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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.

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PERSPECTIVE: This is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

Most models of induced hyperalgesia display stereotyped behavioral responses to modalities such as heat and low threshold mechanical stimuli. Despite this, the nociceptive apparatus and the subsequent subjective experiences that define hyperalgesia differ greatly, as do accompanying symptoms of the associated pain state. Based on several recent in-depth studies of the activation patterns of primary afferents, we predicted that divergent activation paradigms could trigger distinct plastic events at second-order neurons that converge to sensitize the spinal circuits to evoked stimuli. The spinal circuits engaged by inflammatory and post-surgical pain states can be elucidated based on multiplex labeling and time course evaluations of the transcriptional signatures using deep sequencing. These experiments revealed a population of neurons apparently undetected by single-cell and single-nucleus sequencing methods because of its sparse representation in the superficial laminae of the dorsal horn. Investigations into the character and connectivity of these second order spinal neurons revealed that they participate similarly in at least two models of hyperalgesia. These models are driven by different transcriptional machinery at the level of the dorsal spinal cord, and the most transcriptionally evident neurons express very high levels of both the opioid peptide dynorphin and receptors that may be involved in phenotypic switching during hyperalgesia. We identified multiple cell populations, representing opponent processes acting to promote and abrogate hyperalgesia. Surprisingly, both neural and glial substrates of plasticity were divergent between the models, but converged at the pathway level. Examples of convergent pathways included immune-like activation and upregulation of the dynorphin precursor gene, whereas the activation of neuronal genes was divergent. These investigations outline a multidisciplinary strategy for identification of targets for analgesia and hyperalgesia research, and identify a set of neuronal receptors for future analgesic target development.

General anesthesia and monitored sedation are vital to nearly all procedural aspects of modern medicine. Inhaled agents such as halogenated gases have long been an integral aspect of anesthesia. In recent years, the noncompetitive NMDA antagonist and dissociative anesthetic ketamine has seen increasing utilization in general anesthesia, as well as in the treatment of chronic pain and depression. The use of ketamine for such chronic conditions has raised questions into its addictive potential, and its potential role in the treatment of existing addictions. New investigations using high density electrical recordings and other multidimensional techniques have led to a clearer understanding of how these agents work, and how they can be utilized to fill clinical needs. To further study their effects at the molecular level, we used deep RNA sequencing to examine the time course (1hr, 10hr, or 10hr + 24hr recovery) of gene changes induced in the frontal cortex, hippocampus, and amygdala during ketamine or isoflurane administration. When compared to isoflurane, the transcriptional signature of ketamine showed similarities and differences that suggested region-specific gene regulation patterns that highlight the molecular underpinnings of the broad clinical uses of ketamine. The evaluation of brain regions subserving the diverse functions of learning and memory, executive function, and emotional-affective dimensions yielded insight into the neural localization of anesthetic actions and potential vulnerabilities associated with general anesthesia. Our investigation demonstrates unique region-specific and time-dependent transcriptional signatures that bridge molecular and systems level findings to expand current understanding of the shifts in neuronal activity associated with general anesthetics and their downstream effects.