Frequent first-trimester pregnancy loss in rhesus macaques infected with African-lineage Zika virus.

In the 2016 Zika virus (ZIKV) pandemic, a previously unrecognized risk of birth defects surfaced in babies whose mothers were infected with Asian-lineage ZIKV during pregnancy. Less is known about the impacts of gestational African-lineage ZIKV infections. Given high human immunodeficiency virus (HIV) burdens in regions where African-lineage ZIKV circulates, we evaluated whether pregnant rhesus macaques infected with simian immunodeficiency virus (SIV) have a higher risk of African-lineage ZIKV-associated birth defects. Remarkably, in both SIV+ and SIV- animals, ZIKV infection early in the first trimester caused a high incidence (78%) of spontaneous pregnancy loss within 20 days. These findings suggest a significant risk for early pregnancy loss associated with African-lineage ZIKV infection and provide the first consistent ZIKV-associated phenotype in macaques for testing medical countermeasures.

Selective Serotonin Reuptake Inhibitors within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder. The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist on the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity-based, drug-sensing fluorescent reporters targeted to the plasma membrane, cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also used chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for >2.4 h. They inhibit SERT transport-associated currents sixfold or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the therapeutic lag of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the antidepressant discontinuation syndrome.SIGNIFICANCE STATEMENT Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to SERT, which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2-6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands engage their therapeutic target(s).

Multifocal electroretinography increases following experimental glaucoma in nonhuman primates with retinal ganglion cell axotomy.

PURPOSE: To determine whether short-latency changes in multifocal electroretinography (mfERG) observed in experimental glaucoma (EG) are secondary solely to retinal ganglion cell (RGC) loss or whether there is a separate contribution from elevated intraocular pressure (IOP). METHODS: Prior to operative procedures, a series of baseline mfERGs were recorded from six rhesus macaques using a 241-element unstretched stimulus. Animals then underwent hemiretinal endodiathermy axotomy (HEA) by placing burns along the inferior 180° of the optic nerve margin in the right eye (OD). mfERG recordings were obtained in each animal at regular intervals following for 3-4 months to allow stabilization of the HEA effects. Laser trabecular meshwork destruction (LTD) to elevate IOP was then performed; first-order kernel (K1) waveform root-mean-square (RMS) amplitudes for the short-latency segment of the mfERG wave (9-35 ms) were computed for two 7-hexagon groupings-the first located within the superior (non-axotomized) macula and the second within the inferior (axotomized) macula. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was done. RESULTS: By 3 months post HEA, there was marked thinning of the inferior nerve fiber layer as measured by optical coherence tomography. Compared with baseline, no statistically significant changes in 9-35 ms K1 RMS amplitudes were evident in either the axotomized or non-axotomized portions of the macula. Following LTD, mean IOP in HEA eyes rose to 46 ± 9 compared with 20 ± 2 mmHg (SD) in the fellow control eyes. In the HEA + EG eyes, statistically significant increases in K1 RMS amplitude were present in both the axotomized inferior and non-axotomized superior portions of the OD retinas. No changes in K1 RMS amplitude were found in the fellow control eyes from baseline to HEA epoch, but there was a smaller increase from baseline to HEA + EG. Upregulation of GFAP in the Müller cells was evident in both non-axotomized and axotomized retina in eyes with elevated IOP. CONCLUSIONS: The RMS amplitudes of the short-latency mfERG K1 waveforms are not altered following axotomy but undergo marked increases following elevated IOP. This suggests that the increase in mfERG amplitude was not solely a result of RGC loss and may reflect photoreceptor and bipolar cell dysfunction and/or changes in Müller cells.

Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids.

We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBP's second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivity─notably enantioselectivity against R-methadone─for biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.

Chronic Menthol Does Not Change Stoichiometry or Functional Plasma Membrane Levels of Mouse α3ß4-Containing Nicotinic Acetylcholine Receptors.

Heteromeric α3ß4 nicotinic acetylcholine (ACh) receptors (nAChRs) are pentameric ligand-gated cation channels that include at least two α3 and two ß4 subunits. They have functions in peripheral tissue and peripheral and central nervous systems. We examined the effects of chronic treatment with menthol, a major flavor additive in tobacco cigarettes and electronic nicotine delivery systems, on mouse α3ß4 nAChRs transiently transfected into neuroblastoma-2a cells. Chronic menthol treatment at 500 nM, near the estimated menthol concentration in the brain following cigarette smoking, altered neither the [ACh]-response relationship nor Zn2+ sensitivity of ACh-evoked currents, suggesting that menthol does not change α3ß4 nAChR subunit stoichiometry. Chronic menthol treatment failed to change the current density (peak current amplitude/cell capacitance) of 100 µM ACh-evoked currents. Chronic menthol treatment accelerated desensitization of 100 and 200 µM ACh-evoked currents. Chronic nicotine treatment (250 µM) decreased ACh-induced currents, and we found no additional effect of including chronic menthol. These data contrast with previously reported, marked effects of chronic menthol on ß2* nAChRs studied in the same expression system. Mechanistically, the data support the emerging interpretation that both chronic menthol and chronic nicotine act on nAChRs in the early exocytotic pathway, and that this pathway does not present a rate-limiting step to the export of α3ß4 nAChRs; these nAChRs include endoplasmic reticulum (ER) export motifs but not ER retention motifs. Previous reports show that smoking mentholated cigarettes enhances tobacco addiction; but our results show that this effect is unlikely to arise via menthol actions on α3ß4 nAChRs.

Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.

Upregulation of ß2 subunit-containing (ß2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate ß2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(ß2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(ß2)3 nAChRs. Menthol alone also increases the number of α6ß2 receptors that exclude the ß3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway.

Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons.

Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2α, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein. We incubated cultures for 2 weeks with 200 nm nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured TH+ neurons displayed α4, α6, and ß3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the TH+ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by ∼2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.

CRISPR/Cas9 gene editing in Drosophila via visual selection in a summer classroom.

CRISPR/Cas9 methods are a powerful in vivo approach to edit the genome of Drosophila melanogaster. To convert existing Drosophila GAL4 lines to LexA driver lines in a secondary school classroom setting, we applied the CRISPR-based genetic approach to a collection of Gal4 'driver' lines. The integration of the yellow+ coat color marker into homology-assisted CRISPR knock-in (HACK) enabled visual selection of Gal4-to-LexA conversions using brightfield stereo-microscopy available in a broader set of standard classrooms. Here, we report the successful conversion of eleven Gal4 lines with expression in neuropeptide-expressing cells into corresponding, novel LexA drivers. The conversion was confirmed by LexA- and Gal4-specific GFP reporter gene expression. This curriculum was successfully implemented in a summer course running 16 hours/week for seven weeks. The modularity, flexibility, and compactness of this course should enable development of similar classes in secondary schools and undergraduate curricula, to provide opportunities for experience-based science instruction, and university-secondary school collaborations that simultaneously fulfill research needs in the community of science.

Development of Microcystoid Macular Degeneration in the Retina of Nonhuman Primates: Time-Course and Associated Pathologies.

PURPOSE: Microcystoid macular degeneration (MMD) is a condition where cystoid vacuoles develop within the inner nuclear layer of the retina in humans in a variety of disorders. Here we report the occurrence of MMD in non-human primates (NHPs) with various retinal ganglion cell (RGC) pathologies and evaluate the hypothesis that MMD does not precede RGC loss but follows it. METHODS: Morphological studies were performed of the retinas of NHPs, specifically both rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis), in which MMD was identified after induction of experimental glaucoma (EG), hemiretinal endodiathermy axotomy (HEA), and spontaneous idiopathic bilateral optic atrophy. In vivo imaging analyses included fundus photography, fluorescein angiography (FA), optical coherence tomography (OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), light microscopy, and electron microscopy. RESULTS: MMD, like that seen on OCT scans of humans, was found in both rhesus and cynomolgus macaques with EG. Of 13 cynomolgus macaques with chronic EG imaged once with OCT six of 13 animals were noted to have MMD. MMD was also evident in a cynomolgus macaque with bilateral optic atrophy. Following HEA, MMD did not develop until at least 2 weeks following the RNFL loss. CONCLUSION: These data suggest that MMD may be caused by a retrograde trans-synaptic process related to RGC loss. MMD is not associated with inflammation, nor would it be an independent indicator of drug toxicity per se in pre-clinical regulatory studies. Because of its inconsistent appearance and late development, MMD has limited use as a clinical biomarker.

The effect of pentobarbital sodium and propofol anesthesia on multifocal electroretinograms in rhesus macaques.

We compared the suitability of pentobarbital sodium (PB) and propofol (PF) anesthetics for multifocal electroretinograms (mfERGs) in rhesus macaques. mfERGs were collected from 4 ocularly normal rhesus macaques. All animals were pre-anesthetized with intramuscular ketamine (10-15 mg/kg). Intravenous PB induction/maintenance levels were 15 mg/kg/2-10 mg/kg and for PF, 2-5 mg/kg/6-24 mg/kg/h. There were 3 testing sessions with PB anesthesia and 5-7 testing sessions with PF anesthesia. All PB sessions were carried out before PF. First-order (K1) and second-order (first slice) kernels (K2.1) response density amplitude (RDA), implicit time (IT), and root mean square signal-to-noise ratios (RMS SNR) of the low-frequency (LFC) and high-frequency (HFC) components were evaluated. The use of PF or PB anesthesia resulted in robust, replicable mfERGs in rhesus macaques; however, RMS SNR of K1 LFC in ring and quadrant analyses was significantly larger for PF than for PB. Additionally, K1 RDA under PF was significantly larger than under PB for N1, P1, and P2 components (ring and quadrant) and for N2 (quadrant). PF IT was significantly prolonged (<1 ms) relative to PB IT for N1, P1 (ring), and N1 (quadrant), while PB IT was significantly prolonged (0.8-4.2 ms) relative to PF IT for N2 and P2 (ring and quadrant). K1 HFC and K2.1 LFC did not differ significantly between PB and PF in the ring or quadrant analyses. The response differences found with PB and PF anesthesia likely arise from variable relative effects of the anesthetics on retinal γ-aminobutyric acid (GABA(A)) receptors, and in part, on glycine and on glutamate receptors. Given the advantages of a stable anesthetic plane with continuous intravenous infusion and a smoother, more rapid recovery, PF is an appealing alternative for mfERG testing in rhesus macaques.

Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Serial multifocal electroretinograms during long-term elevation and reduction of intraocular pressure in non-human primates.

The purpose of this study was to evaluate the relationship between elevations of intraocular pressure (IOP) and the multifocal electroretinogram (mfERG) in non-human primates. Experimental glaucoma was induced in 4 rhesus and 4 cynomolgus monkeys by laser trabecular meshwork destruction (LTD) in one eye. To evaluate the contribution of ganglion cells to mfERG changes, one monkey of each species had previously underwent unilateral optic nerve transection (ONT). After >or=44 weeks of elevation, the IOP was reduced by trabeculectomy in 2 non-transected animals. In the intact (non-transected) animals, there was an increase in the amplitude of the early mfERG waveforms (N1 and P1) of the first-order kernel (K1) throughout the period of IOP elevation in all of the rhesus, but not all of the cynomolgus monkeys. A species difference was also present as a decrease of the second-order kernel, first slice (K2.1) in all of the cynomolgus monkeys but only in 1 of the rhesus monkeys (the 1 with the ONT). Similar IOP effects on the mfERG were seen in the ONT animals. Surgical lowering of IOP resulted in a return of the elevated K1 amplitudes to baseline levels. However, the depressed K2.1 RMS in the cynomolgus monkeys did not recover. These results demonstrate species-specific changes in cone-driven retinal function during periods of elevated IOP. These IOP-related effects can occur in the absence of retinal ganglion cells and may be reversible.

Quantitative definition of neurobehavior, vision, hearing and brain volumes in macaques congenitally exposed to Zika virus.

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design.

Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors.

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

Biosensors Show the Pharmacokinetics of S-Ketamine in the Endoplasmic Reticulum.

The target for the "rapid" (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug's target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/µM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 µM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

Menthol Enhances Nicotine Reward-Related Behavior by Potentiating Nicotine-Induced Changes in nAChR Function, nAChR Upregulation, and DA Neuron Excitability.

Understanding why the quit rate among smokers of menthol cigarettes is lower than non-menthol smokers requires identifying the neurons that are altered by nicotine, menthol, and acetylcholine. Dopaminergic (DA) neurons in the ventral tegmental area (VTA) mediate the positive reinforcing effects of nicotine. Using mouse models, we show that menthol enhances nicotine-induced changes in nicotinic acetylcholine receptors (nAChRs) expressed on midbrain DA neurons. Menthol plus nicotine upregulates nAChR number and function on midbrain DA neurons more than nicotine alone. Menthol also enhances nicotine-induced changes in DA neuron excitability. In a conditioned place preference (CPP) assay, we observed that menthol plus nicotine produces greater reward-related behavior than nicotine alone. Our results connect changes in midbrain DA neurons to menthol-induced enhancements of nicotine reward-related behavior and may help explain how smokers of menthol cigarettes exhibit reduced cessation rates.

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression.

In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Measurement of regional choroidal blood flow in rabbits and monkeys using fluorescent microspheres.

OBJECTIVE: To develop a quantitative measure of regional variation in choroidal blood flow (ChBF). METHODS: Five million 15-microm fluorescent microspheres were injected into the left ventricles of 4 rabbits and 3 monkeys. The fixed globes were bleached, flat mounted, and photomicrographed. After image analysis to locate each microsphere, regional densities and blood flow were determined. RESULTS: Regional variation in ChBF was clearly evident. In the rabbit, a high density of spheres was seen in the visual streak. This was surrounded by a middle peripheral area of low sphere density and a far peripheral region of moderately high density. In the monkeys, sphere density was markedly greater in the macula compared with the periphery. Contour plots produced lines of constant flow that were oval and extended farther nasally than temporally in the monkeys. The ratio of central to peripheral ChBF was much greater in the monkeys than in the rabbits. CONCLUSION: Quantitative assessment of regional ChBF can be performed using a modification of the fluorescent microsphere impaction method. CLINICAL RELEVANCE: This method of determining regional ChBF will be useful for studying the vascular effects of pharmacologic agents and for characterizing animal models of human disease involving the outer retina.

Multifocal visual evoked potentials in the anesthetized non-human primate.

PURPOSE: To evaluate monkey multifocal visual evoked cortical potentials (mfVEPs) recorded from central and peripheral fields for reliability and isolation from electroretinographic (ERG) activity. METHODS: The mfVEP stimulus consisted of a 7-element hexagonal array that subtended 80 degrees of the central visual field. Recordings were made under intravenous pentobarbital sodium (15 mg/kg) anesthesia. Two monkeys with absent optic nerve and ganglion cell function after combined unilateral optic nerve transection and experimental ocular hypertension (ONT/OHT) were followed longitudinally. In a second study, 16 ophthalmologically normal monkeys were tested once. RESULTS: Testing of the non-transected eye in two transected animals revealed robust first- and second-order kernel, first slice (K1 and K2.1) mfVEPs. Stimulation of the transected eye revealed no contamination of the mfVEP from the concurrently recorded multifocal ERGs. There was complete separation of the root-mean-square (RMS) mfVEP amplitudes from the transected and the fellow eyes tested repeatedly across a 4- to 17- month period. The largest amplitude mfVEP was generated by the central element; however, mfVEPs were recorded from outside the central 20 degrees element. The 16 normal animals showed waveforms similar to the normal eyes of the ONT/OHT animals both in shape and distribution throughout the visual field. A scalar-product measure showed both K1 and K2.1 mfVEPs from central and some peripheral elements were statistically distinct from noise. CONCLUSIONS: mfVEPs can be reliably recorded from non-human primates anesthetized with pentobarbital. Under the recording conditions described, mfVEPs are not contaminated by ERG activity. mfVEPs may be useful in animal models of diseases that differentially affect macular and peripheral visual field responsiveness.