Interoceptive and exteroceptive signals, and the corresponding coordinated control of internal organs and sensory functions, including pain, are received and orchestrated by multiple neurons within the peripheral, central and autonomic nervous systems. A central aim of the present report is to obtain a molecularly informed basis for analgesic drug development aimed at peripheral rather than central targets. We compare three key peripheral ganglia: nodose, sympathetic (superior cervical), and dorsal root ganglia in the rat, and focus on their molecular composition using next-gen RNA-Seq, as well as their neuroanatomy using immunocytochemistry and in situ hybridization. We obtained quantitative and anatomical assessments of transmitters, receptors, enzymes and signaling pathways mediating ganglion-specific functions. Distinct ganglionic patterns of expression were observed spanning ion channels, neurotransmitters, neuropeptides, G-protein coupled receptors (GPCRs), transporters, and biosynthetic enzymes. The relationship between ganglionic transcript levels and the corresponding protein was examined using immunohistochemistry for select, highly expressed, ganglion-specific genes. Transcriptomic analyses of spinal dorsal horn and intermediolateral cell column (IML), which form the termination of primary afferent neurons and the origin of preganglionic innervation to the SCG, respectively, disclosed pre- and post-ganglionic molecular-level circuits. These multimodal investigations provide insight into autonomic regulation, nodose transcripts related to pain and satiety, and DRG-spinal cord and IML-SCG communication. Multiple neurobiological and pharmacological contexts can be addressed, such as discriminating drug targets and predicting potential side effects, in analgesic drug development efforts directed at the peripheral nervous system.
Ketamine, an N-methyl-D-aspartate (NMDA)-receptor antagonist, is a recently revitalized treatment for pain and depression, yet its actions at the molecular level remain incompletely defined. In this molecular-pharmacological investigation in the rat, we used short- and longer-term infusions of high dose ketamine to stimulate neuronal transcription processes. We hypothesized that a progressively stronger modulation of neuronal gene networks would occur over time in cortical and limbic pathways. A continuous intravenous administration paradigm for ketamine was developed in rat consisting of short (1 h) and long duration (10 h, and 10 h + 24 h recovery) infusions of anesthetic concentrations to activate or inhibit gene transcription in a pharmacokinetically controlled fashion. Transcription was measured by RNA-Seq in three brain regions: frontal cortex, hippocampus, and amygdala. Cellular level gene localization was performed with multiplex fluorescent in situ hybridization. Induction of a shared transcriptional regulatory network occurred within 1 h in all three brain regions consisting of (a) genes involved in stimulus-transcription factor coupling that are induced during altered synaptic activity (immediate early genes, IEGs, such as c-Fos, 9–12 significant genes per brain region, p < 0.01 per gene) and (b) the Nrf2 oxidative stress-antioxidant response pathway downstream from glutamate signaling (Nuclear Factor Erythroid-Derived 2-Like 2) containing 12–25 increasing genes (p < 0.01) per brain region. By 10 h of infusion, the acute results were further reinforced and consisted of more and stronger gene alterations reflecting a sustained and accentuated ketamine modulation of regional excitation and plasticity. At the cellular level, in situ hybridization localized up-regulation of the plasticity-associated gene Bdnf, and the transcription factors Nr4a1 and Fos, in cortical layers III and V. After 24 h recovery, we observed overshoot of transcriptional processes rather than a smooth return to homeostasis suggesting an oscillation of plasticity occurs during the transition to a new phase of neuronal regulation. These data elucidate critical molecular regulatory actions during and downstream of ketamine administration that may contribute to the unique drug actions of this anesthetic agent. These molecular investigations point to pathways linked to therapeutically useful attributes of ketamine.
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