Morphological and functional evidence of increased excitatory signaling in the prelimbic cortex during ethanol withdrawal
Introduction
Alcohol use disorders (AUDs) represent a spectrum of pathological patterns of alcoholic beverage consumption, which are notably characterized by loss of control over drinking and the emergence of negative affect during abstinence (American Psychiatric Association, 2013). The transition from moderate alcohol consumption to AUD is characterized by a gradual switch from positive to negative reinforcement, whereby the motivation to drink alcohol is no longer driven by the rewarding effects of intoxication, but by relief from the aversive effects of withdrawal (Cooney et al., 1997, de Castro et al., 2007, Johnson and Fromme, 1994, Lee et al., 2013, Pombo et al., 2016, Sinha, 2013). During abstinence, the heightened incentive salience of alcohol-associated cues combined with deficits in inhibitory control contribute to the reinstatement of drinking (Koob and Volkow, 2016).
Abstinence and cue-induced craving in human alcoholics are associated with abnormal activity in a number of prefrontal cortex (PFC) subregions implicated in reward anticipation, decision making and impulse control, including the dorsolateral PFC, ventromedial PFC, orbitofrontal cortex (OFC) and anterior cingulate cortex (Cyders et al., 2014, Jasinska et al., 2014, Seo et al., 2016). AUDs also cause volume loss and impaired functional connectivity of prefrontal regions (see Dupuy and Chanraud, 2016, Oscar-Berman and Marinkovic, 2007 for reviews). In accordance with these imaging data, AUD subjects exhibit deficits in a number of cognitive processes relevant to decision making and emotional processing (see Le Berre et al., 2017 for review).
Rodents exposed to chronic intermittent ethanol (CIE) also exhibit impaired performance in various cognitive tasks when tested during withdrawal. For instance, they show deficits in behavioral flexibility (attentional set-shifting and reversal learning in discrimination tasks), excessive impulsivity (premature responding in the 5-choice serial reaction time task), and impaired fear extinction retrieval (Badanich et al., 2011, Holmes et al., 2012, Hu et al., 2015, Irimia et al., 2014, Irimia et al., 2015, Kroener et al., 2012, Trantham-Davidson et al., 2014). Some of these cognitive abilities are known to depend upon medial PFC (mPFC) functional integrity (Chudasama and Robbins, 2006, Dalley et al., 2004, Myers-Schulz and Koenigs, 2012, Seamans et al., 2008, Uylings et al., 2003). Accordingly, studies investigating the molecular basis of the behavioral phenotypes induced by chronic ethanol exposure have identified local changes in excitatory and inhibitory neurotransmission, as well as monoaminergic modulation, within the mPFC (Holmes et al., 2012, Irimia et al., 2017, Kroener et al., 2012, Pleil et al., 2015, Trantham-Davidson et al., 2014). These functional changes are paralleled by structural alterations in dendritic arborization and spines of mPFC pyramidal neurons (Holmes et al., 2012, Kim et al., 2015, Kroener et al., 2012, Navarro and Mandyam, 2015).
The rodent mPFC is subdivided into prelimbic (PL, dorsally located) and infralimbic (IL, ventrally located) areas that exhibit differential connectivity, neurochemistry and behavioral functionality (Dolan and Dayan, 2013, George and Koob, 2010, Gourley and Taylor, 2016, Heidbreder and Groenewegen, 2003, Moorman et al., 2015, Vertes, 2004). In instrumental learning the PL and IL cortices promote mutually exclusive behaviors such that goal-directed actions require the PL area, while habitual responding requires the IL area (Balleine and Dickinson, 1998, Coutureau and Killcross, 2003, Killcross and Coutureau, 2003). The antagonistic functions of the PL and IL cortices are best illustrated by fear conditioning experiments, whereby the expression of learned fear is reduced both by PL inactivation or IL stimulation, while the extinction of conditioned freezing response is prevented both by IL inactivation or PL stimulation (Milad and Quirk, 2002, Morgan and LeDoux, 1995, Quirk et al., 2000, Vidal-Gonzalez et al., 2006). A similar distinction between behavioral execution and inhibition being driven by the PL and IL cortices, respectively, has also been proposed for reward/drug seeking, although the dichotomy appears less rigid than for aversive conditioning (Moorman et al., 2015, Peters et al., 2009, Pfarr et al., 2015). Finally, the PL and IL cortices play complementary roles in behavioral flexibility and conflict resolution; the IL facilitates the testing of a previously irrelevant strategy upon rule-shifting and reversal learning, while the PL enables the selection and maintenance of the new strategy (Oualian and Gisquet-Verrier, 2010). In accordance with this functional dichotomy, the few studies that have examined ethanol-induced neuroadaptations in both the PL and IL cortices have observed differential alterations of dendritic spines and synaptic transmission in the two subregions (Gass et al., 2014, Jury et al., 2017b, Pleil et al., 2015).
The mPFC is also laminar, with superficial (2/3) and deeper (5) layers exhibiting differential connectivity (Bitzenhofer et al., 2017, DeNardo et al., 2015, Gabbott et al., 2005). While there is in vitro evidence of layer specificity in the effects of chronic ethanol on mPFC pyramidal neurons (Pava and Woodward, 2014), there is no study to date that has directly compared neuroadaptations occurring in layer 2/3 vs layer 5 neurons in vivo.
Therefore, the present study aimed to: 1) investigate spine alterations in layer 2/3 and layer 5 pyramidal neurons of the PL and IL cortices of CIE-exposed mice, 2) determine whether morphological changes were associated with alterations in intrinsic excitability or excitatory synaptic transmission, and 3) assess whether cellular changes were associated with deficits in cognitive flexibility as measured by reversal learning and strategy switching in the Barnes maze. Data were collected 3–8 days into withdrawal from vapor, a time frame when CIE-exposed mice display escalated ethanol drinking and other behavioral alterations (Becker and Lopez, 2004, Jury et al., 2017a, Kroener et al., 2012).
Section snippets
Animals
Male C57BL/6J mice were obtained from The Scripps Research Institute Rodent Breeding Colony at 8 weeks of age and were acclimated to reverse light cycle (12 h light/dark cycle, lights on at 10:15 p.m.) for at least 10 days before any testing or treatment was performed. Mice were housed in a temperature-controlled vivarium (22 °C). Water (acidified) and food (autoclaved Teklad LM-485/Envigo 7012 diet) were available ad libitum. All procedures were carried out in accordance with the National
CIE increases the maturity of dendritic spines in PL layer 2/3 pyramidal neurons
We examined whether CIE exposure followed by 7 days of withdrawal produced dendritic spine remodeling in layer 2/3 and layer 5 pyramidal neurons of the PL and IL cortices. CIE did not alter spine density in either layer of both subregions (Fig. 1). We further analyzed the morphology of dendritic protrusions, which were classified as mushroom (most mature), stubby, thin and filopodia (least mature) (Fig. 2A). There was a significant interaction between spine type and treatment in PL layer 2/3
Discussion
Our study shows that chronic exposure to ethanol followed by one week of withdrawal elicits morphological and functional changes indicative of enhanced AMPA receptor-mediated excitatory transmission in layer 2/3 pyramidal neurons of the PL subregion of the mouse mPFC. Specifically, we found that subjecting mice to 7 weeks of CIE followed by 6–8 days of withdrawal did not affect dendritic spines in PL layer 5 neurons or IL neurons, but selectively increased spine maturity in PL layer 2/3
Conflicts of interest
None.
Acknowledgement
We thank Dr. Michal Bajo from the Department of Neuroscience at The Scripps Research Institute and Dr. Attila Szucs from the Department of Physiology and Neurobiology at Eötvös Lóránd University and the BioCircuits Institute at University of California San Diego for their assistance with electrophysiological data analysis. This work was supported by National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA) grants K99 AA025408 (FPV), T32 AA007456 (HS), F32 AA024952 (HS), U01 AA013498 (MR),
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These authors contributed equally to the work.