cAMP Response Element-Binding Protein Controls the Appearance of Neuron-Like Traits in Chorion Mesenchymal Cells

Mesenchymal stromal cells (MSC) from bone marrow have been reported to undergo the initial phases of neural differentiation in response to an increase of intracellular cAMP. We investigated the possibility that a similar effect applies to chorion-derived MSC

Therapeutic Induction of Energy Metabolism Reduces Neural Tissue Damage and Increases Microglia Activation in Severe Spinal Cord Injury

: Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged, tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and, neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative, phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a, pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI

All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca(2+) signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors

Ketamine effects on mammalian target of rapamycin signaling in the mouse limbic system depend on functional dopamine D3 receptors

Ketamine is a noncompetitive glutamate N-methyl-D-aspartic acid receptor antagonist. When acutely administered to rodents, it produces a rapid antidepressant effect. There is evidence that N-methyl-D-aspartic acid receptor blockade enhances glutamatergic transmission preferentially engaging α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leading to mTOR (mammalian target of rapamycin) pathways activation, thus resulting into downstream neuroadaptive changes in limbic structures. Recent in-vitro data on primary neuronal cultures showed that ketamine activates mTOR also in dopaminergic neurons, and this activation depends on the presence of functional dopamine D3 receptors. The aim of this work was to study the in-vivo relevance of viable D3 receptors in mediating the effects of acute ketamine administration on the mTOR downstream substrate p70 ribosomal S6 kinase (p70S6K), an obligatory substrate for mTOR. We compared the effects of single ketamine 5 mg/kg, 10 mg/kg, or vehicle injection in wild-type and D3 receptor knockout mice. Animals were killed after 60 min, and their brains were processed for p-p70S6K immunohistochemistry. Ketamine increased p70S6K phosphorylation in prefrontal cortex, nucleus accumbens core and shell, ventral tegmental area, substantia nigra, hippocampal CA1, CA2, and CA3, and basolateral amygdala of wild-type mice but not in mutant mice. Our study demonstrates that ketamine-induced p70S6K phosphorylation is dependent on viable D3R expressed in most of limbic structures

Ketamine Self-Administration Elevates \u3b1CaMKII Autophosphorylation in Mood and Reward-Related Brain Regions in Rats

Modulation of \u3b1CaMKII expression and phosphorylation is a feature shared by drugs of abuse with different mechanisms of action. Accordingly, we investigated whether \u3b1CaMKII expression and activation could be altered by self-administration of ketamine, a non-competitive antagonist of the NMDA glutamate receptor, with antidepressant and psychotomimetic as well as reinforcing properties. Rats self-administered ketamine at a sub-anesthetic dose for 43\ua0days and were sacrificed 24\ua0h after the last drug exposure; reward-related brain regions, such as medial prefrontal cortex (PFC), ventral striatum (vS), and hippocampus (Hip), were used for the measurement of \u3b1CaMKII-mediated signaling. \u3b1CaMKII phosphorylation was increased in these brain regions suggesting that ketamine, similarly to other reinforcers, activates this kinase. We next measured the two main targets of \u3b1CaMKII, i.e., GluN2B (S1303) and GluA1 (S831), and found increased activation of GluN2B (S1303) together with reduced phosphorylation of GluA1 (S831). Since GluN2B, via inhibition of ERK, regulates the membrane expression of GluA1, we measured ERK2 phosphorylation in the crude synaptosomal fraction of these brain regions, which was significantly reduced suggesting that ketamine-induced phosphorylation of \u3b1CaMKII promotes GluN2B (S1303) phosphorylation that, in turn, inhibits ERK 2 signaling, an effect that results in reduced membrane expression and phosphorylation of GluA1. Taken together, our findings point to \u3b1CaMKII autophosphorylation as a critical signature of ketamine self-administration providing an intracellular mechanism to explain the different effects caused by \u3b1CaMKII autophosphorylation on the post-synaptic GluN2B- and GluA1-mediated functions. These data add ketamine to the list of drugs of abuse converging on \u3b1CaMKII to sustain their addictive properties

Generation of mouse hippocampal brain organoids from primary embryonic neural stem cells

: Here we present a protocol to generate standardized cerebral organoids with hippocampal regional specification using morphogen WNT3a. We describe steps for isolating mouse embryonic (E14.5) neural stem cells from the brain subgranular zone, preparing organoids samples for immunofluorescence, calcium imaging, and metabolic profiling. This protocol can be used to generate mouse brain organoids for developmental studies, modeling disease, and drug screening. Organoids can be obtained in one month, thus providing a rapid tool for high-throughput data validation. For complete details on the use and execution of this protocol, please refer to Ciarpella et al. "Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity".1

Reconsolidation of sucrose instrumental memory in rats: the role of retrieval context

Memory reconsolidation enables the update of a previously consolidated memory trace after its reactivation. Although Pavlovian memory reconsolidation has been widely demonstrated, instrumental memory reconsolidation is still debated. The most critical issue on instrumental memory reconsolidation findings have mainly been linked to the presence of specific boundary conditions for reactivation, for instance contextual parameters. In this study, we investigated the role of the spatial context on molecular markers of sucrose instrumental memory reactivation. Following withdrawal, rats previously conditioned to sucrose self-administration underwent either instrumental memory retrieval or no-retrieval in the conditioned context (Context A, AA condition) or in a modified version of the conditioned context (Context B, AB condition). Two hours later, the level of GluA1 and GluN2B receptors, Zif268 and phosphorylated-rpS6 (rpS6P) was measured in key brain areas for memory reactivation. Retrieval in Context A significantly increased GluA1Rs and GluN2BRs in amygdala compared to no-retrieval, indicating that memory successfully reactivated and destabilized. Moreover, Zif268 level was significantly increased after retrieval in Context A in the nucleus accumbens shell, central and basolateral amygdala but not in the hippocampus, while retrieval in Context B significantly increased Zif268 level in all brain areas. On the other hand, rpS6P level was increased in the nucleus accumbens shell and central amygdala, but decreased in the hippocampus, after retrieval in Context A, while retrieval in Context B did not change rpS6P level in brain areas, except for a small but significant decrease in hippocampus. While the increase of Zif268 level indicated that memory reactivation has been triggered in both the conditions, the lack of change in rpS6P levels after retrieval in Context B - in particular in the central amygdala - suggests that the reconsolidation process could not occur after memory reactivation in a context different from the conditioned one

Smokers "Context Reactivity" in Virtual Domestic Environments

Although the effects of proximal smoking cues have been widely studied in smokers, little is known on the features associated with background spatial context effect, that is, "context reactivity." The aim of this study was to investigate context reactivity exhibited by smokers in virtual cue-free domestic scenarios

Immunolocalization of leptin and leptin receptor in colorectal mucosa of ulcerative colitis, Crohn's disease and control subjects with no inflammatory bowel disease

The expression of leptin and leptin receptor (Ob-R) has been partially elucidated in colon of patients with inflammatory bowel diseases (IBDs), even though leptin is involved in angiogenesis and inflammation. We previously reported overexpression of GLUT5 fructose transporter, in aberrant clusters of lymphatic vessels in lamina propria of IBD and controls. Here, we examine leptin and Ob-R expression in the same biopsies. Specimens were obtained from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls who underwent screening for colorectal cancer, follow-up after polypectomy or with a history of lower gastrointestinal symptoms. Immunohistochemistry revealed leptin in apical and basolateral membranes of short epithelial portions, Ob-R on the apical pole of epithelial cells. Leptin and Ob-R were also identified in structures and cells scattered in the lamina propria. In UC, a significant correlation between leptin and Ob-R in the lamina propria was found in all inflamed samples, beyond non-inflamed samples of the proximal tract, while in CD, it was found in inflamed distal samples. Most of the leptin and Ob-R positive areas in the lamina propria were also GLUT5 immunoreactive in inflamed and non-inflamed mucosa. A significant correlation of leptin or Ob-R expression with GLUT5 was observed in the inflamed distal samples from UC. Our findings suggest that there are different sites of leptin and Ob-R expression in large intestine and those in lamina propria do not reflect the status of mucosal inflammation. The co-localization of leptin and/or Ob-R with GLUT5 may indicate concomitance effects in colorectal lamina propria areas