Following discovery of the neuropeptide Tuberoinfundibular Peptide of 39 residues (TIP39) and its receptor, the Parathyroid Hormone Receptor 2 (PTH2R) the section focused on investigation of their biological roles. Neuroanatomical mapping showed synthesis of TIP39 in discrete neuronal groups in the thalamus and brainstem that project to brain areas involved in emotional function. Following its anatomical mapping, laboratory projects turned to investigation of hypotheses derived from the distribution of the PTH2R/TIP39 system. Findings include that TIP39 modulates activation of hypothalamic neuroendocrine neurons including that it is important for an appropriate homeostatic response to cold. These studies lead to a general model for TIP39 action, that activation of PTH2Rs may be necessary for robust and sufficient excitatory transmitter release under high demand conditions. TIP39 was also found to modulate both acute pain sensitivity, and the return to normal sensitivity in chronic pain models. The latter effect appears to involve the locus coeruleus, a brainstem nucleus that contains noradrenergic neurons with modulatory influence throughout the CNS. In combination with other observations this suggests that TIP39 may be one of the modulators involved in the relationship between sensory stimuli and mood. Difficulty distinguishing between effects of ongoing aversive sensory input and its long-term consequences is a significant roadblock to investigating relationships between sensory stimuli and mood. To overcome this limitation we developed a paradigm to compare cellular and behavioral changes during and after reversing neuropathic pain in mice. Tactile allodynia and neuronal activation in the spinal cord dorsal horn produced by a cuff placed around the sciatic nerve resolved within several days following cuff removal. In contrast, changes in elevated O-maze, forced-swim, Y-maze spontaneous alternation and novel-object recognition test performance that developed after nerve cuff placement persisted at least 3 weeks after nerve cuffs were removed. Thus anxiety- and depression-like behaviors persisted following apparent resolution of pain in this paradigm. One contemporary idea regarding the cellular changes underlying depression is that inhibition of normal adult neurogenesis plays a role. Adult hippocampal neurogenesis is inhibited in chronic pain models, suggesting that neurogenesis inhibition may be involved in chronic pain associated depression. Using the reversible neuropathic model we found that adult hippocampal neurogenesis was decreased for at least 3 weeks following pain resolution. Also, FosB, a neuronal activity marker with a long half-life, remained elevated in the basolateral amygdala of mice with resolved nociception and persisting behavioral effects. Thus the reversible neuropathic paradigm points to specific cellular phenotypes that can be used to elucidate links between nociceptive sensory signaling and mood disorders. Previously, we found that mice with inactivating mutations of the genes encoding TIP39 (TIP39-KO) or the PTH2R (PTH2R-KO) have increased anxiety-like behavior under stressful conditions, relative to wild-type. Mice without TIP39 signaling have increased stress-dependent impairment in tests of memory function. Dysfunctional responses to stress are widely thought to contribute to depression, implying that this neuropeptide system plays a role in normal resilience. We investigated TIP39/PTH2R contributions to long-term fear memory using a model of post-traumatic stress disorder (PTSD) in which mice are exposed to a single aversive stimulus (foot-shock), after which fear memory is evaluated by measuring the time spent motionless (freezing, a rodent fear-like response) when the animals are re-exposed to the specific environment (fear context) in which the stimulus was delivered. While absence of TIP39 signaling did not cause a detectable change in fear memory 1 week after shock, both TIP39-KO and PTH2R-KO mice had increased fear-like behavior in the fear context after 2 and 4 weeks. Increasing in fear memory over time following a fear-inducing event is called fear incubation. Based on similarity to delayed symptom onset that frequently occurs in PTSD, fear incubation is used to investigate mechanisms that may contribute to PTSD. Thus, we investigated the neuroanatomical basis for enhanced fear incubation in mice that lack TIP39 signaling. Comparing neuronal activation patterns between mice with and without TIP39 signaling, either immediately after foot-shock or 4 weeks later when animals were returned to the fear context, revealed that TIP39 signaling absense prevented the normal activation of c-Fos in the medial nucleus of the amygdala (MeA). We used a PTH2R antagonist and mice that express Cre-recombinase in neurons that normally contain PTH2Rs, which we previously developed, to investigate whether the TIP39/PTH2R system in the MeA is responsible for the enhanced fear incubation. MeA targeted delivery of a virus encoded PTH2R antagonist reproduced the enhanced fear incubation of the KO mice. Selective ablation of MeA neurons that normally synthesize PTH2Rs following stereotaxic delivery of a virus Cre-dependently encoding a diphtheria toxin receptor also led to enhanced fear incubation. Transient inhibition of MeA PTH2R expressing neurons using a virally delivered Cre-recombinase dependent Designer Receptor Exclusively Activated by Designer Drugs (DREADD), starting just before or just after foot-shock, or at the time of recall testing 1 month later showed that inhibiting PTH2R neurons near the time of the event but not at the time of recall testing caused enhanced fear incubation. Putting these observations together, these experiments showed for the first time that the state of the medial nucleus of the amygdala just after a traumatic event has a major effect on fear-like behavior a month later. It further shows that signaling by a neuropeptide can have a major effect on that state. Ongoing work examines contributions of TIP39 signaling in the medial nucleus of the amygdala to other behaviors. Based on decreased inter-male aggressive behavior and increased social investigative behavior by PTH2R-KO mice, and similar effects following pharmacogenetic inhibition of medial amygdalar PTH2R expressing neurons the system appears to play a significant role in selection of social responses. Several previous studies provide evidence that that TIP39 signaling influences maternal behavior. Work with Arpad Dobolyi (Etvs Lornd University) during this reporting period provided anatomical evidence that TIP39 neurons in the thalamic subparafascicular area relay suckling related information to the hypothalamus to affect maternal behaviors. New work with Richard Gallo (University of California San Diego) showed that both TIP39 and the PTH2R are expressed at high levels in populations of cells within the skin, and implied that the system regulates keratinocyte function. Danny Winder (Vanderbilt University) used a virus we made that Cre-dependently expresses the human diphtheria toxin receptor to selectively ablate corticotrophin-releasing factor (CRF) expressing neurons in the central amygdala and show that CRF dependent effects of ethanol on glutamatergic transmission do not depend on the presence of local CRF neurons. Overall, our data suggest that TIP39 signaling may normally limit detrimental effects of environmental stress on emotional state. Dysfunctional responses to stress are widely thought to contribute to depression, implying that this neuropeptide system plays a role in normal resilience.
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