Age and experience dependent changes in Egr-1 expression during the ontogeny of the context preexposure facilitation effect (CPFE).

The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which acquisition of the contextual representation and association of the retrieved contextual memory with an immediate foot-shock are separated by 24 h. During the CPFE, learning- related expression patterns of the early growth response-1 gene (Egr-1) vary based on training phase and brain sub-region in adult and adolescent rats (Asok, Schreiber, Jablonski, Rosen, & Stanton, 2013; Schreiber, Asok, Jablonski, Rosen, & Stanton, 2014; Chakraborty, Asok, Stanton, & Rosen, 2016). The current experiments extended our previous findings by examining Egr-1 expression in infant (PD17) and juvenile (PD24) rats during the CPFE using preexposure protocols involving single-exposure (SE) or multiple-exposure (ME) to context. Following a 5 min preexposure to the training context (i.e. the SE protocol), Egr-1 expression in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC) and lateral nucleus of the amygdala (LA) was differentially increased in PD24 rats relative to PD17 rats. In contrast, increased Egr-1 expression following an immediate foot-shock (2s, 1.5 mA) did not differ between PD17 and PD24 rats, and was not learning-related. Interestingly, increasing the number of exposures to the training chamber on the preexposure day (i.e. ME protocol) altered training-day expression such that a learning-related increase in expression was observed in the mPFC in PD24 but not PD17 rats. Together, these results illustrate a clear maturation of Egr-1 expression that is both age- and experience-dependent. In addition, the data suggest that regional activity and plasticity within the mPFC on the preexposure but not the training day may contribute to the ontogenetic profile of the effect. Further studies are necessary to elucidate the causal role of sub-region-specific neuroplasticity in the ontogeny of the CPFE.

Antagonism of muscarinic acetylcholine receptors in medial prefrontal cortex disrupts the context preexposure facilitation effect.

Cholinergic function plays a role in a variant of context fear conditioning known as the context preexposure facilitation effect (CPFE; Robinson-Drummer, Dokovna, Heroux, & Stanton, 2016). In the CPFE, acquisition of a context representation, the context-shock association, and expression of context fear occur across successive phases, usually 24h apart. Systemic administration of scopolamine, a muscarinic acetylcholine receptor antagonist, prior to each phase (context preexposure, immediate-shock training, and testing) disrupts the CPFE in juvenile rats (Robinson-Drummer et al., 2016). Dorsal hippocampal (dHPC) cholinergic function contributes significantly to this effect, as local infusion of scopolamine into the dHPC prior to any individual phase of the CPFE produces a disruption identical to systemic administration (Robinson-Drummer et al., 2016). The current experiment extended these findings to another forebrain region implicated in the CPFE, the medial prefrontal cortex (mPFC). Adolescent rats received bilateral infusions of scopolamine (35µg/side) or PBS 10min before all three phases of the CPFE or only prior to a single phase. Intra-mPFC administration of scopolamine prior to all three phases significantly impaired fear conditioning suggesting that mPFC cholinergic function is necessary for successful CPFE performance. Analyses of the individual infusion days revealed a significant impairment of the CPFE when infusions occurred prior to preexposure or training (i.e. immediate footshock) but not prior to testing. In total, these findings suggests a role of mPFC cholinergic function in the acquisition and/or consolidation of a contextual representation and the context-shock association but not in retrieval or expression of fear memory. Implications for mPFC involvement in contextual fear conditioning and neurological dysfunction following neonatal alcohol exposure are discussed.

Effects of exercise and environmental complexity on deficits in trace and contextual fear conditioning produced by neonatal alcohol exposure in rats.

In rodents, voluntary exercise and environmental complexity increases hippocampal neurogenesis and reverses spatial learning and long-term potentiation deficits in animals prenatally exposed to alcohol. The present experiment extended these findings to neonatal alcohol exposure and to delay, trace, and contextual fear conditioning. Rats were administered either 5.25 g/kg/day alcohol via gastric intubation or received sham-intubations (SI) between Postnatal Day (PD) 4 and 9 followed by either free access to a running wheel on PD 30-41 and housing in a complex environment on PD 42-72 (wheel-running plus environmental complexity; WREC) or conventional social housing (SHSH) from PD 30 to 72. Adult rats (PD 80 ± 5) received 5 trials/day of a 10-s flashing-light conditioned stimulus (CS) paired with .8 mA footshock either immediately (delay conditioning) or after a 10-s trace interval (trace conditioning) for 2 days. Neonatal alcohol exposure impaired context and trace conditioning, but not short-delay conditioning. The WREC intervention did not reverse these deficits, despite increasing context-related freezing in ethanol-exposed and SI animals.

USAID program for the prevention and treatment of vaginal fistula.

The cornerstone of the US Agency for International Development (USAID) fistula program is to support and strengthen local capacity for fistula repair. The USAID program includes support to upgrade facilities, enhance local surgical repair capability, allocate equipment and supplies to operating rooms, implement quality improvement systems, and cover the women's transportation costs to and from the treatment facilities. The program also offers training in clinical and counseling skills; transferring skills South-to-South; and monitoring and evaluating the program's effectiveness. As new fistula cases continually increase the backlog of untreated cases, its efforts are also directed toward the prevention of fistula and the reintegration of treated women into their communities. Furthermore, the program challenges the culture of sexual violence against women that leads to traumatic gynecologic fistulas.

Variants of contextual fear conditioning induce differential patterns of Egr-1 activity within the young adult prefrontal cortex.

Contextual fear conditioning is a form of associative learning where animals must experience a context before they can associate it with an aversive stimulus. Single-trial contextual fear conditioning (sCFC) and the context preexposure facilitation effect (CPFE) are two variants of CFC where learning about the context is temporally contiguous (sCFC) with or separated (CPFE) from receiving a footshock in that context. Neural activity within CA1 of the dorsal hippocampus (CA1), amygdala (LA), and prefrontal cortex (PFC) may play a critical role when animals learn to associate a context with a footshock (i.e., training). Previous studies from our lab have found that early-growth-response gene 1 (Egr-1), an immediate early gene, exhibits unique patterns of activity within regions of the PFC following training in sCFC and the CPFE of juvenile rats. In the present study, we extended our studies by examining Egr-1 expression in young adult rats to determine (1) if our previous work reflected changes unique to development or extend into adulthood and (2) to contrast expression profiles between sCFC and the CPFE. Rats that learned context fear with sCFC showed increased Egr-1 in the anterior cingulate, orbitofrontal and infralimbic cortices relative to non-associative controls following training, but expression in prelimbic cortex did not differ between fear conditioned and non-associative controls. In contrast, rats trained in the CPFE also showed increased Egr-1 in all the prefrontal cortex regions, including prelimbic cortex. These findings replicate our previous findings in juveniles and suggest that Egr-1 in specific PFC subregions may be uniquely involved in learning context-fear in the CPFE compared to sCFC.

Neurotoxic effects of neonatal triethyltin (TET) exposure are exacerbated with aging.

Neonatal Long-Evans rats dosed with TET (5 mg/kg; IP) or saline on postnatal day (PND) 10 were examined across the life span for neural damage and performance on spatial learning tasks. A subset of rats were sacrificed to assess early damage with Nissl-staining, Timm's histochemistry, and glial fibrillary acidic protein (GFAP) immunohistochemistry 2, 7, or 14 days after dosing. Littermates were tested behaviorally in a T-maze spatial delayed alternation task on PND 23 or PND 90, and in a Morris water maze place learning task at 3, 12, or 24 months postdosing and then sacrificed for histological analysis. In neonatal rats, histological analysis indicated gliosis in discrete cortical regions, loss of Nissl-stained neurons in the hippocampal formation, entorhinal cortex and piriform cortex, and loss of Timm's staining in the entorhinal cortex. The behavioral assessment at PND 23 indicated a significant impairment in the T-maze. However, no significant impairments were observed in the T-maze at 3 months or the water maze at 3 or 12 months postdosing. At 24 months, TET-treated rats showed significant deficits in acquisition and retention of the water maze task compared with age-matched controls. Both groups of 24 months old rats were significantly impaired compared with young controls. At 24 months, there was a general age-related decrease in the optical density of Timm's staining in cortical regions (9%), compounded by a further decrease in the entorhinal cortex and outer molecular layer of the dentate gyrus of the hippocampus in TET treated rats (30%). These data indicate that early developmental exposure to an organometal resulted in morphological damage that was apparent behaviorally only during early postnatal development and with advanced aging.

Ontogeny of the conditioned eyeblink response in rats: acquisition or expression?

Eyeblink conditioning depends critically on an identified brainstem-cerebellar circuit and is modulated under some circumstances by the hippocampus, amygdala, and other forebrain regions. Developmental studies of eyeblink conditioning could help elucidate questions concerning the behavioral expression of plasticity within these brain circuits and regions, and of their functional interactions, as they unfold during ontogeny. Recently, this laboratory has shown that conditioning of the eyeblink reflex develops dramatically between Postnatal Days (PND) 17 and PND 24 in the rat. The present study asked whether the developmental emergence of the eyeblink conditioned response (CR) occurs gradually or abruptly over this age range, and whether it reflects developmental changes in acquisition or expression of the learned eyeblink reflex. In Experiment 1, rat pups received two consecutive days of training beginning on PND 17, 20, or 24. Conditioned responses occurred at low levels on PND 17-18, intermediate levels on PND 20-21, and high levels on PND 24-25. In Experiment 2, 17-day-old rats received 2 days of training, 72 h apart, so that effects of training on PND 17 could be examined at an age, PND 20, when expression of the eyeblink CR was clearly possible. On PND 20, rat pups that had received paired training on PND 17 showed significantly faster conditioning than controls that had received unpaired training or no training on PND 17. These findings suggest that neural plasticity underlying associative learning developmentally precedes its overt expression in behavior. Hypotheses concerning the nature and locus of this learning are discussed.

Eyeblink conditioning in the infant rat: an animal model of learning in developmental neurotoxicology.

Classical conditioning of the eyeblink reflex is a relatively simple procedure for studying associative learning that was first developed for use with human subjects more than half a century ago. The use of this procedure in laboratory animals by psychologists and neuroscientists over the past 30 years has produced a powerful animal model for studying the behavioral and biological mechanisms of learning. As a result, eyeblink conditioning is beginning to be pursued as a very promising model for predicting and understanding human learning and memory disorders. Among the many advantages of this procedure are (a) the fact that it can be carried out in the same manner in both humans and laboratory animals; (b) the many ways in which it permits one to characterize changes in learning at the behavioral level; (c) the readiness with which hypotheses regarding the neurological basis of behavioral disorders can be formulated and tested; (d) the fact that it can be used in the same way across the life-span; and (e) its ability to distinguish, from normative groups, populations suffering from neurological conditions associated with impaired learning and memory, including those produced by exposure to neurotoxicants. In this article, we argue that these properties of eyeblink conditioning make it an excellent model system for studying early impairments of learning and memory in developmental neurotoxicology. We also review progress that has been made in our laboratory in developing a rodent model of infant eyeblink conditioning for this purpose.

Social influences on conditioned cortisol secretion in the squirrel monkey.

'Social buffering'--the apparent capacity of group membership to reduce the adrenocortical response to stress, was investigated in squirrel monkeys by means of a between-within experimental design. Adult males were assigned to two groups. Group paired (n = 6) received pairings of a conditional stimulus (CS) with footshock. Group control (n = 6) received CS presentations without shock. All animals were then tested with 10 presentations of the CS without shock under three social-housing conditions, in four successive phases of the experiment: individual, dyad, group, and individual housing. Neither group showed a cortisol response to the CS prior to training. Following training, CS-evoked elevations of cortisol were found only in group paired, and only in the individual housing conditions. These results replicate and extend our previous finding that the presence of conspecifics can ameliorate a neuroendocrine response to psychological stressors in squirrel monkeys.

Fimbria-fornix transections disrupt the ontogeny of delayed alternation but not position discrimination in the rat.

In Experiment 1, Long-Evans rat pups received fimbria-fornix transections or sham surgery on Postnatal Day 10 (PND10) and were then trained on PND23 to perform either a discrete-trials delayed alternation (DA) or a simple position discrimination (PD) task in a T maze. Rat pups in both surgical conditions learned the PD task within five 12-trial blocks of training. However, only sham-operated pups learned the DA task. In Experiment 2, performance of DA emerged between PND19 and PND27 in sham-operated pups but failed entirely to develop in pups with early lesions. In Experiment 3, fornix-transected pups that were given extended DA training (132 trials) on PND23-PND24 showed some improvement in performance but remained impaired in relation to sham-operated controls. These findings implicate the limbic system in the postnatal development of DA but not PD and suggest that dual-process theories of memory may be relevant to the psychobiology of cognitive development.

Differential ontogeny of working memory and reference memory in the rat.

In two experiments we examined the ability of 15-, 21-, and 27-day-old rats to perform two spatial working memory problems (delayed alternation and discrete-trials delayed alternation) and a reference memory problem (position habit) in a T-maze. In the delayed alternation problem, each animal was presented with a series of free-choice trials and was rewarded for regularly alternating responses to the left and right arms of the T-maze. In the discrete-trials delayed alternation problem, each animal was forced to one maze arm and rewarded (forced run) and was then placed back into the start box and given a choice of arms (choice run). The direction of forced runs followed an irregular, counterbalanced series, and animals were rewarded for choosing the alternate maze arm on choice runs. In the position habit problem, animals were rewarded for consistently choosing one of the two arms of the T-maze. Performance on these problems was assessed relative to control conditions in which reward was not contingent on choice behavior. At all ages, rat pups learned to perform the delayed alternation and position habit problems (Experiment 1). However, only 21- and 27-day-old rats were able to learn the discrete-trials delayed alternation problem. The 15-day-olds were unable to learn this task (Experiment 2). The results of these experiments show that reference memory capacity is present by at least 15 days of age in the rat and does not develop further at later ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Medial prefrontal cortex lesions and spatial delayed alternation in the developing rat: recovery or sparing?

In Experiment 1, Long-Evans rat pups received medial prefrontal cortex (PFC) aspirations or sham surgery on Postnatal Day 10 (PND10) and were then trained on PND23 to perform one of two T-maze tasks: discrete-trials delayed alternation (DA) or simple position discrimination. Early PFC damage produced a selective failure to learn the DA task. In Experiment 2, pups given the same lesion or sham surgery were trained on DA on PND19, PND27, or PND33. In relation to sham-operated controls, pups with PFC damage were impaired on PND19, somewhat impaired on PND27, and entirely unimpaired when tested on PND33. In Experiment 3, pups given larger lesions of the frontal cortex on PND10 were impaired on DA when tested on PND23 but not when tested on PND33. These findings indicate that early PFC lesions result in a memory deficit around the time of weaning, which then recovers over the next 10-14 days of development. Moreover, the early deficit is selective for a late developing cognitive process (or processes) that is involved in acquisition of DA.

Maternal deprivation potentiates pituitary-adrenal stress responses in infant rats.

In three experiments we examined the effect of maternal deprivation on the pituitary-adrenal response of 12-, 16- and 20-day-old rat pups to novelty stress. Infant rats were either deprived individually in heated incubators or left in the home nest with their mother and then tested for their corticosteroid response to 30-min exposure to a novel test arena (novelty-stress). In Experiment 1 we showed that the magnitude of the stress response was a positively accelerated function of the deprivation interval. Stress responses were not increased after 1 hour of deprivation, were modestly increased after 8 hours of deprivation, and were dramatically increased after 24 hours of deprivation. In Experiment 2 we asked whether potentiation of the stress response resulted from the maternal or the nutritive components of the deprivation procedure. Pups were tested under one of four treatment conditions formed by a 2 (Maternally Deprived vs. Nondeprived) x 2 (Nutritively Deprived vs. Nondeprived) factorial design. At 12 and 16 days of age, potentiation of the stress response was traced to the absence of maternal care and not nutrients. At 20 days of age, both maternal and nutritive deprivation contributed to the potentiated stress response. The results of Experiment 3 showed that this effect was mediated, at least in part, by increased adrenocortical sensitivity to ACTH, because the corticosteroid response to exogenous ACTH administration was also increased by maternal deprivation. These findings add to a growing body of literature that supports the concept of maternal regulation of infant physiology. They also support previous reports from this laboratory indicating that suppression of the pituitary-adrenal system is modulated by maternal variables during the preweaning period in the rat.

Adrenal responses to reinforcement and extinction: role of expectancy versus instrumental responding.

Plasma corticosterone levels were evaluated during operant conditioning in order to determine the effect of reinforced and nonreinforced responding (extinction) on adrenal activation. The influence of instrumental responding was assessed by comparing trained rats with yoked subjects that received a matched reward schedule in the absence of an operant task. Reinforcement sessions resulted in a significant decrease in adrenal secretion at 20 min, but not at 5 min, whereas extinction caused a rapid increase in corticosterone levels at 5 min and an even greater elevation by 20 min. Comparison of the operant and yoked subjects showed that this effect of reinforcement and extinction was not dependent on instrumental responding, but rather on the receipt or withdrawal of the expected reward.

Posterodorsal septal lesions impair performance on both shift and stay working memory tasks.

Two groups of rats were trained preoperatively on either a shift or a stay problem in a T-maze. Training trials consisted of two runs, an "information run" in which a subject was forced to go down one of the two arms of the T-maze, followed immediately by a "choice run" in which the subject could choose either arm. In the shift condition, rats were rewarded with wet mash only for choosing the arm opposite the one they entered on the information run. In the stay condition, rats were rewarded for entering the arm that was entered on the information run. In both conditions, rats ultimately learned to perform with median accuracy of 100%, but the shift group reached this level of performance after fewer trials than the stay group. In a subsequent phase, the delay between information runs and choice runs was increased from 0 to 30, 60, 90, 210 and then decreased back to 0 s. Choice accuracy in both groups declined as the delay increased and returned to 100% at the 0-s delay. Half of the subjects in each condition then received either lesions of the posterodorsal septum-aimed at disconnecting the septum and hippocampus-or control surgery. Postoperative retention deficits resulted from posterodorsal septal lesions in both shift and stay conditions. There was some recovery of performance but no indication of "savings" during postoperative training. These results indicate that deficits in maze performance by rats with septo-hippocampal damage are not restricted to tasks that require alternation of spatial locations. This finding falsifies the notion that maze deficits reflect a spontaneous alternation deficit or changed "spatial strategy," but it supports the hypothesis of a working memory deficit in these animals.

Early cerebellar lesions impair eyeblink conditioning in developing rats: differential effects of unilateral lesions on postnatal day 10 or 20.

Experiment 1 demonstrated that the ipsilateral cerebellar hemisphere is essential for the acquisition of eyeblink conditioning in infant rats and that cerebellar lesions given on Postnatal Day 10 (PND10) produced deficits in eyeblink conditioning when given to either hemisphere. For both hemispheres, lesions that were restricted to the cerebellar cortex produced less severe deficits than lesions that included the deep nuclei. Experiment 2 showed that the age at which the cerebellar lesions occurred determined whether damage to the contralateral cerebellar hemisphere impaired conditioning. Lesions of either the ipsilateral or contralateral hemisphere that included the deep nuclei disrupted eyeblink conditioning when given on PND10. In contrast, when lesions were given on PND20, ipsilateral lesions that included the deep nuclei abolished conditioning, while the same lesion given to the contralateral hemisphere had no effect.

Eyeblink conditioning in the developing rat.

Long-Evans rat pups, 17-18 or 24 days of age, were trained with an eyeblink conditioning (EBC) procedure that was used previously with adult rats (Skelton, 1988). Pups received 3 sessions of delay conditioning in a single day at about 4-hr intervals (100 trials/session). Trials involved pairings of an auditory conditioned stimulus (2.8-kHz, 82-dB tone) and a periocular-shock unconditioned stimulus (US; 100 ms, 2 mA), which were presented 280 ms apart. EBC was observed at both ages, but older pups learned much more rapidly. Subsequent experiments established that this effect is associative (Experiment 2), that age differences in EBC cannot be attributed to differences in ability to respond or in sensitivity to the US (Experiment 3), and that EBC rate can be modulated by motivational state (Experiment 4). This preparation may help elucidate the relation between neural development and the ontogeny of learning.

Disruption of human eyeblink conditioning after central cholinergic blockade with scopolamine.

Human (Homo sapiens) volunteers (N = 72) received saline, a low dose of oral scopolamine (0.6 mg), a high dose of oral scopolamine (1.2 mg), or a peripheral analogue (glycopyrrolate). They then underwent classical conditioning of the eyeblink response to a tone conditioned stimulus (CS) and a corneal airpuff unconditioned stimulus (UCS) in a delay conditioning paradigm. There was a dose-related decline in acquisition of the conditioned response. These drug-induced conditioning deficits were similar to those previously reported in rabbit eyeblink conditioning and could not be attributed to such nonassociative factors as changes in auditory thresholds to the tone CS, magnitude of reflexive blinks to the airpuff UCS, or to changes in spontaneous blink rates.

Memory and the septo-hippocampal cholinergic system in the rat.

This study examined the effects of intrahippocampal injections of scopolamine (a muscarinic antagonist drug) on performance of a working-memory task (contingently reinforced T-maze alternation) and a reference-memory task (visual discrimination) by the same rats in the same maze. Rats in the first shipment were trained in delayed alternation, received bilateral implantation of cannulae aimed at the CA 3 field of the dorsal hippocampus, and were tested for retention with 1 microliter microinjections of scopolamine (35 micrograms) and saline on alternate days. These rats were then trained on visual discrimination and tested alternately under scopolamine or saline as described above. It was found that scopolamine impaired performance of delayed alternation to a greater extent than performance of visual discrimination. Data from rats in the second shipment replicated this finding, with the order of the tasks reversed, and, additionally, showed that delayed alternation, but not visual discrimination, was impaired at a dose of 12 micrograms/microliter. A dose of 4 micrograms/microliter had no effect on either task. It is concluded that performance of a working-memory task is significantly more sensitive to disruption of cholinergic mechanisms in the hippocampus than performance of a reference-memory task.