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.

Female rats display higher methamphetamine-primed reinstatement and c-Fos immunoreactivity than male rats.

Methamphetamine (meth) dependence is often characterized by persistent and chronic relapse (i.e., return to drug use). Previous work suggests females may be at greater risk to relapse. In this study, we extended this limited evidence and identified sex-dependent neural substrates related to meth-triggered reinstatement. Male and female Sprague-Dawley rats were implanted with indwelling jugular catheters. Half of the rats were then trained to self-administer meth (0.05 mg/kg/inf); the other half self-administered saline during 21 daily sessions (2 h). Rats were then given 12 extinction sessions. Twenty-four hours after the last extinction session, rats received reinstatement testing. Half of the rats received a meth-prime (0.3 mg/kg, IP) injection and the remaining rats received a saline injection. This design resulted in 4 separate groups for each sex, allowing for careful investigation of brain regions related to meth-triggered reinstatement. Brains were harvested following the reinstatement session and c-Fos immunoreactivity was measured in multiple brain regions. Meth triggered reinstatement in both sexes and this effect was more robust in females compared to males. Significant sex differences were detected. Females showed greater c-Fos immunoreactivity in the cingulate cortex area 1, lateral orbitofrontal cortex, prelimbic cortex, caudate-putamen, nucleus accumbens core and shell, and central nucleus of the amygdala following meth-primed reinstatement.

Chronic activation of fear engrams induces extinction-like behavior in ethanol-exposed mice.

Alcohol withdrawal directly impacts the brain's stress and memory systems, which may underlie individual susceptibility to persistent drug and alcohol-seeking behaviors. Numerous studies demonstrate that forced alcohol abstinence, which may lead to withdrawal, can impair fear-related memory processes in rodents such as extinction learning; however, the underlying neural circuits mediating these impairments remain elusive. Here, we tested an optogenetic strategy aimed at mitigating fear extinction retrieval impairments in male c57BL/6 mice following exposure to alcohol (i.e., ethanol) and forced abstinence. In the first experiment, extensive behavioral extinction training in a fear-conditioned context was impaired in ethanol-exposed mice compared to controls. In the second experiment, neuronal ensembles processing a contextual fear memory in the dorsal hippocampus were tagged and optogenetically reactivated repeatedly in a distinct context in ethanol-exposed and control mice. Chronic activation of these cells resulted in a context-specific, extinction-like reduction in fear responses in both control and ethanol-exposed mice. These findings suggest that while ethanol can impair the retrieval an extinction memory, optogenetic manipulation of a fear engram is sufficient to induce an extinction-like reduction in fear responses.

Artificially Enhancing and Suppressing Hippocampus-Mediated Memories.

Emerging evidence indicates that distinct hippocampal domains differentially drive cognition and emotion [1, 2]; dorsal regions encode spatial, temporal, and contextual information [3-5], whereas ventral regions regulate stress responses [6], anxiety-related behaviors [7, 8], and emotional states [8-10]. Although previous studies demonstrate that optically manipulating cells in the dorsal hippocampus can drive the behavioral expression of positive and negative memories, it is unknown whether changes in cellular activity in the ventral hippocampus can drive such behaviors [11-14]. Investigating the extent to which distinct hippocampal memories across the longitudinal axis modulate behavior could aid in the understanding of stress-related psychiatric disorders known to affect emotion, memory, and cognition [15]. Here, we asked whether tagging and stimulating cells along the dorsoventral axis of the hippocampus could acutely, chronically, and differentially promote context-specific behaviors. Acute reactivation of both dorsal and ventral hippocampus cells that were previously active during memory formation drove freezing behavior, place avoidance, and place preference. Moreover, chronic stimulation of dorsal or ventral hippocampal fear memories produced a context-specific reduction or enhancement of fear responses, respectively, thus demonstrating bi-directional and context-specific modulation of memories along the longitudinal axis of the hippocampus. Fear memory suppression was associated with a reduction in hippocampal cells active during retrieval, while fear memory enhancement was associated with an increase in basolateral amygdala activity. Together, our data demonstrate that discrete sets of cells throughout the hippocampus provide key nodes sufficient to bi-directionally reprogram both the neural and behavioral expression of memory.

Understanding the contributions of visual stimuli to contextual fear conditioning: A proof-of-concept study using LCD screens.

The precise contribution of visual information to contextual fear learning and discrimination has remained elusive. To better understand this contribution, we coupled the context pre-exposure facilitation effect (CPFE) fear conditioning paradigm with presentations of distinct visual scenes displayed on 4 LCD screens surrounding a conditioning chamber. Adult male Long-Evans rats received non-reinforced context pre-exposure on Day 1, an immediate 1.5mA foot shock on Day 2, and a non-reinforced context test on Day 3. Rats were pre-exposed to either digital Context (dCtx) A, dCtx B, a distinct Ctx C, or no context on Day 1. Digital context A and B were identical except for the visual image displayed on the LCD screens. Immediate shock and retention testing occurred in dCtx A. Rats pre-exposed dCtx A showed the CPFE with significantly higher levels of freezing compared to controls. Rats pre-exposed to Context B failed to show the CPFE, with freezing that did not highly differ from controls. These results suggest that visual information contributes to contextual fear learning and that visual components of the context can be manipulated via LCD screens. Our approach offers a simple modification to contextual fear conditioning paradigms whereby the visual features of a context can be manipulated to better understand the factors that contribute to contextual fear discrimination and generalization.

Advances in Diagnosis and Treatment of Fetal Alcohol Spectrum Disorders: From Animal Models to Human Studies.

Prenatal alcohol exposure can cause a number of physical, behavioral, cognitive, and neural impairments, collectively known as fetal alcohol spectrum disorders (FASD). This article examines basic research that has been or could be translated into practical applications for the diagnosis or treatment of FASD. Diagnosing FASD continues to be a challenge, but advances are being made at both basic science and clinical levels. These include identification of biomarkers, recognition of subtle facial characteristics of exposure, and examination of the relation between face, brain, and behavior. Basic research also is pointing toward potential new interventions for FASD involving pharmacotherapies, nutritional therapies, and exercise interventions. Although researchers have assessed the majority of these treatments in animal models of FASD, a limited number of recent clinical studies exist. An assessment of this literature suggests that targeted interventions can improve some impairments resulting from developmental alcohol exposure. However, combining interventions may prove more efficacious. Ultimately, advances in basic and clinical sciences may translate to clinical care, improving both diagnosis and treatment.

Effects of neonatal alcohol dose and exposure window on long delay and trace eyeblink conditioning in juvenile rats.

Classical eyeblink conditioning has been used to assess learning and memory impairments in both humans and animal model studies of fetal alcohol spectrum disorders (FASD). Gestational exposure to alcohol in humans and its equivalent in rats severely impairs various eyeblink conditioning tasks, but less is known about how these effects are influenced by variables, such as the timing and dose of alcohol exposure. In a series of four experiments, we systematically examine how varying the timing and dose of alcohol exposure impact long delay and trace eyeblink conditioning in juvenile rats, tasks that both depend on a brainstem-cerebellar circuit but differ in that trace conditioning additionally recruits the hippocampus and prefrontal cortex. Using a "third-trimester-equivalent" alcohol exposure model, rats were exposed to a high binge dose of alcohol at one of two alcohol doses over postnatal days (PD) 4-9 or PD 7-9, windows of exposure thought to differentially target the cerebellum and hippocampus. Sham-intubated and untreated rats served as controls. As juveniles, rats from each treatment condition were trained in either a long delay or trace eyeblink conditioning task. Alcohol-exposed rats demonstrated general conditioning impairments compared to controls during long delay conditioning, with more robust impairments in rats exposed to the higher alcohol dose (5.25 g/kg/day) than those that received the lower dose (4.66 g/kg/day). Alcohol-exposed rats showed trace conditioning impairments compared to controls only when the high dose of alcohol was administered over PD 4-9 or PD 7-9. These findings indicate significant learning and memory impairments following neonatal alcohol exposure at both PD 4-9 and PD 7-9. The pattern of impairments across delay and trace conditioning suggest that alcohol disrupts processes that are common to both tasks. These findings are consistent with studies of delay and trace eyeblink conditioning in children with FASD. Future studies of the mechanisms underlying these deficits will further our understanding of brain injury and memory impairments resulting from developmental alcohol exposure.

Malformation of the superior olivary complex in an animal model of autism.

Autism is a neurodevelopmental disorder characterized by social difficulties, impaired communication skills and repetitive behavioral patterns. Additionally, there is evidence that auditory deficits are a common feature of the autism spectrum disorders. Despite the prevalence of autism, the neurobiology of this disorder is poorly understood. However, abnormalities in neuronal morphology, cell number and connectivity have been described throughout the autistic brain. Indeed, we have demonstrated significant dysmorphology in the superior olivary complex (SOC), a collection of auditory brainstem nuclei, in the autistic brain. Prenatal exposure to valproic acid (VPA) in humans has been associated with autism and in rodents prenatal VPA exposure produces many neuroanatomical and behavioral deficits associated with autism. Thus, in an effort to devise an animal model of the autistic auditory brainstem, we have investigated neuronal number and morphology in animals prenatally exposed to valproic acid (VPA). In VPA exposed rats, we find significantly fewer neurons and significant alterations in neuronal morphology. Thus, prenatal VPA exposure in rats appears to produce similar dysmorphology as we have reported in the autistic human brain.

Effects of dose and period of neonatal alcohol exposure on the context preexposure facilitation effect.

BACKGROUND: Alcohol exposure in the rat on postnatal days (PD) 4 to 9 is known to partially damage the hippocampus and to impair hippocampus-dependent behavioral tasks. We previously reported that PD4 to 9 alcohol exposure eliminated the context preexposure facilitation effect (CPFE) in juvenile rats, a hippocampus-dependent variant of contextual fear conditioning. In the CPFE, context exposure and immediate shock occur on successive occasions and this produces conditioned freezing relative to a control group that is not preexposed to the training context. Here, we extend our earlier findings by examining the effects of neonatal alcohol administered at multiple doses or over different neonatal exposure periods. METHOD: Rat pups (male and female) were exposed to a single binge dose of alcohol at one of 3 doses (2.75, 4.00, or 5.25 g/kg/d) over PD4 to 9 (Experiment 1) or to 5.25 g over PD4 to 6 or PD7 to 9 (Experiment 2). Sham-intubated (SI) and undisturbed (UD) rats served as controls. On PD31, rats were preexposed to either the training context (Pre) or an alternate context (No-Pre). On PD32, rats received an immediate unsignaled footshock (1.5 mA, 2 seconds) in the training context. Finally, on PD33, all rats were returned to the training context and tested for contextual freezing over a 5-minute period. RESULTS: Undisturbed- and SI-Pre rats showed the CPFE, i.e., context preexposure facilitated contextual conditioning, relative to their No-Pre counterparts. The immediate shock deficit was present in all No-Pre groups, regardless of previous alcohol exposure. In Experiment 1, blood alcohol level was negatively correlated with contextual freezing. Group 2.75 g-Pre did not differ from controls. Group 4.00 g-Pre froze significantly less than Groups UD- and SI-Pre but more than Group 5.25-Pre, which showed the immediate shock deficit. In Experiment 2, alcohol exposure limited to PD7 to 9, but not PD4 to 6, disrupted the CPFE. CONCLUSIONS: This is the first demonstration of dose-related impairment on a hippocampus-dependent task produced by neonatal alcohol exposure in the rat. Exposure period effects support previous studies of alcohol and spatial learning. The CPFE is a more sensitive behavioral task that can be used to elucidate developmental alcohol-induced deficits over a range of alcohol doses that are more relevant to human exposure levels.

Ontogeny and neural substrates of the context preexposure facilitation effect.

Contextual fear conditioning emerges around post-natal day (PD) 23 in the rat. This is thought to reflect hippocampus-dependent conjunctive learning, which binds the individual features of the context into a unified representation (Rudy, 1993). However, context conditioning can also be supported by hippocampus-independent, feature-based simple associations (Rudy, 2009) and these may operate at PD 23-24 (Pugh & Rudy, 1996). To address this issue, we studied the ontogeny of a variant of contextual fear conditioning, termed the context-preexposure-facilitation-effect (CPFE), in which exposure to context and (immediate) foot shock occur on successive occasions. This variant requires conjunctive as opposed to feature-based simple associations (Rudy, 2009). We tested PD 17, 24, and 31 rats on the CPFE vs. conventional context conditioning (Exp. 1) and on the CPFE with stronger reinforcement (Exp. 2). The CPFE emerged on PD 24 regardless of reinforcer strength and in parallel with context conditioning. Infusions of the NMDA-receptor antagonist, MK-801, into the dorsal hippocampus just before pre-exposure on PD 24 eliminated the CPFE, whereas infusions occurring after pre-exposure had no effect (Exp. 3). These findings demonstrate a role of hippocampal NMDA receptors in the CPFE as early as PD 24 and implicate conjunctive learning mechanisms in the ontogeny of contextual fear conditioning.

Variants of contextual fear conditioning are differentially impaired in the juvenile rat by binge ethanol exposure on postnatal days 4-9.

Neonatal ethanol exposure in the rat is known to partially damage the hippocampus, but such exposure causes only modest or inconsistent deficits on hippocampus-dependent behavioral tasks. This may reflect variable sensitivity of these tasks or residual function following partial hippocampal injury. The context preexposure facilitation effect (CPFE) is a variant of context conditioning in which context exposure and immediate shock occur on successive occasions. During testing, preexposed rats freeze more than non-preexposed controls. The CPFE is more sensitive to anterograde hippocampal injury than standard contextual fear conditioning (e.g., Rudy JW, O'Reilly RC. Conjunctive representations, the hippocampus, and contextual fear conditioning. Cogn Affect Behav Neurosci 2001;1:66-82). We report that rats exposed to a high binge dose of ethanol (5.25g/kg/day) over postnatal days [PD] 4-9 failed to demonstrate the CPFE when preexposed to the conditioning context on PD31, relative to sham-intubated and undisturbed controls (Experiment 1). Neonatal alcohol disrupted conditioned freezing to a much lesser extent relative to controls when context preexposure was followed by a standard context conditioning trial rather than immediate shock (Experiment 2). Fear conditioning to a discrete auditory cue (tone) was unaffected by neonatal alcohol exposure ruling out possible performance effects (Experiment 3). These findings suggest that the CPFE is an especially sensitive task for detecting hippocampal injury produced by neonatal alcohol. Mixed results with other tasks may reflect residual hippocampal function and/or the use of alternate neurobehavioral systems or "strategies" following alcohol-induced brain damage.

Interstimulus interval (ISI) discrimination of the conditioned eyeblink response in a rodent model of autism.

Rats exposed to valproic acid (VPA) on gestational day 12 (GD12) have been advanced as a rodent model of autism [Arndt TL, Stodgell, Rodier PM. The teratology of autism. Int J Dev Neurosci 2005;23: 189-99.]. These rats show cerebellar anomalies and alterations in eyeblink conditioning that are associated with autism. Autistic humans and VPA-exposed rats show normal responses to conditioned and unconditioned stimuli, but they show marked differences from comparison groups in acquisition, magnitude, and timing of the conditioned response (CR). This study examined VPA-induced eyeblink CR timing differences by training rats on an interstimulus interval (ISI) discrimination task, in which two distinct conditioned stimuli (CS; tone and light) are paired with the same unconditioned stimulus (US; periocular shock) at two distinct CS-US intervals. Previous findings suggest that this task would produce abnormally large and prematurely timed CRs for VPA-exposed rats relative to controls. Adult male Long-Evans rats that were exposed to either VPA or saline on GD 12.5 were trained on an ISI discrimination task [Brown KL, Pagani JH, Stanton ME. The ontogeny of interstimulus interval (ISI) discrimination of the conditioned eyeblink response in rats. Behav Neurosci 2006;120: 1057-70.]. In support of earlier findings, we observed early acquisition and enhanced magnitude of the CR in VPA rats compared with controls on long CS trials. VPA rats also showed prematurely timed CRs to long- CS trials, but not to short- CS trials. The ISI discrimination procedure used in the current study reveals differential timed responses in this animal model of autism not previously seen.

Combined blockade of serotonergic and muscarinic transmission disrupts the anterior thalamic head direction signal.

Head direction (HD) cells have been speculated to be part of a network mediating navigational behavior. Previous work has shown that combined administration of serotonergic and muscarinic antagonists eliminates hippocampal theta activity and produces navigational deficits more severe than blockade of either neurotransmitter system alone. The authors sought to assess this effect on the directional characteristics of HD cells. HD cells were recorded from the anterior dorsal thalamus of Long-Evans rats before and after administration of the serotonergic antagonist methiothepin, the muscarinic antagonist scopolamine, both drugs, or saline. Combined drug administration produced HD cells with preferred directions that drifted within recording sessions. In addition, cells showed shifts in the preferred directions at the start of a session relative to the position of the major landmarks, suggesting that combined drug administration led to deficits in landmark control of the HD system. Single drug exposures to methiothepin or scopolamine did not noticeably affect the directional characteristics of HD cells. This finding that navigation-impairing drugs can disrupt the HD signal provides further evidence that this network plays an important role in navigational behavior.