|dc.description.abstract||Alcohol use disorders are chronic relapsing conditions that represent one of the leading causes of
preventable death. The enduring propensity to relapse is one of the fundamental features of alcohol use
disorders and stress is a major precipitant of relapse behaviour. The pharmacological stressor, yohimbine,
has been widely used to elicit relapse-like behaviour in rodents and craving in human addicts, however, the
neurochemical mechanisms in which yohimbine exert its actions are still elusive. Neuropeptides and their
receptors offer potential as novel treatments of psychiatric and other disorders as their signalling powerfully
modulates important functions such as stress responses, anxiety and social behaviours. Therefore I examined
the mechanism in which yohimbine precipitates relapse-like behaviour, and region-specific involvement of
five neuropeptide systems; corticotropin releasing factor (CRF), orexin (OX), dynorphin (DYN), cocaine and
amphetamine regulated transcript (CART) and relaxin-3 in yohimbine-induced reinstatement of alcohol
Firstly, to determine brain regions in which yohimbine-induced relapse to alcohol seeking specifically
activates, the pattern of neural activation following home cage yohimbine (1 mg/kg i.p.) and yohimbineinduced
relapse to alcohol seeking was examined. Fos immunohistochemistry (IHC) across 40 brain regions
revealed a distinct pattern of neural activation. A number of regions were activated specifically following
yohimbine-induced reinstatement of alcohol seeking including the dorsal hippocampus and hypothalamus,
while the prefrontal cortex and extended amygdala were activated by both home cage yohimbine
administration and yohimbine-induced reinstatement of alcohol seeking. These data suggest the prefrontal
cortex and extended amygdala may be activated by yohimbine to drive reinstatement of alcohol seeking,
while other brain regions are recruited during this process. In light of these novel results I examined the
neurochemical phenotype of the activated neurons within the extended amygdala and hypothalamus.
Yohimbine-induced reinstatement of alcohol seeking increased Fos activation in central nucleus of the
amygdala (CeA) CRF, DYN, CART and GABA neurons; bed nucleus of the stria terminalis (BNST) CRF and
CART neurons; and hypothalamic CART and OX neurons compared to naïve and vehicle controls.
The neuropeptide relaxin-3 and its cognate receptor, relaxin family peptide receptor 3 (RXFP3), modulate
stress-induced reinstatement of alcohol seeking in rats and while the BNST has been implicated in this
regard, the CeA also receives a relaxin-3 innervation and CeA neurons densely express RXFP3 mRNA.
Therefore, the role of the relaxin-3/RXFP3 signalling within the CeA in yohimbine-induced reinstatement of
alcohol seeking was examined. Bilateral intra-CeA injections of the selective RXFP3 antagonist, R3(B1-22)R (1
μg/side), attenuated yohimbine-induced reinstatement of alcohol seeking, suggesting relaxin-3/RXFP3
signalling in the CeA as another node that mediates yohimbine-induced reinstatement of alcohol seeking.
The major source of relaxin-3 is confined to a small stress-sensitive hindbrain region, the nucleus incertus
(NI). Relaxin-3 NI neurons express receptors for stress neuropeptides including CRF1, CRF2, OX1 and OX2.
Previous reports implicate OX2, but not OX1 receptors within the NI to mediate yohimbine-induced
reinstatement of alcohol seeking. Although OX neurons directly innervate the NI, the pattern of innervation
is unknown, therefore to determine the identify the OX inputs to the NI that are activated following
yohimbine-induced reinstatement of alcohol seeking, the retrograde tracer cholera toxin subunit β (CTβ; 80
nl) was discretely injected into the NI and activation of OX inputs to the NI examined. Initial findings
revealed back-labelled neurons evenly throughout OX neuron-containing regions of the hypothalamus,
some of which were activated following stress-induced reinstatement of alcohol seeking. To examine the
contribution of NI CRF1 and CRF2 receptors in yohimbine-induced relapse reinstatement of seeking, bilateral
injections of the CRF1 antagonist (CP376395, 500 ng) or CRF2 antagonist (astressin-2B, 200 ng) were
administered discretely within the NI. Intra-NI antagonism of CRF1 receptors attenuated yohimbine-induced
reinstatement of alcohol seeking. In contrast, CRF2 receptor antagonism had no significant effect. In line with
these data, CRF1, but not CRF2, receptor mRNA was upregulated in the NI following chronic ethanol intake.
Our quantitative PCR analysis also identified CRF mRNA within the rat NI, and the existence of a newly
identified population of CRF-containing neurons was subsequently confirmed by detection of CRF
immunoreactivity in rat and mouse NI. These data suggest that NI neurons contribute to reinstatement of
alcohol seeking, via an involvement of CRF1 signalling. Furthermore, chronic ethanol intake leads to
neuroadaptive changes in CRF and relaxin-3 systems within rat NI.
The source of CRF to the NI remains elusive. This in part is hindered by the difficulties in the detection of
CRF peptide immunoreactivity without the prior administration of colchicine. Therefore to examine the
putative CRF inputs to the NI, a Crf-IRES-Cre::Ai14 reporter mouse was employed. Immunohistochemical
evaluation of the Crf-IRES-Cre mouse showed good validity in the distribution and concordance of tdTomato
reporter distribution with CRF immunoreactivity. Further, tdTomato neurons in the PVN were responsive to
both acute forced swim stress and yohimbine administration; while chronic stress led to a baseline increase
in tdTomato activation across several stress sensitive brain regions. Using Allen Brain Institute resources to
determine CRF innervation of the NI revealed that very little CRF innervation to the NI originates in CRF-rich
regions, suggesting that either more diffuse CRF+ regions send projections to the NI, or that other
mechanisms, such as intrinsic CRF release or volume transmission are involved.
These findings represent a major contribution to the field and provide further evidence that the CRF, DYN,
OX and relaxin-3 systems are involved in stress-induced alcohol seeking behaviours in rats. These studies
also provide the first evidence that the CART system may be involved in stress-induced relapse behaviours,
and may provide a novel target to treat relapse behaviours. These studies also warrant further examination
of the relaxin-3/RXFP3 system particularly in stress and reward related disorders.||en_US