Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Progress towards an accurate determination of the Boltzmann constant by Doppler spectroscopy

In this paper, we present significant progress performed on an experiment dedicated to the determination of the Boltzmann constant, k, by accurately measuring the Doppler absorption profile of a line in a gas of ammonia at thermal equilibrium. This optical method based on the first principles of statistical mechanics is an alternative to the acoustical method which has led to the unique determination of k published by the CODATA with a relative accuracy of 1.7 ppm. We report on the first measurement of the Boltzmann constant by laser spectroscopy with a statistical uncertainty below 10 ppm, more specifically 6.4 ppm. This progress results from improvements in the detection method and in the statistical treatment of the data. In addition, we have recorded the hyperfine structure of the probed saQ(6,3) rovibrational line of ammonia by saturation spectroscopy and thus determine very precisely the induced 4.36 (2) ppm broadening of the absorption linewidth. We also show that, in our well chosen experimental conditions, saturation effects have a negligible impact on the linewidth. Finally, we draw the route to future developments for an absolute determination of with an accuracy of a few ppm.Comment: 22 pages, 11 figure

A novel behavioral fish model of nociception for testing analgesics

Pain is a major symptom in many medical conditions, and often interferes significantly with a person's quality of life. Although a priority topic in medical research for many years, there are still few analgesic drugs approved for clinical use. One reason is the lack of appropriate animal models that faithfully represent relevant hallmarks associated with human pain. Here we propose zebrafish (Danio rerio) as a novel short-term behavioral model of nociception, and analyse its sensitivity and robustness. Firstly, we injected two different doses of acetic acid as the noxious stimulus. We studied individual locomotor responses of fish to a threshold level of nociception using two recording systems: a video tracking system and an electric biosensor (the MOBS system). We showed that an injection dose of 10% acetic acid resulted in a change in behavior that could be used to study nociception. Secondly, we validated our behavioral model by investigating the effect of the analgesic morphine. In time-course studies, first we looked at the dose-response relationship of morphine and then tested whether the effect of morphine could be modulated by naloxone, an opioid antagonist. Our results suggest that a change in behavioral responses of zebrafish to acetic acid is a reasonable model to test analgesics. The response scales with stimulus intensity, is attenuated by morphine, and the analgesic effect of morphine is blocked with naloxone. The change in behavior of zebrafish associated with the noxious stimulus can be monitored with an electric biosensor that measures changes in water impedance. © 2011 by the authors; licensee MDPI, Basel, Switzerland

Stimulation of electro-olfactogram responses in the main olfactory epithelia by airflow depends on the type 3 adenylyl cyclase

Cilia of olfactory sensory neurons are the primary sensory organelles for olfaction. The detection of odorants by the main olfactory epithelium (MOE) depends on coupling of odorant receptors to the type 3 adenylyl cyclase (AC3) in olfactory cilia. We monitored the effect of airflow on electro-olfactogram (EOG) responses and found that the MOE of mice can sense mechanical forces generated by airflow. The airflow-sensitive EOG response in the MOE was attenuated when cAMP was increased by odorants or by forskolin suggesting a common mechanism for airflow and odorant detection. In addition, the sensitivity to airflow was significantly impaired in the MOE from AC3−/− mice. We conclude that AC3 in the MOE is required for detecting the mechanical force of airflow, which in turn may regulate odorant perception during sniffing

A roadmap to integrate astrocytes into Systems Neuroscience.

Systems neuroscience is still mainly a neuronal field, despite the plethora of evidence supporting the fact that astrocytes modulate local neural circuits, networks, and complex behaviors. In this article, we sought to identify which types of studies are necessary to establish whether astrocytes, beyond their well-documented homeostatic and metabolic functions, perform computations implementing mathematical algorithms that sub-serve coding and higher-brain functions. First, we reviewed Systems-like studies that include astrocytes in order to identify computational operations that these cells may perform, using Ca2+ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in a time scale of subseconds to seconds in sensory processing, neuromodulation, brain state, memory formation, fear, and complex homeostatic reflexes. Next, we propose a list of actions to gain insight into the outstanding question of which variables are encoded by such computations. The application of statistical analyses based on machine learning, such as dimensionality reduction and decoding in the context of complex behaviors, combined with connectomics of astrocyte-neuronal circuits, is, in our view, fundamental undertakings. We also discuss technical and analytical approaches to study neuronal and astrocytic populations simultaneously, and the inclusion of astrocytes in advanced modeling of neural circuits, as well as in theories currently under exploration such as predictive coding and energy-efficient coding. Clarifying the relationship between astrocytic Ca2+ and brain coding may represent a leap forward toward novel approaches in the study of astrocytes in health and disease

Wakefulness affects synaptic and network activity by increasing extracellular astrocyte-derived adenosine

Loss of sleep causes an increase in sleep drive and deficits in hippocampal-dependent memory. Both of these responses are thought to require activation of adenosine A1 receptors (adorA1Rs) and release of transmitter molecules including ATP, which is rapidly converted to adenosine in the extracellular space, from astrocytes in a process termed gliotransmission. Although it is increasingly clear that astrocyte-derived adenosine plays an important role in driving the homeostatic sleep response and the effects of sleep loss on memory (Halassa et al., 2009; Florian et al., 2011), previous studies have not determined whether the concentration of this signaling molecule increases in response to wakefulness. Here, we show that the level of adorA1R activation increases in response to wakefulness in mice (Mus musculus). We found that this increase affected synaptic transmission in the hippocampus and modulated network activity in the cortex. Direct biosensor-based measurement of adenosine showed that the net extracellular concentration of this transmitter increased in response to normal wakefulness and sleep deprivation. Genetic inhibition of gliotransmission prevented this increase and attenuated the wakefulness-dependent changes in synaptic and network regulation by adorA1R. Consequently, we conclude that wakefulness increases the level of extracellular adenosine in the hippocampus and that this increase requires the release of transmitters from astroctyes

Measurement of the Boltzmann constant by the Doppler broadening technique at a 3,8x10-5 accuracy level

In this paper, we describe an experiment performed at the Laboratoire de Physique des Lasers and dedicated to an optical measurement of the Boltzmann constant. With the proposed innovative technique, determining comes down to an ordinary frequency measurement. The method consists in measuring as accurately as possible the Doppler absorption profile of a rovibrational line of ammonia in thermal equilibrium. This profile is related to the Maxwell-Boltzmann molecular velocity distribution along the laser beam. A fit of the absorption line shape leads to a determination of the Doppler width proportional to sqrt(kT) and thus to a determination of the Boltzmann constant. The laser source is an ultra-stable CO2 laser with a wavelength . The absorption cell is placed in a thermostat keeping the temperature at 273.15 K within 1.4 mK. We were able to measure with a relative uncertainty as small as 3.8x10-5, which represents an improvement of an order of magnitude for an integration time comparable to our previous measurement published in 2007 [1

The Chemokine CCL2 Mediates the Seizure-enhancing Effects of Systemic Inflammation

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures. Brain inflammation is increasingly recognized as a critical factor for seizure precipitation, but the molecular mediators of such proconvulsant effects are only partly understood. The chemokine CCL2 is one of the most elevated inflammatory mediators in patients with pharmacoresistent epilepsy, but its contribution to seizure generation remains unexplored. Here, we show, for the first time, a crucial role for CCL2 and its receptor CCR2 in seizure control. We imposed a systemic inflammatory challenge via lipopolysaccharide (LPS) administration in mice with mesial temporal lobe epilepsy. We found that LPS dramatically increased seizure frequency and upregulated the expression of many inflammatory proteins, including CCL2. To test the proconvulsant role of CCL2, we administered systemically either a CCL2 transcription inhibitor (bindarit) or a selective antagonist of the CCR2 receptor (RS102895). We found that interference with CCL2 signaling potently suppressed LPS-induced seizures. Intracerebral administration of anti-CCL2 antibodies also abrogated LPS-mediated seizure enhancement in chronically epileptic animals. Our results reveal that CCL2 is a key mediator in the molecular pathways that link peripheral inflammation with neuronal hyperexcitability

Single-molecule stochastic resonance

Stochastic resonance (SR) is a well known phenomenon in dynamical systems. It consists of the amplification and optimization of the response of a system assisted by stochastic noise. Here we carry out the first experimental study of SR in single DNA hairpins which exhibit cooperatively folding/unfolding transitions under the action of an applied oscillating mechanical force with optical tweezers. By varying the frequency of the force oscillation, we investigated the folding/unfolding kinetics of DNA hairpins in a periodically driven bistable free-energy potential. We measured several SR quantifiers under varied conditions of the experimental setup such as trap stiffness and length of the molecular handles used for single-molecule manipulation. We find that the signal-to-noise ratio (SNR) of the spectral density of measured fluctuations in molecular extension of the DNA hairpins is a good quantifier of the SR. The frequency dependence of the SNR exhibits a peak at a frequency value given by the resonance matching condition. Finally, we carried out experiments in short hairpins that show how SR might be useful to enhance the detection of conformational molecular transitions of low SNR.Comment: 11 pages, 7 figures, supplementary material (http://prx.aps.org/epaps/PRX/v2/i3/e031012/prx-supp.pdf