Research report
Morphine self-administration into the lateral septum depends on dopaminergic mechanisms: Evidence from pharmacology and Fos neuroimaging

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Abstract

Mice self-administer morphine into the lateral septum (LS), but the neuronal connections underlying this behaviour remain unknown. The present study tested whether the acquisition of intra-LS morphine self-administration depends on dopaminergic mechanisms. Mice were allowed to self-inject morphine (5 or 20 ng/50 nl) or vehicle directly into the LS using a spatial discrimination Y-maze task. Fos imaging was used to evaluate neuronal activation in cerebral structures directly connected to the LS or belonging to the dopaminergic system. The involvement of dopaminergic and opioidergic mechanisms was assessed by pre-treating naive mice peripherally with the D1 antagonist SCH23390, the D2/D3 antagonist sulpiride or the opiate antagonist naloxone before daily self-administration sessions. Mice acquired self-administration behaviour for intra-LS morphine that was associated with increased Fos expression in the ventral tegmental area (VTA), dorsal and ventral striatum and prefrontal cortex. Pre-treating animals with naloxone, SCH23390 or sulpiride completely prevented them from acquiring intra-LS morphine self-administration. All three antagonists consistently blocked Fos expression in the prefrontal cortex, but not in the VTA and striatum. Taken together, our results show that morphine self-administration into the LS depends on dopaminergic (D1 and D2/D3) and opioidergic mechanisms. Furthermore, they suggest that opioid peptides released in the LS could participate in regulating the activity of mesotegmental dopaminergic neurons.

Introduction

The septal region was the first “reward centre” to be identified [50], but its implication in reward processes has been poorly explored further. Rats display intra-septal self-administration behaviour for both morphine [81] and met-enkephalin [82], suggesting that the septum participates in the reinforcing properties of opiates. This hypothesis is supported by anatomical data showing that mu, delta and kappa opioid receptors are expressed throughout the septal region [1], [2], [42]. Mice also self-administer morphine into the septal area, and this behaviour is acquired more rapidly with LS as opposed to MS injections [10]. Thus the lateral septum seems to play an important role within the septal area in supporting local morphine self-administration.

The lateral septum is connected to the ventral tegmental area (VTA) through both direct and indirect reciprocal projections [31], [65], suggesting that the LS could support morphine self-administration by modulating mesolimbic activity [75]. The dopamine (DA) system arising from the VTA has received considerable attention as the major neurobiological substrate involved in mediating the rewarding properties of opiates [3], [17], [35], [76], [86], although not the only one [53], [58]. Neurochemical data have consistently supported the existence of a functional opiate-DA interaction. Systemic injections of heroin [29] or of mu or delta opioid receptor agonists [29], [59], [60], [80] increase the release of mesolimbic extracellular DA. Consistently, electrophysiological studies have identified a stimulant effect of opiates on dopaminergic neurons in the VTA [26], [45]. Electrical stimulation of the LS also regulates the firing rates of VTA neurons [41] suggesting that the rewarding properties of morphine, when injected into the LS, involve connections with the VTA and rely on dopaminergic mechanisms.

Our first experiment was designed to evaluate neuronal activation in brain regions that are sources or terminals of DA neurons in animals self-administering morphine into the LS, using c-fos labelling. A few additional regions were selected for being direct anatomical targets of the LS, in an attempt to assess the activity of LS projection neurons. Fos immunochemistry has been widely used as an indirect technique to map neuronal activation [18], [30], [70]. Furthermore, induction of Fos protein is known to be highly sensitive to morphine treatment under various experimental conditions [6], [19], [23], [85].

Considering the proximity of the lateral septal region and the nucleus accumbens (NAc), specifically the shell part of the NAc (AcbSh), which is also known to support opiate self-administration [13], [14], [25], [51], the reinforcing properties of intra-LS morphine may result from diffusion towards this structure. In the present study, the anatomical specificity of intra-LS morphine self-administration was assessed by comparing the performance and the pattern of brain Fos expression of animals receiving 20 ng morphine into the LS with these of animals receiving the same dose of morphine into the dorsal part of the AcbSh.

If morphine self-administration into the LS involves dopaminergic mechanisms, pre-treating animals with DA receptor antagonists should prevent its acquisition. Among DA receptors, D1 and D2 subtypes might not contribute equally to the rewarding and conditioning properties of opiates [40], [43], [77]. Moreover, in case of local injections, the balance between D1 and D2 mechanisms may differ depending where morphine is infused [14]. We designed a second series of experiments to test the effects of pre-treating mice with either the selective D1 antagonist SCH 233390 or the D2/D3 antagonist sulpiride on the acquisition of intra-LS morphine self-administration, and we compared them with the effects of pre-treatment with an opiate receptors antagonist (naloxone), expected to prevent morphine self-administration. Immunochemistry for Fos protein is particularly sensitive to dopaminergic manipulations [66], [69]. We used this technique in the present study to evaluate the consequences of previous pharmacological treatments on neuronal activity in DA-related cerebral structures and anatomical targets of the LS.

Section snippets

Animals and surgery

Ninety-one male mice of the inbred BALB/cByJICO strain obtained from Iffa-Credo (Lyon, France) were used. At 10 weeks of age, they were housed individually with ad libitum access to food and water in a temperature-controlled room (23 ± 1 °C) maintained on a 12 h light/dark cycle (lights on at 7.00 a.m.). Mice were aged 14–18 weeks and weighed about 28–32 g at the beginning of the experiments.

Under deep anaesthesia (Avertin, 300 mg/kg ip; local Xylocaine, 5%), animals were implanted unilaterally, in a

Histology

Cannula placements within the lateral septum and the shell of the nucleus accumbens were verified histologically on Fos-stained sections by using the track of the guide and the injection cannula. Representative injection sites are shown in Fig. 2. Only animals with injection sites located within the targeted structure were included in the analysis. In experiment I, final group sizes were n = 7–8 for intra-LS as well as intra-AcbSh self-administration. In experiment II, group sizes were n = 5 for

Anatomical specificity of intra-LS morphine self-administration

The present study confirms that morphine injection into the LS induces regular self-administration behaviour in naive mice [10] over a four-fold dose range (5 or 20 ng/50 nl). Therefore, stimulation of opioid receptors into the LS support operant responding, despite anxiogenic effects observed in the elevated plus-maze and four-hole box after forced injections in a comparable dose range [37]. Cocaine similarly supports vigorous self-administration behaviour despite anxiogenic properties [15], [20]

Acknowledgements

We thank Drs. C. Olmstead and A. Pradhan for their helpful comments on a draft of the manuscript. This investigation was supported by grants from the CNRS (UMR 5106).

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