Ashley M. Blouin

Narp immunostaining of human hypocretin (orexin) neurons Loss in narcolepsy

Objective: To investigate whether neuronal activity-regulated pentraxin (Narp) colocalizes with hypocretin (Hcrt or orexin) in the normal human brain and to determine if Narp staining is lost in the narcoleptic human brain. Background: Human narcolepsy is characterized by a loss of the peptide hypocretin in the hypothalamus. This loss could result from the degeneration of neurons containing hypocretin or from a more specific loss of the ability of these neurons to synthesize Hcrt. Narp has been found to colocalize with hypocretin in the rat hypothalamus. Methods: We investigated the distribution of Narp in three normal and four narcoleptic human postmortem brains using immunohistochemistry with an antibody to Narp. Colocalization studies of Narp and hypocretin were also performed in two normal brains using immunohistochemistry with an antibody to Narp and an antibody to hypocretin. Results: We found that Narp colocalizes with hypocretin in the lateral hypothalamic area (LHA), the dorsomedial hypothalamus (DMH), the dorsal hypothalamic area (DHA), and the posterior hypothalamic area (PHA) of the normal human. The number of Narp-positive neurons was reduced by 89% in these areas of the narcoleptic hypothalamus. In contrast, Narp staining in the paraventricular (Pa) and supraoptic nuclei (SO) of the human hypothalamus did not differ between normal and narcoleptic brains. Conclusions: This finding supports the hypothesis that narcolepsy results from the specific loss of hypocretin neurons. Loss of hypothalamic Narp may contribute to the symptoms of narcolepsy.

Human hypocretin and melanin-concentrating hormone levels are linked to emotion and social interaction

The neurochemical changes underlying human emotions and social behavior are largely unknown. Here we report on the changes in the levels of two hypothalamic neuropeptides, hypocretin-1 (Hcrt-1) and melanin concentrating hormone (MCH), measured in the human amygdala. We show that Hcrt-1 levels are maximal during positive emotion, social interaction, and anger, behaviors that induce cataplexy in human narcoleptics. In contrast, MCH levels are minimal during social interaction, but are increased after eating. Both peptides are at minimal levels during periods of postoperative pain despite high levels of arousal. MCH levels increase at sleep onset, consistent with a role in sleep induction, whereas Hcrt-1 levels increase at wake onset, consistent with a role in wake induction. Levels of these two peptides in humans are not simply linked to arousal, but rather to specific emotions and state transitions. Other arousal systems may be similarly emotionally specialized.

Nonmuscle myosin IIB as a therapeutic target for the prevention of relapse to methamphetamine use.

Memories associated with drug use increase vulnerability to relapse in substance use disorder (SUD), and there are no pharmacotherapies for the prevention of relapse. Previously, we reported a promising finding that storage of memories associated with methamphetamine (METH), but not memories for fear or food reward, is vulnerable to disruption by actin depolymerization in the basolateral amygdala complex (BLC). However, actin is not a viable therapeutic target because of its numerous functions throughout the body. Here we report the discovery of a viable therapeutic target, nonmuscle myosin IIB (NMIIB), a molecular motor that supports memory by directly driving synaptic actin polymerization. A single intra-BLC treatment with Blebbistatin (Blebb), a small-molecule inhibitor of class II myosin isoforms, including NMIIB, produced a long-lasting disruption of context-induced drug seeking (at least 30 days). Further, postconsolidation genetic knockdown of Myh10, the heavy chain of the most highly expressed NMII in the BLC, was sufficient to produce METH-associated memory loss. Blebb was found to be highly brain penetrant. A single systemic injection of the compound selectively disrupted the storage of METH-associated memory and reversed the accompanying increase in BLC spine density. This effect was specific to METH-associated memory, as it had no effect on an auditory fear memory. The effect was also independent of retrieval, as METH-associated memory was disrupted 24 h after a single systemic injection of Blebb delivered in the home cage. Together, these results argue for the further development of small-molecule inhibitors of NMII as potential therapeutics for the prevention of SUD relapse triggered by drug associations.

Relation of melanin concentrating hormone levels to sleep, emotion and hypocretin levels.

We thank the editor 1 and commentators 2-5 for their kind remarks on our human microdialysis paper 6 and for integrating the results of all three papers. 6-8 Our findings in the human are quite compatible with Konadhode et al. 7 They found that optogenetic activation of MCH neurons decreased sleep onset latency and increased total sleep time. We see a great increase in MCH levels in the human brain at sleep onset and a smaller elevation of MCH levels throughout sleep. These microdialysis data together with Konadhode et al.’s stimulation data establish a causal link between MCH release and normal human sleep. In contrast, we see that Hcrt level, assessed from the same aliquots of microdialysis fluid, decreases prior to sleep onset. We agree with the commentators’ suggestion that our findings of elevated MCH level after eating may be related to postprandial relaxation, although this increase is clearly occurring in a waking state and may simply be linked to satiety. An important finding of our study was that whereas MCH and Hcrt are often inversely related to each other, this is not always the case. For example, MCH and Hcrt levels were both markedly decreased while the subjects were experiencing pain during waking. We found a strong link between Hcrt release and emotion, especially positive emotion. No such emotional link was seen with MCH levels with the same assays in the same aliquots. These unique human data thus highlight another difference between MCH and Hcrt: Hcrt is strongly related to positive affect, whereas MCH is not related to positive or negative affect within waking, but rather to sleep onset and satiation or postprandial relaxation. We directly addressed the role of Hcrt neurons in a wide range of behaviors in prior studies in the dog, cat, rat, and mouse. 9-14 These studies all point to a role for Hcrt in maintaining arousal during positively motivated behaviors such as play or bar pressing for food or water. Our human data are consistent with that conclusion. CITATION Blouin AM; Siegel JM. Relation of melanin concentrating hormone levels to sleep, emotion and hypocretin levels. SLEEP

The potential of epigenetics in stress-enhanced fear learning models of PTSD

Prolonged distress and dysregulated memory processes are the core features of post-traumatic stress disorder (PTSD) and represent the debilitating, persistent nature of the illness. However, the neurobiological mechanisms underlying the expression of these symptoms are challenging to study in human patients. Stress-enhanced fear learning (SEFL) paradigms, which encompass both stress and memory components in rodents, are emerging as valuable preclinical models of PTSD. Rodent models designed to study the long-term mechanisms of either stress or fear memory alone have identified a critical role for numerous epigenetic modifications to DNA and histone proteins. However, the epigenetic modifications underlying SEFL remain largely unknown. This review will provide a brief overview of the epigenetic modifications implicated in stress and fear memory independently, followed by a description of existing SEFL models and the few epigenetic mechanisms found to date to underlie SEFL. The results of the animal studies discussed here highlight neuroepigenetics as an essential area for future research in the context of PTSD through SEFL studies, because of its potential to identify novel candidates for neurotherapeutics targeting stress-induced pathogenic memories.

Memory disrupting effects of nonmuscle myosin II inhibition depend on the class of abused drug and brain region

Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared to be disrupted with cocaine. Unlike in the amygdala, methamphetamine-associated memory storage was not disrupted by NMIIi in the hippocampus, nucleus accumbens, or orbitofrontal cortex. NMIIi in the hippocampus did appear to disrupt reconsolidation. Identification of the unique mechanisms responsible for NMII-mediated, amygdala-dependent disruption of memory storage associated with the amphetamine class may enable induction of retrieval-independent vulnerability to other pathological memories.

Social stress-potentiated methamphetamine seeking: Stress-potentiated METH seeking

Human studies of substance use disorder show that psychological stress and drug availability interact following rehabilitation, contributing to the high relapse potential. Social stressors trigger particularly strong motivation for drug, but how this affects neuronal function to increase relapse is unknown. Animal models, which allow for the dissection of neural mechanisms, primarily utilize physical stressors to trigger relapse. To recapitulate psychosocial post‐rehabilitation challenges in animals, we developed a model of social stress‐potentiated methamphetamine (METH) seeking. Rats receive a single social defeat (SD) session after completion of self‐administration and extinction of lever pressing. While a reminder of the SD was insufficient to reinstate METH seeking on its own, rats that received a reminder of SD followed by a METH‐priming injection displayed potentiated reinstatement over METH‐priming alone. Examination of neuronal activation patterns of the METH‐primed reinstatement session identified c‐Fos‐immunoreactivity in the basolateral amygdala (BLA) as correlated with SD score, a measure of defeat latency. Rapidly defeated rats showed potentiated METH‐primed reinstatement and elevated BLA c‐Fos compared with controls. Conversely, rats that were undefeated during the social stress did not show potentiated METH‐primed reinstatement or elevated BLA c‐Fos. Interestingly, inactivation of the BLA with baclofen/muscimol prior to the stress reminder and METH‐priming generated a potentiation of METH seeking in the undefeated rats, suggesting the BLA may mediate resilience to the stressor. This model provides a tool for the further dissection of neural mechanisms mediating social stress‐potentiated relapse and for the development of relapse‐reducing therapeutics.