br Methods Adult male B CRHtklee mice g
Methods Adult male B6, 129 CRHtklee mice (25–30g at the beginning of the experiments) that were wild-type (CRF1R+/+) and recessive homozygous or knockout (CRF1R−/−) were housed six per cage. Mice were 3–5months old and derived from mating CRF1R+/− breeders (Jackson Laboratory, California). Wild-type and CRF1R−/− offspring were identified by PCR analysis of tail DNA. Mice colony room was maintained under a 12-h light/dark cycle (lights on from 8a.m. until 8p.m.) at 22±2°C. Food and water were available ad libitum. Animals were handled daily during one week prior the beginning of the experiment to minimize stress. All surgical and experimental procedures were performed in accordance with the European Communities Council Directive 24 November 1986 (86/609EEC) and the local Animal Ethics Committee (REGA ES300305440012).
Results The weight of the animals was checked along the experiments since it is known that chronic morphine treatment induces a decrease in body weight gain due to a lower caloric intake. Two-way ANOVA for body weight gain revealed a main effect of morphine treatment (F1,44=194.57, p<0.0001) but there was neither significant genotype effects (F1,44=0.70, p=0.4060) nor significant interaction between genotype and morphine treatment (F1,44=0.00, p=0.9983). Our results showed that wild-type and CRF1R knockout mice receiving morphine treatment had a significantly (p<0.001) lower body weight than animals receiving saline injection (Fig. 1A). Present results are in agreement with previous studies (Papaleo et al., 2007) which demonstrated that CRF1R deficiency did not affect body weight reduction gain induced by escalating doses of morphine.
Discussion In the current study, morphine withdrawal was precipitated by naloxone administration to morphine-treated wild-type or CRF1R KO mice. Although the spontaneous withdrawal is more similar to the human condition (Cobuzzi and Riley, 2011), in the current assessment we used antagonist-precipitated withdrawal since the effects of spontaneous withdrawal are less pronounced and more difficult to detect and because acute withdrawal occurs more reliably when it is precipitated by an antagonist. Our data indicate a weight loss after naloxone-precipitated withdrawal in wild-type mice; the weight loss in morphine-withdrawn CRF1R knockout animals was significantly attenuated. In general, when naloxone was injected to morphine-treated wild-type mice, animals showed higher motor activity and diarrhea than CRF1R knockout mice. These findings are consistent with previous reports from our laboratory (Navarro-Zaragoza et al., 2010, Garcia-Carmona et al., 2012) and other laboratories (Iredale et al., 2000, Lu et al., 2000). However, our finding ligand gated with previous studies (Papaleo et al., 2007) showing an increase in some signs of opiate withdrawal in CRF1R−/− mice. This discrepancy can be justified by following explanation: Different types of morphine addiction regimens (i.e., doses of morphine, time of treatment) have been used in the latter study regarding present one. In addition, the last study employed spontaneous morphine withdrawal whereas herein we used opioid-antagonist-precipitated morphine withdrawal and this can be responsible of the different results obtained. Accordingly, previous studies showed that challenging opiate-dependent animals with opioid receptor antagonists induced behavioral signs that greatly differed, in both intensity and type, from those observed in animals undergoing spontaneous opiate withdrawal (Cicero et al., 2002, Houshyar et al., 2004, Linseman, 1977, Mucha et al., 1979, Papaleo and Contarino, 2006, Papaleo et al., 2007, Papaleo et al., 2008, Ruiz et al., 1996) suggesting major behavioral and molecular differences between spontaneous and opioid receptor antagonist-precipitated opiate withdrawal. Previous reports have demonstrated that the activation of noradrenergic terminals innervating PVN modulate the HPA axis activity in response to morphine withdrawal. Thus, naloxone-precipitated morphine withdrawal increased NA turnover and c-Fos expression in PVN concomitantly with an increase in the activity of the TH positive neurons in NTS (as reflected by c-Fos expression) (Laorden et al., 2002a, Laorden et al., 2002b, Smith and Aston-Jones, 2008). It is known that PVN is mainly innervated by the A1 in VLM, and A2 in NTS (Cunningham and Sawchenko, 1988), brain areas related with the cardiac activity (Dampney et al., 2000, Kc and Dick, 2010). The present findings demonstrated a significant elevation of NA turnover in the PVN, which projects to NTS and VLM in morphine withdrawn wild-type mice. In addition, we reported that morphine withdrawal is associated with an increase in c-Fos expression in VLM and in the number of TH phosphorylated at serine 40 positive neurons co-expressing c-Fos (a marker of neuronal activity) demonstrating an enhancement of TH activity. There is evidence supporting the idea that CRF neurons in PVN innervate noradrenergic brainstem nuclei (Gray and Magnuson, 1987), and the existence of a NA-CRF loop in which NA would stimulate the release of CRF in PVN. CRF from this nucleus would induce the release of NA in the brainstem noradrenergic areas (Koob, 1999b). In addition, it is known that CeA and BNST contain high density of CRF neurons that are densely innervated by noradrenergic terminals (Delfs et al., 2000, Forray and Gysling, 2004). Retrograde tracing studies have revealed that the majority of NA inputs to extended amygdale and to the hypothalamic PVN arise from the ventral noradrenergic bundle originating in NTS and VLM in the caudal brainstem (Smith and Aston-Jones, 2008). In this regard, previous results from our laboratory showed a significant increase of the number of CRF-containing neurons in PVN and BNST (Nuñez et al., 2010) and an enhanced expression of c-Fos labeling of TH-positive neurons in the NTS (Laorden et al., 2002a) after naloxone-precipitated withdrawal.