A perfect day for zebrafish : neuromodulation of sleep in a diurnal vertebrate

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish (Danio rerio), a diurnal vertebrate, has emerged as a promising model system for sleep and arousal research.

In this thesis, I describe two studies on sleep/arousal pathways that I conducted using zebrafish, and I discuss how the findings can be combined in future projects to advance our understanding of vertebrate sleep/arousal pathways. In the first study, I discovered a neuropeptide that regulates zebrafish sleep and arousal as a result of a large-scale effort to identify molecules that regulate behavioral states. Taking advantage of facile zebrafish genetics, I constructed mutants for the three known receptors of this peptide and identified the one receptor that exclusively mediates the observed behavioral effects. I further show that the peptide exerts its behavioral effects independently of signaling at a key module of a neuroendocrine signaling pathway. This finding contradicts the hypothesis put forth in mammalian systems that the peptide acts through the classical neuroendocrine pathway; our data further generate new testable hypotheses for determining the central nervous system or alternative neuroendocrine pathways involved.

Second, I will present the development of a chemigenetic method to non-invasively manipulate neurons in the behaving zebrafish. I validated this technique by expressing and inducing the chemigenetic tool in a restricted population of sleep-regulating neurons in the zebrafish. As predicted by established models of this vertebrate sleep regulator, chemigenetic activation of these neurons induced hyperactivity, whereas chemigenetic ablation of these neurons induced increased sleep behavior. Given that light is a potent modulator of behavior in zebrafish, our proof-of-principle data provide a springboard for future studies of sleep/arousal and other light-dependent behaviors to interrogate genetically-defined populations of neurons independently of optogenetic tools.

Contributions of parietal cortex to reach planning

Sensory-motor circuits course through the parietal cortex of the human and monkey brain. How parietal cortex manipulates these signals has been an important question in behavioral neuroscience. This thesis presents experiments that explore the contributions of monkey parietal cortex to sensory-motor processing, with an emphasis on the area's contributions to reaching. First, it is shown that parietal cortex is organized into subregions devoted to specific movements. Area LIP encodes plans to make saccadic eye movements. A nearby area, the parietal reach region (PRR), plans reaches. A series of experiments are then described which explore the contributions of PRR to reach planning. Reach plans are represented in an eye-centered reference frame in PRR. This representation is shown to be stable across eye movements. When a sequence of reaches is planned, only the impending movement is represented in PRR, showing that the area is more related to movement planning than to storing the memory of reach targets. PRR resembles area LIP in each of these properties: the two areas may provide a substrate for hand-eye coordination. These findings yield new perspectives on the functions of the parietal cortex and on the organization of sensory-motor processing in primate brains.

Studying conscious and unconscious vision with functional Magnetic Resonance Imaging : the BOLD promise

Waking up from a dreamless sleep, I open my eyes, recognize my wife’s face and am filled with joy. In this thesis, I used functional Magnetic Resonance Imaging (fMRI) to gain insights into the mechanisms involved in this seemingly simple daily occurrence, which poses at least three great challenges to neuroscience: how does conscious experience arise from the activity of the brain? How does the brain process visual input to the point of recognizing individual faces? How does the brain store semantic knowledge about people that we know? To start tackling the first question, I studied the neural correlates of unconscious processing of invisible faces. I was unable to image significant activations related to the processing of completely invisible faces, despite existing reports in the literature. I thus moved on to the next question and studied how recognition of a familiar person was achieved in the brain; I focused on finding invariant representations of person identity – representations that would be activated any time we think of a familiar person, read their name, see their picture, hear them talk, etc. There again, I could not find significant evidence for such representations with fMRI, even in regions where they had previously been found with single unit recordings in human patients (the Jennifer Aniston neurons). Faced with these null outcomes, the scope of my investigations eventually turned back towards the technique that I had been using, fMRI, and the recently praised analytical tools that I had been trusting, Multivariate Pattern Analysis. After a mostly disappointing attempt at replicating a strong single unit finding of a categorical response to animals in the right human amygdala with fMRI, I put fMRI decoding to an ultimate test with a unique dataset acquired in the macaque monkey. There I showed a dissociation between the ability of fMRI to pick up face viewpoint information and its inability to pick up face identity information, which I mostly traced back to the poor clustering of identity selective units. Though fMRI decoding is a powerful new analytical tool, it does not rid fMRI of its inherent limitations as a hemodynamics-based measure.

Aerosol-cloud-precipitation interactions in marine stratocumulus clouds

Marine stratocumulus clouds are generally optically thick and shallow, exerting a net cooling influence on climate. Changes in atmospheric aerosol levels alter cloud microphysics (e.g., droplet size) and cloud macrophysics (e.g., liquid water path, cloud thickness), thereby affecting cloud albedo and Earth’s radiative balance. To understand the aerosol-cloud-precipitation interactions and to explore the dynamical effects, three-dimensional large-eddy simulations (LES) with detailed bin-resolved microphysics are performed to explore the diurnal variation of marine stratocumulus clouds under different aerosol levels and environmental conditions. It is shown that the marine stratocumulus cloud albedo is sensitive to aerosol perturbation under clean background conditions, and to environmental conditions such as large-scale divergence rate and free tropospheric humidity.

Based on the in-situ Eastern Pacific Emitted Aerosol Cloud Experiment (E-PEACE) during Jul. and Aug. 2011, and A-Train satellite observation of 589 individual ship tracks during Jun. 2006-Dec. 2009, an analysis of cloud albedo responses in ship tracks is presented. It is found that the albedo response in ship tracks depends on the mesoscale cloud structure, the free tropospheric humidity, and cloud top height. Under closed cell structure (i.e., cloud cells ringed by a perimeter of clear air), with sufficiently dry air above cloud tops and/or higher cloud top heights, the cloud albedo can become lower in ship tracks. Based on the satellite data, nearly 25% of ship tracks exhibited a decreased albedo. The cloud macrophysical responses are crucial in determining both the strength and the sign of the cloud albedo response to aerosols.

To understand the aerosol indirect effects on global marine warm clouds, multisensory satellite observations, including CloudSat, MODIS, CALIPSO, AMSR-E, ECMWF, CERES, and NCEP, have been applied to study the sensitivity of cloud properties to aerosol levels and to large scale environmental conditions. With an estimate of anthropogenic aerosol fraction, the global aerosol indirect radiative forcing has been assessed.

As the coupling among aerosol, cloud, precipitation, and meteorological conditions in the marine boundary layer is complex, the integration of LES modeling, in-situ aircraft measurements, and global multisensory satellite data analyses improves our understanding of this complex system.

Kinetics and mechanisms of adsorption of Escherichia coli bacteriophage T_4 to activated carbon

A study was conducted on the adsorption of Escherichia coli bacteriophage T₄ to activated carbon. Preliminary adsorption experiments were also made with poliovirus Type III. The effectiveness of such adsorbents as diatomaceous earth, Ottawa sand, and coconut charcoal was also tested for virus adsorption.

The kinetics of adsorption were studied in an agitated solution containing virus and carbon. The mechanism of attachment and site characteristics were investigated by varying pH and ionic strength and using site-blocking reagents.

Plaque assay procedures were developed for bacteriophage T₄ on Escherichia coli cells and poliovirus Type III on monkey kidney cells. Factors influencing the efficiency of plaque formation were investigated.

The kinetics of bacteriophage T₄ adsorption to activated carbon can be described by a reversible second-order equation. The reaction order was first order with respect to both virus and carbon concentration. This kinetic representation, however, is probably incorrect at optimum adsorption conditions, which occurred at a pH of 7.0 and ionic strength of 0.08. At optimum conditions the adsorption rate was satisfactorily described by a diffusion-limited process. Interpretation of adsorption data by a development of the diffusion equation for Langmuir adsorption yielded a diffusion coefficient of 12 X 10^-8 cm²/sec for bacteriophage T₄. This diffusion coefficient is in excellent agreement with the accepted value of 8 X 10^-8 cm²/sec. A diffusion-limited theory may also represent adsorption at conditions other than the maximal. A clear conclusion on the limiting process cannot be made.

Adsorption of bacteriophage T₄ to activated carbon obeys the Langmuir isotherm and is thermodynamically reversible. Thus virus is not inactivated by adsorption. Adsorption is unimolecular with very inefficient use of the available carbon surface area. The virus is probably completely excluded from pores due to its size.

Adsorption is of a physical nature and independent of temperature. Attraction is due to electrostatic forces between the virus and carbon. Effects of pH and ionic strength indicated that carboxyl groups, amino groups, and the virus's tail fibers are involved in the attachment of virus to carbon. The active sites on activated carbon for adsorption of bacteriophage T₄ are carboxyl groups. Adsorption can be completely blocked by esterifying these groups.

Primary cosmic-ray positrons and negatrons in 1968 at energies between 11 and 204 MeV

The cosmic-ray positron and negatron spectra between 11 and 204 MeV have been measured in a series of 3 high-altitude balloon flights launched from Fort Churchill, Manitoba, on July 16, July 21, and July 29, 1968. The detector system consisted of a magnetic spectrometer utilizing a 1000-gauss permanent magnet, scintillation counters, and a lucite Čerenkov counter.

Launches were timed so that the ascent through the 100 g/cm² level of residual atmosphere occurred after the evening geomagnetic cutoff transition. Data gathered during ascent are used to correct for the contribution of atmospheric secondary electrons to the flux measured at float altitude. All flights floated near 2.4 g/cm².

A pronounced morning intensity increase was observed in each flight. We present daytime positron and negatron data which support the interpretation of the diurnal flux variation as a change in the local geomagnetic cutoff. A large diurnal variation was observed in the count rate of positrons and negatrons with magnetic rigidities less than 11 MV and is evidence that the nighttime cutoff was well below this value.

Using nighttime data we derive extraterrestrial positron and negatron spectra. The positron-to-total-electron ratio which we measure indicates that the interstellar secondary, or collision, source contributes ≾50 percent of the electron flux within this energy interval. By comparing our measured positron spectrum with the positron spectrum calculated for the collision source we derive the absolute solar modulation for positrons in 1968. Assuming negligible energy loss during modulation, we derive the total interstellar electron spectrum as well as the spectrum of directly accelerated, or primary, electrons. We examine the effect of adiabatic deceleration and find that many of the conclusions regarding the interstellar electron spectrum are not significantly altered for an assumed energy loss of up to 50 percent of the original energy.