Ethanol interaction with α3ß4 nicotinic acetylcholine receptors in neurons of the laterodorsal tegmentum.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) play a key role in the rewarding effects of ethanol (EtOH), and while several nAChR subtypes have been implicated, attention has recently shifted to a role for the α3ß4 nAChR. The laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic nucleus that sends excitatory projections to the ventral tegmental area, is an Integral part of the brain reward pathway. Here we investigate a potential role for LDTg α3ß4 nAChRs in EtOH self-administration and reward. METHODS: Sprague-Dawley rats were given ad libitum access to a 20% EtOH solution, as part of a two-bottle choice paradigm. Approximately 1 week after removal of EtOH access, we measured LDTg α3ß4 nAChR current responses to focal application of acetylcholine (ACh), using whole-cell patch clamp electrophysiology recordings in acute brain slices. In addition, we used whole-cell electrophysiology to assess the acute effects of EtOH on the sensitivity of LDTg α3ß4 nAChRs. RESULTS: Focal application of ACh onto LDTg neurons resulted in large α3ß4 nAChR-mediated inward currents, the magnitude of which showed a positive correlation with levels of EtOH self-administration. In addition, using brain slices taken from EtOH-naïve rats, bath application of EtOH resulted in a moderate potentiation of LDTg α3ß4 nAChR sensitivity. CONCLUSIONS: Using a rat model, increased α3ß4 nAChR function was associated with greater EtOH self-administration, with α3ß4 nAChR function also acutely potentiated by EtOH. Assuming that similar findings apply to humans, the α3ß4 nAChR could be a therapeutic target in the treatment of EtOH use disorder.

Current Status of Multiple Drug Molecules, and Vaccines: An Update in SARS-CoV-2 Therapeutics.

The coronavirus disease of 2019 (COVID-19) is a pandemic disease that has taken the lives of many around the world. It is caused by severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2). To date, the USA, Italy, Spain, France, Russia, and the UK have been hit the hardest by the virus. However, death counts are still rising. Some nations have managed to "flatten" the death rate via protective measures such physical distancing, quarantine measures, and therapeutic management. The structure of the SARS-CoV-2 virus comprises of S proteins, M proteins, E proteins, hemagglutinin esterases, nucleocapsid proteins, and a 30-kb RNA genome. Viral proteases cleave these polyproteins and RNA-dependent polymerases replicate the genome. Currently, there are no effective therapies against this new disease. Numerous investigators are developing novel protease inhibitors, some of which have made it into clinical trials. Researchers are also attempting to develop a vaccine. In this review paper, we discuss the latest therapeutic developments against COVID-19. Graphical Abstract.

Nicotinic modulation of descending pain control circuitry.

Along with the well-known rewarding effects, activation of nicotinic acetylcholine receptors (nAChRs) can also relieve pain, and some nicotinic agonists have analgesic efficacy similar to opioids. A major target of analgesic drugs is the descending pain modulatory pathway, including the ventrolateral periaqueductal gray (vlPAG) and the rostral ventromedial medulla (RVM). Although activating nAChRs within this circuitry can be analgesic, little is known about the subunit composition and cellular effects of these receptors, particularly within the vlPAG. Using electrophysiology in brain slices from adult male rats, we examined nAChR effects on vlPAG neurons that project to the RVM. We found that 63% of PAG-RVM projection neurons expressed functional nAChRs, which were exclusively of the α7-subtype. Interestingly, the neurons that express α7 nAChRs were largely nonoverlapping with those expressing µ-opioid receptors (MOR). As nAChRs are excitatory and MORs are inhibitory, these data suggest distinct roles for these neuronal classes in pain modulation. Along with direct excitation, we also found that presynaptic nAChRs enhanced GABAergic release preferentially onto neurons that lacked α7 nAChRs. In addition, presynaptic nAChRs enhanced glutamatergic inputs onto all PAG-RVM projection neuron classes to a similar extent. In behavioral testing, both systemic and intra-vlPAG administration of the α7 nAChR-selective agonist, PHA-543,613, was antinociceptive in the formalin assay. Furthermore, intra-vlPAG α7 antagonist pretreatment blocked PHA-543,613-induced antinociception via either administration method. Systemic administration of submaximal doses of the α7 agonist and morphine produced additive antinociceptive effects. Together, our findings indicate that the vlPAG is a key site of action for α7 nAChR-mediated antinociception.

Tolerance to Ethanol or Nicotine Results in Increased Ethanol Self-Administration and Long-Term Depression in the Dorsolateral Striatum.

Ethanol (EtOH) and nicotine are the most widely coabused drugs. Tolerance to EtOH intoxication, including motor impairment, results in greater EtOH consumption and may result in a greater likelihood of addiction. Previous studies suggest that cross-tolerance between EtOH and nicotine may contribute to the abuse potential of these drugs. Here we demonstrate that repeated intermittent administration of either EtOH or nicotine in adult male Sprague Dawley rats results in tolerance to EtOH-induced motor impairment and increased EtOH self-administration. These findings suggest that nicotine and EtOH cross-tolerance results in decreased aversive and enhanced rewarding effects of EtOH. Endocannabinoid signaling in the dorsolateral striatum (DLS) has been implicated in both EtOH tolerance and reward, so we investigated whether nicotine or EtOH pretreatment might modulate endocannabinoid signaling in this region. Using similar EtOH and nicotine pretreatment methods resulted in increased paired-pulse ratios of evoked EPSCs in enkephalin-positive medium spiny neurons in DLS slices. Thus, EtOH and nicotine pretreatment may modulate glutamatergic synapses in the DLS presynaptically. Bath application of the CB1 receptor agonist Win 55,2-212 increased the paired-pulse ratio of evoked EPSCs in control slices, while Win 55,2-212 had no effect on paired-pulse ratio in slices from either EtOH- or nicotine-pretreated rats. Consistent with these effects, nicotine pretreatment occluded LTD induction by high-frequency stimulation of the corticostriatal inputs to the dorsolateral striatum. These results suggest that nicotine and EtOH pretreatment modulates striatal synapses to induce tolerance to the motor-impairing effects of EtOH, which may contribute to nicotine and EtOH coabuse.

Ethanol-Induced Motor Impairment Mediated by Inhibition of α7 Nicotinic Receptors.

UNLABELLED: Nicotine and ethanol (EtOH) are among the most widely co-abused substances, and nicotinic acetylcholine receptors (nAChRs) contribute to the behavioral effects of both drugs. Along with their role in addiction, nAChRs also contribute to motor control circuitry. The α7 nAChR subtype is highly expressed in the laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic center that contributes to motor performance through its projections to thalamic motor relay centers, including the mediodorsal thalamus. We demonstrate that EtOH concentrations just above the legal limits for intoxication in humans can inhibit α7 nAChRs in LDTg neurons from rats. This EtOH-induced inhibition is mediated by a decrease in cAMP/PKA signaling. The α7 nAChR-positive allosteric modulator PNU120596 [N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea], which interferes with receptor desensitization, completely eliminated EtOH modulation of these receptors. These data suggest that EtOH inhibits α7 responses through a PKA-dependent enhancement of receptor desensitization. EtOH also inhibited the effects of nicotine at presynaptic α7 nAChRs on glutamate terminals in the mediodorsal thalamus. In vivo administration of PNU120596 either into the cerebral ventricles or directly into the mediodorsal thalamus attenuated EtOH-induced motor impairment. Thus, α7 nAChRs are likely important mediators of the motor impairing effects of moderate EtOH consumption. SIGNIFICANCE STATEMENT: The motor-impairing effects of ethanol contribute to intoxication-related injury and death. Here we explore the cellular and neural circuit mechanisms underlying ethanol-induced motor impairment. Physiologically relevant concentrations of ethanol inhibit activity of a nicotinic receptor subtype that is expressed in brain areas associated with motor control. That receptor inhibition is mediated by decreased receptor phosphorylation, suggesting an indirect modulation of cell signaling pathways to achieve the physiological effects.

Regenerative therapeutic potential of adipose stromal cells in early stage diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved "b" wave amplitude (as measured by electroretinogram) within 1-3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.

Vascular endothelial growth factor (VEGF) induced proliferation of human fetal derived ciliary epithelium stem cells is mediated by jagged-N cadherin pathway.

The pigmented ciliary epithelium (PCE) of mammalian eye harbors resident population of stem cells that lie in apposition with endothelial cells which release vascular endothelial growth factor (VEGF) that may influence the fate and function of these stem cells in ways that remain unclear. We examined the role of VEGF in proliferation of PCE stem cells and expression of Notch, Jagged, N-Cadherin and ß-Catenin which are known to maintain proliferation state of neural stem cells. We cultured human PCE cells obtained from 12-20 weeks old fetal eyes. The neurospheres were analyzed for the proliferation capacity of PCE stem cells in presence of VEGF on 3,6 and 9 day. Real time PCR was used to quantitate the mRNA expression of above mentioned genes on PCE derived neurospheres on 3,6 and 9 day. We found increased number of neurospheres when PCE stem cells were stimulated with VEGF along with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) than EGF and bFGF. BrdU immunostaining was done to analyze the proliferation of CE cells and presence of neural and retinal progenitor markers such as Nestin and Pax6 were also investigated. An increased Notch and Jagged mRNA was observed on 6(th) day in VEGF, EGF and bFGF treated PCE cells as compared to 0,3 and 9 day. A similar pattern was noticed with N-cadherin and ß-catenin mRNA levels. These findings may clarify the role of VEGF on PCE stem cell proliferation with possible involvement of Notch, Jagged, N-cadherin and ß-Catenin. The data may suggest importance of harvesting 6(th) day neurospheres for transplantation purposes in preclinical investigations pertaining to retinal degenerative diseases, however, additional studies are needed to substantiate the findings.

Ciliary epithelium: an underevaluated target for therapeutic regeneration.

The purpose of stem cells in various organs of vertebrates is to replenish dying cells or to replace damaged tissues. However, a few organs have reasonable, while others have very limited regenerative, capacity. Until the last two decades, the organs such as brain, heart, and kidneys were known to lack regenerative capacity for lack of resident stem cell population. However, with advancement of techniques and an increase in scientific communication, new discoveries have brought novel concepts and data to discover and manipulate these valuable resources. Much focus has been devoted to understanding the regulation and maintenance of these stem cells. We discuss the preclinical data emerging from retino-vascular interactions useful in the exploitation of ciliary epithelium-derived stem cells for therapeutic regeneration.

Vascular endothelial growth factor-A and chemokine ligand (CCL2) genes are upregulated in peripheral blood mononuclear cells in Indian amyotrophic lateral sclerosis patients.

BACKGROUND: We have earlier shown that protein levels of vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) were elevated in Indian amyotrophic lateral sclerosis (ALS) patients. Here, we report the mRNA levels of VEGF-A and CCL2 in Indian ALS patients since they display extended survival after disease onset. METHODS: VEGF-A and CCL2 mRNA levels were measured in peripheral blood mononuclear cells (PBMCs) of 50 sporadic Indian ALS patients using Real Time Polymerase Chain Reaction (PCR) and compared with normal controls (n = 50). Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption. RESULTS: VEGF-A and CCL2 mRNA levels were found to be significantly elevated in PBMCs in ALS patients as compared to controls. PBMCs from definite ALS revealed higher VEGF-A mRNA expression as compared to probable and possible ALS. CCL2 mRNA levels were found to be unaltered when definite, probable and possible ALS were compared. PBMCs from patients with respiratory dysfunction showed much higher VEGF-A and CCL2 elevation when compared to patients without respiratory dysfunction. No association of smoking, alcohol and meat consumption with VEGF-A and CCL2 was observed after analyzing the data with univariate and multivariate analysis. CONCLUSION: VEGF-A and CCL2 mRNA upregulation in PBMCs may have a clinico-pathological/etiological/epidemiological association with ALS pathogenesis. The cross-cultural and cross-ethnic investigations of these molecules could determine if they have any role in enhancing the mean survival time unique to Indian ALS patients.