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Oscillatory output from the suprachiasmatic nuclei (SCN) of the hypothalamus communicates time-of-day information to the brain and body. The SCN's intrinsic ~24-h rhythm can be measured in the neuronal firing rate both in vivo and in vitro, where it continues unperturbed. This robust reporter of endogenous physiology in the SCN brain slice can be widely used to study dynamic changes in SCN physiology, its changing sensitivity to phase-altering signals, and underlying mechanisms. To provide relevant and reproducible data, care must be taken to ensure health of the SCN brain slice. The methods detailed here have been proven to produce healthy, long-lived brain slices.

Multiple scattering and absorption limit the depth at which biological tissues can be imaged with light. In thick unlabeled specimens, multiple scattering randomizes the phase of the field and absorption attenuates light that travels long optical paths. These obstacles limit the performance of transmission imaging. To mitigate these challenges, we developed an epi-illumination gradient light interference microscope (epi-GLIM) as a label-free phase imaging modality applicable to bulk or opaque samples. Epi-GLIM enables studying turbid structures that are hundreds of microns thick and otherwise opaque to transmitted light. We demonstrate this approach with a variety of man-made and biological samples that are incompatible with imaging in a transmission geometry: semiconductors wafers, specimens on opaque and birefringent substrates, cells in microplates, and bulk tissues. We demonstrate that the epi-GLIM data can be used to solve the inverse scattering problem and reconstruct the tomography of single cells and model organisms., Quantitative phase imaging techniques have been limited by multiple scattering of light or its use in transmission mode. Here, the authors show a gradient light interference microscopy method in a reflection geometry which allows for label-free phase imaging of bulk and opaque samples.

This soft machine can pick up and release an ultrathin “living” cell sheet and electronics without crumpling., “Living” cell sheets or bioelectronic chips have great potentials to improve the quality of diagnostics and therapies. However, handling these thin and delicate materials remains a grand challenge because the external force applied for gripping and releasing can easily deform or damage the materials. This study presents a soft manipulator that can manipulate and transport cell/tissue sheets and ultrathin wearable biosensing devices seamlessly by recapitulating how a cephalopod’s suction cup works. The soft manipulator consists of an ultrafast thermo-responsive, microchanneled hydrogel layer with tissue-like softness and an electric heater layer. The electric current to the manipulator drives microchannels of the gel to shrink/expand and results in a pressure change through the microchannels. The manipulator can lift/detach an object within 10 s and can be used repeatedly over 50 times. This soft manipulator would be highly useful for safe and reliable assembly and implantation of therapeutic cell/tissue sheets and biosensing devices.

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 strain-induced 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 arra...

Oxidation-reduction reactions are essential to life as the core mechanisms of energy transfer. A large body of evidence in recent years presents an extensive and complex network of interactions between the circadian and cellular redox systems. Recent advances show that cellular redox state undergoes a ~24-h (circadian) oscillation in most tissues and is conserved across the domains of life. In nucleated cells, the metabolic oscillation is dependent upon the circadian transcription-translation machinery and, vice versa, redox-active proteins and cofactors feed back into the molecular oscillator. In the suprachiasmatic nucleus (SCN), a hypothalamic region of the brain specialized for circadian timekeeping, redox oscillation was found to modulate neuronal membrane excitability. The SCN redox environment is relatively reduced in daytime when neuronal activity is highest and relatively oxidized in nighttime when activity is at its lowest. There is evidence that the redox environment directly modulates SCN K(+) channels, tightly coupling metabolic rhythms to neuronal activity. Application of reducing or oxidizing agents produces rapid changes in membrane excitability in a time-of-day-dependent manner. We propose that this reciprocal interaction may not be unique to the SCN. In this review, we consider the evidence for circadian redox oscillation and its interdependencies with established circadian timekeeping mechanisms. Furthermore, we will investigate the effects of redox on ion-channel gating dynamics and membrane excitability. The susceptibility of myriad different ion channels to modulation by changes in the redox environment suggests that circadian redox rhythms may play a role in the regulation of all excitable cells.

The tumor microenvironment is implicated in orchestrating cancer cell transformation and metastasis. However, specific cell–ligand interactions between cancer cells and the extracellular matrix are difficult to decipher due to a dynamic and multivariate presentation of many signaling molecules. Here we report a versatile peptide microarray platform that is capable of screening for cancer cell phenotypic changes in response to ligand–receptor interactions. Using a screen of 78 peptide combinations derived from proteins present in the melanoma microenvironment, we identify a proteoglycan binding and bone morphogenic protein 7 (BMP7) derived sequence that selectively promotes the expression of several putative melanoma initiating cell markers. We characterize signaling associated with each of these peptides in the activation of melanoma pro-tumorigenic signaling and reveal a role for proteoglycan mediated adhesion and signaling through Smad 2/3. A defined substratum that controls the state of malignant melanoma may prove useful in spatially normalizing a heterogeneous population of tumor cells for discovery of therapeutics that target a specific state and for identifying new drug targets and reagents for intervention., A peptide microarray reveals combinations of surface bound peptides that promote a stem cell-like state in melanoma cells. Proteoglycan mediated adhesion and bone morphogenetic protein signaling are proposed to orchestrate this transformation.

Localization of metabolites using multiplexed mass spectrometry imaging (MSI) provides important chemical information for biological research. In contrast to matrix-assisted laser desorption/ionization (MALDI), TiO2-assisted laser desorption/ionization (LDI) for MSI improves detection of low molecular mass metabolites (

Various neurodegenerative diseases and disorders as well as sleep deprivation seriously impact memory and cognition and can become life-threatening. Current medical techniques attempt to combat these detrimental effects mainly through the administration of neuromedicine. However, drug efficacy is limited by rapid dispersal of the drugs to off-target sites while the site of administration is prone to overdose. Many neuropathological conditions are accompanied by excessive reactive oxygen species (ROS) due to the inflammatory response. Accordingly, ROS-responsive drug delivery systems have emerged as a promising solution. To guide intelligent and comprehensive design of ROS-responsive drug delivery systems, this review article discusses the two following topics: (1) the biology of ROS in both healthy and diseased nervous systems and (2) recent developments in ROS-responsive, drug delivery system design. Overall, this review article would assist efforts to make better decisions about designing ROS-responsive, neural drug delivery systems, including the selection of ROS-responsive functional groups and disease-homing ligands.

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with high spatial resolution and detect optical pathlength changes with nanometer scale sensitivity. We found that optical pathlength fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience., Comment: 20 pages, 5 figures

Behaviors, such as sleeping, foraging, and learning, are controlled by different regions of the rat brain, yet they occur rhythmically over the course of day and night. They are aligned adaptively with the day-night cycle by an endogenous circadian clock in the suprachiasmatic nucleus (SCN), but local mechanisms of rhythmic control are not established. The SCN expresses a ~24-hr oscillation in reduction-oxidation that modulates its own neuronal excitability. Could circadian redox oscillations control neuronal excitability elsewhere in the brain? We focused on the CA1 region of the rat hippocampus, which is known for integrating information as memories and where clock gene expression undergoes a circadian oscillation that is in anti-phase to the SCN. Evaluating long-term imaging of endogenous redox couples and biochemical determination of glutathiolation levels, we observed oscillations with a ~24 hr period that is 180° out-of-phase to the SCN. Excitability of CA1 pyramidal neurons, primary hippocampal projection neurons, also exhibits a rhythm in resting membrane potential that is circadian time-dependent and opposite from that of the SCN. The reducing reagent glutathione rapidly and reversibly depolarized the resting membrane potential of CA1 neurons; the magnitude is time-of-day-dependent and, again, opposite from the SCN. These findings extend circadian redox regulation of neuronal excitability from the SCN to the hippocampus. Insights into this system contribute to understanding hippocampal circadian processes, such as learning and memory, seizure susceptibility, and memory loss with aging.

Intracellular transport is fundamental for neuronal function and development and is dependent on the formation of stable actin filaments. N-cadherin, a cell-cell adhesion protein, is actively involved in neuronal growth and actin cytoskeleton organization. Various groups have explored how neurons behaved on substrates engineered to present N-cadherin; however, few efforts have been made to examine how these surfaces modulate neuronal intracellular transport. To address this issue, we assembled a substrate to which recombinant N-cadherin molecules are physiosorbed using graphene oxide (GO) or reduced graphene oxide (rGO). N-cadherin physisorbed on GO and rGO led to a substantial enhancement of intracellular mass transport along neurites relative to N-cadherin on glass, due to increased neuronal adhesion, neurite extensions, dendritic arborization and glial cell adhesion. This study will be broadly useful for recreating active neural tissues in vitro and for improving our understanding of the development, homeostasis, and physiology of neurons. STATEMENT OF SIGNIFICANCE: Intracellular transport of proteins and chemical cues is extremely important for culturing neurons in vitro, as they replenish materials within and facilitate communication between neurons. Various studies have shown that intracellular transport is dependent on the formation of stable actin filaments. However, the extent to which cadherin-mediated cell-cell adhesion modulates intracellular transport is not heavily explored. In this study, N-cadherin was adsorbed onto graphene oxide-based substrates to understand the role of cadherin at a molecular level and the intracellular transport within cells was examined using spatial light interference microscopy. As such, the results of this study will serve to better understand and harness the role of cell-cell adhesion in neuron development and regeneration.

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.

Three-dimensional (3D) biomimetic systems hold great promise for the study of biological systems in vitro as well as for the development and testing of pharmaceuticals. Here, we test the hypothesis that an intact segment of lumbar rat spinal cord will form functional neuromuscular junctions (NMJs) with engineered, 3D muscle tissue, mimicking the partial development of the peripheral nervous system (PNS). Muscle tissues are grown on a 3D-printed polyethylene glycol (PEG) skeleton where deflection of the backbone due to muscle contraction causes the displacement of the pillar-like “feet.” We show that spinal cord explants extend a robust and complex arbor of motor neurons and glia in vitro. We then engineered a “spinobot” by innervating the muscle tissue with an intact segment of lumbar spinal cord that houses the hindlimb locomotor central pattern generator (CPG). Within 7 days of the spinal cord being introduced to the muscle tissue, functional neuromuscular junctions (NMJs) are formed, resulting in the development of an early PNS in vitro. The newly innervated muscles exhibit spontaneous contractions as measured by the displacement of pillars on the PEG skeleton. Upon chemical excitation, the spinal cord-muscle system initiated muscular twitches with a consistent frequency pattern. These sequences of contraction/relaxation suggest the action of a spinal CPG. Chemical inhibition with a blocker of neuronal glutamate receptors effectively blocked contractions. Overall, these data demonstrate that a rat spinal cord is capable of forming functional neuromuscular junctions ex vivo with an engineered muscle tissue at an ontogenetically similar timescale.

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 desorption/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 to performing IR imaging before MSI. The difference is likely due to fixing the tissue section during MALDI matrix removal, thereby preventing analyte degradation occurring during IR imaging from an unfixed specimen. Leveraging the unique capabilities of each modality, we utilized pan sharpening of MS (mass spectrometry) ion images with selected bands from IR spectroscopy and midlevel data fusion. In comparison to sharpening with histological images, pan sharpening can employ a plethora of IR bands, producing sharpened MS images while retaining the fidelity of the initial ion images. Using Laplacian pyramid sharpening, we determine the localization of several lipids present within the hippocampus with high mass accuracy at 5 μm pixel widths. Further, through midlevel data fusion of the imaging data sets combined with k-means clustering, the combined data set discriminates between additional anatomical structures unrecognized by the individual imaging approaches. Significant differences between molecular ion abundances are detected between relevant structures within the hippocampus, such as the CA1 and CA3 regions. Our methodology provides high quality multiplex and multimodal chemical imaging of the same tissue sample, enabling more advanced data processing and analysis routines.

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 results suggest that SCN physiology is modulated by differential peptide release of both known and unexpected peptides that communicate time-of-day-specific photic signals via previously unreported neuropeptide signatures.

In neural interface platforms, cultures are often carried out on a flat, open, rigid, and opaque substrate, posing challenges to reflecting the native microenvironment of the brain and precise engagement with neurons. Here we present a neuron cell culturing platform that consists of arrays of ordered microtubes (2.7–4.4 μm in diameter), formed by strain-induced self-rolled-up nanomembrane (s-RUM) technology using ultrathin (

Key determinants in the emergence of complex cellular morphologies and functions are cues in the micro-environment. Primary among these is the presence of neighboring cells as networks form. Therefore, 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 (E-PDMS) and hydrothermal annealing create unique conditions that produce high-strength bonds between 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 biocompatibility. Our extraction protocol accelerates oligomer removal from 5 to 2 days. Resulting microfluidic platforms are uniquely suited for cell-network studies owing to high bond strengths, effectively corralling cellular extensions and eliminating harmful oligomers. We demonstrate simple, simultaneous actuation of multiple microfluidic domains for invoking ATP- and glutamate-induced Ca2+ signaling in glial-cell networks. These low-cost, simple E-PMDS modifications and flow manipulations further enable microfluidic technologies for cell-signaling and network studies as well as novel applications.

Although the contrast generated in transmitted light microscopy is due to the elastic scattering of light, multiple scattering scrambles the image and reduces overall visibility. To image both thin and thick samples, we turn to gradient light interference microscopy (GLIM) to simultaneously measure morphological parameters such as cell mass, volume, and surfaces as they change through time. Because GLIM combines multiple intensity images corresponding to controlled phase offsets between laterally sheared beams, incoherent contributions from multiple scattering are implicitly cancelled during the phase reconstruction procedure. As the interfering beams traverse near identical paths, they remain comparable in power and interfere with optimal contrast. This key property lets us obtain tomographic parameters from wide field z-scans after simple numerical processing. Here we show our results on reconstructing tomograms of bovine embryos, characterizing the time-lapse growth of HeLa cells in 3D, and preliminary results on imaging much larger specimen such as brain slices.

Significance: Functional states of organisms vary rhythmically with a period of about a day (i.e., circadian). This endogenous dynamic is shaped by day–night alternations in light and energy. Mammalian circadian rhythms are orchestrated by the hypothalamic suprachiasmatic nucleus (SCN), a brain region specialized for timekeeping. These autonomous ∼24-h oscillations are cell-based, requiring transcription–translation-based regulation. SCN circadian oscillations include the maintenance of intrinsic rhythms, sensitivities to input signals, and generation of output signals. These change predictably as time proceeds from dawn to day, dusk, and through the night. SCN neuronal excitability, a highly energy-demanding process, also oscillates over ∼24 h. The nature of the relationship of cellular metabolism and excitability had been unknown. Recent Advances: Global SCN redox state was found to undergo an autonomous circadian rhythm. Redox state is relatively reduced in daytime, when neuronal activity is high, and oxidized during nighttime, when neurons are relatively inactive. Redox modulates neuronal excitability via tight coupling: imposed reducing or oxidizing shifts immediately alter membrane excitability. Whereas an intact transcription–translation oscillator is necessary for the redox oscillation, metabolic modulation of excitability is too rapid to be under clockwork control. Critical Issues: Our observations lead to the hypothesis that redox state and neuronal activity are coupled nontranscriptional circadian oscillators in SCN neurons. Critical issues include discovering molecular and cellular substrates and functional consequences of this redox oscillator. Future Directions: Understanding interdependencies between cellular energy metabolism, neuronal activity, and circadian rhythms is critical to developing therapeutic strategies for treating neurodegenerative diseases and brain metabolic syndromes. Antioxid. Redox Signal. 20, 2955–2965.

We present phase correlation imaging (PCI) as a novel approach to study cell dynamics in a spatially-resolved manner. PCI relies on quantitative phase imaging time-lapse data and, as such, functions in label-free mode, without the limitations associated with exogenous markers. The correlation time map outputted in PCI informs on the dynamics of the intracellular mass transport. Specifically, we show that PCI can extract quantitatively the diffusion coefficient map associated with live cells, as well as standard Brownian particles. Due to its high sensitivity to mass transport, PCI can be applied to studying the integrity of actin polymerization dynamics. Our results indicate that the cyto-D treatment blocking the actin polymerization has a dominant effect at the large spatial scales, in the region surrounding the cell. We found that PCI can distinguish between senescent and quiescent cells, which is extremely difficult without using specific markers currently. We anticipate that PCI will be used alongside established, fluorescence-based techniques to enable valuable new studies of cell function.

We present a new method, referred to as phase correlation imaging (PCI), to study cell dynamics and function through temporal phase correlation analysis. PCI offers label-free, high-performance, simple-design, as well as suitability for operation in a conventional microscopy setting. PCI works without the need for controlled or synchronized photoactivation and sophisticated acquisition schemes, and only involves taking a sequence of phase images. The PCI image incorporates information on the phase fluctuations induced by both Brownian motion and deterministic motion of intracellular transport across large scales. We employed spatial light interference microscopy (SLIM) recently developed in our laboratory to experimentally measure quantitative phase information which renders the thickness and refractive index of cellular components without adding contrast agents. The acquisition process is repeated to obtain time-lapse phase images. We calculate the correlation time at each pixel for acquired time-lapse phase images and obtain the correlation time map in space. By temporal correlation analysis, PCI reveals cell dynamics information, which is complementary to quantitative phase imaging itself.

Current methods for light delivery in in vivo optogenetic applications are typically accomplished via a single multimode fiber that diffuses light over a large area of the brain, and relies on the spatial distribution of transfected light-sensitive neurons for targeted control. In our investigations, an imaging fiber bundle (Schott) containing 4,500 individual fibers, each with a diameter of 7.5 µm, and an overall outer bundle diameter of 530 µm, served as a conduit for light delivery and optical recording/imaging. The use of this fiber bundle, in contrast to a single multimode fiber, allows for individually-addressable fibers, spatial selectivity at the stimulus site, more precise control of light delivery, and full field-of-view imaging and/or optical recordings of individual neurons in local neural circuits. An objective coupled the two continuous wave diode laser sources (561nm/488nm) (Coherent) for stimulation and imaging into the fiber bundle while a set of galvanometer-scanning mirrors was used to couple the light stimulus to distinct fibers within the proximal end of the imaging fiber bundle. In our study, C1V1(E122T/E162T)-TS-p2A-mCherry (Karl Deisseroth, Stanford) and GCaMP6s transgenic mice (Jackson Labs) were utilized for this all-optical approach. The results of our investigation demonstrate that imaging fiber bundles provide a new level of spatial selectivity and control of light delivery to specific neurons, as well as function as a conduit for optical imaging and recording at the in vivo site of stimulation, in contrast to the use of single multimode fibers that diffusely illuminate neural tissue and lack in vivo imaging capabilities.

Melatonin is released from the pineal gland into the circulatory system at night in the absence of light, acting as "hormone of darkness" to the brain and body. Melatonin also can regulate circadian phasing of the suprachiasmatic nucleus (SCN). During the day-to-night transition, melatonin exposure advances intrinsic SCN neural activity rhythms via the melatonin type-2 (MT2) receptor and downstream activation of protein kinase C (PKC). The effects of melatonin on SCN phasing have not been linked to daily changes in the expression of core genes that constitute the molecular framework of the circadian clock. Using real-time RT-PCR, we found that melatonin induces an increase in the expression of two clock genes, Period 1 (Per1) and Period 2 (Per2). This effect occurs at CT 10, when melatonin advances SCN phase, but not at CT 6, when it does not. Using anti-sense oligodeoxynucleotides (α ODNs) to Per 1 and Per 2, as well as to E-box enhancer sequences in the promoters of these genes, we show that their specific induction is necessary for the phase-altering effects of melatonin on SCN neural activity rhythms in the rat. These effects of melatonin on Per1 and Per2 were mediated by PKC. This is unlike day-active non-photic signals that reset the SCN clock by non-PCK signal transduction mechanisms and by decreasing Per1 expression. Rather, this finding extends roles for Per1 and Per2, which are critical to photic phase-resetting, to a nonphotic zeitgeber, melatonin, and suggest that the regulation of these clock gene transcripts is required for clock resetting by diverse regulatory cues.

Thalamocortical neurons in dorsal lateral geniculate nucleus (dLGN) dynamically convey visual information from retina to the neocortex. Activation of metabotropic glutamate receptors (mGluRs) exerts multiple effects on neural integration in dLGN; however, their direct influence on the primary sensory input, namely retinogeniculate afferents, is unknown. In the present study, we found that pharmacological or synaptic activation of type 1 mGluRs (mGluR1s) significantly depresses glutamatergic retinogeniculate excitation in rat thalamocortical neurons. Pharmacological activation of mGluR1s attenuates excitatory synaptic responses in thalamocortical neurons at a magnitude sufficient to decrease suprathreshold output of these neurons. The reduction in both NMDA and AMPA receptor-dependent synaptic responses results from a presynaptic reduction in glutamate release from retinogeniculate terminals. The suppression of retinogeniculate synaptic transmission and dampening of thalamocortical output was mimicked by tetanic activation of retinogeniculate afferent in a frequency-dependent manner that activated mGluR1s. Retinogeniculate excitatory synaptic transmission was also suppressed by the glutamate transport blocker TBOA (dl-threo-β-benzyloxyaspartic acid), suggesting that mGluR1s were activated by glutamate spillover. The data indicate that presynaptic mGluR1contributes to an activity-dependent mechanism that regulates retinogeniculate excitation and therefore plays a significant role in the thalamic gating of visual information.

Daily rhythms of mammalian physiology, metabolism, and behavior parallel the day-night cycle. They are orchestrated by a central circadian clock in the brain, the suprachiasmatic nucleus (SCN). Transcription of clock genes is sensitive to metabolic changes in reduction and oxidation (redox); however, circadian cycles in protein oxidation have been reported in anucleate cells, where no transcription occurs. We investigated whether the SCN also expresses redox cycles and how such metabolic oscillations might affect neuronal physiology. We detected self-sustained circadian rhythms of SCN redox state that required the molecular clockwork. The redox oscillation could determine the excitability of SCN neurons through nontranscriptional modulation of multiple potassium (K(+)) channels. Thus, dynamic regulation of SCN excitability appears to be closely tied to metabolism that engages the clockwork machinery.

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca2+ increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca2+-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC–MS/MS and CE–MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1, were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca2+-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion.

The elucidation of the genomes of a large number of mammalian species has produced a huge amount of data on which to base physiological studies. These endeavours have also produced surprises, not least of which has been the revelation that the number of protein coding genes needed to make a mammal is only 22 333 (give or take). However, this small number belies an unanticipated complexity that has only recently been revealed as a result of genomic studies. This complexity is evident at a number of levels: (i) cis-regulatory sequences; (ii) noncoding and antisense mRNAs, most of which have no known function; (iii) alternative splicing that results in the generation of multiple, subtly different mature mRNAs from the precursor transcript encoded by a single gene; and (iv) post-translational processing and modification. In this review, we examine the steps being taken to decipher genome complexity in the context of gene expression, regulation and function in the hypothalamic-neurohypophyseal system (HNS). Five unique stories explain: (i) the use of transcriptomics to identify genes involved in the response to physiological (dehydration) and pathological (hypertension) cues; (ii) the use of mass spectrometry for single-cell level identification of biological active peptides in the HNS, and to measure in vitro release; (iii) the use of transgenic lines that express fusion transgenes enabling (by cross-breeding) the generation of double transgenic lines that can be used to study vasopressin (AVP) and oxytocin (OXT) neurones in the HNS, as well as their neuroanatomy, electrophysiology and activation upon exposure to any given stimulus; (iv) the use of viral vectors to demonstrate that somato-dendritically released AVP plays an important role in cardiovascular homeostasis by binding to V1a receptors on local somata and dendrites; and (v) the use of virally-mediated optogenetics to dissect the role of OXT and AVP in the modulation of a wide variety of behaviours.

Fourier transform light scattering (FTLS) has been recently developed as a novel, ultrasensitive method for studying light scattering from inhomogeneous and dynamic structures. FTLS relies on quantifying the optical phase and amplitude associated with a coherent image field and propagating it numerically to the scattering plane. In this paper, we review the principle and applications of FTLS to static and dynamic light scattering from biological tissues and live cells. Compared with other existing light scattering techniques, FTLS has significant benefits of high sensitivity, speed, and angular resolution. We anticipate that FTLS will set the basis for disease diagnosis based on intrinsic tissue optical properties and provide an efficient tool for quantifying cell structures and dynamics.

Dopamine (DA) receptors are the principal targets of drugs used in the treatment of schizophrenia. Among the five DA receptor subtypes, the D4 subtype is of particular interest because of the relatively high affinity of the atypical neuropleptic clozapine for D4 compared with D2 receptors. GABA-containing neurons in the thalamic reticular nucleus (TRN) and globus pallidus (GP) express D4 receptors. TRN neurons receive GABAergic afferents from globus pallidus (GP), substantia nigra pars reticulata (SNr), and basal forebrain as well as neighboring TRN neuron collaterals. In addition, TRN receives dopaminergic innervations from substantia nigra pars compacta (SNc); however, the role of D4 receptors in neuronal signaling at inhibitory synapses is unknown. Using whole cell recordings from in vitro pallido-thalamic slices, we demonstrate that DA selectively suppresses GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked by GP stimulation. The D2-like receptor (D2,3,4) agonist, quinpirole, and selective D4 receptor agonist, PD168077, mimicked the actions of DA. The suppressive actions of DA and its agonists were associated with alterations in paired pulse ratio and a decrease in the frequency of miniature IPSCs, suggesting a presynaptic site of action. GABAA receptor agonist, muscimol, induced postsynaptic currents in TRN neurons were unaltered by DA or quinpirole, consistent with the presynaptic site of action. Finally, DA agonists did not alter intra-TRN inhibitory signaling. Our data demonstrate that the activation of presynaptic D4 receptors regulates GABA release from GP efferents but not TRN collaterals. This novel and selective action of D4 receptor activation on GP-mediated inhibition may provide insight to potential functional significance of atypical antipsychotic agents. These findings suggest a potential heightened TRN neuron activity in certain neurological conditions, such as schizophrenia and attention deficit hyperactive disorders.

We review the principle and application of Fourier transform light scattering (FTLS), a new technique developed in our laboratory to study static and dynamic light scattering (DLS) from the biological tissues and live cells. The results demonstrate that FTLS has significant benefits over existing light scattering techniques in terms of sensitivity and resolution. We anticipate that FTLS will set the basis for disease diagnosis based on the intrinsic tissue optical properties and provide an efficient tool for quantifying cell structures and dynamics.

Photic information embedded in the alternation between days and nights directly affects circadian systems, ensuring that organisms are in tune with the period imposed by the light:dark (LD) cycle (Pittendrigh and Daan, 1976). This entrainment is a fundamental property of circadian clocks and of rhythms they regulate. In mammals, the master circadian clock is localized within the cells of the hypothalamic suprachiasmatic nucleus (SCN) (Klein et al., 1991). The SCN receives photic information directly from the retina via a retinohypothalamic tract (RHT) projection that contains glutamate and pituitary adenylate cyclase-activating peptide (PACAP) (Hannibal et al., 2000; Moore and Lenn, 1972). The current model for light-induced clock resetting proposes that glutamate and PACAP are co-released in the SCN upon photic stimulation. Whereas the role of glutamate in clock resetting has received considerable attention (Ding et al., 1994; Ebling, 1996; Gillette and Mitchell, 2002), the contribution of PACAP to the photic response of the SCN is still unclear (Morin and Allen, 2006). Multiple animal models have been designed to clarify the contribution of PACAP to circadian function. Three independently developed strains of transgenic mice with null mutations for PACAP expression have been created, as well as a knockout of PAC1R, the primary PACAP receptor (reviewed in Hannibal, 2006) (Hannibal et al., 1997; Kopp et al., 1999; von Gall et al., 1998). Circadian behavioral responses to light of two of these mouse models differ, likely due to experimental idiosyncracies and subtleties of genetic backgrounds and lesions. Specifically, a PACAP peptide-null mouse, developed in the Institute of Cancer Research (ICR) mouse eliminating exon 5 of the PACAP gene-coding sequence (Kawaguchi et al., 2003), exhibits significant attenuations of light-induced phase advance at CT 21, but no significant alteration in behavioral responses to light during the early night (Kawaguchi et al., 2003). These PACAP-null mice also express lower light-induced c-Fos at CT15 compared to WT, but, interestingly, show identical light-induced c-Fos at CT 21, where the behavioral deficits are observed. The PACAP peptide-null mouse developed by Colwell and colleagues (Colwell et al., 2004) lacks exons 3, 4, and 5a of the PACAP gene, eliminating not only PACAP, but also the PACAP-related peptide (PRP) encoded by exons 3 and 4. Significant attenuations of light-induced phase resetting are seen in both phase delays and advances, in contrast to the reports of Kawaguchi et al. (2003). However, in spite of significant deficits in the response to the acute effects of light, these PACAP-null mice re-entrain to 8-h shifts in the LD cycle at normal rates (Colwell et al., 2004). The third PACAP-null mouse model has not been studied in a circadian context, and is the subject of the present report (Hamelink et al., 2002). To more thoroughly investigate the role of PACAP in circadian resetting, we evaluated the circadian responses to light of this third PACAP-null animal model. This animal was generated in the C57BL/6 strain with only the PACAP-coding sequence deleted, eliminating a potential source of confound in the results. Details and specificity of the genetic lesion, complete loss of PACAP expression and functional deficits in PACAP-mediated adrenal function have been described previously (Hamelink et al., 2002). We paid particular attention to the phase-advance portion of the circadian cycle, a time where models studied previously displayed common deficits in light-induced resetting. We report here that mice lacking PACAP show specific impairment in their ability to generate phase advances to a single brief light exposure when housed in darkness, whereas endogenous free-running period and phase-delaying capabilities are intact. PACAP-null mice are able to entrain to a 23-h T-cycle, although with longer phase-angle of entrainment, and with slower rates of entrainment following a 6-h step advance of the LD cycle. These data indicate that PACAP contributes to normal integration of the phase-advancing signal required for acute phase shifts and for entrainment.

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characterization of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not immediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected., Mammalian peptidome studies are extended to smaller, well-defined brain structures ranging from an individual brain nucleus to individual neurons. Specifically, we characterize the peptidome of the supraoptic nucleus and individual magnocellular neurons within the nucleus. With the use of a coronal brain slice preparation and optimized sample preparation strategies to reduce postmortem degradation, 85 peptides have been characterized, including 20 peptides previously not reported.

The emergent self-organization of a neuronal network in a developing nervous system is the result of a remarkably orchestrated process involving a multitude of chemical, mechanical and electrical signals. Little is known about the dynamic behavior of a developing network (especially in a human model) primarily due to a lack of practical and non-invasive methods to measure and quantify the process. Here we demonstrate that by using a novel optical interferometric technique, we can non-invasively measure several fundamental properties of neural networks from the sub-cellular to the cell population level. We applied this method to quantify network formation in human stem cell derived neurons and show for the first time, correlations between trends in the growth, transport, and spatial organization of such a system. Quantifying the fundamental behavior of such cell lines without compromising their viability may provide an important new tool in future longitudinal studies.

An outstanding challenge for humanity is to continue to understand how our brain works and invent technology to restore neural circuit functionalities. Many neural interfaces used for neuron cell cultures are flat, open, rigid, and opaque, posing challenges to reflecting the native microenvironment of the brain and precise engagement with neurons. Here we present a novel neural interface consisting of silicon nitride microtube arrays formed by a new nanotechnology platform that simply relies on strain-induced self-rolled-up membrane (S-RUM) mechanism [1]. We have found that these microtubes provide robust physical confinement and unprecedented guidance effect towards outgrowth of primary cortical neurons [2]. What is more surprising is that a dramatic increase (20x) of the growth rate inside the microtube compared to regions outside the microtubes has been observed [2]. The outgrowth of hippocampal neurons is also explored using identical platforms with the intent of integrating both networks on the same chip. The unique characteristics of these S-RUM microtubes that enabled the cell guidance and acceleration will be discussed in detail: (1) True 3D geometrical confinement: the perfect cylindrical geometry of the S-RUM microtubes with tunable diameters and ultra-thin walls, the same type of small and confined spaces neurons grow in vivo, provides conformal 3D adhesion tailored to different kinds of cells. (2) Electrostatic adhesion: the deposited SiN x thin films are found to have a high density of trapped positive charges that naturally attract the axon towards the microtube opening. The ability of the microtube array to control the speed and direction of axonal extension provides a key element in arranging patterned neural networks that have both short and long range connections. (3) Optically transparent: having the ability to see through both the tube and the underlying substrate enabled direct observation of how cells transition from the flat regions to different parts of the tube.

White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique. Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead. WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.