Papers by Martha Gillette
APL Bioengineering, 2018
Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent... more Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of “multi-cellular engineered living systems,” which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.
ACS Chemical Neuroscience, Aug 1, 2018
A systems approach to regulation of neuronal excitation in the mollusc Pleurobranchaea has descri... more A systems approach to regulation of neuronal excitation in the mollusc Pleurobranchaea has described novel interactions of cyclic AMP-gated cation current (I Na,cAMP), Ca 2+ , pH i , and NO. I Na,cAMP appears in many neurons of feeding and locomotor neuronal networks. It is likely one of the family of hyperpolarization-activated, cyclic-nucleotidegated currents (h-current) of vertebrate and invertebrate pacemaker networks. There are two isoforms. Ca 2+ regulates both voltage dependence and depolarization-sensitive inactivation in both isoforms. The Type 1 I Na,cAMP of the feeding network is enhanced by intracellular acidification. A direct dependence of I Na,cAMP on cAMP allows the current to be used as a reporter on cAMP concentrations in the cell, and from there to the intrinsic activities of the synthetic adenyl cyclase and the degradative phosphodiesterase. Type 2 I Na,cAMP of the locomotor system is activated by serotonergic inputs, while Type 1 of the feeding network is thought to be regulated peptidergically. NO synthase activity is high in the CNS, where it differs from standard neuronal NO synthase in not being Ca 2+ sensitive. NO acidifies pH i , potentiating Type 1, and may act to open proton channels. A cGMP pathway does not mediate NO effects as in other systems. Rather, nitrosylation likely mediates its actions. An integrated model of the action of cAMP, Ca 2+ , pH i , and NO in the feeding network postulates that NO regulates proton conductance to cause neuronal excitation in the cell body on the one hand, and relief of activity-induced hyperacidification in fine dendritic processes on the other.
Journal of Proteome Research, Jan 11, 2013
In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light i... more In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, ten endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (FDR adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.
Understanding function in healthy and diseased brains requires knowledge of the biochemistry occu... more Understanding function in healthy and diseased brains requires knowledge of the biochemistry occurring within the neurons and supporting cells making up the brain. What is the cell-to-cell variation of the neurotransmitters and neuromodulators in the nervous system and how does this change in an activity-dependent manner? For many compounds, the answer is unknown. Here we use a range of unique sampling protocols, capillary-scale separations, and microfluidic devices with both freshly isolated and cultured primary neurons to probe neurotransmitter and neuropeptide content and release from neurons in well-defined networks and from well-controlled microenvironments.
Analytical chemistry, Jan 6, 2018
The brain functions through chemical interactions between many different cell types, including ne... more The brain functions through chemical interactions between many different cell types, including neurons and glia. Acquiring comprehensive information on complex, heterogeneous systems requires multiple analytical tools, each of which have unique chemical specificity and spatial resolution. Multimodal imaging generates complementary chemical information via spatially localized molecular maps, ideally from the same sample, but requires method enhancements that span from data acquisition to interpretation. We devised a protocol for performing matrix-assisted laser desorp-tion/ionization (MALDI)-Fourier transform ion cyclotron resonance-mass spectrometry imaging (MSI), followed by infrared (IR) spectroscopic imaging on the same specimen. Multimodal measurements from the same tissue provide precise spatial alignment between modalities, enabling more advanced image processing such as image fusion and sharpening. Performing MSI first produces higher quality data from each technique compared...
Molecular Neuroendocrinology, 2016
MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nature Methods, 4, 8... more MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nature Methods, 4, 828-833. [The state of MALDI mass spectrometry as an information-rich imaging technique with broad current and future application possibilities.]
Micromachines
Cues in the micro-environment are key determinants in the emergence of complex cellular morpholog... more Cues in the micro-environment are key determinants in the emergence of complex cellular morphologies and functions. Primary among these is the presence of neighboring cells that form networks. For high-resolution analysis, it is crucial to develop micro-environments that permit exquisite control of network formation. This is especially true in cell science, tissue engineering, and clinical biology. We introduce a new approach for assembling polydimethylsiloxane (PDMS)-based microfluidic environments that enhances cell network formation and analyses. We report that the combined processes of PDMS solvent-extraction and hydrothermal annealing create unique conditions that produce high-strength bonds between solvent-extracted PDMS (E-PDMS) and glass—properties not associated with conventional PDMS. Extraction followed by hydrothermal annealing removes unbound oligomers, promotes polymer cross-linking, facilitates covalent bond formation with glass, and retains the highest biocompatibili...
ABSTRACTComplex brain functions, including learning and memory, arise in part from the modulatory... more ABSTRACTComplex brain functions, including learning and memory, arise in part from the modulatory role of astrocytes on neuronal circuits. Functionally, the dentate gyrus (DG) exhibits differences in the acquisition of long-term potentiation (LTP) between day and night. We hypothesize that the dynamic nature of astrocyte morphology plays an important role in the functional circuitry of hippocampal learning and memory, specifically in the DG. Standard microscopy techniques, such as differential interference contrast (DIC), present insufficient contrast for detecting changes in astrocyte structure and function and are unable to inform on the intrinsic structure of the sample in a quantitative manner. Recently, gradient light interference microscopy (GLIM) has been developed to upgrade a DIC microscope with quantitative capabilities such as single-cell dry mass and volume characterization. Here, we present a methodology for combining GLIM and electrophysiology to quantify the astrocyte...
ABSTRACTComplex brain functions, including learning and memory, arise in part from the modulatory... more ABSTRACTComplex brain functions, including learning and memory, arise in part from the modulatory role of astrocytes on neuronal circuits. Functionally, the dentate gyrus (DG) exhibits differences in the acquisition of long-term potentiation (LTP) between day and night. We hypothesize that the dynamic nature of astrocyte morphology plays an important role in the functional circuitry of hippocampal learning and memory, specifically in the DG. Standard microscopy techniques, such as differential interference contrast (DIC), present insufficient contrast for detecting changes in astrocyte structure and function and are unable to inform on the intrinsic structure of the sample in a quantitative manner. Recently, gradient light interference microscopy (GLIM) has been developed to upgrade a DIC microscope with quantitative capabilities such as single-cell dry mass and volume characterization. Here, we present a methodology for combining GLIM and electrophysiology to quantify the astrocyte...
ABSTRACTComplex brain functions, including learning and memory, arise in part from the modulatory... more ABSTRACTComplex brain functions, including learning and memory, arise in part from the modulatory role of astrocytes on neuronal circuits. Functionally, the dentate gyrus (DG) exhibits differences in the acquisition of long-term potentiation (LTP) between day and night. We hypothesize that the dynamic nature of astrocyte morphology plays an important role in the functional circuitry of hippocampal learning and memory, specifically in the DG. Standard microscopy techniques, such as differential interference contrast (DIC), present insufficient contrast for detecting changes in astrocyte structure and function and are unable to inform on the intrinsic structure of the sample in a quantitative manner. Recently, gradient light interference microscopy (GLIM) has been developed to upgrade a DIC microscope with quantitative capabilities such as single-cell dry mass and volume characterization. Here, we present a methodology for combining GLIM and electrophysiology to quantify the astrocyte...
The driving principle behind man-made robots is force actuation leading to a form of directed mov... more The driving principle behind man-made robots is force actuation leading to a form of directed movement or locomotion. Natural systems can motivate the design and development of robots that replicate or enhance many basic locomotive strategies—such as climbing, crawling,1 walking,2 jumping,3,4 or swimming5–9— with novel solutions. Biological soft robotics derives inspiration and design principles from organic systems to facilitate engineering approaches to challenges that have historically plagued conventional robotic actuators. Traditional hard skeletons (made of high stiffness metals or plastics) and electromagnetic actuators can
Sleep Medicine Clinics, 2012
The daily transition from light to darkness has significantly shaped the evolution of most living... more The daily transition from light to darkness has significantly shaped the evolution of most living species, from unicellular organisms to mammals. Adaptation to this environmental constraint occurred through the emergence of a circadian system capable of adjusting both behavioral and physiological processes to this light-dark cycle. Superimposed upon the daily light-dark cycle is a seasonal influence that modifies the relative durations of day and night over the course of a year. Be they day-active or night-active, all organisms need a means of keeping time in a 24-hour world as well to adapt to the availability of food, and to avoid predators. In addition, they require a means of adjusting to changes in day length or transition times that may occur. Interestingly, rather than simply reflecting the external day-night cycle, these rhythms in behaviors persist in the absence of exogenous timing cues such as light, food availability, or social cues. Every organism expresses an endogenous rhythm that varies slightly from 24 h, making it circadian, or 'about a day.' Uninterrupted, this circadian rhythm persists. These circadian rhythms can be observed in outputs such as the patterning of the sleep-wake cycle, and in humans, core body temperature is often used as a marker of circadian phase. In addition, numerous endogenous hormones can be used as markers (reviewed by Van Cauter 1). Although hormonal rhythms exhibit complex waveforms due to combined effects of the circadian pacemaker, organismic state, such as activity level, sleep and feeding, and the pulsatile nature of secretion, clear diurnal patterns of secretion have been reported 2. Plasma melatonin 3, 4 , growth hormone 5 , prolactin 6 , thyrotropin-releasing hormone 7 , luteinizing
Background: Neuropeptides are critical integrative elements within the central circadian clock in... more Background: Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood. Methodology/Principal Findings: Little SAAS localization and co-expression with established SCN neuropeptides were evaluated by immunohistochemistry using highly specific antisera and stereological analysis. Functional context was assessed relative to c-FOS induction in light-stimulated animals and on neuronal circadian rhythms in glutamate-stimulated brain slices. We found that little SAAS-expressing neurons comprise the third most abundant neuropeptidergic class (16.4%) with unusual functional circuit contexts. Little SAAS is localized within the densely retinorecipient central SCN of both rat and...
ACS Applied Materials & Interfaces
Directing neurons to form predetermined circuits with the intention of treating neurological diso... more Directing neurons to form predetermined circuits with the intention of treating neurological disorders and neurodegenerative diseases is a fundamental goal and current challenge in neuroengineering. Until recently, only neuronal aggregates were studied and characterized in culture, which can limit information gathered to populations of cells. In this study, we use a substrate constructed of arrays of straininduced self-rolled-up membrane 3D architectures. This results in changes in the neuronal architecture and altered growth dynamics of neurites. Hippocampal neurons from postnatal rats were cultured at low confluency (∼250 cells mm −2) on an array of transparent rolled-up microtubes (μ-tubes; 4−5 μm diameter) of varying topographical arrangements. Neurite growth on the μ-tubes was characterized and compared to controls in order to establish a baseline for alignment imposed by the topography. Compared to control substrates, neurites are significantly more aligned toward the 0°reference on the μ-tube array. Pitch (20−60 and 100 μm) and μ-tube length (30−80 μm) of array elements were also varied to investigate their impact on neurite alignment. We found that alignment was improved by the gradient pitch arrangement and with longer μ-tubes. Application of this technology will enhance the ability to construct intentional neural circuits through array design and manipulation of individual neurons and can be adapted to address challenges in neural repair, reinnervation, and neuroregeneration.
Neuromethods, 2015
Key determinants in the development of complex morphology and function are the cues present in a ... more Key determinants in the development of complex morphology and function are the cues present in a cell's environment and its response to them. Primary among these extracellular factors is the presence and infl uence of neighboring cells. It is crucial, therefore, in studying development to be able to replicate in vitro these network-like conditions. This is especially true of neuroscience, tissue engineering, and clinical biology, where network formation and function are critical aspects of any investigation. Here we describe an easy and inexpensive technique based on microfl uidics that provides a high degree of control in positioning and guiding cells, thereby enabling the laying down of desired cellular networks. This approach facilitates the study of synaptic connections where information is communicated between neurons. Such microscale devices are increasingly being employed for studying neurons in highly controlled environments wherein different regions of a network, or even a cell, are cultured in fl uidically isolated compartments. Enhanced strategies such as highly regulated manipulations of fl uid fl ow and physical guidance cues when combined with this compartmentalization provide an unparalleled degree of spatiotemporal control over the conformation of the neural network and the stimulation of synapses. This facilitates high-resolution investigations despite the cellular complexity. Consequently, the microfl uidic culture platform presents an unparalleled context for unraveling the changes occurring at the microscale and nanoscale of synaptic connections, thereby aiding elucidation of the nuances of neuronal development, wiring, and function.
ACS chemical neuroscience, Jan 20, 2018
Daily oscillations of brain and body states are under complex temporal modulation by environmenta... more Daily oscillations of brain and body states are under complex temporal modulation by environmental light and the hypothalamic suprachiasmatic nucleus (SCN), the master circadian clock. To better understand mediators of differential temporal modulation, we characterize neuropeptide releasate profiles by nonselective capture of secreted neuropeptides in an optic nerve horizontal SCN brain slice model. Releasates are collected following electrophysiological stimulation of the optic nerve/retinohypothalamic tract under conditions that alter the phase of the SCN activity state. Secreted neuropeptides are identified by intact mass via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found time-of-day-specific suites of peptides released downstream of optic nerve stimulation. Peptide release was modified differentially with respect to time-of-day by stimulus parameters and by inhibitors of glutamatergic or PACAPergic neurotransmission. The res...
The brain is the most intricate, energetically active, and plastic organ in the body. These featu... more The brain is the most intricate, energetically active, and plastic organ in the body. These features extend to its cellular elements, the neurons and glia. Understanding neurons, or nerve cells, at the cellular and molecular levels is the cornerstone of modern neuroscience. The complexities of neuron structure and function require unusual methods of culture to determine how aberrations in or between cells give rise to brain dysfunction and disease. Here we review the methods that have emerged over the past century for culturing neurons in vitro, from the landmark finding by Harrison (1910) - that neurons can be cultured outside the body - to studies utilizing culture vessels, micro-islands, Campenot and brain slice chambers, and microfluidic technologies. We conclude with future prospects for neuronal culture and considerations for advancement. We anticipate that continued innovation in culture methods will enhance design capabilities for temporal control of media and reagents (chem...
Soc. Neurosci. Abstr, 1994
Pituitary adenylate cyclase-activating peptide (PACAP) is involved in autonomous regulation, incl... more Pituitary adenylate cyclase-activating peptide (PACAP) is involved in autonomous regulation, including timekeeping, by its action on the suprachiasmatic nucleus and on neuroendocrine secretion, energy metabolism, and transmitter release. In particular, the interactions between PACAP and the glutamatergic system are well recognized. We compared the effect of intravenously administered PACAP to that of placebo in eight healthy male subjects. PACAP in a concentration of 4 ϫ 12.5 g was administered in a pulsatile fashion hourly between 2200 and 0100. Sleep EEG was recorded from 2300 to 1000, which was also the time when subjects were allowed to sleep. Blood samples were taken every 20 min between 2200 and 0700 for the determination of cortisol, GH, and prolactin. PACAP administration led to no changes in the macrosleep structure as assessed according to standard criteria. Spectral analysis revealed a significant reduction in the-frequency range in the first 4-h interval and of the spindle frequency range in the second 4-h interval of the registration period. This was accompanied by an increase in the time constant of the physiological ␦-power decline in the course of the night, i.e., a less pronounced dynamic of the reduction of ␦-power with time. This was accompanied by a trend (P Ͻ 0.1) toward decreased prolactin secretion in the first 4-h period of the night. No other changes in endocrine secretion were observed. We concluded that PACAP leads to a reduction of the dynamics of homeostatic sleep regulation and prolactin secretion. Both effects are the opposite of those seen after sleep deprivation but similar to the changes after napping, i.e., a reduced sleep propensity. This implies that PACAP might be involved in homeostatic sleep regulation. cortisol; growth hormone; prolactin THE PRESENCE of pituitary adenylate cyclase-activating polypeptide (PACAP), named after its stimulation of adenylate cyclase activity, and hence, cAMP formation in the pituitary, has been localized in the mammalian brain, including the hypothalamus (3). PACAP can induce pulses of growth hormone (GH), ACTH, prolactin, and LH release, possibly via PACAP-I receptors that are present on anterior pituitary cells (2).
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Papers by Martha Gillette