Retinal waves prime visual motion detection by simulating future optic flow.

The ability to perceive and respond to environmental stimuli emerges in the absence of sensory experience. Spontaneous retinal activity prior to eye opening guides the refinement of retinotopy and eye-specific segregation in mammals, but its role in the development of higher-order visual response properties remains unclear. Here, we describe a transient window in neonatal mouse development during which the spatial propagation of spontaneous retinal waves resembles the optic flow pattern generated by forward self-motion. We show that wave directionality requires the same circuit components that form the adult direction-selective retinal circuit and that chronic disruption of wave directionality alters the development of direction-selective responses of superior colliculus neurons. These data demonstrate how the developing visual system patterns spontaneous activity to simulate ethologically relevant features of the external world and thereby instruct self-organization.

Transverse sinus injections drive robust whole-brain expression of transgenes.

Convenient, efficient and fast whole-brain delivery of transgenes presents a persistent experimental challenge in neuroscience. Recent advances demonstrate whole-brain gene delivery by retro-orbital injection of virus, but slow and sparse expression and the large injection volumes required make this approach cumbersome, especially for developmental studies. We developed a novel method for efficient gene delivery across the central nervous system in neonatal mice and rats starting as early as P1 and persisting into adulthood. The method employs transverse sinus injections of 2-4 µL of AAV9 at P0. Here, we describe how to use this method to label and/or genetically manipulate cells in the neonatal rat and mouse brain. The protocol is fast, simple, can be readily adopted by any laboratory, and utilizes the widely available AAV9 capsid. The procedure is adaptable for diverse experimental applications ranging from biochemistry, anatomical and functional mapping, gene expression, silencing, and editing.

Simultaneous mesoscopic and two-photon imaging of neuronal activity in cortical circuits.

Spontaneous and sensory-evoked activity propagates across varying spatial scales in the mammalian cortex, but technical challenges have limited conceptual links between the function of local neuronal circuits and brain-wide network dynamics. We present a method for simultaneous cellular-resolution two-photon calcium imaging of a local microcircuit and mesoscopic widefield calcium imaging of the entire cortical mantle in awake mice. Our multi-scale approach involves a microscope with an orthogonal axis design where the mesoscopic objective is oriented above the brain and the two-photon objective is oriented horizontally, with imaging performed through a microprism. We also introduce a viral transduction method for robust and widespread gene delivery in the mouse brain. These approaches allow us to identify the behavioral state-dependent functional connectivity of pyramidal neurons and vasoactive intestinal peptide-expressing interneurons with long-range cortical networks. Our imaging system provides a powerful strategy for investigating cortical architecture across a wide range of spatial scales.

An NMDA receptor-dependent mechanism for subcellular segregation of sensory inputs in the tadpole optic tectum.

In the vertebrate CNS, afferent sensory inputs are targeted to specific depths or layers of their target neuropil. This patterning exists ab initio, from the very beginning, and therefore has been considered an activity-independent process. However, here we report that, during circuit development, the subcellular segregation of the visual and mechanosensory inputs to specific regions of tectal neuron dendrites in the tadpole optic tectum requires NMDA receptor activity. Blocking NMDARs during the formation of these sensory circuits, or removing the visual set of inputs, leads to less defined segregation, and suggests a correlation-based mechanism in which correlated inputs wire to common regions of dendrites. This can account for how two sets of inputs form synapses onto different regions of the same dendrite. Blocking NMDA receptors during later stages of circuit development did not disrupt segregation, indicating a critical period for activity-dependent shaping of patterns of innervation.

Early development and function of the Xenopus tadpole retinotectal circuit.

The retinotectal circuit is the major component of the amphibian visual system. It is comprised of the retinal ganglion cells (RGCs) in the eye, which project their axons to the optic tectum and form synapses onto postsynaptic tectal neurons. The retinotectal circuit is relatively simple, and develops quickly: Xenopus tadpoles begin displaying retinotectal-dependent visual avoidance behaviors by approximately 7-8 days post-fertilization, early larval stage. In this review we first provide a summary of the dynamic development of the retinotectal circuit, including the microcircuitry formed by local tectal-tectal connections within the tectum. Second, we discuss the basic visual avoidance behavior generated specifically by this circuit, and how this behavior is being used as an assay to test visual system function.

A population of gap junction-coupled neurons drives recurrent network activity in a developing visual circuit.

In many regions of the vertebrate brain, microcircuits generate local recurrent activity that aids in the processing and encoding of incoming afferent inputs. Local recurrent activity can amplify, filter, and temporally and spatially parse out incoming input. Determining how these microcircuits function is of great interest because it provides glimpses into fundamental processes underlying brain computation. Within the Xenopus tadpole optic tectum, deep layer neurons display robust recurrent activity. Although the development and plasticity of this local recurrent activity has been well described, the underlying microcircuitry is not well understood. Here, using a whole brain preparation that allows for whole cell recording from neurons of the superficial tectal layers, we identified a physiologically distinct population of excitatory neurons that are gap junctionally coupled and through this coupling gate local recurrent network activity. Our findings provide a novel role for neuronal coupling among excitatory interneurons in the temporal processing of visual stimuli.

The horizontal brain slice preparation: a novel approach for visualizing and recording from all layers of the tadpole tectum.

The Xenopus tadpole optic tectum is a multisensory processing center that receives direct visual input as well as nonvisual mechanosensory input. The tectal neurons that comprise the optic tectum are organized into layers. These neurons project their dendrites laterally into the neuropil where visual inputs target the distal region of the dendrite and nonvisual inputs target the proximal region of the same dendrite. The Xenopus tadpole tectum is a popular model to study the development of sensory circuits. However, whole cell patch-clamp electrophysiological studies of the tadpole tectum (using the whole brain or in vivo preparations) have focused solely on the deep-layer tectal neurons because only neurons of the deep layer are visible and accessible for whole cell electrophysiological recordings. As a result, whereas the development and plasticity of these deep-layer neurons has been well-studied, essentially nothing has been reported about the electrophysiology of neurons residing beyond this layer. Hence, there exists a large gap in our understanding about the functional development of the amphibian tectum as a whole. To remedy this, we developed a novel isolated brain preparation that allows visualizing and recording from all layers of the tectum. We refer to this preparation as the "horizontal brain slice preparation." Here, we describe the preparation method and illustrate how it can be used to characterize the electrophysiology of neurons across all of the layers of the tectum as well as the spatial pattern of synaptic input from the different sensory modalities.

Region-specific regulation of voltage-gated intrinsic currents in the developing optic tectum of the Xenopus tadpole.

Across the rostrocaudal (RC) axis of the Xenopus tadpole optic tectum exists a developmental gradient. This gradient has served as a useful model to study many aspects of synapse and dendrite maturation. To compliment these studies, we characterized how the intrinsic excitability, the ease in which a neuron can fire action potentials, might also be changing across the same axis. Whole-cell recordings from tectal neurons at different points along the RC axis revealed a graded increase in intrinsic excitability: compared with neurons at the caudal end of the tectum, neurons at the rostral end fired more action potentials in response to current injection and expressed greater peak Na⁺ and K⁺ currents, the major intrinsic currents in these neurons that underlie the action potential. We also observed, along the same axis and in the same direction, a previously described increase in the amount of synaptic drive received by individual neurons (Wu GY, Malinow R, Cline HT. Science 274: 972-976, 1996). Thus as synaptic activity ramps up across the RC axis, so does intrinsic excitability. The reduction of overall circuit activity induced a compensatory scaling up of peak Na⁺ and K⁺ currents only in the caudal portion of the tectum, suggesting a region-specific, compensatory form of plasticity.