Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.National Institute of Mental Health (U.S.) (R01 MH084966)United States. Army Research Office (United States. Defense Advanced Research Projects Agency Grant W911NF-10-1-0059

Amygdala-ventral striatum circuit activation decreases long-term fear

In humans, activation of the ventral striatum, a region associated with reward processing, is associated with the extinction of fear, a goal in the treatment of fear-related disorders. This evidence suggests that extinction of aversive memories engages reward-related circuits, but a causal relationship between activity in a reward circuit and fear extinction has not been demonstrated. Here, we identify a basolateral amygdala (BLA)-ventral striatum (NAc) pathway that is activated by extinction training. Enhanced recruitment of this circuit during extinction learning, either by pairing reward with fear extinction training or by optogenetic stimulation of this circuit during fear extinction, reduces the return of fear that normally follows extinction training. Our findings thus identify a specific BLA-NAc reward circuit that can regulate the persistence of fear extinction and point toward a potential therapeutic target for disorders in which the return of fear following extinction therapy is an obstacle to treatment.National Institute of Mental Health (U.S.) (R01 MH084966)United States. Army Research OfficeUnited States. Defense Advanced Research Projects Agency (grant W911NF-10-1-0059

Growth hormone biases amygdala network activation after fear learning

Prolonged stress exposure is a risk factor for developing posttraumatic stress disorder, a disorder characterized by the ‘over-encoding’ of a traumatic experience. A potential mechanism by which this occurs is through upregulation of growth hormone (GH) in the amygdala. Here we test the hypotheses that GH promotes the over-encoding of fearful memories by increasing the number of neurons activated during memory encoding and biasing the allocation of neuronal activation, one aspect of the process by which neurons compete to encode memories, to favor neurons that have stronger inputs. Viral overexpression of GH in the amygdala increased the number of amygdala cells activated by fear memory formation. GH-overexpressing cells were especially biased to express the immediate early gene c-Fos after fear conditioning, revealing strong autocrine actions of GH in the amygdala. In addition, we observed dramatically enhanced dendritic spine density in GH-overexpressing neurons. These data elucidate a previously unrecognized autocrine role for GH in the regulation of amygdala neuron function and identify specific mechanisms by which chronic stress, by enhancing GH in the amygdala, may predispose an individual to excessive fear memory formation.National Institute of Mental Health (U.S.) (NIMH R01 MH084966)United States. Defense Advanced Research Projects Agency (DARPA grant W911NF-10-1-0059)United States. Army Research Offic

Changes in Brain MicroRNAs Contribute to Cholinergic Stress Reactions

Mental stress modifies both cholinergic neurotransmission and alternative splicing in the brain, via incompletely understood mechanisms. Here, we report that stress changes brain microRNA (miR) expression and that some of these stress-regulated miRs regulate alternative splicing. Acute and chronic immobilization stress differentially altered the expression of numerous miRs in two stress-responsive regions of the rat brain, the hippocampal CA1 region and the central nucleus of the amygdala. miR-134 and miR-183 levels both increased in the amygdala following acute stress, compared to unstressed controls. Chronic stress decreased miR-134 levels, whereas miR-183 remained unchanged in both the amygdala and CA1. Importantly, miR-134 and miR-183 share a common predicted mRNA target, encoding the splicing factor SC35. Stress was previously shown to upregulate SC35, which promotes the alternative splicing of acetylcholinesterase (AChE) from the synapse-associated isoform AChE-S to the, normally rare, soluble AChE-R protein. Knockdown of miR-183 expression increased SC35 protein levels in vitro, whereas overexpression of miR-183 reduced SC35 protein levels, suggesting a physiological role for miR-183 regulation under stress. We show stress-induced changes in miR-183 and miR-134 and suggest that, by regulating splicing factors and their targets, these changes modify both alternative splicing and cholinergic neurotransmission in the stressed brain

Conditional plasticity in the amygdala: Substrates, molecular mechanisms, and the relationship to fear behavior.

The present studies investigated some of the mechanisms involved in establishing long-term fear memories within the amygdala. First, we sought to determine whether the acquisition of contextual and auditory fear conditioning was dissociable within amygdaloid nuclei. We observed that both forms of fear conditioning required intact lateral (LA), central (CE), and anterior basal nuclei (BA) of the amygdala, suggesting that the acquisition of contextual and auditory fear conditioning rely on similar anatomical substrates. We also investigated the effects of pre-training NMDA receptor (NMDAR) blockade of the basolateral complex (LA and BA) or CE on savings (learning that is not present during an initial testing session) of fear conditioning. We found that NMDAR antagonism in either amygdaloid region prevented behavioral savings of both contextual and auditory fear conditioning, indicating that plasticity in both amygdaloid regions is critical for the acquisition of fear conditioning. In a related set of experiments, we investigated the effects of NMDAR antagonism on the acquisition and expression of conditional spike firing in LA. We found that NMDAR antagonism abolished the acquisition of behavioral and neuronal indices of associative learning, while having minimal effects on the expression of such learning. These studies indicate that amygdaloid NMDARs make an essential contribution to the acquisition of associative fear memories, but play a comparatively limited role in the expression of those memories. We also attempted to dissociate conditioning-related changes in amygdaloid spike firing and performance of fear behaviors. We found that amygdaloid spike firing increased for conditional stimuli paired with aversive unconditional stimuli, regardless of overt behavioral indices of fear during extinction testing, while spike firing to non-paired conditional stimuli did not increase. This suggests that the expression of conditional spike firing is regulated by the associative history of a conditional stimulus, and not by behavior-related factors. Lastly, we explored the contribution of amygdaloid protein kinases A and C to the acquisition of fear conditioning, and found that these kinases make significant contributions to the formation of conditional fear memories. Collectively, these studies provide novel information about the establishment of long-term fear memories within the amygdala.Ph.D.Behavioral psychologyBiological SciencesNeurosciencesPhysiological psychologyPsychologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123178/2/3068871.pd

Hippocampal regulation of aversive memories

For many years, the hippocampal formation has been implicated in the regulation of negative emotion, yet the nature of this link has remained elusive. Recent studies have made important links between the hippocampus and regulation of stress hormones that affect aversive memory. Additional studies have shown that the hippocampus regulates the gating of fear by contextual information. An emerging literature also links the hippocampus to prediction errors during fear learning and extinction. The mechanisms by which the hippocampus regulates negative emotion are clearly complicated, but suggest that interventions aimed at restoring normal hippocampal function may help with disorders of negative affect, such as depression or post-traumatic stress disorder and depression

Restoration of hippocampal growth hormone reverses stress-induced hippocampal impairment

Though growth hormone (GH) is synthesized by hippocampal neurons, where its expression is influenced by stress exposure, its function is poorly characterized. Here, we show that a regimen of chronic stress that impairs hippocampal function in rats also leads to a profound decrease in hippocampal GH levels. Restoration of hippocampal GH in the dorsal hippocampus via viral-mediated gene transfer completely reversed stress-related impairment of two hippocampus-dependent behavioral tasks, auditory trace fear conditioning and contextual fear conditioning, without affecting hippocampal function in unstressed control rats. GH overexpression reversed stress-induced decrements in both fear acquisition and long-term fear memory. These results suggest that loss of hippocampal GH contributes to hippocampal dysfunction following prolonged stress and demonstrate that restoring hippocampal GH levels following stress can promote stress resilience

A ghrelin–growth hormone axis drives stress-induced vulnerability to enhanced fear

Hormones in the hypothalamus–pituitary–adrenal (HPA) axis mediate many of the bodily responses to stressors, yet there is no clear relationship between the levels of these hormones and stress-associated mental illnesses such as posttraumatic stress disorder (PTSD). Therefore, other hormones are likely to be involved in this effect of stress. Here we used a rodent model of PTSD in which rats repeatedly exposed to a stressor display heightened fear learning following auditory Pavlovian fear conditioning. Our results show that stress-related increases in circulating ghrelin, a peptide hormone, are necessary and sufficient for stress-associated vulnerability to exacerbated fear learning and these actions of ghrelin occur in the amygdala. Importantly, these actions are also independent of the classic HPA stress axis. Repeated systemic administration of a ghrelin receptor agonist enhanced fear memory but did not increase either corticotropin-releasing factor (CRF) or corticosterone. Repeated intraamygdala infusion of a ghrelin receptor agonist produced a similar enhancement of fear memory. Ghrelin receptor antagonism during repeated stress abolished stress-related enhancement of fear memory without blunting stress-induced corticosterone release. We also examined links between ghrelin and growth hormone (GH), a major downstream effector of the ghrelin receptor. GH protein was upregulated in the amygdala following chronic stress, and its release from amygdala neurons was enhanced by ghrelin receptor stimulation. Virus-mediated overexpression of GH in the amygdala was also sufficient to increase fear. Finally, virus-mediated overexpression of a GH receptor antagonist was sufficient to block the fear-enhancing effects of repeated ghrelin receptor stimulation. Thus, ghrelin requires GH in the amygdala to exert fear-enhancing effects. These results suggest that ghrelin mediates a novel branch of the stress response and highlight a previously unrecognized role for ghrelin and growth hormone in maladaptive changes following prolonged stress.Massachusetts Institute of Technology. Undergraduate Research Opportunities ProgramHugh Hampton Young Memorial FellowshipSchoemaker FellowshipSingleton Presidential FundJames R. Killian FellowshipNational Institute of Mental Health (U.S.) (R01 MH084966)United States. Army Research OfficeUnited States. Defense Advanced Research Projects Agency (Grant W911NF-10-1-0059

Tracking the Time-Dependent Role of the Hippocampus in Memory Recall Using DREADDs

The hippocampus is critical for the storage of new autobiographical experiences as memories. Following an initial encoding stage in the hippocampus, memories undergo a process of systems-level consolidation, which leads to greater stability through time and an increased reliance on neocortical areas for retrieval. The extent to which the retrieval of these consolidated memories still requires the hippocampus is unclear, as both spared and severely degraded remote memory recall have been reported following post-training hippocampal lesions. One difficulty in definitively addressing the role of the hippocampus in remote memory retrieval is the precision with which the entire volume of the hippocampal region can be inactivated. To address this issue, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), a chemical-genetic tool capable of highly specific neuronal manipulation over large volumes of brain tissue. We find that remote (>7 weeks after acquisition), but not recent (1–2 days after acquisition) contextual fear memories can be recalled after injection of the DREADD agonist (CNO) in animals expressing the inhibitory DREADD in the entire hippocampus. Our data demonstrate a time-dependent role of the hippocampus in memory retrieval, supporting the standard model of systems consolidation.National Institute of Mental Health (U.S.) (NIMH (5R01MH061976))National Institute of Mental Health (U.S.) (NIMH (R01 MH084966))United States. Defense Advanced Research Projects Agency (Grant W911NF-10-1-0059)European Research Council (ERC) (Horizon 2020: ERC starter grant-CHIME

Basolateral amygdala regulation of adult hippocampal neurogenesis and fear-related activation of newborn neurons

Impaired regulation of emotional memory is a feature of several affective disorders, including depression, anxiety and post-traumatic stress disorder. Such regulation occurs, in part, by interactions between the hippocampus and the basolateral amygdala (BLA). Recent studies have indicated that within the adult hippocampus, newborn neurons may contribute to support emotional memory, and that regulation of hippocampal neurogenesis is implicated in depressive disorders. How emotional information affects newborn neurons in adults is not clear. Given the role of the BLA in hippocampus-dependent emotional memory, we investigated whether hippocampal neurogenesis was sensitive to emotional stimuli from the BLA. We show that BLA lesions suppress adult neurogenesis, while lesions of the central nucleus of the amygdala do not. Similarly, we show that reducing BLA activity through viral vector-mediated overexpression of an outwardly rectifying potassium channel suppresses neurogenesis. We also show that BLA lesions prevent selective activation of immature newborn neurons in response to a fear-conditioning task. These results demonstrate that BLA activity regulates adult hippocampal neurogenesis and the fear context-specific activation of newborn neurons. Together, these findings denote functional implications for proliferation and recruitment of new neurons into emotional memory circuits.Brain & Behavior Research Foundation (Young Investigator Award)National Institutes of Health (U.S.) (R01MH849662