a-surgical-protocol-for-infraorbital-nerve-chronic-constriction

A Surgical Protocol for Infraorbital Nerve Chronic Constriction Injury in Mice

Trigeminal neuropathic pain arises from injury to sensory pathways of the trigeminal system and is associated with persistent changes in neuronal ...

National Institutes of Health

Kisspeptin (encoded by KISS1) is a crucial activator of reproductive function. The role of kisspeptin has been studied extensively within the hypothalamus but little is known about its significance in other areas of the brain. KISS1 and its cognate receptor are expressed in the amygdala, a key limbic brain structure with inhibitory projections to hypothalamic centers involved in gonadotropin secretion. We therefore hypothesized that kisspeptin has effects on neuronal activation and reproductive pathways beyond the hypothalamus and particularly within the amygdala. To test this, we mapped brain neuronal activity (using manganese-enhanced MRI) associated with peripheral kisspeptin administration in rodents. We also investigated functional relevance by measuring the gonadotropin response to direct intra-medial amygdala (MeA) administration of kisspeptin and kisspeptin antagonist. Peripheral kisspeptin administration resulted in a marked decrease in signal intensity in the amygdala compared to vehicle alone. This was associated with an increase in luteinizing hormone (LH) secretion. In addition, intra-MeA administration of kisspeptin resulted in increased LH secretion, while blocking endogenous kisspeptin signaling within the amygdala by administering intra-MeA kisspeptin antagonist decreased both LH secretion and LH pulse frequency. We provide evidence for the first time that neuronal activity within the amygdala is decreased by peripheral kisspeptin administration and that kisspeptin signaling within the amygdala contributes to the modulation of gonadotropin release and pulsatility. Our data suggest that kisspeptin is a 'master regulator' of reproductive physiology, integrating limbic circuits with the regulation of gonadotropin-releasing hormone neurons and reproductive hormone secretion.

Kisspeptin signaling in the amygdala modulates reproductive hormone secretion.

The medial amygdala (MeA) is crucial for sexual behavior; kisspeptin (Kiss1) also plays a role in sexual function. Kisspeptin receptor (Kiss1r) knockout mice display no sexual behavior. Recently Kiss1 and Kiss1r have been discovered in the posterodorsal subnucleus of the medial amygdala (MePD). We hypothesised that Kiss1 in the MePD may have an influence on male sexual behavior. To test this we bilaterally cannulated the MePD and infused kisspeptin-10 in male rats. This caused the rats to have multiple erections, an effect specific to Kiss1 receptor activation, because Kiss1r antagonism blocked the erectile response. When Kiss1 was infused into the lateral cerebroventricle, there were no observed erections. We also measured the plasma levels of LH when Kiss1 is infused into the MePD or lateral cerebroventricle; Kiss1 increased plasma LH to comparable levels when infused into both sites. We conclude that Kiss1 has a role in male sexual behavior, which is specific to the MePD.

Kisspeptin in the medial amygdala and sexual behavior in male rats.

Cdk5 is a key neuronal kinase necessary for proper brain development, which has recently been implicated in modulating nociception. Conditional deletion of Cdk5 in pain-sensing neurons attenuates pain responses to heat in both the periphery and orofacial regions. Cdk5 activity is regulated by binding to the activators p35 and p39, both of which possess a cyclin box. Our previous examination of the nociceptive role of the well-characterized Cdk5 activator p35 using mice that either lack or overexpress this regulatory subunit demonstrated that Cdk5/p35 activity affects mechanical, chemical, and thermal nociception. In contrast, the nociceptive role of Cdk5’s other less-studied activator p39 is unknown. Here, we report that the knockout of p39 in mice did not affect orofacial and peripheral nociception. The lack of any algesic response to nociceptive stimuli in the p39 knockout mice contrasts with the hypoalgesic effects that result from the deletion of p35. Our data demonstrate different and nonoverlapping roles of Cdk5 activators in the regulation of orofacial as well as peripheral nociception with a crucial role for Cdk5/p35 in pain signaling.

Peripheral and orofacial pain sensation is unaffected by the loss of p39.

Visualization of dynamic cellular activity has greatly expanded our understanding of brain function. Recently, there has been an increasing number of studies imaging rodent brain activity in real time. However, traditional in vivo calcium imaging technology has been limited to superficial brain structures. Because the trigeminal ganglion (TG) is located deep within the cranial cavity of mice, few studies have been able to access to it. To circumvent this limitation, overlying brain tissue must be removed to expose the TG so that optical recording can access deep brain neural ensembles. This unit describes a procedure for conducting non-survival surgery to visualize the TG in live mice. Obtaining large ensembles of direct, real-time readouts of sensory neuron signaling, providing temporal and spatial information across the TG, will help to define the cellular basis of orofacial somatic sensing and pain perception. © 2019 by John Wiley & Sons, Inc.

Visualization of Trigeminal Ganglion Neuronal Activities in Mice.

The mechanisms underlying facial pain are still incompletely understood, posing major therapeutic challenges. Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase involved in pain signaling. However, the regulatory roles of Cdk5 in facial pain signaling and the possibility of therapeutic intervention at the level of mouse trigeminal ganglion primary neurons remain elusive. In this study, we use optimized intravital imaging to directly compare trigeminal neuronal activities after mechanical, thermal, and chemical stimulation. We then test whether facial inflammatory pain in mice could be alleviated by the Cdk5 inhibitor peptide TFP5. We demonstrate regulation of total Ca intensity by Cdk5 activity using transgenic and knockout mouse models. In mice with vibrissal pad inflammation, application of TFP5 specifically decreases total Ca intensity in response to noxious stimuli. It also alleviates inflammation-induced allodynia by inhibiting activation of trigeminal peripheral sensory neurons. Cdk5 inhibitors may provide promising non-opioid candidates for pain treatment.

Visualization of trigeminal ganglion sensory neuronal signaling regulated by Cdk5.

Diabetic peripheral neuropathy (DPN) is characterized by spontaneous pain in the extremities. Incidence of DPN continues to rise with the global diabetes epidemic. However, there remains a lack of safe, effective analgesics to control this chronic painful condition. Dorsal root ganglia (DRG) contain soma of sensory neurons and modulate sensory signal transduction into the central nervous system. In this study, we aimed to gain a deeper understanding of changes in molecular pathways in the DRG of DPN patients with chronic pain. We recently reported transcriptomic changes in the DRG with DPN. Here, we expand upon those results with integrated metabolomic, proteomic, and phospho-proteomic analyses to compare the molecular profiles of DRG from DPN donors and DRG from control donors without diabetes or chronic pain. Our analyses identified decreases of select amino acids and phospholipid metabolites in the DRG from DPN donors, which are important for cellular maintenance. Additionally, our analyses revealed changes suggestive of extracellular matrix (ECM) remodeling and altered mRNA processing. These results reveal new insights into changes in the molecular profiles associated with DPN.

Integrative multiomic analyses of dorsal root ganglia in diabetic neuropathic pain using proteomics, phospho-proteomics, and metabolomics.

Proteomics and phosphoproteomics play crucial roles in elucidating the dynamics of post-transcriptional processes. While experimental methods and workflows have been established in this field, a persistent challenge arises when dealing with small samples containing a limited amount of protein. This limitation can significantly impact the recovery of peptides and phosphopeptides. In response to this challenge, we have developed a comprehensive experimental workflow tailored specifically for small-scale samples, with a special emphasis on neuronal tissues like the trigeminal ganglion. Our proposed workflow consists of seven steps aimed at optimizing the preparation of limited tissue samples for both proteomic and phosphoproteomic analyses. One noteworthy innovation in our approach involves the utilization of a dual enrichment strategy for phosphopeptides. Initially, we employ Fe-NTA Magnetic beads, renowned for their specificity and effectiveness in capturing phosphopeptides. Subsequently, we complement this approach with the TiO-based method, which offers a broader spectrum of phosphopeptide recovery. This innovative workflow not only overcomes the challenges posed by limited sample sizes but also establishes a new benchmark for precision and efficiency in proteomic investigations. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein extraction and digestion Basic Protocol 2: TMT labeling and peptide cleanup Basic Protocol 3: IMAC Fe-NTA magnetic beads phosphopeptide enrichment Basic Protocol 4: TiO2 enrichment Basic Protocol 5: Fe-NTA phosphopeptide Enrichment Basic Protocol 6: High pH peptide fractionation Basic protocol 7: LC-MS/MS analysis and database search.

Article	Year
Kisspeptin signaling in the amygdala modulates reproductive hormone secretion. Brain structure & function\| PubMed ID: 25758403	2016
Kisspeptin in the medial amygdala and sexual behavior in male rats. Neuroscience letters\| PubMed ID: 27233219	2016
Peripheral and orofacial pain sensation is unaffected by the loss of p39. Molecular pain\| PubMed ID: 28969475	2017
Visualization of Trigeminal Ganglion Neuronal Activities in Mice. Current protocols in cell biology\| PubMed ID: 30724481	2019
Visualization of trigeminal ganglion sensory neuronal signaling regulated by Cdk5. Cell reports\| PubMed ID: 35263573	2022
Integrative multiomic analyses of dorsal root ganglia in diabetic neuropathic pain using proteomics, phospho-proteomics, and metabolomics. Scientific reports\| PubMed ID: 36220867	2022
Optimized Workflow for Proteomics and Phosphoproteomics With Limited Tissue Samples. Current protocols\| PubMed ID: 38646944	2024

Minghan Hu

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