Research Proposal (non-funded; 2015)
Background and Significance. Corticotropin releasing factor (CRF) may play a critical role in the motivational effects of continued alcohol abuse [1]. While CRF has been intensively studied for its function in stress and anxiety within the HPA axis, it its function in key limbic areas position this peptide to regulate affective behaviors. Two regions that are critical to learning and affective control are the lateral portions of the central nucleus of the amygdala (LCeA) and bed nucleus of the stria terminalis (LBNST). CRF+ cells project to other brain regions where they can influence post-synaptic cells containing CRF receptors. Modulation of the CRF system in animal models of alcohol use disorder will elucidate the neurocircuitry underlying behavioral aspects of the disorder and highlight potential therapeutic approaches to its treatment.
CRF and alcohol. Repeated ethanol exposure in adulthood has a significant impact on CRF in the extended amygdala. For example, repeated ethanol exposure significantly elevates CRF mRNA in the LCeA and type-1 CRF receptor (CRFr1) mRNA in the basolateral amygdala (BLA) and medial division of the CeA (MCeA) in rats [2, 3]. In contrast, such exposure leads to a decrease in CRFr2 mRNA in the BLA [3]. CRFr1s and CRFr2s typically have apposing effects on stress, anxiety, and fear; CRFr1 activation is associated with increases in anxiety while activation of CRFr2 decreases anxiety [4]. Alcohol-induced alterations to the CRF system within the extended amygdala are hypothesized to lead to anxiety-like and alcohol-seeking behaviors during alcohol withdrawal [1]. During ethanol withdrawal, CRF release increases within the CeA and the BNST of alcohol0-dependent rats [5, 6], with increases evident as early as 6-8hrs post-alcohol cessation [1]. CRFr1 antagonist treatment during alcohol-withdrawal reduces alcohol self-administration [2, 7] and anxiety-like behavior [8] in dependent rats. Likewise, treatment with CRFr2 agonists in the CeA during withdrawal also reduces alcohol self-administration in dependent rats [see 1]. Withdrawal-induced changes in the CRF system may persist for extended periods. For example, in rodents subjected to repeated cycles of alcohol intoxication and withdrawal, increased levels of CRF and CRFr1 persisted for weeks following alcohol cessation [3].
CRF and fear conditioning. In addition to its role in stress and anxiety-like behaviors, CRF modulates fear conditioning, a learning paradigm whereby a previously neutral conditioning stimulus (CS; e.g., light or context) is paired with an aversive unconditioned stimulus (US; e.g., foot shock). After repeated CS-US pairings, presentation of the CS alone will elicit a species-typical fear response (i.e., freezing). Rats administered CRF via ICV show increases in fear conditioning [9] and injections of CRFr1 antagonists impair long-term, but not short-term context fear memories [10]. Rats subjected to repeated ethanol exposure followed by a withdrawal period prior to fear conditioning show elevated fear acquisition and impaired fear extinction [11-13]. Likewise, rats fear conditioned prior to receiving repeated alcohol exposure followed by withdrawal show elevated fear responses and impaired fear extinction, suggesting that alcohol exposure also affects fear retrieval [14]. An examination of neural activity following fear conditioning demonstrated elevated c-Fos expression in the prelimbic (but not the infralimbic) cortex, the BLA, and the MCeA in rats that received repeated alcohol exposure compared to controls [14]. Since repeated alcohol exposure increases CRF or CRFr1s in the CeA, BLA, and the mPFC [1, 4], and because alcohol-related changes in CRF may underlie increased glutamatergic transmission in the CeA following repeated alcohol exposure [15], fear conditioning impairments in alcohol-exposed rats may result indirectly from altered glutamatergic activity within the amygdala. The proposed experiments will examine the role of CRF in impairments in fear conditioning and extinction in Cre-CRF rats repeatedly exposed to alcohol. In Crf-Cre rats, Cre is expressed in 95% of CRF-positive cells in the LCeA and LBNST [16]. By injecting Crf-Cre rats with an adeno-associated virus (AAV) construct that targets Cre+ cells (AAV-Ef1α-DIO-εYFP), CRF+ cells can be infected with light-sensitive (e.g., ArchT) or CNO-sensitive (e.g., hM4Di) receptors. Application of green laser light or CNO can therefore selectively inactivate CRF+ cells during specific periods of learning or during alcohol withdrawal (see below). Utilizing these methods, this proposal asks:
AIM 1: Does optogenetic inactivation of CRF-containing neurons in the LCeA or LBNST during conditioning mitigate fear conditioning impairments in rats following withdrawal of repeated alcohol exposure?
AIM2: Does chemogenetic inactivation of CRF-containing neurons in the extended amygdala during alcohol withdrawal mitigate subsequent alcohol-induced impairments in fear conditioning?
Design and Procedures. The subjects will be adult male Crf-Cre Wistar rats as previously described [16]. Rats will undergo stereotaxic infusions of AAV and optic fiber implantation targeted bilaterally at the LCeA (AP -1.88 from Bregma, ML ± 3.8, DV -8.0). For experiments in AIM 1 (Exps. 1.1, 1.2), rats will be infused with AAV-Ef1α-DIO-εArchT3.0-εYFP (CRF-ArchT) or AAV-Ef1α-DIO-εYFP (CRF-YFP). Both CRF-ArchT and CRF-YFP rats will receive green light illumination (10mW at tip, 532 nm) at specified times. For experiments in Aim 2 (Exps. 2.1, 2.2), rats will be infused with AAV-DIO-hM4Di-mCherry (Cre-hM4Di). Rats will be injected with 2 mg/kg CNO or saline (ip) at specified times (see below). Following one month of recovery, rats will receive daily injections of ethanol (EtOH; 1.5 g/kg) or saline (Sal) over 5 days (Days 1-5) and allowed three days of withdrawal (Days 6-8) prior to behavioral testing as previously described [14]. Rats will undergo fear conditioning (Day 9), context test (Day 10), CS extinction (Day 11), and extinction retention (Day 12) following modified procedures previously described [14]. Fear conditioning and context testing will occur in a conditioning chamber designated Context A (to be determined), while CS extinction and extinction retention will occur in a distinct context (Context B). During fear conditioning, rats will receive three tone (30 s, 4 kHz, 75 dB) CS presentations that co-terminate with a foot shock US (1.0 s, 0.5 mA). The first tone onset will occur 120s into the conditioning session with a variable intertrial interval (ITI) averaging 110s. Twenty-four hours following conditioning, rats will be returned to Context A where they will undergo a stimulus-free 32-minute context test. The following day, rats will be placed into Context B and, following a 120s baseline period, receive 20 CS presentations (60s ITI) over a 32-minute session. During the extinction retention test, rats will be placed into Context B and receive 3 CS presentations (60s ITI) following a 120s baseline period. Following behavioral testing, rats will be transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA in PBS). Brain sections (40 µm) will be collected for verification of cannula placement and immunohistochemical analysis as previously described [16]. Experiments 1.1 and 1.2. Does inactivation of CRF+ cells in the LCeA or LBNST during fear conditioning prevent enhanced fear acquisition in EtOH rats? Experiment 1.1 will involve a 2 (EtOH vs. Sal) x 2 (CRF-ArchT vs. CRF-YFP) design. All rats will receive light stimulation targeted at the LCeA only during CS-US presentations during fear conditioning on Day 9 to determine if CRF+ cells in the CeA contribute to alcohol-induced enhancements of fear conditioning and impaired CS extinction previously described [11, 12, 14]. Previous data demonstrate normal CS acquisition in EtOH rats and we hypothesize no differences between EtOH and Sal. Under illumination, CS acquisition is also hypothesized not to affect conditioning in any group. EtOH rats show impaired contextual fear extinction [13]. We hypothesize that this impairment will be mitigated in the ETOH Crf-ArchT group, with freezing levels similar to Sal Crf-YFP rats. Previous studies demonstrate that knockdown of CRF disrupts the consolidation of contextual fear memories [10]. It is therefore likely that Sal Crf-ArchT will show impaired contextual fear conditioning relative to Sal Crf-YFP rats, providing evidence that Crf+ CeA cells are involved in contextual fear conditioning. We also expect EtOH Crf-YFP rats to show impaired CS extinction and extinction retention relative to Sal Crf-YFP rats. This effect should be mitigated in EtOH Crf-ArchT rats. A previous study showed that CNO inhibition of Crf+ cells in Crf-Cre via hM4Di prior to extinction had no effect on CS extinction and retention [16]. Given these data, light inhibition of Crf+ cells in Sal-ArchT rats is not expected to affect CS extinction or retention. Experiment 1.2 will follow the design of Exp. 1.1 except that light stimulation will target the LBNST. The LCeA and LBNST both express CRF and are highly interconnected, and lesions to both regions disrupt contextual fear conditioning. It is unclear how activity in one of these regions affects activity in the other. The parallel nature of Exp. 1.1 and 1.2 may highlight essential differences between these structures in the modulation of fear conditioning in EtOH rats. Experiment 1.3 and 1.4. Does inactivation of CRF+ cells in the LCeA or LBNST during CS extinction mitigate extinction impairments in EtOH rats? Experiment 1.3 and 1.4 will follow the design of Exp. 1.1 and 1.2 except that light illumination in the LCeA (1.3) or LBNDS (1.4) will occur during the 32 minute context test and again during the 32 minute CS extinction session. If impaired context and CS extinction results from alcohol-induced increases in the CRF system in the extended amygdala, than optogenetic inhibition of Crf+ cells in the CeA should mitigate these effects. Inactivation of Crf+ cells in Sal Crf-ArchT rats is not expected to affect contextual or CS fear retrieval or extinction. Experiment 2.1. Does inactivation of CRF+ cells in the extended amygdala during ethanol withdrawal prevent fear conditioning impairments in EtOH rats? Alcohol-induced changes to the CRF system are evident during the ethanol withdrawal period [1]. Experiment 2.1 will be a 2 (EtOH vs. Sal) x 2 (CNO vs. saline) design. Rats will receive daily injections of CNO or saline during the 3 day withdrawal period following repeated ethanol exposure. Starting on Day 9, rats will undergo fear conditioning as described in Exps. 1.1 & 1.2. If alcohol-induced changes to the CRF system occur during the withdrawal period, than blocking Crf+ cells in the extended amygdala via CNO should mitigate any alcohol-induced fear conditioning impairments. CNO injections should not affect conditioning in Sal rats. Immunohistological Analysis. Brains will undergo immunohistological analysis for Cre, CRF, CRFr1, and c-Fos throughout the extended amygdala (MCeA, LCeA, BLA, and BNDS). Following previous results, we expect high levels of overlap of Cre+ and CRF+ cells in the LCeA and the LBNST [16]. EtOH rats are expected to show behavior-induced elevated c-Fos expression in the MCeA relative to Sal controls, an effect that should be mitigated by inactivation of CRF+ cells in both the LCeA and LBNST. Ethanol treatments are expected to increase CRF in the LCeA and LBNST and increase CRFr1s in the MCeA, BLA, and LBNST as previously described [2, 3], with CNO-mediated inhibition of CRF+ cells during the alcohol-withdrawal period mitigating these effects. Additional Studies. Functional imaging in awake fear conditioned rats has been previously described [17, 18]. Slight modifications to the fear conditioning protocol could allow for functional imaging in EtOH rats during CRF manipulations. For example, by using a flashing light LED as the CS, rats could be trained under light CS-US pairings. The light CS could then be presented during fMRI to examine brain activity during fear retrieval.
References
1. Koob, G.F., A role for brain stress systems in addiction. Neuron, 2008. 59(1): p. 11-34.
2. Roberto, M., et al., Corticotropin releasing factor-induced amygdala gamma-aminobutyric Acid release plays a key role in alcohol dependence. Biol Psychiatry, 2010. 67(9): p. 831-9.
3. Sommer, W.H., et al., Upregulation of voluntary alcohol intake, behavioral sensitivity to stress, and amygdala crhr1 expression following a history of dependence. Biol Psychiatry, 2008. 63(2): p. 139-45.
4. Zorrilla, E.P., et al., Behavioral, biological, and chemical perspectives on targeting CRF(1) receptor antagonists to treat alcoholism. Drug Alcohol Depend, 2013. 128(3): p. 175-86.
5. Funk, C.K., et al., Corticotropin-releasing factor within the central nucleus of the amygdala mediates enhanced ethanol self-administration in withdrawn, ethanol-dependent rats. J Neurosci, 2006. 26(44): p. 11324-32.
6. Olive, M.F., et al., Elevated extracellular CRF levels in the bed nucleus of the stria terminalis during ethanol withdrawal and reduction by subsequent ethanol intake. Pharmacol Biochem Behav, 2002. 72(1-2): p. 213-20.
7. Funk, C.K., et al., Corticotropin-releasing factor 1 antagonists selectively reduce ethanol self-administration in ethanol-dependent rats. Biol Psychiatry, 2007. 61(1): p. 78-86.
8. Overstreet, D.H., D.J. Knapp, and G.R. Breese, Modulation of multiple ethanol withdrawal-induced anxiety-like behavior by CRF and CRF1 receptors. Pharmacol Biochem Behav, 2004. 77(2): p. 405-13.
9. Skorzewska, A., et al., The effect of CRF and alpha-helical CRF((9-41)) on rat fear responses and amino acids release in the central nucleus of the amygdala. Neuropharmacology, 2009. 57(2): p. 148-56.
10. Pitts, M.W., et al., The central nucleus of the amygdala and corticotropin-releasing factor: insights into contextual fear memory. J Neurosci, 2009. 29(22): p. 7379-88.
11. Bertotto, M.E., et al., Influence of ethanol withdrawal on fear memory: Effect of D-cycloserine. Neuroscience, 2006. 142(4): p. 979-90.
12. Holmes, A., et al., Chronic alcohol remodels prefrontal neurons and disrupts NMDAR-mediated fear extinction encoding. Nat Neurosci, 2012. 15(10): p. 1359-61.
13. Broadwater, M. and L.P. Spear, Consequences of ethanol exposure on cued and contextual fear conditioning and extinction differ depending on timing of exposure during adolescence or adulthood. Behav Brain Res, 2013. 256: p. 10-9.
14. Quinones-Laracuente, K., et al., The effect of repeated exposure to ethanol on pre-existing fear memories in rats. Psychopharmacology (Berl), 2015. 232(19): p. 3615-22.
15. Roberto, M., N.W. Gilpin, and G.R. Siggins, The central amygdala and alcohol: role of gamma-aminobutyric acid, glutamate, and neuropeptides. Cold Spring Harb Perspect Med, 2012. 2(12): p. a012195.
16. Pomrenze, M., et al., A Crh-Cre rat for studying the anatomy and function of CRF circuits. Society for Neuroscience (Poster Abstract), 2015.
17. Harris, A.P., et al., Imaging learned fear circuitry in awake mice using fMRI. Eur J Neurosci, 2015. 42(5): p. 2125-34.
18. Brydges, N.M., et al., Imaging conditioned fear circuitry using awake rodent fMRI. PLoS One, 2013. 8(1): p. e54197.
Nathen Murawski 