Investigation of the physiological functions of the neuropeptide Tuberoinfundibular Peptide of 39 residues (TIP39) and its receptor, the Parathyroid Hormone 2 (PTH2) receptor has been a recent focus of the laboratory. These molecules were discovered in this laboratory several years ago. In previous project years we mapped the neuroanatomical distributions of TIP39 and the PTH2 receptor. TIP39 is synthesized by 3 discrete groups of neurons, 2 at the caudal border of the thalamus and one in the brainstem. TIP39 synthesizing neurons project to several brain areas that are involved in the regulation of emotional function. These areas contain a matching distribution of the PTH2 receptor, and neurons in these regions project to the areas containing TIP39 neurons. Thus the system is ideally positioned to coordinate and modulate functions relevant to mental disorders. Following this anatomical mapping, laboratory projects turned to investigation of hypotheses derived from the distribution of TIP39 and the PTH2R. The hypothalamus contains a relatively high density of PTH2Rs and TIP39 containing terminals. In previous project years we found that TIP39 modulates activation of neurons in the hypothalamic paraventricular nucleus, which controls several neuroendocrine functions, including release of glucocorticoid stress hormones from the adrenal gland (corticosterone in rodents). TIP39 does this by acting on the terminals of neurons within the paraventricular nucleus that release the classic fast-acting transmitter glutamate. Thus TIP39 modulates excitatory inputs to neuroendocrine cells. We also found that TIP39 signaling in another hypothalamic region, the median preoptic nucleus, contributes to thermoregulation. Specifically, an appropriate homeostatic response to cold exposure required TIP39 signaling, while maintenance of a normal baseline temperature in an environment to which mice were adapted did not. These studies lead to the general model that TIP39 action on presynaptic PTH2Rs on some populations of glutamatergic neurons may be necessary for robust and sufficient excitatory transmitter release under particular high demand conditions. In previous years of the project we found that mice with genetic deletion of the gene encoding TIP39 (TIP39-KO) have increased anxiety-like behavior that depends upon the level of stress created by the testing conditions. Under conditions of minimal stress, loss of TIP39 had little effect, while under increased stress animals without TIP39 exhibited significantly greater anxiety-like behavior than mice with normal TIP39 function. We also investigated the role of TIP39 in modulating the effects of stress on cognitive function. Mice lacking TIP39 signaling because of either ligand or receptor loss or acute receptor blockade showed impaired performance in behavioral tests that depend on memory function (object recognition and social recognition tests and spontaneous alternation in a Y-maze), under conditions of novelty-induced arousal but not when acclimated to the testing environment. We also found that TIP39 signaling modulates long-term emotional memory. In a mouse model of post-traumatic stress disorder in which the animals are exposed to a single traumatic event (electric foot-shock) after which fear memory is evaluated by re-exposing them to the context of the traumatic event and measuring the time spent motionless (freezing, a rodent fear-like response) the lack of TIP39 signaling did not cause a detectable change in fear memory one week after the shock. However, both mice lacking the peptide gene as well as mice with null mutation in the PTH2-R gene exhibited greater fear-like behavior than wild-type mice two weeks following the shock. Preliminary data suggests that PTH2-R signaling in the medial amygdalar region contributes to this phenomenon. Thus our data suggest that TIP39 signaling may normally limit the detrimental effects of environmental stress on emotional state. Dysfunctional responses to stress are widely thought to contribute to depression. We also found that TIP39 modulates acute pain sensitivity, acting primarily within the brain to affect the processing of painful sensory information. Some of the brain areas where TIP39 appears to modulate pain are also implicated in affective disorders. There is a significant association between chronic pain and depression. The extents to which pain affects mood and to which mood affects pain are not currently clear, nor is the extent to which shared vulnerability factors contribute to the development of both chronic pain and mood disorders. Because of TIP39s involvement in acute pain, the localization of TIP39 signaling to brain regions involved in affective dimensions of pain, its involvement is stress responses, and the significant societal burden of comorbid chronic pain and depression, we began investigating the involvement of TIP39 signaling in chronic pain, with the long-term goal of assessing its contribution to the relationships between pain and depression. Using a nerve injury as a model of neuropathic pain, we found that as compared to controls, mice lacking TIP39 signaling because of mutation in either the PTH2R or TIP39 genes developed less tactile and thermal hypersensitivity, and more rapidly returned to baseline sensory thresholds following the injury. Effects of inflammatory injury were similarly decreased in knockout mice. Blockade of α-2 adrenergic receptors increased the tactile and thermal sensitivity of apparently recovered knockout mice, returning it to levels of neuropathic controls. The locus coeruleus (LC), a brainstem nucleus, is the most likely source of the neuromodulator, norepinephrine, that could have this effect. Supporting this hypothesis, mice with LC area injection of lentivirus encoding a secreted PTH2R antagonist had a rapid, α-2 reversible, apparent recovery from neuropathic injury similar to the knockout mice. Ablation of LC area glutamatergic neurons led to local PTH2R-ir loss, and barley lectin (a transneuronal tracing reagent) was transferred from local glutamatergic neurons to GABA interneurons that surround the LC. These results suggest that TIP39 signaling modulates sensory thresholds via effects on glutamatergic transmission to brainstem noradrenergic neurons via GABAergic interneurons. The data suggest that TIP39 may normally inhibit release of hypoalgesic amounts of norepinephrine during chronic pain. A speculation on the physiological adaptiveness of TIP39s contribution to maintenance of central sensitization is that it enhances guarding behavior, thus contributing to healing. A next step in this line of investigation will be to evaluate the effects of TIP39 signaling on mood and anxiety related behavior in these chronic pain models. Because of the Sections long-term interest in neuromodulator influences on mood disorders we initiated a project aimed at increased understanding of the contributions of LC noradrenergic signaling to mood and anxiety related behaviors. Correlative observations suggest that LC function is involved in a variety of brain disorders but there is little direct evidence addressing its specific role(s). Recently developed reagents, such as transgenic mice that express Cre-recombinase in defined neuronal populations and viruses encoding receptors with Cre-dependent expression should make mechanistic studies feasible. During this review period we initiated this research direction using transgenic mice and recombinant viruses to map the major forebrain projections to the LC in mouse. One significant accomplishment was production of strong evidence for direct innervation of noradrenergic LC neurons by GABA neurons of the central amygdalar nucleus. Future studies will use this approach to examine the effects of changes in LC function on mood and anxiety related behaviors.

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Cservenák, Melinda; Kis, Viktor; Keller, Dávid et al. (2016) Maternally involved galanin neurons in the preoptic area of the rat. Brain Struct Funct :
Sato, Emi; Muto, Jun; Zhang, Ling-Juan et al. (2016) The Parathyroid Hormone Second Receptor PTH2R and its Ligand Tuberoinfundibular Peptide of 39 Residues TIP39 Regulate Intracellular Calcium and Influence Keratinocyte Differentiation. J Invest Dermatol 136:1449-59
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Dimitrov, Eugene L; Kuo, Jonathan; Kohno, Kenji et al. (2013) Neuropathic and inflammatory pain are modulated by tuberoinfundibular peptide of 39 residues. Proc Natl Acad Sci U S A 110:13156-61
Dimitrov, Eugene L; Yanagawa, Yuchio; Usdin, Ted B (2013) Forebrain GABAergic projections to locus coeruleus in mouse. J Comp Neurol 521:2373-97
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Dobolyi, Arpad; Dimitrov, Eugene; Palkovits, Miklos et al. (2012) The neuroendocrine functions of the parathyroid hormone 2 receptor. Front Endocrinol (Lausanne) 3:121
Coutellier, Laurence; Usdin, Ted B (2011) Enhanced long-term fear memory and increased anxiety and depression-like behavior after exposure to an aversive event in mice lacking TIP39 signaling. Behav Brain Res 222:265-9
Gao, Ji-Liang; Schneider, Erich H; Dimitrov, Eugene L et al. (2011) Reduced fear memory and anxiety-like behavior in mice lacking formylpeptide receptor 1. Behav Genet 41:724-33

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