Elsevier

Brain Research

Volume 1726, 1 January 2020, 146505
Brain Research

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

https://doi.org/10.1016/j.brainres.2019.146505Get rights and content

Highlights

  • Minimal sensitization protocol increases both 50 kHz USVs and ergometric movements.

  • AMPH sensitization of 50 kHz USV and ergometric movement are not correlated.

  • No observed difference in amount of Zif-268 staining between rats grouped by USV.

  • Observed regional correlations in rats grouped by duration of USV calling.

  • Zif-268 expression suggests association of medial corticostriatal regions with USVs.

Abstract

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.

Introduction

The measurement of ultrasonic vocalizations (USVs) in adult rats is thought to reflect internal motivational and emotional states (Brudzynski, 2009, Brudzynski, 2013). Recording of rat USVs has been utilized effectively as a quantifiable metric of emotional states in a wide variety of experimental settings (Wöhr and Schwarting, 2013, Rippberger et al., 2015). The two primary USV categories (the 22 kHz and 50 kHz USV types) in the rat are differentiated by general sonographic character (emitted sound frequency, call duration, etc.), the context their emission occurs in (negative versus positive valence contexts), and their underlying neurochemical systems (for reviews see Brudzynski, 2013, Brudzynski, 2015, Brudzynski et al., 2018). Emission of 50 kHz USVs is generally attributed to activity of the ascending mesolimbic dopamine system as both direct and indirect modulations to the activity of this system produce profound effects on calling behaviour (Knutson et al., 1998, Knutson et al., 1999, Burgdorf et al., 2007, Ciucci et al., 2007, Ciucci et al., 2009, Scardochio et al., 2015). The degree of call induction has been postulated as reflective of an underlying positive emotional arousal and associated level of developed emotional state (Burgdorf et al., 2011a, Barker et al., 2015, Brudzynski et al., 2018, Simola and Brudzynski, 2018).

A similar behavioural measure of this arousal and emotional state often employed in the rat beside recording of USVs, is locomotor activity (Taracha et al., 2012, Taracha et al., 2014, Garcia and Cain, 2016). Both of these behaviours are increased after application of drugs that directly stimulate the mesolimbic dopamine system. Moreover, these behaviours share a functional association with medial aspects of cortico-striatal topography, whereby the ‘limbic’ or ‘motivational’ basal ganglia circuits are popularly believed to reside (for helpful review see Ikemoto et al., 2015). Neural changes within such medial cortico-striatal brain regions occurs upon repeated exposure to evocative stimuli (whether pharmacological or contextual). Such changes are believed to underly part of an organism’s adaptive response to emotionally salient contexts (Ikemoto et al., 2015). As such, USVs and motor activity feature prominently in many experimental paradigms centered around models of addiction and sensitization (Hooks et al., 1991, Hooks et al., 1992, Panksepp et al., 2002, Browning et al., 2011, Maier et al., 2012, Mahler et al., 2013, Simola et al., 2014, Barker et al., 2015).

Several such studies have recently found that these two behavioural measurements respond differently to psychostimulant sensitization (Taracha et al., 2012, Taracha et al., 2014, Ahrens et al., 2013, Costa et al., 2015, Garcia and Cain, 2016). In addition to a dissociation between calling and locomotor behaviours, this sensitization research has revealed extensive inter-individual variability in their expression (Taracha et al., 2012, 2014, 2016). In several studies that employed psychostimulants to investigate 50 kHz calling, the initial individual variability was used to screen out or categorize subjects (Wright et al., 2010, Wright et al., 2012, Taracha et al., 2012, Taracha et al., 2014, Taracha et al., 2015, Taracha et al., 2016, Ahrens et al., 2013).

The psychostimulant D-amphetamine (AMPH) has been used extensively to investigate expression and sensitization of 50 kHz USVs (Ahrens et al., 2009, Simola and Morelli, 2015, Taracha et al., 2016, Simola and Costa, 2018). Most knowledge about the possible brain areas involved in AMPH-induced 50 kHz calling has been inferred from targeted pharmacological studies (i.e., AMPH microinjections into the shell of the nucleus accumbens, NAcSh; Burgdorf et al., 2001, Thompson et al., 2006).

The degree of involvement among various forebrain regions with this AMPH-induced USV sensitization has begun to be investigated using changes in the expression of transcription factors associated with immediate early genes (IEGs). The detection of well characterized IEG-associated inducible transcription factors such as Fos or Zif-268 has been reliably used to index levels of neural activation associated with psychostimulant administration (Beckmann and Wilce, 1997, Steiner, 2010). The research into psychostimulant-induced brain activation has extended the findings of dissociation in behavioural sensitization between locomotor and 50 kHz calling activities (Costa et al., 2015, Hamed et al., 2016, Kaniuga et al., 2016).

These studies have revealed a complex network of associated brain systems possibly involved in the expression and sensitization of 50 kHz USVs. Importantly, a role of both cortical and subcortical forebrain structures in promoting the AMPH-induced 50 kHz USVs has been highlighted (Costa et al., 2015, Hamed et al., 2016, Kaniuga et al., 2016). In line with this, Costa and colleagues (2015) established a critical role of glutamatergic signaling for the acute and conditioned effects of AMPH-induced 50 kHz calling and AMPH-induced Zif expression. However, this research has either used extensive sensitization protocols (>2 AMPH injections) or overlooked possible inter-individual variation.

The purpose of the present study was to complement this earlier work by investigating the relationships between brain regions of interest and 50 kHz calling in minimally sensitized rats (two injections). Using this approach, it was hoped that the possible forebrain networks associated with the individual variability observed in 50 kHz calling after AMPH could be explored and identified by Zif as a molecular marker. We also sought to determine whether the dissociation of AMPH-induced behavioural sensitization observed between 50 kHz USVs and locomotor activity could be extended to a more general measure of ergometric activity that, in addition to locomotor activity, includes all major muscular activity of the body. We assessed behavioural augmentation between two injections of AMPH for small and large movements, 50 kHz USV call rate, and mean sound frequency of emitted 50 kHz USVs. Differences between injections in favour of augmentation were taken as indicative that they represented behavioural forms of sensitization to the drug. The current research also ultimately aimed at determining if patterns of AMPH-induced gene regulation would correlate with 50 kHz USV behaviour across cortical and striatal forebrain regions. These regions included the medial prefrontal cortex (prelimbic, PL; and infralimbic, IL), ventral striatum (nucleus accumbens core, NAcC; and nucleus accumbens shell, NAcSh), medial portion of the dorsal striatum (DMS), premotor cortex (M1), and basolateral amygdala (BLA). We predicted that 50 kHz calling and general ergometric activity would be separable behavioural measures although both would show sensitization to two-injections of AMPH.

Section snippets

Amphetamine administration and the sensitization of measured ergometric activity

As anticipated, a (2 × 2) repeated measures ANOVA on large movements across the entire cohort found a significant main effect of drug injection (first versus second injection, INJ1 vs INJ2; F1,23 = 28.9, p < .001), significant main effect of timepoint (pre-injection versus post-injection, Pre-Post; F1,23 = 164.8, p < .001), and a significant interaction effect (INJ × PrePost; F1, 23 = 22.1, p < .001). Observed large movements (large scores of ergometric activity) were greater in INJ2 recordings

Discussion

In the present study we obtained measures of ergometric activity, 50 kHz calling, and expression of Zif protein across several cortical and subcortical brain regions after a TIPS protocol using AMPH. The results suggested that although both movement activity and 50 kHz USV measures did show aspects of sensitization after the TIPS protocol, these two behavioural measures were distinct, dissociable and not positively correlated. Moreover, the present study identified a measure of 50 kHz USV

Conclusions

In the present study we demonstrated that the effect of a two-injection sensitization with AMPH differentially affects measures of ergometric activity and 50 kHz USV emission. Moreover, we found that after the second AMPH injection, there was considerable inter-individual variability in time spent calling which was dissociable from other measures of sensitization. Within the whole experimental cohort, following the second AMPH injection, there was correlative evidence of induced

Subjects

A total of 31 male Long Evans rats (Charles River Laboratories, Saint-Constant, QC, Canada) were used in this study. Rats were 50 days old at their entry into the experiment with an average weight of 231 g at the start and an average weight of 321 g at the end of the experiment. In accordance with Brock University protocols for laboratory handling, all rats were housed in pairs in polycarbonate cages (48 × 27 × 20 cm) with a plastic tube inside for hiding and an aspen block of wood for

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada via Discovery Development Grant to S.M.B. The paper is part of the doctoral dissertation of K.G.M.

Compliance with ethical standards

All animal use procedures were conducted in compliance with guidelines and policies set forth by the Canadian Council on Animal Care and were approved by the institutional ethical committee for animal experimentation.

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      In this line, lisdexamfetamine (LDX), a pro-drug of amphetamine, induces increased locomotor activity and 50-kHz USV that are prevented by lithium or valproate administration (Macêdo et al., 2013; de Souza et al., 2015; Wendler et al., 2016; Bristot et al., 2019). According to their spectrogram shapes, 50-kHz USV can be classified into different subtypes (Costa et al., 2020; Mulvihill and Brudzynski, 2020; Wright et al., 2010) and we have applied a rather common classification into four subtypes: trill, step, mixed and flat (e.g. Pereira et al., 2014; Wöhr et al., 2015). The trill subtype has been related to positive affect, while the flat subtype is probably associated to social communication (Wöhr and Schwarting, 2013; Simola and Brudzynski, 2018).

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