Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle pool in <0.5 millisecond. A comparison with action potential-evoked release suggested that a brief increase of [Ca2+]i to approximately 10 micromolar would be sufficient to reproduce the physiological release pattern. Thus, the calcium sensitivity of release at this synapse is high, and the distinction between phasic and delayed release is less pronounced than previously thought.

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted approximately 35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised approximately 5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 microm(2), and the nearest neighbors were separated, on average, by 0.59 microm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of approximately 60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were "anatomically docked." Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) approximately 35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP.

A basic question in the field of motor control is how different actions are represented by activity in spinal projection neurons. We used a new behavioral assay to identify visual stimuli that specifically drive basic motor patterns in zebrafish. These stimuli evoked consistent patterns of neural activity in the neurons projecting to the spinal cord, which we could map throughout the entire population using in vivo two-photon calcium imaging. We found that stimuli that drive distinct behaviors activated distinct subsets of projection neurons, consisting, in some cases, of just a few cells. This stands in contrast to the distributed activation seen for more complex behaviors. Furthermore, targeted cell by cell ablations of the neurons associated with evoked turns abolished the corresponding behavioral response. This description of the functional organization of the zebrafish motor system provides a framework for identifying the complete circuit underlying a vertebrate behavior.In a behaving animal, the brain communicates its intentions to muscles via the pattern of activity in descending projection neurons 1 . In vertebrates, these cells respond to the detection and processing of sensory stimuli and transmit their motor command to the local networks of the spinal cord, which in turn initiate and coordinate muscle contraction 2 . A fundamental question in neuroscience is how the commands that initiate behaviors are encoded in the activation of these projection neurons 3-5 .The spinal projection system of fish provides an excellent model for studying this code 6 . A diverse range of swimming behaviors can be seen in 6-d-old zebrafish 7-9 that are mediated by a descending projection to the spinal cord consisting of fewer than 300 neurons 10,11 . These neurons are easily labeled with fluorescent indicators 12 , optically accessible with modern imaging techniques and arranged in a stereotyped pattern such that the same cells or groups of cells can easily be identified from one fish to the next 13 . These neurons are morphologically diverse, with distinct dendritic fields and axonal projection patterns 13,14 , suggesting that they serve different behavioral functions. Nevertheless, determining how differing patterns of activity in these spinal projection neurons produce different motor outputs has proved to be difficult. 23,24 , the actual command is encoded in distributed activity throughout the population of control neurons 2 .The swimming behaviors of zebrafish, including the complex sequence of turns and swims that make up the escape response, can be broken down into basic kinematic elements 8 . It is possible that the motor commands for these basic behaviors originate from distinct populations of neurons, which when combined would appear as a `distributed command'. We found that whole-field visual motion, with the direction dynamically locked to the fish's body axis, is able to selectively evoke some of these basic swim patterns. Calcium imaging of stimulus-evoked responses in the complete population of n...

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.

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.

Transmitter release is triggered by highly localized, transient increases in the presynaptic Ca(2+) concentration ([Ca(2+)]). Rapidly decaying [Ca(2+)] elevations were generated using Ca(2+) uncaging techniques, and [Ca(2+)] was measured with a low-affinity Ca(2+) indicator in a giant presynaptic terminal, the calyx of Held, in rat brain slices. The rise time and amplitude of evoked excitatory postsynaptic currents (EPSCs) depended on the half-width of the fluorescence transient, which was predicted by a five-binding site model of a Ca(2+) sensor having relatively high affinity (K(d) approximately 13 microM). Very fast [Ca(2+)] transients (half-width <0.5 ms) evoked EPSCs similar to those elicited by a single action potential (AP) in the same synapse. Triggering release with dual [Ca(2+)] transients of variable amplitudes demonstrated the supralinear transfer function of the sensor. The sensitivity of release to the time course of the [Ca(2+)] transient may contribute to mechanisms by which the presynaptic AP waveform controls synaptic strength.

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.

SUMMARY Neural pathways projecting from sensory organs to higher brain centers form topographic maps in which neighbor relationships are preserved from a sending to a receiving neural population. Sensory input can generate compartmentalized electrical and biochemical activity in the dendrites of a receiving neuron. Here, we show that in the developing retinotectal projection of young Xenopus tadpoles, visually driven Ca2+ signals are topographically organized at the subcellular, dendritic scale. Functional in vivo two-photon Ca2+ imaging revealed that the sensitivity of dendritic Ca2+ signals to stimulus location in visual space is correlated with their anatomical position within the dendritic tree of individual neurons. This topographic distribution was dependent on NMDAR activation, whereas global Ca2+ signals were mediated by Ca2+ influx through dendritic, voltage-dependent Ca2+ channels. These findings suggest a framework for plasticity models that invoke local dendritic Ca2+ signaling in the elaboration of neural connectivity and dendrite-specific information storage.