Maternal continuous oral oxycodone self-administration alters pup affective/social communication but not spatial learning or sensory-motor function.

BACKGROUND: The broad use/misuse of prescription opioids during pregnancy has resulted in a surge of infants with Neonatal Opioid Withdrawal Syndrome (NOWS). Short-term irritability and neurological complications are its hallmarks, but the long-term consequences are unknown. METHODS: A newly-developed preclinical model of oxycodone self-administration enables adult female rats to drink oxycodone (∼10/mg/kg/day) before and during pregnancy, and after delivery, and to maintain normal liquid intake, titrate dosing, and avoid withdrawal. RESULTS: Oxycodone was detected in the serum of mothers and pups. Growth parameters in dams and pups and litter mass and size were similar to controls. There were no differences in paw retraction latency to a thermal stimulus between Oxycodone and Control pups at postnatal (PN) 2 or PN14. Oxycodone and Control pups had similar motor coordination, cliff avoidance, righting time, pivoting, and olfactory spatial learning from PN3 through PN13. Separation-induced ultrasonic vocalizations at PN8 revealed higher call frequency in Oxycodone pups relative to Control pups (p<0.031; Cohen's d=1.026). Finally, Oxycodone pups displayed withdrawal behaviors (p's<0.029; Cohen's d's>0.806), and Oxycodone males only vocalized more than Control pups in the first minute of testing (p's<0.050; Cohen's d's>.866). Significant effects were corroborated by estimation plots. CONCLUSIONS: Our rat model of oral oxycodone self-administration in pregnancy shows exacerbated affect/social communication in pups in a sex-dependent manner but spared cognition and sensory-motor behaviors. This preclinical model reproduces selective aspects of human opioid use during pregnancy, enabling longitudinal analysis of how maternal oxycodone changes emotional behavior in the offspring.

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.

Infant Trauma Alters Social Buffering of Threat Learning: Emerging Role of Prefrontal Cortex in Preadolescence.

Within the infant-caregiver attachment system, the primary caregiver holds potent reward value to the infant, exhibited by infants' strong preference for approach responses and proximity-seeking towards the mother. A less well-understood feature of the attachment figure is the caregiver's ability to reduce fear via social buffering, commonly associated with the notion of a "safe haven" in the developmental literature. Evidence suggests this infant system overlaps with the neural network supporting social buffering (attenuation) of fear in the adults of many species, a network known to involve the prefrontal cortex (PFC). Here, using odor-shock conditioning in young developing rats, we assessed when the infant system transitions to the adult-like PFC-dependent social buffering of threat system. Rat pups were odor-shock conditioned (0.55 mA-0.6 mA) at either postnatal day (PN18; dependent on mother) or 28 (newly independent, weaned at PN23). Within each age group, the mother was present or absent during conditioning, with PFC assessment following acquisition using 14C 2-DG autoradiography and cue testing the following day. Since the human literature suggests poor attachment attenuates the mother's ability to socially buffer the infants, half of the pups at each age were reared with an abusive mother from PN8-12. The results showed that for typical control rearing, the mother attenuated fear in both PN18 and PN28 pups, although the PFC [infralimbic (IL) and ventral prelimbic (vPL) cortices] was only engaged at PN28. Abuse rearing completely disrupted social buffering of pups by the mother at PN18. The results from PN28 pups showed that while the mother modulated learning in both control and abuse-reared pups, the behavioral and PFC effects were attenuated after maltreatment. Our data suggest that pups transition to the adult-like PFC social support circuit after independence from the mother (PN28), and this circuit remains functional after early-life trauma, although its effectiveness appears reduced. This is in sharp contrast to the effects of early life trauma during infancy, where social buffering of the infant is more robustly impacted. We suggest that the infant social buffering circuit is disengaged by early-life trauma, while the adolescent PFC-dependent social buffering circuit may use a safety signal with unreliable safety value.

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.

Impairment of the context preexposure facilitation effect in juvenile rats by neonatal alcohol exposure is associated with decreased Egr-1 mRNA expression in the prefrontal cortex.

The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which learning about the context (preexposure) and associating the context with a shock (training) occur on separate occasions. The CPFE is sensitive to a range of neonatal alcohol doses (Murawski & Stanton, 2011). The current study examined the impact of neonatal alcohol on Egr-1 mRNA expression in the infralimbic (IL) and prelimbic (PL) subregions of the mPFC, the CA1 of dorsal hippocampus (dHPC), and the lateral nucleus of the amygdala (LA), following the preexposure and training phases of the CPFE. Rat pups were exposed to a 5.25 g/kg/day single binge-like dose of alcohol (Group EtOH) or were sham intubated (SI; Group SI) over postnatal days (PD) 7-9. In behaviorally tested rats, alcohol administration disrupted freezing. Following context preexposure, Egr-1 mRNA was elevated in both EtOH and SI groups compared with baseline control animals in all regions analyzed. Following both preexposure and training, Group EtOH displayed a significant decrease in mPFC Egr-1 mRNA expression compared with Group SI. However, this decrease was greatest after training. Training day decreases in Egr-1 expression were not found in LA or CA1 in Group EtOH compared with Group SI. A second experiment confirmed that the EtOH-induced training-day deficits in mPFC Egr-1 mRNA expression were specific to groups which learned contextual fear (vs. nonassociative controls). Thus, memory processes that engage the mPFC during the context-shock association may be most susceptible to the teratogenic effects of neonatal alcohol. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

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.

Using the context preexposure facilitation effect to study long-term context memory in preweanling, juvenile, adolescent, and adult rats.

The present study used the context preexposure facilitation effect (CPFE) to examine long-term retention of incidental context learning in periweanling, adolescent and adult rats. The CPFE is a variant of contextual fear conditioning in which encoding the context representation, associating this representation with shock, and expressing the context-shock association each occur on separate occasions. Experiment 1 manipulated the retention interval-1d, 8d, 15d, or 22d-between context preexposure and training with immediate shock to determine how long the encoded context could be remembered (testing always occurred 24h following training). The other factors were age-postnatal day (PND) 24 vs 31-and training group-Preexposed to the training context (Pre) vs. an alternate context (Alt-Pre). At both ages, significantly more freezing was evident in the Pre vs. Alt Pre Groups at the 24h, 8d and 15d retention intervals but not at the 22d interval, indicating that juvenile-adolescent rats remember the context for up to 15d. In contrast, context memory persists for 22days in adult rats (Experiment 2); and is not evident after 24h, 8d, or 15d retention intervals in PND 17 rats (Experiment 3). The present study illustrates the value of the CPFE paradigm for investigations of long-term context memory in developing rats. Implications for the neurobiology of infantile amnesia are discussed.