Antipsychotics increase steroidogenic enzyme gene expression in the rat brainstem

Background Neurosteroids are involved in several important brain functions and have recently been considered novel players in the mechanic actions of neuropsychiatric drugs. There are no reports of murine studies focusing on the effect of chronic neurosteroid treatment in parallel with antipsychotics on key steroidogenic enzyme expression and we therefore focused on steroidogenic enzyme gene expression in the brainstem of rats chronically treated with olanzapine and haloperidol. Methods and results Studies were carried out on adult, male Sprague–Dawley rats which were divided into 3 groups: control and experimental animals treated with olanzapine or haloperidol. Total mRNA was isolated from homogenized brainstem samples for RealTime-PCR to estimate gene expression of related aromatase, 3β-HSD and P450scc. Long-term treatment with the selected antipsychotics was reflected in the modulation of steroidogenic enzyme gene expression in the examined brainstem region; with both olanzapine and haloperidol increasing aromatase, 3β-HSD and P450scc gene expression. Conclusions The present findings shed new light on the pharmacology of antipsychotics and suggest the existence of possible regulatory interplay between neuroleptic action and steroidogenesis at the level of brainstem neuronal centres.

Modulatory effect of olanzapine on SMIM20/phoenixin, NPQ/spexin and NUCB2/nesfatin-1 gene expressions in the rat brainstem

Background Phoenixin, spexin and nesfatin-1 belong to a family of newly discovered multifunctional neuropeptides that play regulatory roles in several brain structures and modulate the activity of important neural networks. However, little is known about their expression and action at the level of brainstem. The present work was, therefore, focused on gene expression of the aforementioned peptides in the brainstem of rats chronically treated with olanzapine, a second generation antipsychotic drug. Methods Studies were carried out on adult, male Sprague–Dawley rats that were divided into 2 groups: control and experimental animals treated with olanzapine (28-day-long intraperitoneal injection, at dose 5 mg/kg daily). All individuals were killed under anesthesia and the brainstem excised. Total mRNA was isolated from homogenized samples of both structures and the RT-PCR method was used for estimation of related SMIM20/phoenixin, NPQ/spexin and NUCB2/nesfatin-1 gene expression. Results Long-term treatment with olanzapine is reflected in qualitatively different changes in expression of examined neuropeptides mRNA in the rat brainstem. Olanzapine significantly decreased NPQ/spexin mRNA expression, but increased SMIM20/phoenixin mRNA level in the rat brainstem; while NUCB2/nesfatin-1 mRNA expression remained unchanged. Conclusions Olanzapine can affect novel peptidergic signaling in the rat brainstem. This may cautiously suggest the presence of an alternative mode of its action.

Exogenous ketone salts inhibit superoxide production in the rat caudal solitary complex during exposure to normobaric and hyperbaric hyperoxia

The use of hyperbaric oxygen (HBO2) in hyperbaric and undersea medicine is limited by the risk of seizures (i.e., CNS oxygen toxicity, CNS-OT) resulting from increased production of reactive oxygen species (ROS) in the CNS. Importantly, ketone supplementation has been shown to delay onset of CNS-OT in rats by ~600% in comparison to control groups (D'Agostino et al., 2013). We have tested the hypothesis that ketone body supplementation inhibits ROS production during exposure to hyperoxygenation in rat brainstem cells. We measured the rate of cellular superoxide (.O2‑) production in the caudal Solitary Complex (cSC) in rat brain slices using a fluorogenic dye, dihydroethidium (DHE), during exposure to control O2 (0.4 ATA) followed by 1-2 hr of normobaric oxygen (NBO2) (0.95 ATA) and HBO2 (1.95, and 4.95 ATA) hyperoxia, with and without a 50:50 mixture of ketone salts (KS) DL-b-hydroxybutyrate (BHB + acetoacetate (AcAc)). All levels of hyperoxia tested stimulated .O2- production similarly in cSC cells, and co-exposure to 5 mM KS during hyperoxia significantly blunted the rate of increase in DHE fluorescence intensity during exposure to hyperoxia. Not all cells tested produced .O2- at the same rate during exposure to control O2 and hyperoxygenation; cells that increased .O2‑ production by >25% during hyperoxia in comparison to baseline were inhibited by KS, whereas cells that did not reach that threshold during hyperoxia were unaffected by KS. These findings support the hypothesis that ketone supplementation decreases the steady state concentrations of superoxide produced during exposure to NBO2 and HBO2 hyperoxia.

Rabies virus matrix protein targets host actin cytoskeleton: A Protein-Protein interaction analysis

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris (P. pastoris) and was partially purified. Subsequently, 2D-Far-WB determined six rat brainstem proteins interacted with recombinant M protein which were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process, and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation (Co-IP), was only successful for M-actin cytoplasmic1 interaction. Totally, our study revealed actin cytoplasmic1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

Signaling in Rat Brainstem via Gpr160 is Required for the Anorexigenic and Antidipsogenic Actions of Cocaine- and Amphetamine-Regulated Transcript Peptide

Recent work identified Gpr160 as a candidate receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp) and described its role in pain modulation. The aims of the present study were to determine if Gpr160 is required for the CARTp's ability to reduce food and water intake and to initially identify the distribution of Gpr160-like immunoreactivity (Gpr160ir) in the rat brain. A passive immunoneutralization approach targeting Gpr160 was used to block the behavioral effects of a pharmacologic dose of CARTp in the fourth cerebroventricle (4V) of rats and to determine the importance of endogenously produced CARTp in the control of ingestive behaviors. Passive immunoneutralization of Gpr160 in the 4V blocked the actions of CARTp to inhibit food and water intakes. Blockade of Gpr160 in the 4V, independent of pharmacologic CART treatment, caused an increase in both overnight food and water intakes. The decrease in food, but not water intake, caused by central injection of CARTp was demonstrated to be interrupted by prior administration of a GLP-1 receptor antagonist. Gpr160ir was observed in several, distinct sites throughout the rat brain, where CARTp staining has been described. Importantly, Gpr160ir was observed to be present in both neuronal and non-neuronal cell types. These data support the hypothesis that Gpr160 is required for the anorexigenic actions of central CARTp injection, and extend these findings to water drinking. Gpr160ir was observed in both neuronal and non-neuronal cell types in regions known to be important in the multiple pharmacologic effects of CARTp, identifying those areas as targets for future compromise of function studies.

Isoflurane inhibits a Kir4.1/5.1-like conductance in neonatal rat brainstem astrocytes and recombinant Kir4.1/5.1 channels in a heterologous expression system

An unwanted side effect of isoflurane anesthesia is suppression of breathing. Despite this clinical significance, effects of isoflurane on cellular and molecular elements of respiratory control are not well understood. Here, we show that isoflurane inhibits heteromeric Kir4.1/5.1 channels in a mammalian expression system, and a Kir4.1/5.1-like conductance in astrocytes in a brainstem respiratory center. These results identify astrocyte Kir4.1/5.1 channels as novel targets of isoflurane and potential substrates for altered respiratory control during isoflurane anesthesia.