Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges.

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-ß, the toxic driver of Alzheimer's disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.

The Severity of Acute Stress Is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons.

The hypothalamo-pituitary-adrenocortical (HPA) axis regulates stress physiology and behavior. To achieve an optimally tuned adaptive response, it is critical that the magnitude of the stress response matches the severity of the threat. Corticotropin-releasing hormone (CRH) released from the paraventricular nucleus of the hypothalamus is a major regulator of the HPA axis. However, how CRH-producing neurons in an intact animal respond to different stressor intensities is currently not known. Using two-photon calcium imaging on intact larval zebrafish, we recorded the activity of CRH cells, while the larvae were exposed to stressors of varying intensity. By combining behavioral and physiological measures, we first determined how sudden alterations in environmental conditions lead to different levels of stress axis activation. Then, we measured changes in the frequency and amplitude of Ca(2+) transients in individual CRH neurons in response to such stressors. The response magnitude of individual CRH cells covaried with stressor intensity. Furthermore, stressors caused the recruitment of previously inactive CRH neurons in an intensity-dependent manner, thus increasing the pool of responsive CRH cells. Strikingly, stressor-induced activity appeared highly synchronized among CRH neurons, and also across hemispheres. Thus, the stressor strength-dependent output of CRH neurons emerges by a dual mechanism that involves both the increased activity of individual cells and the recruitment of a larger pool of responsive cells. The synchronicity of CRH neurons within and across hemispheres ensures that the overall output of the HPA axis matches the severity of the threat. SIGNIFICANCE STATEMENT: Stressors trigger adaptive responses in the body that are essential for survival. How the brain responds to acute stressors of varying intensity in an intact animal, however, is not well understood. We address this question using two-photon Ca(2+) imaging in larval zebrafish with transgenically labeled corticotropin-releasing hormone (CRH) cells, which represent a major regulator of the stress axis. We show that stressor strength-dependent responses of CRH neurons emerge via an intensity-dependent increase in the activity of individual CRH cells, and by an increase in the pool of responsive CRH cells at the population level. Furthermore, we report striking synchronicity among CRH neurons even across hemispheres, which suggests tight intrahypothalamic and interhypothalamic coordination. Thus, our work reveals how CRH neurons respond to different levels of acute stress in vivo.

Stimulation-evoked Ca2+ signals in astrocytic processes at hippocampal CA3-CA1 synapses of adult mice are modulated by glutamate and ATP.

To date, it has been difficult to reveal physiological Ca(2+) events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca(2+) signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca(2+) indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca(2+) signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca(2+) signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca(2+) transients by 30-40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca(2+) signals in processes and failed to affect the somatic Ca(2+) response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca(2+) signals that mimicked the stimulation-evoked astrocytic Ca(2+) responses. We conclude that stimulation-evoked Ca(2+) signals in astrocytic processes at CA3-CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP.

A synaptic corollary discharge signal suppresses midbrain visual processing during saccade-like locomotion.

In motor control, the brain not only sends motor commands to the periphery, but also generates concurrent internal signals known as corollary discharge (CD) that influence sensory information processing around the time of movement. CD signals are important for identifying sensory input arising from self-motion and to compensate for it, but the underlying mechanisms remain unclear. Using whole-cell patch clamp recordings from neurons in the zebrafish optic tectum, we discovered an inhibitory synaptic signal, temporally locked to spontaneous and visually driven locomotion. This motor-related inhibition was appropriately timed to counteract visually driven excitatory input arising from the fish's own motion, and transiently suppressed tectal spiking activity. High-resolution calcium imaging revealed localized motor-related signals in the tectal neuropil and the upstream torus longitudinalis, suggesting that CD enters the tectum via this pathway. Together, our results show how visual processing is suppressed during self-motion by motor-related phasic inhibition. This may help explain perceptual saccadic suppression observed in many species.

Fully automated deconvolution method for on-line analysis of time-resolved fluorescence spectroscopy data based on an iterative Laguerre expansion technique.

Time-resolved fluorescence spectroscopy (TRFS) is a powerful analytical tool for quantifying the biochemical composition of organic and inorganic materials. The potential of TRFS for tissue diagnosis has been recently demonstrated. To facilitate the translation of TRFS to the clinical arena, algorithms for online TRFS data analysis are essential. A fast model-free TRFS deconvolution algorithm based on the Laguerre expansion method has previously been introduced. One limitation of this method, however, is the need to heuristically select two parameters that are crucial for the accurate estimation of the fluorescence decay: the Laguerre parameter alpha and the expansion order. Here, a new implementation of the Laguerre deconvolution method is introduced, in which a nonlinear least-square optimization of the Laguerre parameter alpha is performed, and the optimal expansion order is selected based on a minimum description length criterion (MDL). In addition, estimation of the zero-time delay between the recorded instrument response and fluorescence decay is also performed based on normalized mean square error criterion (NMSE). The method is validated on experimental data from fluorescence lifetime standards, endogenous tissue fluorophores, and human tissue. The proposed automated Laguerre deconvolution method will facilitate online applications of TRFS, such as real-time clinical tissue diagnosis.

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.

Classification of object size in retinotectal microcircuits.

BACKGROUND: A principal task of the visual system is to detect and classify moving objects in the visual environment. Information about the size of an object is critical for selecting appropriate behavioral responses. Object size is encoded in retinal ganglion cell (RGC) activity. Little is known, however, about how inputs from the multitude of RGC subtypes are distributed to higher visual centers and how information is combined from these feature-selective inputs. RESULTS: Here we show that in the zebrafish optic tectum, prey- or predator-like moving targets evoke activity in distinct groups of RGC fibers dependent on target size, demonstrating a retinal origin of tectal size classification. Small-size-selective retinal inputs are relatively more frequent in the most superficial layer of the tectal neuropil, whereas large-size-selective inputs predominate in deeper layers. Monostratified superficial interneurons (SINs) process large-size- and small-size-selective signals dependent on their dendritic target layer, consistent with the retinal input organization. Further downstream, small- and large-sized objects are encoded in population activity of separate sets of tectal neurons. CONCLUSIONS: Ethologically relevant size classes are preferentially processed in different layers of the tectal neuropil. The tectum categorizes visual targets on the basis of retinally computed size information, suggesting a critical role in visually guided response selection.

Visually driven chaining of elementary swim patterns into a goal-directed motor sequence: a virtual reality study of zebrafish prey capture.

Prey capture behavior critically depends on rapid processing of sensory input in order to track, approach, and catch the target. When using vision, the nervous system faces the problem of extracting relevant information from a continuous stream of input in order to detect and categorize visible objects as potential prey and to select appropriate motor patterns for approach. For prey capture, many vertebrates exhibit intermittent locomotion, in which discrete motor patterns are chained into a sequence, interrupted by short periods of rest. Here, using high-speed recordings of full-length prey capture sequences performed by freely swimming zebrafish larvae in the presence of a single paramecium, we provide a detailed kinematic analysis of first and subsequent swim bouts during prey capture. Using Fourier analysis, we show that individual swim bouts represent an elementary motor pattern. Changes in orientation are directed toward the target on a graded scale and are implemented by an asymmetric tail bend component superimposed on this basic motor pattern. To further investigate the role of visual feedback on the efficiency and speed of this complex behavior, we developed a closed-loop virtual reality setup in which minimally restrained larvae recapitulated interconnected swim patterns closely resembling those observed during prey capture in freely moving fish. Systematic variation of stimulus properties showed that prey capture is initiated within a narrow range of stimulus size and velocity. Furthermore, variations in the delay and location of swim triggered visual feedback showed that the reaction time of secondary and later swims is shorter for stimuli that appear within a narrow spatio-temporal window following a swim. This suggests that the larva may generate an expectation of stimulus position, which enables accelerated motor sequencing if the expectation is met by appropriate visual feedback.

Layer-specific targeting of direction-selective neurons in the zebrafish optic tectum.

Direction selectivity (DS) is an important neuronal property in the visual system, but how DS is generated beyond the retina remains controversial. Here, we report a close correspondence between the preferred direction (PD) and the morphology of DS cells in the optic tectum. Ca(2+) imaging in cells expressing the genetically encoded Ca(2+) indicator GCaMP3 and multiphoton-targeted patch-clamp recordings allowed us to compare structure and function in single neurons. The arbors of differently tuned cell types showed stereotypic differences in shape and laminar profile within the tectal neuropil. Excitatory synaptic inputs were directionally tuned and matched the PD of spike output in these cells, while inhibitory inputs were selective for nonpreferred directions. Functional Ca(2+) imaging in afferent axons showed a matching laminar distribution of DS presynaptic activity. Hence, different directions are represented in different layers, which suggests a simple mechanism for how tectal neurons acquire directional tuning in a nascent circuit.