Jose Amat

Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus.

The degree of behavioral control that an organism has over a stressor is a potent modulator of the stressors impact; uncontrollable stressors produce numerous outcomes that do not occur if the stressor is controllable. Research on controllability has focused on brainstem nuclei such as the dorsal raphe nucleus (DRN). Here we find that the infralimbic and prelimbic regions of the ventral medial prefrontal cortex (mPFCv) in rats detect whether a stressor is under the organisms control. When a stressor is controllable, stress-induced activation of the DRN is inhibited by the mPFCv, and the behavioral sequelae of uncontrollable stress are blocked. This suggests a new function for the mPFCv and implies that the presence of control inhibits stress-induced neural activity in brainstem nuclei, in contrast to the prevalent view that such activity is induced by a lack of control.

Escapable and inescapable stress differentially alter extracellular levels of 5-HT in the basolateral amygdala of the rat.

The effects of escapable and yoked inescapable electric tailshocks on extracellular levels of serotonin (5-HT) in the basolateral amygdala were measured by in vivo microdialysis. Inescapable, but not escapable, shock increased extracellular 5-HT in the amygdala relative to restrained controls. Basal levels of 5-HT were elevated 24 h after inescapable shock, and previously inescapably shocked subjects exhibited an exaggerated 5-HT response to two brief footshocks. Levels of extracellular 5-HIAA did not follow any particular pattern and were not correlated with the changes in 5-HT.

Previous experience with behavioral control over stress blocks the behavioral and dorsal raphe nucleus activating effects of later uncontrollable stress: role of the ventral medial prefrontal cortex.

Previous experience with stressors over which the subject has behavioral control blocks the typical behavioral consequences of subsequent exposure to stressors over which the organism has no behavioral control. The present experiments explored the involvement of the ventral medial prefrontal cortex (mPFCv) in mediating this “immunizing” or resilience producing effect of an initial experience with control. Behavioral immunization was blocked by inactivation of the mPFCv with muscimol at the time of the initial experience with control, as well as at the time of the later exposure to uncontrollable stress. Inhibition of protein synthesis within the mPFCv by anisomycin also blocked immunization when administered at the time of the initial controllable stress but had no effect when administered at the time of the later uncontrollable stress. Additional experiments found that the initial experience with control blocks the intense activation of serotonergic cells in the dorsal raphe nucleus that would normally be produced by uncontrollable stress, providing a mechanism for behavioral immunization. Furthermore, mPFCv activity during the initial controllable stressor was required for this effect to occur. These results suggest that the mPFCv is needed both to process information about the controllability of stressors and to utilize such information to regulate responses to subsequent stressors. Moreover, the mPFCv may be a site of storage or plasticity concerning controllability information. These results are consistent with recent research in other domains that explore the functions of the mPFCv.

5-Hydroxytryptamine 2C Receptors in the Basolateral Amygdala Are Involved in the Expression of Anxiety After Uncontrollable Traumatic Stress

BACKGROUNDnExposure to uncontrollable stressors often increases anxiety-like behavior in both humans and rodents. In rat, this effect depends on stress-induced activity within the dorsal raphe nucleus (DRN). However, the role of serotonin in DRN projection regions is largely unknown. The goals of this study were to 1) assess the effect of uncontrollable stress on extracellular serotonin in the basolateral amygdala during the anxiety test, 2) determine whether DRN activity during a poststress anxiety test is involved in anxiety-like behavior, and 3) determine the role of the serotonin 2C receptor (5-HT(2C)) in uncontrollable stress-induced anxiety.nnnMETHODnRats were exposed to tail shocks that were either controllable or uncontrollable. On the following day, anxiety-like behavior was assessed in a Juvenile Social Exploration (JSE) test. Basolateral amygdala (BLA) extracellular serotonin concentrations were assessed during JSE by in vivo microdialysis 24 hours after uncontrollable stress, controllable stress, or no stress. In separate experiments, drugs were administered before the JSE test to inhibit the DRN or to block 5-HT(2C) receptors.nnnRESULTSnExposure to uncontrollable shock reduced later social exploration. Prior uncontrollable stress potentiated serotonin efflux in the BLA during social exploration, but controllable stress did not. Intra-DRN 8-OH-DPAT and systemic and intra-BLA 5-HT(2C) receptor antagonist SB 242,084 prevented the expression of potentiated anxiety in uncontrollably stressed rats. Intra-BLA injection of the 5-HT(2C) agonist CP 809,101 mimicked the effect of stress.nnnCONCLUSIONSnThese results suggest that the anxiety-like behavior observed after uncontrollable stress is mediated by exaggerated 5-HT acting at BLA 5-HT(2C) receptors.

The role of the habenular complex in the elevation of dorsal raphe nucleus serotonin and the changes in the behavioral responses produced by uncontrollable stress

Previous research indicates that the serotonergic neurons of the caudal dorsal raphe nucleus (DRN) are activated to a greater degree by inescapable shock (IS) as compared to escapable shock (ES), causing a greater release of serotonin (5-HT) in the DRN and in target regions. This differential activation is necessary for the behavioral changes that occur after exposure to IS, but not to ES (i.e. learned helplessness/behavioral depression). Although the critical role of the DRN in learned helplessness is clear, the neural inputs to the caudal DRN which result in this selective activation are unknown. One structure that may be involved in the activation of the DRN and the induction of learned helplessness/behavioral depression is the habenular complex. In experiment 1, habenula lesions eliminated the differential rise in DRN extracellular 5-HT levels in response to IS and ES exposure by severely attenuating the rise in 5-HT for both groups. In experiment 2, sham operated and habenula lesioned rats were exposed to either ES, IS or no stress (home cage control; HCC). Twenty-four hours later, sham rats previously exposed to IS exhibited longer escape latencies as compared to both ES and HCC rats (i.e. learned helplessness). The habenular lesion eliminated the differences in escape latency between groups, thus eliminating the induction of learned helplessness/behavioral depression. These results suggest that the habenula is necessary for the differential activation of the DRN and the escape deficits produced by IS.

Escapable and inescapable stress differentially and selectively alter extracellular levels of 5-HT in the ventral hippocampus and dorsal periaqueductal gray of the rat

The effects of escapable and yoked inescapable electric tailshocks on extracellular levels of serotonin (5-HT) in the ventral hippocampus and dorsal periaqueductal gray (dPAG) were measured by in vivo microdialysis. Inescapable, but not escapable shock increased extracellular 5-HT in the ventral hippocampus relative to restrained controls. Basal levels of 5-HT were elevated 24 h after inescapable shock, and previously inescapably shocked subjects exhibited an exaggerated 5-HT response to 2 brief footshocks. In contrast, escapable, but not inescapable shock, increased extracellular 5-HT in the dPAG, increased basal 5-HT in the dPAG 24 h later, and led to an enhanced 5-HT response to subsequent brief footshock.

Opioid Activation of Toll-Like Receptor 4 Contributes to Drug Reinforcement

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4−/− mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

Microinjection of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala.

The intra dorsal raphe nucleus (DRN) administration of corticotropin releasing hormone (CRF) inhibits serotonergic (5-HT) activity in this structure, an effect blocked by antagonists selective for the type 1 CRF receptor (CRF1). The DRN has a high density of the type 2 receptor (CRF2), and so the present experiments explored the impact of CRF2 activation within the DRN on 5-HT function. The intra-DRN administration of the selective CRF2 agonist urocortin 2 (Ucn 2) dose dependently increased 5-HT efflux in the basolateral amygdala, a projection region of the DRN. Intra-DRN Ucn 2 also increased c-fos expression in labeled 5-HT neurons. Both of these effects of Ucn 2 were completely blocked by intra-DRN antisauvagine-30 (ASV-30), a relatively selective CRF2 antagonist. These data suggest that CRF1 and CRF2 activation within the DRN affect 5-HT neurons in opponent fashion. Implications of these results for understanding the behavioral effects of CRF and other CRF-like ligands are discussed.

Stressor Controllability Modulates Stress-Induced Dopamine and Serotonin Efflux and Morphine-Induced Serotonin Efflux in the Medial Prefrontal Cortex

It has previously been shown that inescapable (IS) but not escapable (ES) stress potentiates the rewarding properties of morphine as measured by conditioned place preference and psychomotor activation, and that this potentiation may be mediated by dorsal raphe nucleus (DRN) serotonin (5-HT) neurons. The medial prefrontal cortex (mPFC) has been implicated in both reward and stress, and is a projection region of the DRN. The mPFC also contains dopaminergic afferents from the ventral tegmental area, which has been the focus of many studies exploring both the rewarding properties of drugs and the aversive properties of stress. The role of the mPFC in stress/drug reactivity interactions is largely unknown. The present study usedin vivo microdialysis to examine 5-HT and dopamine (DA) efflux in the mPFC of rats during IS, ES or no stress (NS). IS and ES rats received the stressor in yoked pairs. The stressor consisted of tailshocks that could be terminated for both rats by the ES rats. Large increases in 5-HT and DA levels were observed during IS but not ES or NS. DA and 5-HT efflux were also measured 24u2009h later in the same rats in response to morphine (3u2009mg/kg) or saline. Sustained increases in 5-HT levels were observed after morphine in rats that had previously received IS but not in rats that had received ES or NS. No changes in DA efflux were observed after morphine. Thus, 5-HT and DA in the mPFC may be involved in stressor controllability effects, and the sensitization of 5-HT neurons by IS extends to the mPFC and to morphine as a challenge.

Controllable versus uncontrollable stressors bi-directionally modulate conditioned but not innate fear

Fear conditioning and fear extinction play key roles in the development and treatment of anxiety-related disorders, yet there is little information concerning experiential variables that modulate these processes. Here we examined the impact of exposure to a stressor in a different environment on subsequent fear conditioning and extinction, and whether the degree of behavioral control that the subject has over the stressor is of importance. Rats received a session of either escapable (controllable) tail shock (ES), yoked inescapable (uncontrollable) tail shock (IS), or control treatment (home cage, HC) 7 days before fear conditioning in which a tone and foot shock were paired. Conditioning was measured 24 h later. In a second experiment rats received ES, IS or HC 24 h after contextual fear conditioning. Extinction then occurred every day beginning 7 days later until a criterion was reached. Spontaneous recovery of fear was assessed 14 days after extinction. IS potentiated fear conditioning when given before fear conditioning, and potentiated fear responding during extinction when given after conditioning. Importantly, ES potently interfered with later fear conditioning, decreased fear responding during fear extinction, and prevented spontaneous recovery of fear. Additionally, we examined if the activation of the ventral medial prefrontal cortex (mPFCv) by ES is critical for the protective effects of ES on later fear conditioning. Inactivation of the mPFCv with muscimol at the time of the initial experience with control prevented ES-induced reductions in later contextual and auditory fear conditioning. Finally, we explored if the protective effects of ES extended to an unconditioned fear stimulus, ferret odor. Unlike conditioned fear, prior ES increased the fear response to ferret odor to the same degree as did IS.