Social and olfactory experiences modify neuronal morphology of orbital frontal cortex.

Structural modifications in the dendritic morphology of neurons occur following many forms of experience, including exposure to drugs, complex housing, and training in specific behavioral tasks. The present study examined morphological changes in orbitofrontal (OFC) and medial prefrontal cortex (mPFC) neurons of female rats following experience with a variety of social partners or nonsocial olfactory stimuli. We reasoned that experience with various social partners or olfactory stimuli, and the associated behavioral adaptations, would drive structural modifications in prefrontal cortex neurons engaged by these stimuli. Social experience was manipulated by providing rats with a novel cage-mate or housing the animal with the same cage-mate throughout the study. Similarly, olfactory experience was manipulated by introducing novel, nonsocial odors in the home cage or exposing the animals to the same home-cage odor throughout the study. Both forms of experience resulted in altered dendritic morphology in OFC neurons, whereas morphological changes in mPFC were comparatively small and limited to changes in spine density. These observations indicate that OFC and mPFC neurons respond differently to social and nonsocial olfactory stimulation in adulthood and join the growing body of data illustrating differential effects of experience on structural plasticity in OFC and mPFC. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Moderate prenatal alcohol exposure reduces parvalbumin expressing GABAergic interneurons in the dorsal hippocampus of adult male and female rat offspring.

Prenatal alcohol exposure (PAE) negatively impacts hippocampal development and impairs hippocampal-sensitive learning and memory. However, hippocampal neural adaptations in response to moderate PAE are not completely understood. To explore the effects of moderate PAE on GABAergic interneuron expression, this study used a rat model of moderate PAE to examine the effects of PAE on parvalbumin (PARV)-positive cells in fields CA1, CA3 and the dentate gyrus (DG) of the dorsal hippocampus (dHC). Long-Evans dams were given daily access to 5 % (vol/vol) ethanol or saccharine (SAC) control solutions throughout the course of gestation. Offspring were divided into four separate groups: PAE (n = 7) or SAC (n = 7) males, or PAE (n = 8) or SAC (n = 8) females. All rats were aged to adulthood and, following testing in the Morris water task, their brains were analyzed for the expression of the GABAergic neuronal marker PARV. We report a main effect of PAE on GABAergic expression, with significant reductions in PARV-positive cells in area CA3 for males and the DG for females. There was also a trend for a reduction in PARV expressing neurons in fields CA1 and CA3 in females. The results are discussed in relation to hippocampal GABAergic interneuron function, PAE and behavior.

Addition of ketamine to standard-of-care countermeasures for acute organophosphate poisoning improves neurobiological outcomes.

Rats poisoned with sarin enter into ahyper-cholinergic crisis characterized by excessive salivation, respiratory distress, tremors, seizures, and death. Through the use of rescue medications and an anticonvulsant, death can be avoided in many animals, with the long-term consequences of poisoning partly ameliorated, especially when countermeasures are made available immediately after exposure. However, when anticonvulsant measures are delayed by as little as 30 min, clinical, neurological, cognitive, and psychiatric abnormalities may persist long after the initial exposure. This study sought to determine if the addition of the NMDA receptor antagonist Ketamine to human standard-of-care countermeasures consisting of two rescue medications (2-PAM and atropine) and an anti-convulsant (Midazolam), would afford protection against persistent neurobiological compromise. Rats were exposed to sarin (105 µg/kg via subcutaneous injection), and treated 1 min later with 2-PAM and Atropine Methyl Nitrate (IM) to minimize mortality. One of four anti-convulsant protocols was then initiated at 50 min postsarin:Midazolam alone (MDZ, a single injection (IM) at 0.66 mg/kg); Ketamine alone (KET, a series of five injections (IM) of Ketamine at 7.5 mg/kg, 90 min apart); Midazolam + low dose Ketamine (MDZ + lowKET, a single injection of Midazolam (IM) at 0.66 mg/kg, plus five sequential doses of ketamine (IM) at 2.5 mg/kg, starting at the time of Midazolam dosing and then 90 min apart); Midazolam + high dose Ketamine (MDZ + highKET, a single injection of Midazolam (IM) at 0.66 mg/kg, plus five sequential injections of 7.5 mg/kg Ketamine (IM), starting at the time of Midazolam dosing and then 90 min apart). Animals were preassigned to groups culled at post-exposure Days 1, 7 or 30, for histopathology. For all surviving animals, EEG activity was monitored through skull electrodes for 24-h beginning immediately after sarin exposure. Surviving animals also underwent 24-h EEG monitoring on Days 6, 13, and/or 29, post-sarin. Memory assessment using the Morris Water Maze was performed on Days 1, 4, 7, 14 and 30. Following sarin exposure, 85% of surviving animals demonstrated status epilepticus within 20 min. Each of the anti-convulsant protocols was sufficient to stop convulsions within 1 h of anti-convulsant administration, but all of the animals still showed signs of electrographic status for an additional 2-12 h, without substantial differentiation between treatment groups. However, for post-sarin hours 13-24, the MDZ + highKET group showed significantly less severe EEG abnormalities than the MDZ and KET groups (Mood's Median Test, p < 0.005). At one month post-exposure, 90% of animals that had received Midazolam alone still showed evidence of some epileptiform activity. In contrast, 90% of animals that had received Midazolam + high dose Ketamine combination therapy had EEG profiles that were within normal limits. This difference in EEG outcomes was highly significant (Mood's Median Test, p < 0.001). Likewise, on the water maze, the majority of animals that had received Midazolam combined with either high or low dose Ketamine therapy returned to near baseline levels of mnemonic performance within 2 weeks, whereas the majority of the animals that had received midazolam alone or ketamine alone demonstrated persistent and significant memory impairments even at one month postexposure (Mood's Median Test, p < 0.005). With respect to neuronal necrosis, animals in the MDZ + highKET group showed significantly less overall damage than animals in other treatment groups (Mood's Median Test, p < 0.001). Of special note were findings in the hippocampus, where only 12% of animals in the MDZ + highKET group showed evidence of necrosis on H&E staining, whereas 100% of animals in the KET group, 70% of animals in the MDZ group, and 40% of animals in the MDZ + lowKET group showed evidence of hippocampal necrosis. Overall, the data demonstrate that Ketamine augmentation of an atropine, 2PAM, and Midazolam standard-ofcare for sarin exposure provides clinically-relevant additional protection against the negative neurobiological consequences of sarin, even when initiation of the anti-convulsant countermeasures is delayed by 50 min.

Social Order: Using The Sequential Structure of Social Interaction to Discriminate Abnormal Social Behavior in the Rat.

Social interactions form the basis of a broad range of functions related to survival and mating. The complexity of social behaviors and the flexibility required for normal social interactions make social behavior particularly susceptible to disruption. The consequences of developmental insults in the social domain and the associated neurobiological factors are commonly studied in rodents. Though methods for investigating social interactions in the laboratory are diverse, animals are typically placed together in an apparatus for a brief period (under 30 min) and allowed to interact freely while behavior is recorded for subsequent analysis. A standard approach to the analysis of social behavior involves quantification of the frequency and duration of individual social behaviors. This approach provides information about the allocation of time to particular behaviors within a session, which is typically sufficient for detection of robust alterations in behavior. Virtually all social species, however, display complex sequences of social behavior that are not captured in the quantification of individual behaviors. Sequences of behavior may provide more sensitive indicators of disruptions in social behavior. Sophisticated analysis systems for quantification of behavior sequences have been available for many years; however, the required training and time to complete these analyses represent significant barriers to high-throughput assessments. We present a simple approach to the quantification of behavioral sequences that requires minimal additional analytical steps after individual behaviors are coded. We implement this approach to identify altered social behavior in rats exposed to alcohol during prenatal development, and show that the frequency of several pairwise sequences of behavior discriminate controls from ethanol-exposed rats when the frequency of individual behaviors involved in those sequences does not. Thus, the approach described here may be useful in detecting subtle deficits in the social domain and identifying neural circuits involved in the organization of social behavior.

Ifenprodil infusion in agranular insular cortex alters social behavior and vocalizations in rats exposed to moderate levels of ethanol during prenatal development.

Moderate exposure to alcohol during development leads to subtle neurobiological and behavioral effects classified under the umbrella term fetal alcohol spectrum disorders (FASDs). Alterations in social behaviors are a frequently observed consequence of maternal drinking, as children with FASDs display inappropriate aggressive behaviors and altered responses to social cues. Rodent models of FASDs mimic the behavioral alterations seen in humans, with rats exposed to ethanol during development displaying increased aggressive behaviors, decreased social investigation, and altered play behavior. Work from our laboratory has observed increased wrestling behavior in adult male rats following prenatal alcohol exposure (PAE), and increased expression of GluN2B-containing NMDA receptors in the agranular insular cortex (AIC). This study was undertaken to determine if ifenprodil, a GluN2B preferring negative allosteric modulator, has a significant effect on social behaviors in PAE rats. Using a voluntary ethanol exposure paradigm, rat dams were allowed to drink a saccharin-sweetened solution of either 0% or 5% ethanol throughout gestation. Offspring at 6-8 months of age were implanted with cannulae into AIC. Animals were isolated for 24h before ifenprodil or vehicle was infused into AIC, and after 15min they were recorded in a social interaction chamber. Ifenprodil treatment altered aspects of wrestling, social investigatory behaviors, and ultrasonic vocalizations in rats exposed to ethanol during development that were not observed in control animals. These data indicate that GluN2B-containing NMDA receptors in AIC play a role in social behaviors and may underlie alterations in behavior and vocalizations observed in PAE animals.

Effects of sex and housing on social, spatial, and motor behavior in adult rats exposed to moderate levels of alcohol during prenatal development.

Persistent deficits in social behavior, motor behavior, and behavioral flexibility are among the major negative consequences associated with exposure to ethanol during prenatal development. Prior work from our laboratory has linked moderate prenatal alcohol exposure (PAE) in the rat to deficits in these behavioral domains, which depend upon the ventrolateral frontal cortex (Hamilton et al., 2014) [20]. Manipulations of the social environment cause modifications of dendritic morphology and experience-dependent immediate early gene expression in ventrolateral frontal cortex (Hamilton et al., 2010) [19], and may yield positive behavioral outcomes following PAE. In the present study we evaluated the effects of housing PAE rats with non-exposed control rats on adult behavior. Rats of both sexes were either paired with a partner from the same prenatal treatment condition (ethanol or saccharin) or from the opposite condition (mixed housing condition). At four months of age (∼3 months after the housing manipulation commenced), social behavior, tongue protrusion, and behavioral flexibility in the Morris water task were measured as in (Hamilton et al., 2014) [20]. The behavioral effects of moderate PAE were primarily limited to males and were not ameliorated by housing with a non-ethanol exposed partner. Unexpectedly, social behavior, motor behavior, and spatial flexibility were adversely affected in control rats housed with a PAE rat (i.e., in mixed housing), indicating that housing with a PAE rat has broad behavioral consequences beyond the social domain. These observations provide further evidence that moderate PAE negatively affects social behavior, and underscore the importance of considering potential negative effects of housing with PAE animals on the behavior of critical comparison groups.

Moderate prenatal alcohol exposure enhances GluN2B containing NMDA receptor binding and ifenprodil sensitivity in rat agranular insular cortex.

Prenatal exposure to alcohol affects the expression and function of glutamatergic neurotransmitter receptors in diverse brain regions. The present study was undertaken to fill a current gap in knowledge regarding the regional specificity of ethanol-related alterations in glutamatergic receptors in the frontal cortex. We quantified subregional expression and function of glutamatergic neurotransmitter receptors (AMPARs, NMDARs, GluN2B-containing NMDARs, mGluR1s, and mGluR5s) by radioligand binding in the agranular insular cortex (AID), lateral orbital area (LO), prelimbic cortex (PrL) and primary motor cortex (M1) of adult rats exposed to moderate levels of ethanol during prenatal development. Increased expression of GluN2B-containing NMDARs was observed in AID of ethanol-exposed rats compared to modest reductions in other regions. We subsequently performed slice electrophysiology measurements in a whole-cell patch-clamp preparation to quantify the sensitivity of evoked NMDAR-mediated excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons of AID to the GluN2B negative allosteric modulator ifenprodil. Consistent with increased GluN2B expression, ifenprodil caused a greater reduction in NMDAR-mediated EPSCs from prenatal alcohol-exposed rats than saccharin-exposed control animals. No alterations in AMPAR-mediated EPSCs or the ratio of AMPARs/NMDARs were observed. Together, these data indicate that moderate prenatal alcohol exposure has a significant and lasting impact on GluN2B-containing receptors in AID, which could help to explain ethanol-related alterations in learning and behaviors that depend on this region.

Postnatal alterations in GABAB receptor tone produce sensorimotor gating deficits and protein level differences in adulthood.

The GABA transmitter system plays a vital role in modulating synaptic formation and activity during development. The GABAB receptor subtype in particular has been implicated in cell migration, promotion of neuronal differentiation, neurite outgrowth, and synapse formation but it's role in development is not well characterized. In order to investigate the effects of brief alterations in GABAB signaling in development, we administered to rats the GABAB agonist baclofen (2.0mg/kg) or antagonist phaclofen (0.3mg/kg) on postnatal days 7, 9, and 12, and evaluated sensorimotor gating in adulthood. We also examined tissue for changes in multiple proteins associated with GABAB receptor function and proteins associated with synapse formation. Our data indicate that early postnatal alterations to GABAB receptor-mediated signaling produced sex differences in sensorimotor gating in adulthood. Additionally, we found differences in GABAB receptor subunits and kalirin protein levels in the brain versus saline treated controls. Our data demonstrate that a subtle alteration in GABAB receptor function in early postnatal life induces changes that persist into adulthood.

Moderate prenatal alcohol exposure and quantification of social behavior in adult rats.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE(1), and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

Effects of moderate prenatal ethanol exposure and age on social behavior, spatial response perseveration errors and motor behavior.

Persistent deficits in social behavior are among the major negative consequences associated with exposure to ethanol during prenatal development. Prior work from our laboratory has linked deficits in social behavior following moderate prenatal alcohol exposure (PAE) in the rat to functional alterations in the ventrolateral frontal cortex [21]. In addition to social behaviors, the regions comprising the ventrolateral frontal cortex are critical for diverse processes ranging from orofacial motor movements to flexible alteration of behavior in the face of changing consequences. The broader behavioral implications of altered ventrolateral frontal cortex function following moderate PAE have, however, not been examined. In the present study we evaluated the consequences of moderate PAE on social behavior, tongue protrusion, and flexibility in a variant of the Morris water task that required modification of a well-established spatial response. PAE rats displayed deficits in tongue protrusion, reduced flexibility in the spatial domain, increased wrestling, and decreased investigation, indicating that several behaviors associated with ventrolateral frontal cortex function are impaired following moderate PAE. A linear discriminant analysis revealed that measures of wrestling and tongue protrusion provided the best discrimination of PAE rats from saccharin-exposed control rats. We also evaluated all behaviors in young adult (4-5 months) or older (10-11 months) rats to address the persistence of behavioral deficits in adulthood and possible interactions between early ethanol exposure and advancing age. Behavioral deficits in each domain persisted well into adulthood (10-11 months), however, there was no evidence that aging enhances the effects of moderate PAE within the age ranges that were studied.

Baclofen administration alters fear extinction and GABAergic protein levels.

The investigation of GABAergic systems in learning and extinction has principally focused on ionotropic GABA(A) receptors. Less well characterized is the metabotropic GABA(B) receptor, which when activated, induces a more sustained inhibitory effect and has been implicated in regulating oscillatory activity. Few studies have been carried out utilizing GABA(B) ligands in learning, and investigations of GABA(B) in extinction have primarily focused on interactions with drugs of abuse. The current study examined changes in GABA(B) receptor function using the GABA(B) agonist baclofen (2 mg/mL) or the GABA(B) antagonist phaclofen (0.3 mg/mL) on trace cued and contextual fear conditioning and extinction. The compounds were either administered during training and throughout extinction in Experiment 1, or starting 24 h after training and throughout extinction in Experiment 2. All drugs were administered 1 mL/kg via intraperitoneal injection. These studies demonstrated that the administration of baclofen during training and extinction trials impaired animals' ability to extinguish the fear association to the CS, whereas the animals that were administered baclofen starting 24 h after training (Experiment 2) did display some extinction. Further, contextual fear extinction was impaired by baclofen in both experiments. Tissue analyses suggest the cued fear extinction deficit may be related to changes in the GABA(B2) receptor subunit in the amygdala. The data in the present investigation demonstrate that GABA(B) receptors play an important role in trace cued and contextual fear extinction, and may function differently than GABA(A) receptors in learning, memory, and extinction.

Deficits in emotional learning and memory in an animal model of schizophrenia.

Alterations in N-methyl-D-aspartate (NMDA) receptor function have been linked to numerous behavioral deficits and neurochemical alterations. Recent investigations have begun to explore the role of NMDA receptor function on principally inhibitory neurons and their role in network function. One of the prevailing models of schizophrenia proposes a reduction in NMDA receptor function on inhibitory interneurons and the resulting disinhibition may give rise to aspects of the disorder. Studies using NMDA receptor antagonists such as PCP and ketamine have induced schizophrenia-like behavioral deficits in animal model systems as well as changes in inhibitory circuits. The current study investigated whether the administration of a subanesthetic dose of ketamine (8 mg/kg subcutaneously), that disrupts sensorimotor gating, also produces impairments in a Pavlovian emotional learning and memory task. We utilized both standard delay and trace cued and contextual fear conditioning (CCF) paradigms to examine if ketamine produces differential effects when the task is more difficult and relies on connectivity between specific brain regions. Rats administered ketamine displayed no significant deficits in cued or contextual fear following the delay conditioning protocol. However, ketamine did produce a significant impairment in the more difficult trace conditioning protocol. Analyses of tissue from the hippocampus and amygdala indicated that the administration of ketamine produced an alteration in GABA receptor protein levels differentially depending on the task. These data indicate that 8 mg/kg of ketamine impairs learning in the more difficult emotional classical conditioning task and may be related to altered signaling in GABAergic systems.