHz USVs compared with rats treated with vehicle (Figure 4).
Fig. 4. Effect of repeated treatment with amphetamine on the emission of 50-kHz USVs. Rats received
five amphetamine administrations on either consecutive or alternate days. USVs were recorded during the first and fifth amphetamine administration. *p<0.05 compared with vehicle-treated rats; adm. =
administration
Two-way ANOVA revealed a significant effect of treatment (F1,10 = 12.42, p<0.05), but no effect of time (F1,10 = 1.31, p>0.05) and no interaction treatment × time (F1,10 = 1.23, p>0.05) in the rats that received amphetamine on consecutive days.
Similarly, two-way ANOVA showed a significant effect of treatment (F1,10 = 11.37, p<0.05), but no effect of time (F1,10 = 1.18, p>0.05), and no interaction treatment x time (F1,10 = 1.59, p>0.05) in the rats that were treated with amphetamine on alternate days.
Acute challenge with amphetamine (2 mg/kg, i.p.) seven days after treatment discontinuation elicited a stronger emission of 50-kHz USVs compared with that induced by vehicle challenge (Figure 5). T-test showed that this effect occurred both in rats treated by amphetamine on consecutive days (t = 4.78, df = 10, p<0.05) and rats treated with amphetamine on alternate days (t = 4.49, df = 10, p<0.05).
Fig. 5. Emission of 50-kHz USVs after drug challenge recorded from rats previously treated with amphetamine or morphine. Rats treated on either consecutive or alternate days received a challenge with
their respective drug at 7 days from treatment discontinuation, and drug-stimulated 50-kHz USVs were recorded. *p<0.05 compared with vehicle-treated rats
6.2.2 Morphine
Rats treated with morphine (7.5 mg/kg, s.c.) emitted a number of 50-kHz USVs similar to that recorded from rats treated with vehicle (Figure 6).
Fig. 6. Effect of repeated treatment with morphine on the emission of 50-kHz USVs. Rats
received five morphine administrations on either consecutive or alternate days. USVs were recorded
during the first and fifth morphine administration. #p<0.05 compared with morphine-treated rats. adm. =
administration
Two-way ANOVA revealed no significant effect of treatment (F1,12 = 0.13, p>0.05) in rats that received morphine on consecutive days, although both a significant effect of time (F1,12 = 4.63, p<0.05) and a significant interaction treatment × time (F1,12 = 7.06, p<0.05) were present.
With regard to the rats that received morphine on alternate days, two-way ANOVA revealed no significant effect of treatment (F1,12 = 0.02, p>0.05) and no interaction treatment × time (F1,12 = 0.53, p>0.05), although it showed a significant effect of time (F1,12 = 7.87, p<0.05).
Subsequent Tukey’s post-hoc test revealed that rats treated with morphine, on either consecutive or alternate days, emitted a significant higher number of 50-kHz USVs on the fifth drug administration, compared with first administration (Figure 6).
Acute challenge with morphine (7.5 mg/kg, s.c.) seven days after treatment discontinuation elicited an emission of 50-kHz USVs similar to that displayed by vehicle challenge (Figure 5).
6.3 Emission of 50-kHz USVs recorded on re-exposure to the drug-paired
environment
6.3.1 Amphetamine
Rats that received amphetamine (2 mg/kg, i.p.) on consecutive days showed an emission of 50-kHz USVs similar to that of vehicle-treated rats when re-exposed to the test cage 7 days after treatment discontinuation. Conversely, rats treated with amphetamine on alternate days displayed a more marked emission of 50-kHz USVs than vehicle-treated rats when re-exposed to the test cage 7 days after treatment discontinuation, as indicated by t-test (t = 2.31, df = 10, p<0.05) (Figure 7).
Fig. 7. Emission of 50-kHz USVs after re-exposure to the test cage by rats previously treated with amphetamine or morphine. Rats treated on either consecutive or alternate days were re-exposed to
the test cage, 7 days after treatment discontinuation, and vocalizations were recorded. *p<0.05 compared with vehicle-treated rats
No differences were observed between rats treated with amphetamine, on either consecutive or alternate days, and rats treated with vehicle when the emission of 50-kHz USVs and their extinction were
evaluated starting at 7 days after amphetamine challenge (14 days after repeated treatment discontinuation) (Figure 8). Moreover, two-way ANOVA showed that the extinction of 50-kHz USVs recorded on repeated test cage re-exposure was similar between rats treated with amphetamine on consecutive days and rats treated with amphetamine on alternate days.
Fig. 8. Emission of 50-kHz USVs by amphetamine-treated rats during repeated test-cage re-exposure. Starting at 7 days after drug challenge, rats previously treated with amphetamine on either
consecutive or alternate days were re-exposed to the test cage (10 min) once a day for 5 consecutive days, and vocalizations were recorded on the first and fifth exposure. exp. = exposure
6.3.2 Morphine
Rats treated with morphine (7.5 mg/kg, s.c.) on consecutive days displayed an emission of 50-kHz USVs similar to that exhibited by vehicle-treated rats when re-exposed to the test cage starting 7 days after treatment discontinuation (Figure 7).
Similar results were obtained in the same rats with regard to the emission and extinction of 50-kHz USVs recorded upon repeated test cage re-exposure starting 7 days after acute morphine challenge (14 days after repeated treatment discontinuation) (Figure 9).
At variance to what observed in rats that received morphine on consecutive days, rats treated with morphine (7.5 mg/kg, s.c.) on alternate days exhibited a more marked emission of 50-kHz USVs than vehicle-treated rats, when re-exposed to the test cage starting 7 days after treatment discontinuation, as indicated by t-test (t=2.69; df=12; p<0.05) (Figure 7). Furthermore, significant differences were observed between rats treated with morphine on alternate days and vehicle-treated rats with regard to the extinction of 50-kHz USVs emitted in response to repeated re-exposure to the test cage. Two-way ANOVA revealed a significant effect of treatment (F1,12 = 4.77, p<0.05) and time (F1,12 = 6.00, p<0.05), although no significant interaction treatment × time (F1,12 = 1.6, p>0.05) was observed. Subsequent Tukey’s post-hoc test demonstrated that rats treated with morphine exhibited a more pronounced vocalization during the first session of extinction (Figure 9).
Fig. 9. Emission of 50-kHz USVs by morphine-treated rats during repeated test-cage re-exposure. Starting at 7 days after drug challenge, rats previously treated with morphine on either
consecutive or alternate days were re-exposed to the test cage (10 min) once a day for 5 consecutive days, and vocalizations were recorded on the first and fifth exposure. *p<0.05 compared with vehicle-treated
Finally, two-way ANOVA indicated a significant effect of time (F1,14 = 9.18, p<0.05), when the extinction of 50-kHz USVs measured in rats treated with morphine on consecutive days was compared with that observed in rats treated with morphine on alternate days.
6.4 Emission of “trill” subtype of 50-kHz ultrasonic vocalizations during the
repeated and intermittent administration of drugs and following test cage
re-exposure
Repeated treatment with amphetamine (2 mg/kg, i.p.) and subsequent amphetamine challenge, 7 days after treatment discontinuation, have proven to be the only conditions able to stimulate the emission of 50-kHz USVs of the “trill” subtype, compared with the repeated administration of vehicle (Table 1). Two-way ANOVA applied to rats repeatedly administered amphetamine on alternate days revealed a significant effect of treatment (F1,10 = 9.44, p<0.05), but no effect of time (F1,10 = 3.5, p>0.05) and no interaction treatment × time (F1,10 = 3.52, p>0.05). Moreover, t-test indicated a significant effect of amphetamine challenge on the emission of “trill” 50-kHz USVs (t=3.03; df = 10; p<0.05).
Table 1. Emission of 50kHz “trill” ultrasonic vocalizations in response to repeated drug treatment, drug challenge and test cage re-exposure; *p<0.05 compared with vehicle-treated rats
Emission of “trill” 50-kHz ultrasonic vocalizations after repeated treatment with amphetamine or morphine and drug challenge
First administration Fifth administration Challenge
Treatment Number Percentage Number Percentage Number Percentage Amphetamine 109.7±41.9* 9.6±3.2 27±13.6* 5.2±3.0 49.8±16.4* 5.6±2.5
Morphine 0.1±0.1 2.1±2.1 2.3±1.3 3.1±1.2 0.1±0.1 0.2±0.2
Vehicle 0.3±0,2 3±2.1 0.5±0.5 0.5±0.5 0.2±0.2 0.3±0.3
Emission of “trill” 50-kHz ultrasonic vocalizations after re-exposure to the test cage Challenge day First re-exposure Fifth re-exposure Treatment Number Percentage Number Percentage Number Percentage
Amphetamine 3±1.2 3.9±2.5 0.3±0.3 0.4±0.4 1.7±1.7 1.4±1.4
Morphine 8.5±4.4 3.7±1.5 5.1±2.2 5.5±1.7 3±1.2 6.5±2.4
7. DISCUSSION
Several studies have demonstrated that rats emit 50-kHz USVs in response to rewarding stimuli of various nature, and therefore it has been suggested that these vocalizations could be a useful tool to study the effects of psychoactive substances with rewarding properties. However, a previous comprehensive analysis of the effects of various substances with abuse potential on the emission of 50-kHz USVs has demonstrated that some of these substances fail to stimulate such vocalizations when administered acutely [11]. Based on these considerations, this study evaluated the direct and conditioned effects elicited by the repeated administration of amphetamine and morphine on 50-kHz USVs. This approach was followed in order to understand the similarities and differences in the effects of two drugs with marked abuse potential on the emission of 50-kHz USVs by rats, and to further understand how this behavioral response can be affected by pharmacological manipulations.
7.1 Direct effects of amphetamine and morphine on 50-kHz ultrasonic vocalizations
The results of this study have demonstrated a significant emission of 50-kHz USVs only by rats subjected to the repeated administration of amphetamine and challenge with the drug 7 days after treatment discontinuation. Conversely, morphine failed to induce the emission of 50-kHz USVs, even after repeated administration. Moreover, the analysis of the “trill” subtype of 50-kHz USVs revealed significant changes only during amphetamine treatment and challenge.
The different ability of amphetamine and morphine to stimulate the emission of 50-kHz USVs immediately following their administration can be explained based on the different mechanisms of action of these substances with regard to the stimulation of the dopaminergic transmission. In fact, previous studies have shown that the stimulation of the dopaminergic receptors in the NAc shell is crucial for the emission of 50-kHz USVs [12,13,48,51]. While both amphetamine and morphine promote the release of dopamine in the NAc shell, morphine induces a less intense effect compared with amphetamine [68]. Moreover, repeated administration of morphine has been found associated with a reduction, rather than an increase, in the release of dopamine in NAc shell [72,73]. Based on these data, it is possible to hypothesize that 50-kHz USVs are emitted only when the release of dopamine and/or the occupation of dopamine receptors in the NAc shell is above a threshold value. In addition to this hypothesis, it should be considered that the emission of 50-kHz USVs is as well modulated by neurotransmitters other than dopamine, such as glutamate and norepinephrine [9,74]. Therefore, the differences between the effects of
amphetamine and morphine on the emission of 50-kHz USVs found in this study can be explained based on both the modulation of dopaminergic transmission and the influence of other neurotransmitters.
In consideration of the fact that 50-kHz USVs are an index of pleasure and reward [75,76] and that drugs of abuse stimulate the circuits involved in reward and produce sensitization to some of their effects [77,78], the occurrence of a progressive increase in the emission of 50-kHz USVs would be conceivable during repeated treatment with drugs of abuse. This study seems to indicate the presence of this effect for the repeated administration of morphine, as morphine-treated rats emitted a higher number of 50-kHz USVs on the fifth administration, compared with the first administration. However, a more correct interpretation of these results indicates the occurrence of tolerance to the inhibitory effects of morphine on the emission of 50-kHz USVs, rather than the development of sensitization [11]. In fact, the number of vocalizations recorded following the fifth administration of morphine was found similar to that emitted by rats repeatedly treated with vehicle.
In a similar way, the experiments performed in rats treated with amphetamine have shown that while this substance stimulates a marked emission of 50-kHz USVs, the total number of vocalizations did not significantly change over repeated drug treatment and challenge administered 7 days after drug discontinuation. This finding is in line with recent data showing that rats repeatedly treated with amphetamine do not develop sensitization to the emission of 50-kHz USVs, even when the substance is administered in conditions (e.g., in a novel environment) known to promote the behavioral sensitization [79].
As mentioned above, previous studies have shown that substances of abuse which have a major dopaminergic mechanism of action (amphetamine, cocaine, methylphenidate) are those which most efficiently stimulate the emission of 50-kHz USVs [11,50,58,74,80]. In this regard, it is however important to mention that while the ability to release dopamine is important for the reinforcing properties of drugs of abuse [81], the increased levels of this neurotransmitter that may be associated with the emission of 50-kHz USVs [12,13], are not necessarily indicative of the ability of drugs of abuse to induce reward and euphoria [82,83]. These consideration, may therefore contribute to explain the different effects of amphetamine and morphine observed in this study.
7.2 Conditioned effects of amphetamine and morphine on 50-kHz ultrasonic
vocalizations
Another interesting result of this study is that the direct and conditioned effects of drugs of abuse on 50-kHz USVs are regulated in a distinct manner. In fact, re-exposure of rats to the test cage, which was previously associated with drug administration, provoked a significant emission of 50-kHz USVs not only in rats treated with amphetamine, which vocalized during the repeated drug treatment, but also in rats treated with morphine, which did not. It is interesting to notice that the 50-kHz USVs emitted by rats during the re-exposure to the test cage appear to be conditioned in nature. In fact, previous experiments performed according to the same experimental protocol used in this study have shown that rats treated with morphine in their home cage, alternated with vehicle administration in the test cage, emitted a modest number of 50-kHz USVs when re-exposed to the test cage 7 days after treatment discontinuation (N.Simola, unpublished observations). Finally, it is interesting to point out that rats treated with morphine exhibited an emission of conditioned 50-kHz USVs that persisted even after 7 days from the last administration of the substance. This result is in accordance with the marked conditioning properties of opiates, and with the elevated potential of abuse of these substances [84].
The emission of conditioned vocalizations by rats previously treated with amphetamine or morphine and subsequently re-exposed to the environment previously associated with the drug was observed by some earlier studies, although others failed to observe this effect in rats treated with morphine [53,74,85]. These discrepant results can be due to the rats’ interindividual variability in the emission of vocalizations (which has been reported by numerous studies), to the use of different rat strains, or to other experimental factors, like drug doses and different protocols of drug administration. Nevertheless, the results of this study confirm that rats previously treated with either amphetamine or morphine emit 50-kHz USVs when re-exposed to the environment previously associated with the drug, and suggest that ultrasonic vocalizations can be a very useful tool for the study of the conditioned properties of drugs.
7.3 Direct and conditioned effects of amphetamine and morphine on “trill” 50-kHz
ultrasonic vocalizations
Previous studies have suggested that the “trill” subtype of 50-kHz USVs could be a specific index of reward and euphoria, as rats preferably emit these vocalizations in response to rewarding stimuli,
such as homospecific or heterospecific (“tickling”) social contacts and administration of amphetamine [49,50,59]. In this study, amphetamine was the only substance that stimulated a significant emission of “trill” vocalizations, although repeated treatment with this drug did not result in sensitization to this effect, but rather in its attenuation. The lack of sensitization in 50-kHz “trill” USVs in response to repeated treatment with amphetamine can appear in contrast with the hypothesis that such vocalizations would specifically reflect the hedonic properties of substances of abuse. In this regard, it is interesting to mention that a previous study reported the development of a sensitized emission of “trill” 50-kHz USVs in Long-Evans rats repeatedly treated with amphetamine [59]. Similar to what described above for the total number of amphetamine-stimulated 50-kHz USVs, the discrepancy between the present and previous studies could be due to methodological issues, like the use of a particular rat strain, drug dose, USVs recording times, and criteria used to classify 50-kHz USVs as “trill”. However, it is also important to highlight that the results of the present study are in accordance with other studies that have observed no sensitization in the emission of 50-kHz “trill” USVs during both self-administration of methamphetamine and acquisition of conditioned place preference to morphine [74,86]. Therefore, similarly to what observed in this study for the total number of 50-kHz USVs, the presence/absence of “trill” vocalizations after the administration of drugs with abuse potential can indicate effects not necessarily related to the hedonic properties of these substances. In this regard, it is important to underline that this study has observed the emission of conditioned “trill” 50-kHz USVs during the re-exposure of rats treated with either amphetamine or morphine to the test cage, although this effect did not reach statistical significance.
7.4 Role of the administration protocol in the emission of 50-kHz ultrasonic
vocalizations
This study found that the emission of 50-kHz USVs in response to the administration of either amphetamine or morphine did not significantly change with the protocol of drug administration, being comparable between rats that have received either substance on consecutive days and rats treated on alternate days. However, rats that received drug treatment on alternate days, showed a stronger emission of conditioned 50-kHz USVs following the re-exposure to the test cage 7 days after repeated treatment discontinuation. In this regard, it is interesting to highlight that previous studies have shown how the emission of conditioned 50-kHz USVs by rats previously treated with substances of abuse can indicate the animal’s expectation towards these substances [14,15,16]. Moreover, a previous study in rats treated with
cocaine has shown that the expectation towards the substance is greater in rats treated in intermittent manner, compared to rats treated in continuous manner [87]. Based on these considerations, the results of this study could be explained by assuming that rats treated with either amphetamine or morphine on alternate days experience a greater expectation towards these substances compared with rats that received the same substances on consecutive days. This greater expectation towards the substances of abuse could be then translated in a more marked emission of conditioned 50-kHz USVs following the re-exposure to the environment previously associated with drug administration. In this regard, it is interesting to highlight that this study found a markedly persistent emission of conditioned 50-kHz USVs in rats treated with morphine, a substance that has significant conditioning effects and high propensity to induce dependence [84]. Based on these considerations, it can be hypothesized that 50-kHz USVs could represent a useful tool for studying the factors that are responsible of compulsive search for substances of abuse, although, a more detailed study of the factors that promote conditioned vocalizations is necessary.
8. CONCLUSIONS
1. This study has demonstrated a significant (p<0.05) emission of 50-kHz USVs by rats repeatedly administered amphetamine, and challenge with the same drug 7 days after treatment discontinuation. Conversely, morphine failed to induce the emission of 50-kHz USVs, even after repeated administration.
2. Re-exposure of rats to the test cage, which was previously associated with drug administration, provoked a significant (p<0.05) emission of 50-kHz USVs in rats treated with either amphetamine or morphine. However, only rats treated with morphine exhibited an emission of conditioned 50-kHz USVs that persisted even after 7 days from the last administration of the substance (p<0.05).
3. The emission of 50-kHz USVs in response to the administration of amphetamine or morphine was not significantly (p>0.05) affected by the protocol of drug administration (continuous or intermittent). Conversely, the emission of conditioned 50-kHz USVs following test cage re-exposure 7 days after repeated treatment discontinuation was stronger in those rats treated on alternate days (p<0.05).
4. Amphetamine was the only substance that stimulated a significant emission of the “trill” subtype of vocalizations, although repeated treatment with this drug did not result in sensitization to this effect, but rather in its attenuation.
9. PRACTICAL RECOMMENDATIONS
This study suggests that investigating the modifications in the emission of 50-kHz USVs by rats treated with substances of abuse can be a useful experimental model to evaluate the expectation towards these substances that may be triggered by the presence of environmental stimuli associated with their intake. Hence, the emission of 50-kHz USVs could be used to implement new experimental models to study the psychological dependence that pushes an individual to the use and search of addictive substances. Substances of abuse induce long-term modifications in the brain which involve second messengers and pathways of signal transduction, and are the basis of drug dependence and relapse. In this regard, it is interesting to notice that the likelihood of relapse is higher in those situations when the individual comes into close contact with environmental cues previously associated with the intake of the drug to which he or she was addicted. The results of this study are in line with this evidence, by showing that rats re-exposed to an environment previously associated with the administration of drugs of abuse (amphetamine or morphine in this particular case), display a marked emission of 50-kHz USVs. Finally, the results of this study indicate that the use of 50-kHz USVs is better suited to evaluate the conditioned effects of drugs of abuse, rather than the acute, direct, rewarding properties of these substances.
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