Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

Long-term dopamine neurochemical monitoring in primates.

Many debilitating neuropsychiatric and neurodegenerative disorders are characterized by dopamine neurotransmitter dysregulation. Monitoring subsecond dopamine release accurately and for extended, clinically relevant timescales is a critical unmet need. Especially valuable has been the development of electrochemical fast-scan cyclic voltammetry implementing microsized carbon fiber probe implants to record fast millisecond changes in dopamine concentrations. Nevertheless, these well-established methods have only been applied in primates with acutely (few hours) implanted sensors. Neurochemical monitoring for long timescales is necessary to improve diagnostic and therapeutic procedures for a wide range of neurological disorders. Strategies for the chronic use of such sensors have recently been established successfully in rodents, but new infrastructures are needed to enable these strategies in primates. Here we report an integrated neurochemical recording platform for monitoring dopamine release from sensors chronically implanted in deep brain structures of nonhuman primates for over 100 days, together with results for behavior-related and stimulation-induced dopamine release. From these chronically implanted probes, we measured dopamine release from multiple sites in the striatum as induced by behavioral performance and reward-related stimuli, by direct stimulation, and by drug administration. We further developed algorithms to automate detection of dopamine. These algorithms could be used to track the effects of drugs on endogenous dopamine neurotransmission, as well as to evaluate the long-term performance of the chronically implanted sensors. Our chronic measurements demonstrate the feasibility of measuring subsecond dopamine release from deep brain circuits of awake, behaving primates in a longitudinally reproducible manner.

Prolonged dopamine signalling in striatum signals proximity and value of distant rewards.

Predictions about future rewarding events have a powerful influence on behaviour. The phasic spike activity of dopamine-containing neurons, and corresponding dopamine transients in the striatum, are thought to underlie these predictions, encoding positive and negative reward prediction errors. However, many behaviours are directed towards distant goals, for which transient signals may fail to provide sustained drive. Here we report an extended mode of reward-predictive dopamine signalling in the striatum that emerged as rats moved towards distant goals. These dopamine signals, which were detected with fast-scan cyclic voltammetry (FSCV), gradually increased or--in rare instances--decreased as the animals navigated mazes to reach remote rewards, rather than having phasic or steady tonic profiles. These dopamine increases (ramps) scaled flexibly with both the distance and size of the rewards. During learning, these dopamine signals showed spatial preferences for goals in different locations and readily changed in magnitude to reflect changing values of the distant rewards. Such prolonged dopamine signalling could provide sustained motivational drive, a control mechanism that may be important for normal behaviour and that can be impaired in a range of neurologic and neuropsychiatric disorders.

A system for recording neural activity chronically and simultaneously from multiple cortical and subcortical regions in nonhuman primates.

A major goal of neuroscience is to understand the functions of networks of neurons in cognition and behavior. Recent work has focused on implanting arrays of ∼100 immovable electrodes or smaller numbers of individually adjustable electrodes, designed to target a few cortical areas. We have developed a recording system that allows the independent movement of hundreds of electrodes chronically implanted in several cortical and subcortical structures. We have tested this system in macaque monkeys, recording simultaneously from up to 127 electrodes in 14 brain regions for up to one year at a time. A key advantage of the system is that it can be used to sample different combinations of sites over prolonged periods, generating multiple snapshots of network activity from a single implant. Used in conjunction with microstimulation and injection methods, this versatile system represents a powerful tool for studying neural network activity in the primate brain.

Ipsilateral cortical fMRI responses after peripheral nerve damage in rats reflect increased interneuron activity.

In the weeks following unilateral peripheral nerve injury, the deprived primary somatosensory cortex (SI) responds to stimulation of the ipsilateral intact limb as demonstrated by functional magnetic resonance imaging (fMRI) responses. The neuronal basis of these responses was studied by using high-resolution fMRI, in vivo electrophysiological recordings, and juxtacellular neuronal labeling in rats that underwent an excision of the forepaw radial, median, and ulnar nerves. These nerves were exposed but not severed in control rats. Significant bilateral increases of fMRI responses in SI were observed in denervated rats. In the healthy SI of the denervated rats, increases in fMRI responses were concordant with increases in local field potential (LFP) amplitude and an increased incidence of single units responding compared with control rats. In contrast, in the deprived SI, increases in fMRI responses were associated with a minimal change in LFP amplitude but with increased incidence of single units responding. Based on action potential duration, juxtacellular labeling, and immunostaining results, neurons responding to intact forepaw stimulation in the deprived cortex were identified as interneurons. These results suggest that the increases in fMRI responses in the deprived cortex reflect increased interneuron activity.

Eye-Drop Abstinence in Glaucoma Patients Admitted to the Hospital Service: A Single Institution Assessment.

PURPOSE: Anecdotally, many patients admitted to an inpatient general medicine service do not have their glaucoma eye drops started. The purpose of this study was to determine the extent of eye-drop abstinence after inpatient admission. DESIGN: Retrospective, cross-sectional, hospital-based study. PARTICIPANTS: Four hundred seventy-five patients admitted to the general medicine regional hospital service from January 2016 through February 2018 with a known past medical diagnosis of or active outpatient medications for glaucoma. METHODS: The combination of an administrative database and cross-referenced patient charts were reviewed for demographic data, past medical problems, inpatient orders, intake history and physical, length of stay, and admitting diagnosis. MAIN OUTCOME MEASURES: The effect of (1) outpatient glaucoma drops reconciliation and (2) recognition of glaucoma as a pertinent past medical problem in a patient's intake history and physical on inpatient eye-drop administration. The overall rate of eye-drop abstinence also was recorded. RESULTS: Of 475 patients, 46.3% were women, with an average age of 80.2 years (standard deviation, 11.1 years). Average length of stay was 4.61 days (standard deviation, 3.7 days). In total, 63.8% achieved successful administration of medication on the hospital floor, resulting in a 36.2% eye-drop abstinence rate during the hospital stay. Three hundred eighty-six of 475 patients (81.3%) achieved successful glaucoma medication reconciliation. Patients with successful reconciliation had a significantly different rate of eye-drop administration (73.3% vs. 21.0%; P ≤ 0.001). Recognition of glaucoma in the history and physical occurred in only 42.5% of patients. There was a significant difference in eye-drop administration when glaucoma was included in the history and physical (75.7% vs. 55.0%; P ≤ 0.001). CONCLUSIONS: Glaucoma treatment incurs a high rate of medication noncompliance, especially in the elderly. The present study demonstrates that more than one third of patients admitted to an academic medical center do not receive their glaucoma medications. Patients discharged to nursing homes, subacute rehabilitation, and assisted living facilities rely on appropriate discharge medication reconciliation, resulting in forced abstinence during transition of care. An emphasis on appropriate medical reconciliation and recognition of glaucoma as a pertinent past medical problem will improve rates of eye-drop discontinuation on inpatient admission significantly.

α5 nAChR modulation of the prefrontal cortex makes attention resilient.

A loss-of-function polymorphism in the α5 nicotinic acetylcholine receptor (nAChR) subunit gene has been linked to both drug abuse and schizophrenia. The α5 nAChR subunit is strategically positioned in the prefrontal cortex (PFC), where a loss-of-function in this subunit may contribute to cognitive disruptions in both disorders. However, the specific contribution of α5 to PFC-dependent cognitive functions has yet to be illustrated. In the present studies, we used RNA interference to knockdown the α5 nAChR subunit in the PFC of adult rats. We provide evidence that through its contribution to cholinergic modulation of cholinergic modulation of neurons in the PFC, the α5 nAChR plays a specific role in the recovery of attention task performance following distraction. Our combined data reveal the potent ability of this subunit to modulate the PFC and cognitive functions controlled by this brain region that are impaired in disease.

Quantifying Direct DNA Damage in the Basal Layer of Skin Exposed to UV Radiation from Sunbeds.

Nonmelanoma and melanoma skin cancers are attributable to DNA damage caused by ultraviolet (UV) radiation exposure. One DNA photoproduct, the cyclobutane pyrimidine dimer (CPD), is believed to lead to DNA mutations caused by UV radiation. Using radiative transfer simulations, we compare the number of CPDs directly induced by UV irradiation from artificial and natural UV sources (a standard sunbed and the midday summer Mediterranean sun) for skin types I and II on the Fitzpatrick scale. We use Monte Carlo radiative transfer (MCRT) modeling to track the progression of UV photons through a multilayered three dimensional (3D) grid that simulates the upper layers of the skin. By recording the energy deposited in the DNA-containing cells of the basal layer, the number of CPDs formed can be quantified. The aim of this work was to compare the number of CPDs formed in the basal layer of the skin and by implication the risk of developing cancer, as a consequence of irradiation by artificial and natural sources. Our simulations show that the number of CPDs formed per second during sunbed irradiation is almost three times that formed during solar irradiation.

Impaired ß-arrestin recruitment and reduced desensitization by non-catechol agonists of the D1 dopamine receptor.

Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of ß-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of ß-arrestin to D1Rs.

A heated vapor cell unit for dichroic atomic vapor laser lock in atomic rubidium.

The design and performance of a compact heated vapor cell unit for realizing a dichroic atomic vapor laser lock (DAVLL) for the D(2) transitions in atomic rubidium is described. A 5 cm long vapor cell is placed in a double-solenoid arrangement to produce the required magnetic field; the heat from the solenoid is used to increase the vapor pressure and correspondingly the DAVLL signal. We have characterized experimentally the dependence of important features of the DAVLL signal on magnetic field and cell temperature. For the weaker transitions both the amplitude and gradient of the signal are increased by an order of magnitude.

MAM (E17) rodent developmental model of neuropsychiatric disease: disruptions in learning and dysregulation of nucleus accumbens dopamine release, but spared executive function.

RATIONALE: Gestational day 17 methylazoxymethanol (MAM) treatment has been shown to reproduce, in rodents, some of the alterations in cortical and mesolimbic circuitries thought to contribute to schizophrenia. OBJECTIVE: We characterized the behavior of MAM animals in tasks dependent on these circuitries to see what behavioral aspects of schizophrenia the model captures. We then characterized the integrity of mesolimbic dopamine neurotransmission in a subset of animals used in the behavioral experiments. METHODS: MAM animals' capacity for working memory, attention, and resilience to distraction was tested with two different paradigms. Cue-reward learning and motivation were assayed with Pavlovian conditioned approach. Measurements of electrically stimulated phasic and tonic DA release in the nucleus accumbens with fast-scan cyclic voltammetry were obtained from the same animals used in the Pavlovian task. RESULTS: MAM animals' basic attentional capacities were intact. MAM animals took longer to acquire the working memory task, but once learned, performed at the same level as shams. MAM animals were also slower to develop a Pavlovian conditioned response, but otherwise no different from controls. These same animals showed alterations in terminal DA release that were unmasked by an amphetamine challenge. CONCLUSIONS: The predominant behavioral-cognitive feature of the MAM model is a learning impairment that is evident in acquisition of executive function tasks as well as basic Pavlovian associations. MAM animals also have dysregulated terminal DA release, and this may contribute to observed behavioral differences. The MAM model captures some functional impairments of schizophrenia, particularly those related to acquisition of goal-directed behavior.

A "Ciclovia" in San Francisco: Characteristics and physical activity behavior of Sunday Streets participants.

BACKGROUND: Temporary parks such as the monthly event, Sunday Streets SF, support public health goals by using existing infrastructure and street closures to provide physical activity in neighborhoods underserved for recreational resources. Sunday Streets creates routes to enhance community connection. METHODS: Six hundred and thirty-nine participants at 3 Sunday Streets events were surveyed using a 36-item instrument of open- and closed-ended questions about overall physical activity behavior, physical activity while at Sunday Streets, experience of the events, and demographic data. RESULTS: Overall, Sunday Streets participants are physically active (79% engage in activity 3-7 days/week) and approximately represent the ethnic minority distribution of the city. There were significant differences between first-time attendees and multiple-event attendees by duration of physical activity at the event (55.83 minutes vs. 75.13 minutes) and by frequency of physical activity bouts per week (3.69 vs. 4.22). Both groups emphasized the positive experience and safe environment as reasons to return to the event; for first-time attendees, the social environment was another reason to return. CONCLUSIONS: Temporary parks like Sunday Streets have the potential to provide healthful, population-wide physical activity using existing streets. The trend toward increased activity by multiple-event attendees suggests the importance of a regular schedule of events.

Coherent theta oscillations and reorganization of spike timing in the hippocampal- prefrontal network upon learning.

To study the interplay between hippocampus and medial prefrontal cortex (Pfc) and its importance for learning and memory consolidation, we measured the coherence in theta oscillations between these two structures in rats learning new rules on a Y maze. Coherence peaked at the choice point, most strongly after task rule acquisition. Simultaneously, Pfc pyramidal neurons reorganized their phase, concentrating at hippocampal theta trough, and synchronous cell assemblies emerged. This synchronous state may result from increased inhibition exerted by interneurons on pyramidal cells, as measured by cross-correlation, and could be modulated by dopamine: we found similar hippocampal-Pfc theta coherence increases and neuronal phase shifts following local administration of dopamine in Pfc of anesthetized rats. Pfc cell assemblies emerging during high coherence were preferentially replayed during subsequent sleep, concurrent with hippocampal sharp waves. Thus, hippocampal/prefrontal coherence could lead to synchronization of reward predicting activity in prefrontal networks, tagging it for subsequent memory consolidation.

Influence of the hippocampus on interneurons of the rat prefrontal cortex.

The hippocampus and prefrontal cortex (PFC), two structures implicated in learning and memory processes, are linked by a direct hippocampo-prefrontal pathway. It has been shown that PFC pyramidal cells receive monosynaptic excitatory inputs from the hippocampus and, in this study, we sought to determine the influence of the hippocampus on PFC interneurons in anesthetized rats. Extracellular recordings were coupled to juxtacellular injections of neurobiotin or biotinylated dextran amine to morphologically differentiate interneurons from pyramidal cells. In all cases, the action potentials of labeled interneurons were of shorter duration (< 0.70 ms) than those of identified pyramidal cells (> 0.70 ms). Single pulse stimulation of the hippocampal CA1/subiculum region induced an excitatory response in 70% of recorded interneurons in the prelimbic and medial-orbital areas of the PFC. In contrast to the one to two action potentials generated by pyramidal cells, an important group of interneurons fired a burst of action potentials in response to hippocampal stimulation. A large proportion of these excitatory responses was probably monosynaptic as their latency is consistent with the conduction time of the hippocampo-prefrontal pathway. In addition, when both a pyramidal cell and an interneuron were simultaneously recorded and both responded to stimulation, the interneuron consistently fired before the pyramidal cell. In conclusion, the hippocampus exerts a direct excitatory influence on PFC interneurons and is thus capable of feedforward inhibition of pyramidal cells. Hippocampal output is spatially and temporally focalized via this inhibitory process and consequently could facilitate the synchronization of a specific subset of PFC neurons with hippocampal activity.

Multisecond periodicities in basal ganglia firing rates correlate with theta bursts in transcortical and hippocampal EEG.

Multisecond oscillations in firing rate with periods in the range of 2-60 s (mean, 20-35 s) are present in 50-90% of spike trains from basal ganglia neurons recorded from locally anesthetized, immobilized rats. To determine whether these periodic oscillations are associated with similar periodicities in cortical activity, transcortical electroencephalographic (EEG) activity was recorded in conjunction with single- or dual-unit neuronal activity in the subthalamic nucleus (STN) or the globus pallidus (GP), and the data were analyzed with spectral and wavelet analyses. Multisecond oscillations in firing rates of 31% of the STN neurons and 46% of the GP neurons with periodicities significantly correlated with bursts of theta (4-7 Hz) activity in transcortical EEG. Further recordings of localized field potentials in the hippocampus and frontal or parietal cortices simultaneously with GP unit activity showed field potentials from the hippocampus, but not from the frontal or parietal cortices, exhibited bursts of theta rhythm that were correlated with GP firing rate oscillations. These results demonstrate a functional connectivity between basal ganglia neuronal activity and theta band activity in the hippocampus.