BDNF signaling in correlation-dependent structural plasticity in the developing visual system.

During development, patterned neural activity instructs topographic map refinement. Axons with similar patterns of neural activity converge onto target neurons and stabilize their synapses with these postsynaptic partners, restricting exploratory branch elaboration (Hebbian structural plasticity). On the other hand, non-correlated firing in inputs leads to synapse weakening and increased exploratory growth of axons (Stentian structural plasticity). We used visual stimulation to control the correlation structure of neural activity in a few ipsilaterally projecting (ipsi) retinal ganglion cell (RGC) axons with respect to the majority contralateral eye inputs in the optic tectum of albino Xenopus laevis tadpoles. Multiphoton live imaging of ipsi axons, combined with specific targeted disruptions of brain-derived neurotrophic factor (BDNF) signaling, revealed that both presynaptic p75NTR and TrkB are required for Stentian axonal branch addition, whereas presumptive postsynaptic BDNF signaling is necessary for Hebbian axon stabilization. Additionally, we found that BDNF signaling mediates local suppression of branch elimination in response to correlated firing of inputs. Daily in vivo imaging of contralateral RGC axons demonstrated that p75NTR knockdown reduces axon branch elongation and arbor spanning field volume.

Stentian structural plasticity in the developing visual system.

In a small fraction of Xenopus tadpoles, a single retinal ganglion cell (RGC) axon misprojects to the ipsilateral optic tectum. Presenting flashes of light to the ipsilateral eye causes that ipsilateral axon to fire, whereas stimulating the contralateral eye excites all other RGC inputs to the tectum. We performed time-lapse imaging of individual ipsilaterally projecting axons while stimulating either the ipsilateral or contralateral eye. Stimulating either eye alone reduced axon elaboration by increasing branch loss. New branch additions in the ipsi axon were exclusively increased by contralateral eye stimulation, which was enhanced by expressing tetanus neurotoxin (TeNT) in the ipsilateral axon, to prevent Hebbian stabilization. Together, our results reveal the existence of a non-cell-autonomous "Stentian" signal, engaged by activation of neighboring RGCs, that promotes exploratory axon branching in response to noncorrelated firing.

DNA hypomethylation of Synapsin II CpG islands associates with increased gene expression in bipolar disorder and major depression.

BACKGROUND: The Synapsins (SYN1, SYN2, and SYN3) are important players in the adult brain, given their involvement in synaptic transmission and plasticity, as well as in the developing brain through roles in axon outgrowth and synaptogenesis. We and others previously reported gene expression dysregulation, both as increases and decreases, of Synapsins in mood disorders, but little is known about the regulatory mechanisms leading to these differences. Thus, we proposed to study DNA methylation at theses genes' promoter regions, under the assumption that altered epigenetic marks at key regulatory sites would be the cause of gene expression changes and thus part of the mood disorder etiology. METHODS: We performed CpG methylation mapping focusing on the three genes' predicted CpG islands using the Sequenom EpiTYPER platform. DNA extracted from post-mortem brain tissue (BA10) from individuals who had lived with bipolar disorder (BD), major depressive disorder (MDD), as well as psychiatrically healthy individuals was used. Differences in methylation across all CpGs within a CpG island and between the three diagnostic groups were assessed by 2-way mixed model analyses of variance. RESULTS: We found no significant results for SYN1 or SYN3, but there was a significant group difference in SYN2 methylation, as well as an overall pattern of hypomethylation across the CpG island. Furthermore, we found a significant inverse correlation of DNA methylation with SYN2a mRNA expression. CONCLUSIONS: These findings contribute to previous work showing dysregulation of Synapsins, particularly SYN2, in mood disorders and improve our understanding of the regulatory mechanisms that precipitate these changes likely leading to the BD or MDD phenotype.

Tonically active GABAergic neurons in the dorsal periaqueductal gray control instinctive escape in mice.

Escape behavior is a set of locomotor actions that move an animal away from threat. While these actions can be stereotyped, it is advantageous for survival that they are flexible.1,2,3 For example, escape probability depends on predation risk and competing motivations,4,5,6,7,8,9,10,11 and flight to safety requires continuous adjustments of trajectory and must terminate at the appropriate place and time.12,13,14,15,16 This degree of flexibility suggests that modulatory components, like inhibitory networks, act on the neural circuits controlling instinctive escape.17,18,19,20,21,22 In mice, the decision to escape from imminent threats is implemented by a feedforward circuit in the midbrain, where excitatory vesicular glutamate transporter 2-positive (VGluT2+) neurons in the dorsal periaqueductal gray (dPAG) compute escape initiation and escape vigor.23,24,25 Here we tested the hypothesis that local GABAergic neurons within the dPAG control escape behavior by setting the excitability of the dPAG escape network. Using in vitro patch-clamp and in vivo neural activity recordings, we found that vesicular GABA transporter-positive (VGAT+) dPAG neurons fire action potentials tonically in the absence of synaptic inputs and are a major source of inhibition to VGluT2+ dPAG neurons. Activity in VGAT+ dPAG cells transiently decreases at escape onset and increases during escape, peaking at escape termination. Optogenetically increasing or decreasing VGAT+ dPAG activity changes the probability of escape when the stimulation is delivered at threat onset and the duration of escape when delivered after escape initiation. We conclude that the activity of tonically firing VGAT+ dPAG neurons sets a threshold for escape initiation and controls the execution of the flight action.

Are extrasynaptic GABAA receptors important targets for sedative/hypnotic drugs?

High-affinity extrasynaptic GABA(A) receptors are persistently activated by the low ambient GABA levels that are known to be present in extracellular space. The resulting tonic conductance generates a form of shunting inhibition that is capable of altering cellular and network behavior. It has been suggested that this tonic inhibition will be enhanced by neurosteroids, antiepileptics, and sedative/hypnotic drugs. However, we show that the ability of sedative/hypnotic drugs to enhance tonic inhibition in the mouse cerebellum will critically depend on ambient GABA levels. For example, we show that the intravenous anesthetic propofol enhances tonic inhibition only when ambient GABA levels are <100 nm. More surprisingly, the actions of the sleep-promoting drug 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) are attenuated at ambient GABA levels of just 20 nm. In contrast, our data suggest that neurosteroid enhancement of tonic inhibition will be greater at high ambient GABA concentrations. We present a model that takes into account realistic estimates of ambient GABA levels and predicted extrasynaptic GABA(A) receptor numbers when considering the ability of sedative/hypnotic drugs to enhance tonic inhibition. These issues will be important when considering drug strategies designed to target extrasynaptic GABA(A) receptors in the treatment of sleep disorders and other neurological conditions.

TORC1 selectively regulates synaptic maturation and input convergence in the developing visual system.

Newly synthesized proteins support the development of functional neural circuits and previous work has suggested that dysregulated translation mediates certain forms of autism spectrum disorder (ASD). Here, we investigated the role of Target of Rapamycin Complex 1 (TORC1) in synaptic and dendritic development in vivo in the retinotectal system of Xenopus laevis tadpoles. We found that TORC1 signaling regulates dendritic growth and branching and that acute over-activation of TORC1 by Rheb overexpression drove enhanced maturation of excitatory synapses by recruiting AMPA receptors. Interestingly, TORC1 over-activation did not affect inhibitory transmission, resulting in a significant imbalance in the excitatory-to-inhibitory ratio. Rheb overexpression also enlarged excitatory visual input fields in tectal neurons, consistent with dysregulation of retinotopic input refinement and integration of the cell into the circuit. In contrast to other reports that mainly found impairments in synaptic inhibition using broad systemic deletion or mutation of TORC1 regulatory proteins, our findings from acute, local manipulation of TORC1 reveal its critical role in selectively regulating the number and maturity of excitatory, but not inhibitory, synapses in the developing brain.

Exploring the significance of morphological diversity for cerebellar granule cell excitability.

The relatively simple and compact morphology of cerebellar granule cells (CGCs) has led to the view that heterogeneity in CGC shape has negligible impact upon the integration of mossy fibre (MF) information. Following electrophysiological recording, 3D models were constructed from high-resolution imaging data to identify morphological features that could influence the coding of MF input patterns by adult CGCs. Quantification of MF and CGC morphology provided evidence that CGCs could be connected to the multiple rosettes that arise from a single MF input. Predictions from our computational models propose that MF inputs could be more densely encoded within the CGC layer than previous models suggest. Moreover, those MF signals arriving onto the dendrite closest to the axon will generate greater CGC excitation. However, the impact of this morphological variability on MF input selectivity will be attenuated by high levels of CGC inhibition providing further flexibility to the MF → CGC pathway. These features could be particularly important when considering the integration of multimodal MF sensory input by individual CGCs.

Rules for Shaping Neural Connections in the Developing Brain.

It is well established that spontaneous activity in the developing mammalian brain plays a fundamental role in setting up the precise connectivity found in mature sensory circuits. Experiments that produce abnormal activity or that systematically alter neural firing patterns during periods of circuit development strongly suggest that the specific patterns and the degree of correlation in firing may contribute in an instructive manner to circuit refinement. In fish and amphibians, unlike amniotic vertebrates, sensory input directly drives patterned activity during the period of initial projection outgrowth and innervation. Experiments combining sensory stimulation with live imaging, which can be performed non-invasively in these simple vertebrate models, have provided important insights into the mechanisms by which neurons read out and respond to activity patterns. This article reviews the classic and recent literature on spontaneous and evoked activity-dependent circuit refinement in sensory systems and formalizes a set of mechanistic rules for the transformation of patterned activity into accurate neuronal connectivity in the developing brain.

A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.

The introduction of calcium ion (Ca(2+)) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca(2+) dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650-1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca(2+) indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca(2+) indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca(2+) and glutamate imaging in organotypic cultures.

Surface chemistry study of the interactions of benzalkonium chloride with films of meibum, corneal cells lipids, and whole tears.

PURPOSE: To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents. METHODS: The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the sample's lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%. RESULTS: In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration. CONCLUSIONS: The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.