Probes for INS

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pro-nociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here we demonstrate the different contributions of genetically-defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.

Significance Statement The PAG is a midbrain region critical for the modulation of pain. However, the roles played by the distinct cell types within the PAG in nociceptive processing are poorly understood. This work addresses the divergent roles of glutamatergic and GABAergic PAG neuronal subpopulations in nociceptive processing. We demonstrate that activation of glutamatergic neurons or inhibition of GABAergic neurons suppresses nociception. Whereas inhibition of glutamatergic neuronal activity or activation of GABAergic neuronal activity potentiates nociception. This report identifies distinct roles for these neuronal populations in modulating nociceptive processing.

Abstract

BACKGROUND:

Non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are well-recognized post-transcriptional regulators of gene expression. This study examines the expression of microRNA-21 (miR-21) and lncRNA MALAT1 in medullary thyroid carcinomas (MTCs) and their effects on tumor behavior.

METHODS:

Tissue microarrays (TMAs) were constructed using normal thyroid (n=39), primary tumors (N=39) and metastatic MTCs (N=18) from a total of 42 MTC cases diagnosed between 1987 and 2016. In situ hybridization with probes for miR-21 and MALAT1 was performed. PCR quantification of expression was performed in a subset of normal thyroid (N=10) and primary MTCs (N=32). An MTC-derived cell line (MZ-CRC-1) was transfected with small interfering RNAs (siRNAs) targeting miR-21 and MALAT1 to determine the effects on cell proliferation and invasion.

RESULTS:

In situ hybridization (ISH) showed strong (2+ to 3+) expression of miR-21 in 17 (44%) primary MTCs and strong MALAT1 expression in 37 (95%) primary MTCs. Real-time PCR expression of miR-21 (P<0.001) and MALAT1 (P=0.038) in primary MTCs were significantly higher than in normal thyroid, supporting the ISH findings. Experiments with siRNAs showed inhibition of miR-21 and MALAT1 expression in the MTC-derived cell line, leading to significant decreases in cell proliferation (P<0.05) and invasion (P<0.05).

CONCLUSION:

There is increased expression of miR-21 and MALAT1 in MTCs. This study also showed an in vitro pro-oncogenic effect of MALAT1 and miR-21 in MTCs. The results suggest that overexpression of miR-21 and MALAT1 may regulate MTC progression.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several compounds based on ASOs are currently being tested in clinical trials for various neurological disorders. Here we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that the activity of ASOs lasts up to 2 months after a single intrathecal bolus dose in the spinal cord. Interestingly, comparison of subcutaneous, central intracerebroventricular and intrathecal administration shows DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.