Role of dorsal hippocampal muscarinic receptor activity in acquisition and retention of single- versus multiple-trial contextual fear conditioning in adolescent rats.

The present study examined the effects of the muscarinic acetylcholine receptor (mAChR) antagonist, scopolamine, on standard contextual fear conditioning (sCFC). It compared effects of the drug on acquisition (post-shock freezing) versus 24-hr retention of a context-shock association acquired after one or three pairings of a context with unsignaled shock. During single-trial sCFC, systemic scopolamine (0.5 mg/kg, i.p.) prior to training abolished both post-shock and retention freezing (Experiment 1). This same injection during multiple-trial sCFC also abolished post-shock freezing and impaired 24-hr retention freezing (Experiment 2). These results indicate that cholinergic signaling mediates both acquisition and 24-hr retention of a context-shock association across different trial parameters. Experiment 3 further explored these effects by infusing scopolamine (35 µg per side) into the dorsal hippocampus (dHPC) prior to training in single versus multiple-trial sCFC. This infusion spared post-shock but abolished retention test freezing in single-trial sCFC (Experiment 3A), and had no effect on multiple-trial sCFC (Experiment 3B). The current findings suggest that brain-wide cholinergic signaling mediates acquisition and retention of single-trial sCFC. Despite this, while muscarinic cholinergic signaling in the dHPC does mediate retention of single-trial sCFC, it is not required for acquisition of either variant, or retention of multiple-trial sCFC. These findings also rule out impaired sensory processing of contextual cues as a mechanism of impaired context learning by dHPC scopolamine. The results are discussed in relation to the role of cholinergic function across multiple brain memory systems in elemental versus configural forms of contextual fear conditioning (Fanselow, 2010; Rudy, 2009). (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Prefrontal NMDA-receptor antagonism disrupts encoding or consolidation but not retrieval of incidental context learning.

The Context Preexposure Facilitation Effect (CPFE) is a variant of contextual fear conditioning in which learning about the context, acquiring a context-shock association, and retrieval of this association occur separately across three phases (context preexposure, immediate-shock training, and retention). We have shown that prefrontal inactivation or muscarinic-receptor antagonism prior to any phase disrupts retention test freezing during the CPFE in adolescent rats (Heroux et al., 2017; Robinson-Drummer et al., 2017). Furthermore, the medial prefrontal cortex (mPFC) is the only region in which robust learning-related expression of the immediate early genes c-Fos, Arc, Egr-1 and Npas4 is observed during immediate-shock training in the CPFE (Asok et al., 2013; Heroux et al., 2018; Schreiber et al., 2014). However, the role of prefrontal NMDA-receptor plasticity in supporting preexposure- and training-day processes of the CPFE is not known. Therefore, the current study examined the effects of intra-mPFC infusion of the NMDA-receptor antagonist MK-801 or saline vehicle prior to context preexposure (Experiment 1) or immediate-shock training (Experiment 2) in adolescent Long-Evans male and female rats. This infusion given prior to context preexposure but not training abolished retention test freezing, with no difference between MK-801-infused rats and non-associative controls preexposed to an alternative context (pooled across drug). These results demonstrate a role of prefrontal NMDA-receptor plasticity in the acquisition and/or consolidation of incidental context learning (i.e., encoded in the absence of reinforcement). In contrast, this plasticity is not required for context retrieval, or acquisition, expression, or consolidation of a context-shock association during immediate-shock training in the CPFE. These experiments add to a growing body of work implicating the mPFC in Pavlovian contextual fear conditioning processes in rodents.

Mechanisms of context conditioning in the developing rat.

The article reviews our studies of contextual fear conditioning (CFC) in rats during a period of development---Postnatal Day (PND) 17-33---that represents the late-infant, juvenile, and early-adolescent stages. These studies seek to acquire 'systems level' knowledge of brain and memory development and apply it to a rodent model of Fetal Alcohol Spectrum Disorder (FASD). This rodent model focuses on alcohol exposure from PND4-9, a period of brain development equivalent to the human third trimester, when neocortex, hippocampus, and cerebellum are especially vulnerable to adverse effects of alcohol. Our research emphasizes a variant of CFC, termed the Context Preexposure Facilitation Effect (CPFE, Fanselow, 1990), in which context representations incidentally learned on one occasion are retrieved and associated with immediate shock on a subsequent occasion. These representations can be encoded at the earliest developmental stage but seem not to be retained or retrieved until the juvenile period. This is associated with developmental differences in context-elicited expression, in prefrontal cortex, hippocampus, and amygdala, of immediate early genes (IEGs) that are implicated in long-term memory. Loss-of-function studies establish a functional role for these regions as soon as the CPFE emerges during ontogeny. In our rodent model of FASD, the CPFE is much more sensitive to alcohol dose than other commonly used cognitive tasks. This impairment can be reversed by acute administration during behavioral testing of drugs that enhance cholinergic function. This effect is associated with normalized IEG expression in prefrontal cortex during incidental context learning. In summary, our findings suggest that long-term memory of incidentally-learned context representations depends on prefrontal-hippocampal circuitry that is important both for the normative development of context conditioning and for its disruption by developmental alcohol exposure.

Role of dorsal and ventral hippocampal muscarinic receptor activity in acquisition and retention of contextual fear conditioning.

The current study further examined the effect of the muscarinic acetylcholine antagonist, scopolamine, on the Context Preexposure Facilitation Effect (CPFE; Robinson-Drummer, Dokovna, Heroux, & Stanton, 2016). In the CPFE, context representations formed during the preexposure phase are retrieved and associated with immediate shock during the training phase and expressed as freezing during a 24-hr retention phase. Scopolamine abolished postshock and retention freezing when administered systemically prior to preexposure (Experiment 1A) or immediate-shock training (Experiment 1B). Pretraining infusion of scopolamine into dorsal hippocampus (dHPC) disrupted both postshock and retention freezing (Experiments 2A) and retention freezing when the postshock freezing test was omitted (Experiment 2B) but did not alter expression of freezing behavior to an auditory fear stimulus (Experiment 2C). Finally, pretraining scopolamine infusion into ventral hippocampus (vHPC) also abolished postshock and retention test freezing (Experiment 3). These findings suggest similar roles for muscarinic receptor activity in both the dHPC and vHPC in the CPFE. This study advances understanding of the neurobiology of the CPFE by showing that context-shock associations are not learned following disruption of the cholinergic and/or hippocampal function on either the preexposure or training day. Existing theories of the CPFE (Rudy, 2009) have inferred this effect based on impaired 24-hr retention observed in previous studies (Matus-Amat, Higgins, Barrientos, & Rudy, 2004; Robinson-Drummer et al., 2016). However, the present study is the first to demonstrate it directly by including a postshock freezing measure. Further, this study is the first to identify vHPC as another important region necessary for context-shock learning during the CPFE paradigm. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Infant rats can acquire, but not retain contextual associations in object-in-context and contextual fear conditioning paradigms.

Context learning in postnatal day (PD) 16-18 rats has been taken by Revillo, Cotella, Paglini, and Arias (2015, Physiology & Behavior, 148, 6-21) to challenge the view that the ontogeny of contextual learning is related to the development of the hippocampal system (Rudy, 1993, Behavioral Neuroscience, 107(5), 887-891; Schiffino, Murawski, Rosen, & Stanton, 2011 Neurobiology of Learning and Memory, 95(2), 190-198). Whether context learning is "incidental" or "reinforcement-driven" may determine the ontogeny and neural systems involved (Rudy, 2009, Learning & Memory (Cold Spring Harbor, N.Y.), 16, 573-585). However, we have shown differential ontogeny of two different forms of incidental context learning, the context pre-exposure facilitation effect (CPFE; Jablonski, Schiffino, & Stanton, 2012, Developmental Psychobiology, 54(7), 714-722), which emerges between PD 17 and 21; and object-in-context recognition (OiC, Ramsaran, Westbrook, & Stanton, 2016, Developmental Psychobiology, 58(7), 883-895; Ramsaran, Sanders, & Stanton, 2016, Behavioural Brain Research, 298, 37-47), which is present on PD17. We investigated whether this task-dissociation reflects an encoding or a retention deficit, by varying the sample-to-testing intervals for both tasks. Experiment 1A found that PD17 rats were able to perform the OiC task after short (5 min) but not long (24 hr) sample-to-test intervals. Experiments 1B and 1C found that PD17 rats trained on the CPFE are able to acquire and express context-shock associations after short but not long retention intervals. These findings suggest that pre-weanling rats encode contexts but show poor consolidation or retrieval after longer retention intervals.

NMDA receptors and the ontogeny of post-shock and retention freezing during contextual fear conditioning.

The ontogeny and NMDA-receptor (NMDAR) mechanisms of context conditioning were examined during standard contextual fear conditioning (sCFC) - involving context and context-shock learning in the same trial - as a comparison with our previous reports on the Context Preexposure Facilitation Effect (CPFE), which separates these two types of learning by 24 hr. In Experiment 1, systemic administration of the NMDAR antagonist, MK-801, prior to conditioning disrupted retention but not post-shock freezing during sCFC in PD31 rats. Experiment 2 replicated and extended this effect to PD17 versus PD31 rats. Consistent with Experiment 1, pre-training MK-801 spared post-shock freezing but impaired retention freezing in PD31 rats. In contrast, pre-training MK-801 disrupted post-shock freezing in PD17 rats, which showed no retention freezing regardless of drug. These results reveal developmental differences in the role of NMDAR activity in the acquisition versus retention of a context-shock association during sCFC in pre-weanling and adolescent rats.

Medial prefrontal and ventral hippocampal contributions to incidental context learning and memory in adolescent rats.

The Context Preexposure Facilitation Effect (CPFE) is a contextual fear conditioning (CFC) paradigm in which context learning, context-shock learning, and retrieval of contextual fear occur in three distinct phases. The medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and ventral hippocampus (vHPC) are required for the acquisition and/or consolidation of a context representation during incidental context exposure (Heroux et al., 2017; Robinson-Drummer et al., 2016; Rudy & Matus-Amat, 2006). This exposure also induces the expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 in these regions (Heroux et al., 2018, 2019). Despite these studies, it is still unclear how mPFC and vHPC contribute to incidental context learning and memory. The current study examined whether prefrontal or ventral hippocampal inactivation during context preexposure interferes with long-term context memory and IEG activity in the mPFC, vHPC, dHPC and the ventral midline thalamus (VMT, a region connected to both the mPFC and HPC). Adolescent Long-Evans rats were given intra-mPFC (Experiment 1) or intra-vHPC (Experiment 2) infusions of the GABAA receptor agonist muscimol or PBS prior to context preexposure, and then were sacrificed 30 min later and whole mPFC, dHPC, vHPC, and VMT were collected and assayed for IEG mRNA expression via qPCR. Prefrontal or ventral hippocampal inactivation during context exposure abolished subsequent post-shock and retention test freezing in behaviorally-tested littermates of the sacrificed groups. In Experiment 1, prefrontal inactivation reduced expression of c-Fos, Arc, Egr-1, and Npas4 in the mPFC, c-Fos, Arc, and Npas4 in the vHPC, and c-Fos in the VMT, to the level of behaviorally-naïve home-cage controls. Prefrontal inactivation did not alter IEG expression in the dHPC during context exposure. In Experiment 2, ventral hippocampal inactivation impaired expression of all IEGs in the mPFC, dHPC, and vHPC, with no effect in the VMT. Taken together, these results suggest that context memory processes on the preexposure day of the CPFE may depend on mPFC-vHPC circuitry not typically emphasized in studies of incidental or configural learning and memory.

Cholinergic rescue of neurocognitive insult following third-trimester equivalent alcohol exposure in rats.

Neonatal ethanol exposure during the third trimester equivalent of human pregnancy in the rat significantly impairs hippocampal and prefrontal neurobehavioral functioning. Postnatal day [PD] 4-9 ethanol exposure in rats disrupts long-term context memory formation, resulting in abolished post-shock and retention test freezing in a variant of contextual fear conditioning called the Context Preexposure Facilitation Effect (CPFE). This behavioral impairment is accompanied by disrupted medial prefrontal, but not dorsal hippocampal expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 (Heroux, Robinson-Drummer, Kawan, Rosen, & Stanton, 2019). The current experiment examined if systemic administration of the acetylcholinesterase inhibitor physostigmine (PHY) prior to context learning would rescue prefrontal IEG expression and freezing in the CPFE. From PD4-9, Long-Evans rats received oral intubation of ethanol (EtOH; 5.25 g/kg/day) or sham-intubation (SI). Rats received a systemic injection of saline (SAL) or PHY (0.01 mg/kg) prior to all three phases (Experiment 1) or just context exposure (Experiment 2) in the CPFE from PD31-33. A subset of rats were sacrificed 30 min after context learning to assay changes in IEG expression in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and ventral hippocampus (vHPC). Administration of PHY prior to all three phases or just context learning rescued both post-shock and retention test freezing in the CPFE in EtOH rats without altering performance in SI rats. EtOH-SAL rats had significantly reduced mPFC but not dHPC expression of c-Fos, Arc, Egr-1, and Npas4. EtOH-PHY treatment rescued mPFC expression of c-Fos in ethanol-exposed rats and increased Arc and Npas4 regardless of dosing condition. While there was no effect of PHY on dHPC or vHPC expression of Arc, Egr-1, or Npas4, this treatment significantly boosted hippocampal expression of c-Fos regardless of ethanol treatment. These findings implicate impaired cholinergic and prefrontal function in cognitive deficits arising from 3rd-trimester equivalent alcohol exposure.

Differential involvement of amygdalar NMDA receptors across variants of contextual fear conditioning in adolescent rats.

In standard contextual fear conditioning (sCFC), learning of the context and formation of the context-shock association occur in the same training session whereas in the context preexposure facilitation effect (CPFE) learning the context (preexposure) and the context-shock association (training) are separated by 24 h. In both procedures conditioned freezing can be measured immediately (post-shock test) or during a 24-hour retention test. In adult rats, disrupting basolateral amygdala (BLA) activity or plasticity during training on sCFC impairs both post-shock and retention freezing [Maren et al, 1996; 1]. This manipulation on the training day of the CPFE disrupts retention freezing but effects on post-shock freezing are unknown [Matus-Amat et al, 2007; 2]. Experiment 1 extended this literature from adult to adolescent rats and to the role of BLA activity and plasticity in post-shock freezing during the CPFE. Intra-BLA infusions of muscimol prior to the training day of the CPFE disrupted both post-shock and retention freezing in Postnatal Day (PD) 31-33 rats. In the second two experiments, intra-BLA infusions of APV prior to the training day of sCFC disrupted retention but not post-shock freezing, while infusions of APV prior to training of the CPFE disrupt both post-shock and retention freezing. Our findings suggest that the BLA plasticity plays a different role in the CPFE vs. sCFC. Its role in the CPFE is similar in both adolescent and adult rats, while the role of the BLA in post-shock freezing during sCFC may differ across age or across studies that employ different procedures or parameters.

Neonatal ethanol exposure impairs long-term context memory formation and prefrontal immediate early gene expression in adolescent rats.

Fetal alcohol exposure leads to severe disruptions in learning and memory involving the hippocampus and prefrontal cortex in humans. Animal model research on FASD has documented impairment of hippocampal neuroanatomy and function but animal studies of cognition involving the prefrontal cortex are sparse. We have found that a variant of contextual fear conditioning in which both the hippocampus and prefrontal cortex is required, the Context Preexposure Facilitation Effect (CPFE), is particularly sensitive to neurobehavioral disruption caused by neonatal ethanol exposure during the third trimester equivalent of human pregnancy in the rat (i.e., PD4-9). In the CPFE, learning about the context, acquiring a context-shock association, and retrieving contextual fear are temporally separated across three days. The current study asked whether neonatal alcohol exposure impairs context learning, consolidation, or retrieval and examined prefrontal and hippocampal molecular signaling as correlates of this impairment. Long-Evans rats that received oral intubation of ethanol (AE; 5.25 g/kg/day, split into two doses) or underwent sham-intubation (SI) from PND4-9 were tested on the CPFE on PD31-33. Extending our previous reports, ethanol abolished both post-shock and retention test freezing in the CPFE. Assays (qPCR) of immediate early gene expression revealed that ethanol disrupted prefrontal but not hippocampal expression of c-Fos, Arc, Egr-1, and Npas4 during context learning. Finally, ethanol-exposed animals were unimpaired in a standard contextual fear conditioning procedure in which learning about the context and acquiring a context-shock association occurs concurrently. These findings implicate impaired prefrontal function in cognitive deficits arising from 3rd-trimester equivalent alcohol exposure in the rat.

Differential expression of the immediate early genes c-Fos, Arc, Egr-1, and Npas4 during long-term memory formation in the context preexposure facilitation effect (CPFE).

The context preexposure facilitation effect (CPFE) is a contextual fear conditioning paradigm in which learning about the context, acquiring the context-shock association, and retrieving/expressing contextual fear are temporally dissociated into three distinct phases (context preexposure, immediate-shock training, and retention). The current study examined changes in the expression of plasticity-associated immediate early genes (IEGs) during context and contextual fear memory formation on the preexposure and training days of the CPFE, respectively. Using adolescent Long-Evans rats, preexposure and training day expression of the IEGs c-Fos, Arc, Egr-1, and Npas4 in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and basolateral amygdala (BLA) was analyzed using qPCR as an extension of previous studies from our lab examining Egr-1 via in situ hybridization (Asok, Schreiber, Jablonski, Rosen, & Stanton, 2013; Schreiber, Asok, Jablonski, Rosen, & Stanton, 2014). In Expt. 1, context preexposure induced expression of c-Fos, Arc, Egr-1 and Npas4 significantly above that of home-cage (HC) controls in all three regions. In Expt. 2, immediate-shock was followed by a post-shock freezing test, resulting in increased mPFC c-Fos expression in a group preexposed to the training context but not a control group preexposed to an alternate context, indicating expression related to associative learning. This was not seen with other IEGs in mPFC or with any IEG in dHPC or BLA. Finally, when the post-shock freezing test was omitted in Expt. 3, training-related increases were observed in prefrontal c-Fos, Arc, Egr-1, and Npas4, hippocampal c-Fos, and amygdalar Egr-1 expression. These results indicate that context exposure in a post-shock freezing test re-engages IEG expression that may obscure associatively-induced expression during contextual fear conditioning. Additionally, these studies suggest a key role for long-term synaptic plasticity in the mPFC in supporting the CPFE.

Differential involvement of the medial prefrontal cortex across variants of contextual fear conditioning.

The context preexposure facilitation effect (CPFE) is a contextual fear conditioning paradigm in which learning about the context, acquiring the context-shock association, and retrieving/expressing contextual fear are temporally dissociated into three distinct phases. In contrast, learning about the context and the context-shock association happens concurrently in standard contextual fear conditioning (sCFC). By infusing the GABAA receptor agonist muscimol into medial prefrontal cortex (mPFC) in adolescent Long-Evans rats, the current set of experiments examined the functional role of the mPFC in each phase of the CPFE and sCFC. In the CPFE, the mPFC is necessary for the following: acquisition and/or consolidation of context memory (Experiment 1), reconsolidation of a context memory to include shock (Experiment 2), and expression of contextual fear memory during a retention test (Experiment 3). In contrast to the CPFE, inactivation of the mPFC prior to conditioning in sCFC has no effect on acquisition, consolidation, or retention of a contextual fear memory (Experiment 4). Interestingly, the mPFC is not required for acquiring a context-shock association (measured by post-shock freezing) in the CPFE or sCFC (Experiment 2b and 4). Taken together, these results indicate that the mPFC is differentially recruited across stages of learning and variants of contextual fear conditioning (CPFE versus sCFC). More specifically, separating out learning about the context and the context-shock association necessitates activation of the medial prefrontal cortex during early learning and/or consolidation.

Cholinergic mechanisms of the context preexposure facilitation effect in adolescent rats.

The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which context learning, context-shock association, and expression of context conditioning occur in 3 separate phases-preexposure, training, and testing. During the preexposure phase, the CPFE is disrupted by hippocampal NMDA receptor blockade in juvenile rats (Schiffino et al., 2011), and a similar deficit is seen with a subcutaneous injection of the muscarinic receptor antagonist, scopolamine, in adult mice (Brown, Kennard, Sherer, Comalli, & Woodruff-Pak, 2011). As a foundation for further developmental research, the present study examined the role of cholinergic function in the CPFE in adolescent rats during each phase of the CPFE protocol. In Experiment 1, an i.p injection of either 0.5 or 1.0 mg/kg dose of scopolamine administered prior to all 3 phases of the CPFE protocol impaired the CPFE. Experiment 2 further showed that a 0.5 mg/kg injection prior to just 1 of the 3 phases of the CPFE also disrupted contextual fear conditioning. We further showed that the CPFE is impaired by localized scopolamine infusions into dorsal hippocampus on the preexposure day (Experiment 3a), training day (Experiment 3b), and test day (Experiment 3c). These findings demonstrate a role of cholinergic signaling in hippocampus during each of the 3 phases of the CPFE in adolescent rats. Implications for the development and neural basis of the CPFE are discussed. (PsycINFO Database Record

NMDA receptor antagonism disrupts acquisition and retention of the context preexposure facilitation effect in adolescent rats.

The context preexposure facilitation effect (CPFE) is a contextual fear conditioning paradigm in which learning about the context, acquiring the context-shock association, and retrieving/expressing contextual fear are temporally dissociated. The current study investigated the involvement of NMDA receptors in contextual fear acquisition, retention, and expression across all phases of the CPFE in adolescent rats. In Experiment 1 systemic injections of 0.1mg/kg MK-801, a non-competitive NMDA receptor antagonist, given before multiple context preexposure disrupted the acquisition of a context representation. In Experiment 2, pre-training MK-801 disrupted both immediate acquisition of contextual fear measured by postshock freezing, as well as retention test freezing 24h later. Experiment 3 showed that expression of contextual fear via a 24h retention freezing test does not depend on NMDA receptors, indicating that MK-801 disrupts learning rather than performance of freezing behavior. In Experiment 4, consolidation of contextual information was partially disrupted by post-preexposure MK-801 whereas consolidation of contextual fear was not disrupted by post-training MK-801. Finally, Experiment 5 employed a dose-response design and found that a pre-training dose of 0.1mg/kg MK-801 disrupted both postshock and retention test freezing while lower pre-training doses of MK-801 (0.025 or 0.05mg/kg) only disrupted retention freezing. This is the first study to distinguish the role of NMDA receptors in acquisition (post-shock freezing), retention, expression, and consolidation of context vs. context-shock learning using the CPFE paradigm in adolescent rats. The findings provide a foundation for similar developmental studies examining these effects from early ontogeny through adulthood.

Lack of reliability in the disruption of cognitive performance following exposure to protons.

A series of three replications were run to determine the reliability with which exposure to protons produces a disruption of cognitive performance, using a novel object recognition task and operant responding on an ascending fixed-ratio task. For the first two replications, rats were exposed to head-only exposures to 1000 MeV/n protons at the NASA Space Radiation Laboratory. For the third replication, subjects were given head-only or whole-body exposures to both 1000 and 150 MeV/n protons. The results were characterized by a lack of consistency in the effects of exposure to protons on the performance of these cognitive tasks, both within and between replications. The factors that might influence the lack of consistency and the implications for exploratory class missions are discussed.