Background: Prolonged exposure to ketamine results in accelerated neurodegeneration and neurocognitive deficits in the neonatal rats. Experimental models of neurodegeneration have implicated reentry of postmitotic neurons into the cell cycle, leading to cell death. The authors hypothesize that the ketamine-induced neuroapoptosis is partially due to aberrant cycle cell reentry. To explore this hypothesis, the authors characterized the effect of ketamine on the cell cycle signaling pathway in the developing rodent brain in vivo and in vitro. Methods: Postnatal day 7 rat pups and primary neurons were used for the experiments. Each rat pup received five intraperitoneal doses of either saline or ketamine (5, 10, and 20 mg/kg/dose) at 90-min intervals over 6 h. Primary neurons were exposed to varying concentrations of ketamine to determine the dose and duration effects. The expression of cell cycle proteins (cyclin D1, cyclin-dependent kinase 4, and E2F1), Bcl2-interacting mediator of cell death (Bim), and activated caspase-3 was determined. The effect of cyclin D1 knockdown by small interfering RNA was also examined in primary neurons incubated in ketamine. Results: Ketamine mediated a dose- and time-dependent increase in expression of cell cycle proteins and activated caspase-3. Cyclin D1, cyclin-dependent kinase 4, E2F1, Bim, and cleaved caspase-3 expression increased at 12 h and peaked at 24 h in vitro. Knockdown of cyclin D1 by small interfering RNA attenuated Bim and cleaved caspase-3 expression. Conclusion: These findings support a model in which ketamine induces aberrant cell cycle reentry, leading to apoptotic cell death in the developing rat brain.

Orexins (hypocretins) are recently discovered excitatory transmitters implicated in arousal and sleep. Yet, their ionic and signal transduction mechanisms have not been fully clarified. Here we show that orexins suppress G-protein–coupled inward rectifier (GIRK) channel activity, and this suppression is likely to lead to neuronal excitation. Cultured neurons from the locus coeruleus (LC) and the nucleus tuberomammillaris (TM) were used, as well as HEK293A cells transfected with GIRK1 and 2, either human orexin receptor type 1 (OX₁R) or type 2 (OX₂R), mu opioid receptor and GFP cDNAs. In GTPγS-loaded cells, orexin A (OXA, 3 μM) inhibited GIRK currents that had previously been activated by somatostatin (in LC cells), nociceptin (TM cells), or the mu opioid agonist DAMGO (HEK cells). In guanosine triphosphate (GTP)–loaded HEK cells, in which GIRK currents were not preactivated, OXA induced a biphasic response through both types of orexin receptors: an initial current increase and a subsequent decrease to below resting levels. Current–voltage (I–V) relationships revealed that both the OXA-induced and suppressed currents are inwardly rectifying with reversal potentials around E _K. The OXA-induced initial current was partially pertussis toxin (PTX) sensitive and partially PTX insensitive, whereas the OXA-suppressed current was PTX insensitive. These data suggest that orexin receptors couple with more than one type of G-protein, including PTX-sensitive (such as G_i/o) and PTX-insensitive (such as G_q/11) G-proteins. The modulation of GIRK channels by orexins may be one of the cellular mechanisms for the regulation of brain nuclei (e.g., LC and TM) that are crucial for arousal, sleep, and appetite.

Rodent models have opened the door to a better understanding of the neurobiology of brain disorders and increased our ability to evaluate novel treatments. Resting-state functional magnetic resonance imaging (rs-fMRI) allows for in vivo exploration of large-scale brain networks with high spatial resolution. Its application in rodents affords researchers a powerful translational tool to directly assess/explore the effects of various pharmacological, lesion, and/or disease states on known neural circuits within highly controlled settings. Integration of animal and human research at the molecular-, systems-, and behavioral-levels using diverse neuroimaging techniques empowers more robust interrogations of abnormal/ pathological processes, critical for evolving our understanding of neuroscience. We present a comprehensive protocol to evaluate resting-state brain networks using Independent Component Analysis (ICA) in rodent model. Specifically, we begin with a brief review of the physiological basis for rs-fMRI technique and overview of rs-fMRI studies in rodents to date, following which we provide a robust step-by-step approach for rs-fMRI investigation including data collection, computational preprocessing, and brain network analysis. Pipelines are interwoven with underlying theory behind each step and summarized methodological considerations, such as alternative methods available and current consensus in the literature for optimal results. The presented protocol is designed in such a way that investigators without previous knowledge in the field can implement the analysis and obtain viable results that reliably detect significant differences in functional connectivity between experimental groups. Our goal is to empower researchers to implement rs-fMRI in their respective fields by incorporating technical considerations to date into a workable methodological framework.

Neuroplasticity in the mesolimbic dopaminergic system is critical for behavioral adaptations associated with opioid reward and addiction. These processes may be influenced by cholinergic transmission arising from the laterodorsal tegmental nucleus (LDTg), a main source of acetylcholine to mesolimbic dopaminergic neurons. To examine this possibility we asked if chronic systemic morphine administration affects expression of genes in ventral and ventrolateral periaqueductal gray at the level of the LDTg using rtPCR. Specifically, we examined gene expression changes in the area of interest using Neurotransmitters and Receptors PCR array between chronic morphine and saline control groups. Analysis suggested that chronic morphine administration led to changes in expression of genes associated, in part, with cholinergic neurotransmission. Furthermore, using a quantitative immunofluorescent technique, we found that chronic morphine treatment produced a significant increase in immunolabeling of the cholinergic marker (vesicular acetylcholine transporter) in neurons of the LDTg. Finally, systemic administration of the nonselective and noncompetitive neuronal nicotinic antagonist mecamylamine (0.5 or 2 mg/kg) dose-dependently blocked the expression, and to a lesser extent the development, of locomotor sensitization. The same treatment had no effect on acute morphine antinociception, antinociceptive tolerance or dependence to chronic morphine. Taken together, the results suggest that endogenous nicotinic cholinergic neurotransmission selectively contributes to behavioral sensitization to morphine and this process may, in part, involve cholinergic neurons within the LDTg.