Maternal separation with neonatal pain influences later-life fear conditioning and somatosenation in male and female rats.

Early life adversity, including that which occurs in a medical setting, has been increasingly shown to have lasting consequences on both physical and mental health. In order to understand the lasting effects of early-life adversity, such as that might occur in the neonatal intensive care unit (NICU), several rodent models have been developed including maternal separation, neonatal handling, and repeated needle prick pain. However, in the clinical scenario, these stressors are often combined. Thus, the current study seeks to observe the lasting impacts of both neonatal pain and maternal separation in a rodent model. Rats were separated from their dam for 6 h per day during the first 7 days of life, during which they were subjected to repeated needle prick pain or handling. A separate group was left undisturbed. All rats were subsequently tested for threat processing using a 3-day Pavlovian fear conditioning model and for somatosensation using measures of mechanical and thermal thresholds. Results indicated that rats subjected to maternal separation and pain had enhanced fear conditioning in adolescence as well as displaying a modest age-independent tactile hypersensitivity compared to undisturbed controls. These data show that experiencing combined neonatal pain and maternal separation may create a latent vulnerability to subsequent stressors.

Inflammatory neonatal pain disrupts maternal behavior and subsequent fear conditioning in a rodent model.

Infants spending extended time in the neonatal intensive care unit are at greater risk of developing a variety of mental health problems later in life, possibly due to exposure to painful/stressful events. We used a rodent model of inflammatory neonatal pain to explore effects on fear conditioning, somatosensory function and maternal behavior. Hindpaw injections of 2% λ-carrageenan on postnatal days 1 and 4 produced an attenuation in conditioned freezing during the postweaning period, similar to our previous work with acute pain, but did not cause lasting impacts on contextual freezing nor somatosensory function. Additionally, we assessed maternal behavior to observe dam-pup interactions during the neonatal period. Results showed dams of litters which experienced pain spent similar amounts of time with pups as undisturbed controls. However, the specific behaviors differed per condition. Dams of pain litters exhibited less time licking/grooming, but more time nursing than controls. These results suggest changes in maternal care following pain could be a contributing factor underlying the long-term effects of neonatal trauma. Furthermore, our laboratory has previously shown acute, but not inflammatory pain, disrupted conditioned freezing; the current experiment observed the long-term effects of neonatal inflammatory pain on conditioned fear using a weak conditioning protocol.

Neonatal pain and stress disrupts later-life pavlovian fear conditioning and sensory function in rats: Evidence for a two-hit model.

Early life trauma has been linked to increased risks for anxiety, depression, and chronic pain. We used rodent models of acute and inflammatory neonatal pain to explore effects on fear conditioning and somatosensory function. Hindpaw needle pricks or handling on postnatal days (PNDs) 1-7 caused lasting impacts on affective and somatosensory function when assessed at later ages, PNDs 24 (postweaning), 45 (adolescence), or 66 (adulthood). First, auditory, but not contextual, freezing was mildly disrupted regardless of age. Second, a profound postfear conditioning tactile hypersensitivity was observed in neonatally stressed, postweaning rats. In the absence of fear conditioning, the mechanical hypersensitivity was not observed, consistent with a two-hit model of psychopathology. Injections of 2% α-carrageenan did not have the same lasting impact but was slightly protective against observed effects of neonatal vehicle injections. Basal and elicited corticosterone levels postweaning were not altered by neonatal pain or handling. These data demonstrate that neonatal adversity can have lasting impacts on affective and somatosensory function that differs regardless of age.

K-12 Neuroscience Education Outreach Program: Interactive Activities for Educating Students about Neuroscience.

The University of New England's Center for Excellence in the Neurosciences has developed a successful and growing K-12 outreach program that incorporates undergraduate and graduate/professional students. The program has several goals, including raising awareness about fundamental issues in neuroscience, supplementing science education in area schools and enhancing undergraduate and graduate/professional students' academic knowledge and skill set. The outreach curriculum is centered on core neuroscience themes including: Brain Safety, Neuroanatomy, Drugs of Abuse and Addiction, Neurological and Psychiatric Disorders, and Cognition and Brain Function. For each theme, lesson plans were developed based upon interactive, small-group activities. Additionally, we've organized our themes in a "Grow-up, Grow-out" approach. Grow-up refers to returning to a common theme, increasing in complexity as we revisit students from early elementary through high school. Grow-out refers to integrating other scientific fields into our lessons, such as the chemistry of addiction, the physics of brain injury and neuronal imaging. One of the more successful components of our program is our innovative team-based model of curriculum design. By creating a team of undergraduate, graduate/professional students and faculty, we create a unique multi-level mentoring opportunity that appears to be successful in enhancing undergraduate students' skills and knowledge. Preliminary assessments suggest that undergraduates believe they are enhancing their content knowledge and professional skills through our program. Additionally, we're having a significant, short-term impact on K-12 interest in science. Overall, our program appears to be enhancing the academic experience of our undergraduates and exciting K-12 students about the brain and science in general.

Evidence for hippocampus-dependent contextual learning at postnatal day 17 in the rat.

Long-term memory for fear of an environment (contextual fear conditioning) emerges later in development (postnatal day; PD 23) than long-term memory for fear of discrete stimuli (PD 17). As contextual, but not explicit cue, fear conditioning relies on the hippocampus; this has been interpreted as evidence that the hippocampus is not fully developed until PD 23. Alternatively, the hippocampus may be functional prior to PD 23, but unable to cooperate with the amygdala for fearful learning. The current experiments investigate this by separating the phases of conditioning across developmental stages. Rats were allowed to learn about the context on one day and to form the fearful association on another. Rats exposed to the context on PD 17 exhibited significant fear only when trained and tested a week later (PD 23, 24), but not on consecutive days (PD 18, 19), demonstrating that rats can learn about a context as early as PD 17. Further experiments clarify that it is associative mechanisms that are developing between PD 18 and 23. Finally, the hippocampus was lesioned prior to training to ensure the task is being solved in a hippocampus-dependent manner. These data provide compelling evidence that the hippocampus is functional for contextual learning as early as PD 17, however, its connection to the amygdala or other relevant brain structures may not yet be fully developed.

Amygdalar Corticotropin-Releasing Factor Signaling Is Required for Later-Life Behavioral Dysfunction Following Neonatal Pain.

Neonatal pain such as that experienced by infants in the neonatal intensive care unit is known to produce later-life dysfunction including heightened pain sensitivity and anxiety, although the mechanisms remain unclear. Both chronic pain and stress in adult organisms are known to influence the corticotropin-releasing factor (CRF) system in the Central Nucleus of the Amygdala, making this system a likely candidate for changes following neonatal trauma. To examine this, neonatal rats were subjected to daily pain, non-painful handling or left undisturbed for the first week of life. Beginning on postnatal day, 24 male and female rats were subjected to a 4-day fear conditioning and sensory testing protocol. Some subjects received intra-amygdalar administration of either Vehicle, the CRF receptor 1 (CRF1) receptor antagonist Antalarmin, or the CRF receptor 2 (CRF2) receptor antagonist Astressin 2B prior to fear conditioning and somatosensory testing, while others had tissue collected following fear conditioning and CRF expression in the CeA and BLA was assessed using fluorescent in situ hybridization. CRF1 antagonism attenuated fear-induced hypersensitivity in neonatal pain and handled rats, while CRF2 antagonism produced a general antinociception. In addition, neonatal pain and handling produced a lateralized sex-dependent decrease in CRF expression, with males showing a diminished number of CRF-expressing cells in the right CeA and females showing a similar reduction in the number of CRF-expressing cells in the left BLA compared to undisturbed controls. These data show that the amygdalar CRF system is a likely target for alleviating dysfunction produced by early life trauma and that this system continues to play a major role in the lasting effects of such trauma into the juvenile stage of development.

The Effects of Acute Neonatal Pain on Expression of Corticotropin-Releasing Hormone and Juvenile Anxiety in a Rodent Model.

Premature infants in the neonatal intensive care unit (NICU) may be subjected to numerous painful procedures without analgesics. One necessary, though acutely painful, procedure is the use of heel lances to monitor blood composition. The current study examined the acute effects of neonatal pain on maternal behavior as well as amygdalar and hypothalamic activation, and the long-term effects of neonatal pain on later-life anxiety-like behavior, using a rodent model. Neonatal manipulations consisted of either painful needle pricks or non-painful tactile stimulation in subjects' left plantar paw surface which occurred four times daily during the first week of life [postnatal day (PND)1-PND7]. Additionally, maternal behaviors in manipulated litters were compared against undisturbed litters via scoring of videotaped interactions to examine the long-term effects of pain on dam-pup interactions. Select subjects underwent neonatal brain collection (PND6) and fluorescent in situ hybridization (FISH) for corticotropin-releasing hormone (CRH) and the immediate early gene c-fos. Other subjects were raised to juvenile age (PND24 and PND25) and underwent innate anxiety testing utilizing an elevated plus maze (EPM) protocol. FISH indicated that neonatal pain influenced amygdalar CRH and c-fos expression, predominately in males. No significant increase in c-fos or CRH expression was observed in the hypothalamus. Additionally, neonatal pain altered anxiety behaviors independent of sex, with neonatal pain subjects showing the highest frequency of exploratory behavior. Neonatal manipulations did not alter maternal behaviors. Overall, neonatal pain drives CRH expression and produces behavioral changes in anxiety that persist until the juvenile stage.

Limbic system development underlies the emergence of classical fear conditioning during the third and fourth weeks of life in the rat.

Classical fear conditioning creates an association between an aversive stimulus and a neutral stimulus. Although the requisite neural circuitry is well understood in mature organisms, the development of these circuits is less well studied. The current experiments examine the ontogeny of fear conditioning and relate it to neuronal activation assessed through immediate early gene (IEG) expression in the amygdala, hippocampus, perirhinal cortex, and hypothalamus of periweanling rats. Rat pups were fear conditioned, or not, during the third or fourth weeks of life. Neuronal activation was assessed by quantifying expression of FBJ osteosarcoma oncogene (FOS) using immunohistochemistry (IHC) in Experiment 1. Fos and early growth response gene-1 (EGR1) expression was assessed using qRT-PCR in Experiment 2. Behavioral data confirm that both auditory and contextual fear continue to emerge between PD 17 and 24. The IEG expression data are highly consistent with these behavioral results. IHC results demonstrate significantly more FOS protein expression in the basal amygdala of fear-conditioned PD 23 subjects compared to control subjects, but no significant difference at PD 17. qRT-PCR results suggest specific activation of the amygdala only in older subjects during auditory fear expression. A similar effect of age and conditioning status was also observed in the perirhinal cortex during both contextual and auditory fear expression. Overall, the development of fear conditioning occurring between the third and fourth weeks of life appears to be at least partly attributable to changes in activation of the amygdala and perirhinal cortex during fear conditioning or expression. (PsycINFO Database Record

FAAH inhibitor OL-135 disrupts contextual, but not auditory, fear conditioning in rats.

Anxiety disorders are among the most prevalent psychological disorders, have significant negative impacts on quality of life and the healthcare system, and yet effective treatments remain elusive. Manipulating the endocannabinoid system has demonstrated potential for treating anxiety, although the side effects of direct manipulations of cannabinoid receptors keeps them from widespread clinical use. Disrupting the degradation enzyme fatty acid amide hydrolase (FAAH) enhances endogenous signaling and may produce similar efficacy without the side effects. The current experiments examine the effects of low (5.6mg/kg) or moderate (10.0mg/kg) doses of OL-135, a FAAH inhibitor, on the acquisition and consolidation of classical fear conditioning, a common model of trauma-induced anxiety. The acquisition of contextual, but not auditory, fear conditioning was disrupted by both doses of OL-135. Shock reactivity was not affected. Due to the additional neural circuitry required for contextual, but not auditory, fear conditioning, these data suggest that endocannabinoid signaling outside the amygdala may be critical for a subset of fearful memories.

Use of experimenter-given cues in visual co-orienting and in an object-choice task by a new world monkey species, cotton top tamarins (Saguinus oedipus).

Two methods assessed the use of experimenter-given directional cues by a New World monkey species, cotton top tamarins (Saguinus oedipus). Experiment 1 used cues to elicit visual co-orienting toward distal objects. Experiment 2 used cues to generate responses in an object-choice task. Although there were strong positive correlations between monkey pairs to co-orient, visual co-orienting with a human experimenter occurred at a low frequency to distal objects. Human hand pointing cues generated more visual co-orienting than did eye gaze to distal objects. Significant accurate choices of baited cups occurred with human point and tap cues and human look cues. Results highlight the importance of head and body orientation to induce shared attention in cotton top tamarins, both in a task that involved food getting and a task that did not.

Developing and validating trace fear conditioning protocols in C57BL/6 mice.

BACKGROUND: Classical fear conditioning is commonly used to study the biology of fear, anxiety and memory. Previous research demonstrated that delay conditioning requires a neural circuit involving the amygdala, but not usually the hippocampus. Trace and contextual fear conditioning require the amygdala and hippocampus. While these paradigms were developed primarily using rat models, they are increasingly being used in mice. NEW METHOD: The current studies develop trace fear conditioning and control paradigms to allow for the assessment of trace and delay fear conditioning in C57BL/6N mice. Our initial protocol yielded clear delay and contextual conditioning. However, trace conditioning failed to differentiate from an unpaired group and was not hippocampus-dependent. These results suggested that the protocol needed to be modified to specifically accommodate trace conditioning the mice. In order to reduce unconditioned freezing and increase learning, the final protocol was developed by decreasing the intensity of the tone and by increasing the inter-trial interval. RESULTS: Our final protocol produced trace conditioned freezing that was significantly greater than that followed unpaired stimulus exposure and was disrupted by hippocampus lesions. COMPARISON WITH EXISTING METHODS: A review of the literature produced 90 articles using trace conditioning in mice. Few of those articles used any kind of behavioral control group, which is required to rule out non-associative factors causing fearful behavior. Fewer used unpaired groups involving tones and shocks within a session, which is the optimal control group. CONCLUSIONS: Our final trace conditioning protocol can be used in future studies examining genetically modified C57BL/6N mice.

Contextual and auditory fear conditioning continue to emerge during the periweaning period in rats.

Anxiety disorders often emerge during childhood. Rodent models using classical fear conditioning have shown that different types of fear depend upon different neural structures and may emerge at different stages of development. For example, some work has suggested that contextual fear conditioning generally emerges later in development (postnatal day 23-24) than explicitly cued fear conditioning (postnatal day 15-17) in rats. This has been attributed to an inability of younger subjects to form a representation of the context due to an immature hippocampus. However, evidence that contextual fear can be observed in postnatal day 17 subjects and that cued fear conditioning continues to emerge past this age raises questions about the nature of this deficit. The current studies examine this question using both the context pre-exposure facilitation effect for immediate single-shock contextual fear conditioning and traditional cued fear conditioning using Sprague-Dawley rats. The data suggest that both cued and contextual fear conditioning are continuing to develop between PD 17 and 24, consistent with development occurring the in essential fear conditioning circuit.

Role of corticosterone in trace and delay conditioned fear-potentiated startle in rats.

Emotional events often lead to particularly strong memory formation. Corticosterone, the final product of hypothalamic-pituitary-adrenal (HPA)-axis activation, has been suggested to play a critical role in this effect. Although a great deal of work has implicated the amygdala as a necessary structure for the effects of corticosterone, other studies have suggested a critical role for the hippocampus in determining the involvement of corticosterone. The current experiments examined this question by disrupting corticosterone synthesis with administration of metyrapone (25 or 100 mg/kg) prior to training in either dorsal hippocampus-independent delay fear conditioning or dorsal hippocampus-dependent trace fear conditioning. Metyrapone administration 2 hrs prior to training significantly attenuated corticosterone secretion during training, but these effects were transient as corticosterone levels were similar to control subjects following the test session. As hypothesized, only trace fear conditioning was impaired. This suggests that only fear conditioning tasks that are dependent on the dorsal hippocampus require HPA-axis activation in order to be learned.

Neonatal binge alcohol exposure produces dose dependent deficits in interstimulus interval discrimination eyeblink conditioning in juvenile rats.

Alcohol consumption in neonatal rats produces cerebellar damage and is widely used to model 3rd-trimester human fetal alcohol exposure. Neonatal "binge-like" exposure to high doses of alcohol (5 g/kg/day or more) impairs acquisition of eyeblink classical conditioning (EBC), a cerebellar-dependent Pavlovian motor learning task. We have recently found impairments in interstimulus interval (ISI) discrimination--a complex task variant of EBC--in adult rats following postnatal day (PD) 4-9 alcohol exposure at doses of 3, 4, and 5 g/kg/day. Because robust developmental differences in conditioned response (CR) generation and CR latency measures are present between untreated juveniles and adults in this task, we sought to extend alcohol findings to juvenile rats (PD30). Five neonatal treatment groups were used: (1) undisturbed controls, (2) sham intubation controls, (3) 3 g/kg/day of alcohol (blood alcohol concentration {BAC}=139.9 mg/dl), (4) 4 g/kg/day of alcohol (BAC=237.3 mg/dl), or (5) 5 g/kg/day of alcohol (BAC=301.8 mg/dl). Intubations occurred over PD4-9. ISI discrimination training in juveniles (PD30-33) revealed dose-dependent CR deficits in all three alcohol-exposed groups relative to controls. Contrary to expected outcomes, CR latency measures were not significantly affected as a function of neonatal treatment. Comparison of these findings with our recent study in adults suggests that alcohol-induced impairments in ISI discrimination EBC may be greater in adults relative to juveniles. The present findings provide further evidence that ISI discrimination may provide greater sensitivity to functional deficits resulting from moderate levels of neonatal alcohol exposure relative to single-cue EBC paradigms.

Hippocampal activity, but not plasticity, is required for early consolidation of fear conditioning with a short trace interval.

The dorsal hippocampus is required for explicit cue fear conditioning only when a temporal gap is inserted between conditioned stimulus (CS) termination and unconditioned stimulus (US) onset (trace fear conditioning). To examine the role of the dorsal hippocampus in associating temporally discontiguous stimuli and to minimize the potential contribution of contextual cues, fear conditioning was conducted using a relatively short (3-s) trace interval. Inactivation of the dorsal hippocampus using the AMPA receptor antagonist NBQX (3 microg/hemisphere) or the GABA(A) agonist muscimol (5 microg/hemisphere) disrupted trace fear conditioning when conducted immediately following training. Trace conditioning was not disrupted significantly when NBQX was infused either before or 2 h after training. Similarly, NBQX infusions were not effective when the CS and US overlapped (delay conditioning). Moreover, trace conditioning was not impaired by intrahippocampal infusion of either the NMDA receptor antagonist AP5 (5 microg/hemisphere) or the L-type voltage-gated calcium channel (VGCC) blocker diltiazem (20 or 40 microg/hemisphere). These data suggest that the involvement of the dorsal hippocampus in short trace interval fear conditioning is largely restricted to the early period of memory consolidation, during which time it mediates the storage of long-term memory in other brain regions.

Dissociable effects of hippocampus lesions on expression of fear and trace fear conditioning memories in rats.

The role of the hippocampus in memory is commonly investigated by comparing fear conditioning paradigms that differ in their reliance on the hippocampus. For example, the dorsal (septal) portion of the hippocampus is involved in trace, but not delay fear conditioning, two Pavlovian paradigms in which only the relative timing of stimulus presentation is varied. However, a growing literature implicates the ventral (temporal) portion of the hippocampus in the expression of fear, irrespective of prior training. The current experiments evaluated the relative contributions of the dorsal and ventral portions of the hippocampus to trace fear conditioning specifically vs. the expression of conditioned fear in general. Lesions restricted to the dorsal hippocampus blocked acquisition of trace fear conditioning. Larger lesions, also including an adjacent portion of the ventral hippocampus, were required to impair retrieval of trace fear conditioning. Delay fear conditioning was not disrupted in either case. In contrast, lesions that encompassed almost the entire dorsal and ventral hippocampus disrupted expression of both trace and delay fear conditioning. The current data suggest distinct roles in fear conditioning for three regions of the hippocampus: the septal zone is required for acquisition of trace fear conditioning, a larger portion of the hippocampus is critical for memory retrieval, and a region including the temporal zone is required for expression of both trace and delay fear conditioning. These findings are consistent with evidence suggesting the neuroanatomical and functional segregation of the hippocampus into three zones along its septal-temporal axis.

Timing of fear expression in trace and delay conditioning measured by fear-potentiated startle in rats.

In two experiments, the time course of the expression of fear in trace (hippocampus-dependent) versus delay (hippocampus-independent) conditioning was characterized with a high degree of temporal specificity using fear-potentiated startle. In experiment 1, groups of rats were given delay fear conditioning or trace fear conditioning with a 3- or 12-sec trace interval between conditioned stimulus (CS) offset and unconditioned stimulus (US) onset. During test, the delay group showed fear-potentiated startle in the presence of the CS but not after its offset, whereas the trace groups showed fear-potentiated startle both during the CS and after its offset. Experiment 2 compared the time course of fear expression after trace conditioning with the time course in two delay conditioning groups: one matched to the trace conditioning group with respect to CS duration, and the other with respect to ISI. In all groups, fear was expressed until the scheduled occurrence of the US and returned to baseline rapidly thereafter. Thus, in both trace and delay fear conditioning, ISI is a critical determinant of the time course of fear expression. These results are informative as to the possible role of neural structures, such as the hippocampus, in memory processes related to temporal information.