Multispectral tracing in densely labeled mouse brain with nTracer.

SUMMARY: This note describes nTracer, an ImageJ plug-in for user-guided, semi-automated tracing of multispectral fluorescent tissue samples. This approach allows for rapid and accurate reconstruction of whole cell morphology of large neuronal populations in densely labeled brains. AVAILABILITY AND IMPLEMENTATION: nTracer was written as a plug-in for the open source image processing software ImageJ. The software, instructional documentation, tutorial videos, sample image and sample tracing results are available at https://www.cai-lab.org/ntracer-tutorial. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A neuropeptide speeds circadian entrainment by reducing intercellular synchrony.

Shift work or transmeridian travel can desynchronize the body's circadian rhythms from local light-dark cycles. The mammalian suprachiasmatic nucleus (SCN) generates and entrains daily rhythms in physiology and behavior. Paradoxically, we found that vasoactive intestinal polypeptide (VIP), a neuropeptide implicated in synchrony among SCN cells, can also desynchronize them. The degree and duration of desynchronization among SCN neurons depended on both the phase and the dose of VIP. A model of the SCN consisting of coupled stochastic cells predicted both the phase- and the dose-dependent response to VIP and that the transient phase desynchronization, or "phase tumbling", could arise from intrinsic, stochastic noise in small populations of key molecules (notably, Period mRNA near its daily minimum). The model also predicted that phase tumbling following brief VIP treatment would accelerate entrainment to shifted environmental cycles. We tested this using a prepulse of VIP during the day before a shift in either a light cycle in vivo or a temperature cycle in vitro. Although VIP during the day does not shift circadian rhythms, the VIP pretreatment approximately halved the time required for mice to reentrain to an 8-h shifted light schedule and for SCN cultures to reentrain to a 10-h shifted temperature cycle. We conclude that VIP below 100 nM synchronizes SCN cells and above 100 nM reduces synchrony in the SCN. We show that exploiting these mechanisms that transiently reduce cellular synchrony before a large shift in the schedule of daily environmental cues has the potential to reduce jet lag.

Network-mediated encoding of circadian time: the suprachiasmatic nucleus (SCN) from genes to neurons to circuits, and back.

The transcriptional architecture of intracellular circadian clocks is similar across phyla, but in mammals interneuronal mechanisms confer a higher level of circadian integration. The suprachiasmatic nucleus (SCN) is a unique model to study these mechanisms, as it operates as a ∼24 h clock not only in the living animal, but also when isolated in culture. This "clock in a dish" can be used to address fundamental questions, such as how intraneuronal mechanisms are translated by SCN neurons into circuit-level emergent properties and how the circuit decodes, and responds to, light input. This review addresses recent developments in understanding the relationship between electrical activity, [Ca(2+)]i, and intracellular clocks. Furthermore, optogenetic and chemogenetic approaches to investigate the distinct roles of neurons and glial cells in circuit encoding of circadian time will be discussed, as well as the epigenetic and circuit-level mechanisms that enable the SCN to translate light input into coherent daily rhythms.

Representation of ethological events by basolateral amygdala neurons.

The accurate interpretation of ethologically relevant stimuli is crucial for survival. While basolateral amygdala (BLA) neuronal responses during fear conditioning are well studied, little is known about how BLA neurons respond during naturalistic events. We recorded from the rat BLA during interaction with ethological stimuli: male or female rats, a moving toy, and rice. Forty-two percent of the cells reliably respond to at least one stimulus, with over half of these exclusively identifying one of the four stimulus classes. In addition to activation during interaction with their preferred stimulus, these cells signal micro-behavioral interactions like social contact. After stimulus removal, firing activity persists in 30% of responsive cells for several minutes. At the micro-circuit level, information flows from highly tuned event-specific neurons to less specific neurons, and connection strength increases after the event. We propose that individual BLA neurons identify specific ethological events, with event-specific neurons driving circuit-wide activity during and after salient events.

Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis.

The sepsis syndrome represents an improper immune response to infection and is associated with unacceptably high rates of mortality and morbidity. The interactions between T cells and the innate immune system while combating sepsis are poorly understood. In this report, we observed that treatment with the potent, antiapoptotic cytokine interleukin-7 (IL-7) accelerated neutrophil recruitment and improved bacterial clearance. We first determined that T cells were necessary for the previously observed IL-7-mediated enhanced survival. Next, IL-7 increased Bcl-2 expression in T cells isolated from septic mice as early as 3 h following treatment. This treatment resulted in increased gamma interferon (IFN-γ) and IP-10 production within the septic peritoneum together with local and systemic increases of IL-17 in IL-7-treated mice. We further demonstrate that the increase in IL-17 was largely due to increased recruitment and production by γδ T cells, which express CXCR3. Consistent with increased IL-17 production, IL-7 treatment increased CXCL1/KC production, neutrophil recruitment, and bacterial clearance. Significantly, end-organ tissue injury was not significantly different between vehicle- and IL-7-treated mice. Collectively, these data illustrate that IL-7 can mediate the cross talk between Th1 and Th17 lymphocytes during sepsis such that neutrophil recruitment and bacterial clearance is improved while early tissue injury is not increased. All together, these observations may underlay novel potential therapeutic targets to improve the host immune response to sepsis.

Different Roles for VIP Neurons in the Neonatal and Adult Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) drives circadian rhythms in locomotion through coupled, single-cell oscillations. Global genetic deletion of the neuropeptide Vip or its receptor Vipr2 results in profound deficits in daily synchrony among SCN cells and daily rhythms in locomotor behavior and glucocorticoid secretion. To test whether this phenotype depends on vasoactive intestinal polypeptide (VIP) neurons in the SCN, we ablated VIP SCN neurons in vivo in adult male mice through Caspase3-mediated induction of the apoptotic pathway in cre-expressing VIP neurons. We found that ablation of VIP SCN neurons in adult mice caused a phenotype distinct from Vip- and Vipr2-null mice. Mice lacking VIP neurons retained rhythmic locomotor activity with a shortened circadian period, more variable onsets, and decreased duration of daily activity. Circadian hormonal outputs, specifically corticosterone rhythms, were severely dampened. In contrast, deletion of neonatal SCN VIP neurons dramatically reduced circadian gene expression in the cultured SCN, mimicking the effects of global deletion of Vip or Vipr2. These results suggest that SCN VIP neurons play a role in lengthening circadian period and stimulating the daily surge in glucocorticoids in adults and in synchronizing and sustaining daily rhythms among cells in the developing SCN.

Entrainment of Circadian Rhythms Depends on Firing Rates and Neuropeptide Release of VIP SCN Neurons.

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker, integrating environmental input to align physiological and behavioral rhythms to local time cues. Approximately 10% of SCN neurons express vasoactive intestinal polypeptide (VIP); however, it is unknown how firing activity of VIP neurons releases VIP to entrain circadian rhythms. To identify physiologically relevant firing patterns, we optically tagged VIP neurons and characterized spontaneous firing over 3 days. VIP neurons had circadian rhythms in firing rate and exhibited two classes of instantaneous firing activity. We next tested whether physiologically relevant firing affected circadian rhythms through VIP release. We found that VIP neuron stimulation with high, but not low, frequencies shifted gene expression rhythms in vitro through VIP signaling. In vivo, high-frequency VIP neuron activation rapidly entrained circadian locomotor rhythms. Thus, increases in VIP neuronal firing frequency release VIP and entrain molecular and behavioral circadian rhythms. VIDEO ABSTRACT.

Circadian rhythms: to sync or not to sync.

Using real-time imaging of circadian gene expression, a new study reveals how a light pulse briefly desynchronizes clock neurons in the fly brain before they settle into a new, synchronized daily rhythm.

Measuring synchrony in the mammalian central circadian circuit.

Circadian clocks control daily rhythms in physiology and behavior across all phyla. These rhythms are intrinsic to individual cells that must synchronize to their environment and to each other to anticipate daily events. Recent advances in recording from large numbers of cells for many circadian cycles have enabled researchers to begin to evaluate the mechanisms and consequences of intercellular circadian synchrony. Consequently, methods have been adapted to estimate the period, phase, and amplitude of individual circadian cells and calculate synchrony between cells. Stable synchronization requires that the cells share a common period. As a result, synchronized cells maintain constant phase relationships to each (e.g., with cell 1 peaking an hour before cell 2 each cycle). This chapter reviews how circadian rhythms are recorded from single mammalian cells and details methods for measuring their period and phase synchrony. These methods have been useful, for example, in showing that specific neuropeptides are essential to maintain synchrony among circadian cells.

Pulmonary contusion is associated with toll-like receptor 4 upregulation and decreased susceptibility to pseudomonas pneumonia in a mouse model.

Pulmonary contusion is a major cause of respiratory failure in trauma patients. This injury frequently leads to immune suppression and infectious complications such as pneumonia. The mechanism whereby trauma leads to an immune-suppressed state is poorly understood. To further study this phenomenon, we developed an animal model of pulmonary contusion (PC) complicated by pneumonia and assessed the effect of PC and pneumonia on toll-like receptor expression in alveolar macrophages. Using a mouse model, PC was induced on the right lung, and pneumonia was induced with Pseudomonas aeruginosa (Pa) injected intratracheally 48 h after injury. Susceptibility to pneumonia was assessed by mortality at 7 days. Uninjured animals were used as controls. Bronchoalveolar lavage fluid and blood were assayed 48 h after injury and 24 h after Pa instillation to look at markers of systemic inflammation. Toll-like receptor expression in the initial inflammatory response was analyzed by flow cytometry. Unexpectedly, injured animals subjected to intratracheal injection of Pa at 48 h after PC demonstrated increased survival compared with uninjured animals. Bronchoalveolar lavage cytokine expression was increased significantly after Pa administration but not after PC alone. Toll-like receptor 4 expression on alveolar macrophages was significantly elevated in the injured group compared with sham but not in neutrophils. Animals subjected to PC are more resistant to mortality from infection with Pa and display an enhanced cytokine response when subsequently subjected to Pa. Increased expression of toll-like receptor 4 on alveolar macrophages and enhanced innate immunity are a possible mechanism of increased cytokine production and decreased susceptibility to pneumonia.