Intrinsic and Synaptic Contributions to Repetitive Spiking in Dentate Granule Cells.

Repetitive firing of granule cells (GCs) in the dentate gyrus (DG) facilitates synaptic transmission to the CA3 region. This facilitation can gate and amplify the flow of information through the hippocampus. High-frequency bursts in the DG are linked to behavior and plasticity, but GCs do not readily burst. Under normal conditions, a single shock to the perforant path in a hippocampal slice typically drives a GC to fire a single spike, and only occasionally more than one spike is seen. Repetitive spiking in GCs is not robust, and the mechanisms are poorly understood. Here, we used a hybrid genetically encoded voltage sensor to image voltage changes evoked by cortical inputs in many mature GCs simultaneously in hippocampal slices from male and female mice. This enabled us to study relatively infrequent double and triple spikes. We found GCs are relatively homogeneous and their double spiking behavior is cell autonomous. Blockade of GABA type A receptors increased multiple spikes and prolonged the interspike interval, indicating inhibitory interneurons limit repetitive spiking and set the time window for successive spikes. Inhibiting synaptic glutamate release showed that recurrent excitation mediated by hilar mossy cells contributes to, but is not necessary for, multiple spiking. Blockade of T-type Ca2+ channels did not reduce multiple spiking but prolonged interspike intervals. Imaging voltage changes in different GC compartments revealed that second spikes can be initiated in either dendrites or somata. Thus, pharmacological and biophysical experiments reveal roles for both synaptic circuitry and intrinsic excitability in GC repetitive spiking.

Velocity of conduction between columns and layers in barrel cortex reported by parvalbumin interneurons.

Inhibitory interneurons expressing parvalbumin (PV) play critical roles throughout the brain. Their rapid spiking enables them to control circuit dynamics on a millisecond time scale, and the timing of their activation by different excitatory pathways is critical to these functions. We used a genetically encoded hybrid voltage sensor to image PV interneuron voltage changes with sub-millisecond precision in primary somatosensory barrel cortex (BC) of adult mice. Electrical stimulation evoked depolarizations with a latency that increased with distance from the stimulating electrode, allowing us to determine conduction velocity. Spread of responses between cortical layers yielded an interlaminar conduction velocity and spread within layers yielded intralaminar conduction velocities in different layers. Velocities ranged from 74 to 473 µm/ms depending on trajectory; interlaminar conduction was 71% faster than intralaminar conduction. Thus, computations within columns are more rapid than between columns. The BC integrates thalamic and intracortical input for functions such as texture discrimination and sensory tuning. Timing differences between intra- and interlaminar PV interneuron activation could impact these functions. Imaging of voltage in PV interneurons reveals differences in signaling dynamics within cortical circuitry. This approach offers a unique opportunity to investigate conduction in populations of axons based on their targeting specificity.

Unitary synaptic responses of parvalbumin interneurons evoked by excitatory neurons in the mouse barrel cortex.

The mammalian cortex integrates and processes information to transform sensory inputs into perceptions and motor outputs. These operations are performed by networks of excitatory and inhibitory neurons distributed through the cortical layers. Parvalbumin interneurons (PVIs) are the most abundant type of inhibitory cortical neuron. With axons projecting within and between layers, PVIs supply feedforward and feedback inhibition to control and modulate circuit function. Distinct populations of excitatory neurons recruit different PVI populations, but the specializations of these synapses are poorly understood. Here, we targeted a genetically encoded hybrid voltage sensor to PVIs and used fluorescence imaging in mouse somatosensory cortex slices to record their voltage changes. Stimulating a single visually identified excitatory neuron with small-tipped theta-glass electrodes depolarized multiple PVIs, and a common threshold suggested that stimulation elicited unitary synaptic potentials in response to a single excitatory neuron. Excitatory neurons depolarized PVIs in multiple layers, with the most residing in the layer of the stimulated neuron. Spiny stellate cells depolarized PVIs more strongly than pyramidal cells by up to 77%, suggesting a greater role for stellate cells in recruiting PVI inhibition and controlling cortical computations. Response half-width also varied between different excitatory inputs. These results demonstrate functional differences between excitatory synapses on PVIs.

Synaptophysin transmembrane domain III controls fusion pore dynamics in Ca2+-triggered exocytosis.

Synaptophysin (syp) is a major protein of secretory vesicles with four transmembrane domains (TMDs) and a large cytoplasmic C-terminus. Syp has been shown to regulate exocytosis, vesicle cycling, and synaptic plasticity through its C-terminus. However, the roles of its TMDs remain unclear. The TMDs of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are thought to line initial fusion pores, and structural work together with sequence analysis suggest that TMD III of syp may play a similar role. To test this hypothesis, we performed tryptophan scanning experiments of TMD III in chromaffin cells and used amperometry to evaluate fusion pores. In contrast to SNARE TMDs, tryptophan substitutions in syp TMD III had no effect on the flux through initial fusion pores. However, a number of these mutants increased the fraction of kiss-and-run events and decreased the initial fusion pore lifetime. These results indicate that TMD III stabilizes the initial fusion pore and controls the initial choice between kiss and run and full fusion. Late-stage fusion pores were not impacted by TMD III mutations. These results indicate that syp TMD III does not line the initial fusion pore. However, its impact on pore dynamics suggests that it interacts with a SNARE protein implicated as a part of the fusion pore that forms at the onset of exocytosis.

Synaptophysin Regulates Fusion Pores and Exocytosis Mode in Chromaffin Cells.

Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but the role of syp in the process of membrane fusion during Ca2+-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin.SIGNIFICANCE STATEMENT The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca2+-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.

Direct synaptic excitation between hilar mossy cells revealed with a targeted voltage sensor.

The dentate gyrus not only gates the flow of information into the hippocampus, it also integrates and processes this information. Mossy cells (MCs) are a major type of excitatory neuron strategically located in the hilus of the dentate gyrus where they can contribute to this processing through networks of synapses with inhibitory neurons and dentate granule cells. Some prior work has suggested that MCs can form excitatory synapses with other MCs, but the role of these synapses in the network activity of the dentate gyrus has received little attention. Here, we investigated synaptic inputs to MCs in mouse hippocampal slices using a genetically encoded hybrid voltage sensor (hVOS) targeted to MCs by Cre-lox technology. This enabled optical recording of voltage changes from multiple MCs simultaneously. Stimulating granule cells and CA3 pyramidal cells activated well-established inputs to MCs and elicited synaptic responses as expected. However, the weak blockade of MC responses to granule cell layer stimulation by DCG-IV raised the possibility of another source of excitation. To evaluate synapses between MCs as this source, single MCs were stimulated focally. Stimulation of one MC above its action potential threshold evoked depolarizing responses in neighboring MCs that depended on glutamate receptors. Short latency responses of MCs to other MCs did not depend on release from granule cell axons. However, granule cells did contribute to the longer latency responses of MCs to stimulation of other MCs. Thus, MCs transmit their activity to other MCs both through direct synaptic coupling and through polysynaptic coupling with dentate granule cells. MC-MC synapses can redistribute information entering the dentate gyrus and thus shape and modulate the electrical activity underlying hippocampal functions such as navigation and memory, as well as excessive excitation during seizures.

Fusion Pore Expansion and Contraction during Catecholamine Release from Endocrine Cells.

Amperometry recording reveals the exocytosis of catecholamine from individual vesicles as a sequential process, typically beginning slowly with a prespike foot, accelerating sharply to initiate a spike, reaching a peak, and then decaying. This complex sequence reflects the interplay between diffusion, flux through a fusion pore, and possibly dissociation from a vesicle's dense core. In an effort to evaluate the impacts of these factors, a model was developed that combines diffusion with flux through a static pore. This model accurately recapitulated the rapid phase of a spike but generated relations between spike shape parameters that differed from the relations observed experimentally. To explore the possible role of fusion pore dynamics, a transformation of amperometry current was introduced that yields fusion pore permeability divided by vesicle volume (g/V). Applying this transform to individual fusion events yielded a highly characteristic time course. g/V initially tracks the current, increasing ∼15-fold from the prespike foot to the spike peak. After the peak, g/V unexpectedly declines and settles into a plateau that indicates the presence of a stable postspike pore. g/V of the postspike pore varies greatly between events and has an average that is ∼3.5-fold below the peak value and ∼4.5-fold above the prespike value. The postspike pore persists and is stable for tens of milliseconds, as long as catecholamine flux can be detected. Applying the g/V transform to rare events with two peaks revealed a stepwise increase in g/V during the second peak. The g/V transform offers an interpretation of amperometric current in terms of fusion pore dynamics and provides a, to our knowledge, new frameworkfor analyzing the actions of proteins that alter spike shape. The stable postspike pore follows from predictions of lipid bilayer elasticity and offers an explanation for previous reports of prolonged hormone retention within fusing vesicles.

Hebbian and non-Hebbian timing-dependent plasticity in the hippocampal CA3 region.

The timing between synaptic inputs has been proposed to play a role in the induction of plastic changes that enable neural circuits to store information. In the case of spike timing-dependent plasticity (STDP), this relates to the interval between a synaptic input and a postsynaptic spike, thus providing a conceptual link to the Hebb learning rule. Experiments have documented STDP in many synapses and brain regions, and computational models have tested its utility in many neural network functions. However, questions remain about whether timing plays a role in plasticity during natural activity, and whether it can function in information storage. The present study used imaging with voltage sensitive dye to investigate the effectiveness of input timing in the plasticity of responses in the CA3 region of hippocampal slices. Plasticity was induced by sequential dual-site stimulation at 10 ms intervals of either synaptic inputs and cell bodies (synaptic-somatic induction) or of two sets of synaptic inputs (synaptic-synaptic induction). Both protocols potentiated responses, with greater potentiation of responses to the first stimulation of the sequence than the second. Neither of these protocols induced depression. Synaptic-somatic stimulation was much more effective than synaptic-synaptic stimulation in evoking somatic action potentials, but both protocols potentiated responses equally well. This suggests that sequential dual-site stimulation can potentiate equally well with very different degrees of somatic action potential firing. With synaptic-somatic induction, potentiation was focused at the sites of stimulation. In contrast, with synaptic-synaptic induction, the distribution of potentiation varied greatly. Changes in the spatial distribution of responses indicated that sequential dual-site stimulation functions poorly in the storage of activity patterns. These results suggest that in the hippocampal CA3 region, timed sequential activation of two inputs is less effective than theta bursts, both in the induction of LTP and in the storage of information.

The Transmembrane Domain of Synaptobrevin Influences Neurotransmitter Flux through Synaptic Fusion Pores.

The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca2+-triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDs form the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-clamp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores. TMD mutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca2+-triggered exocytosis in mammalian cells.SIGNIFICANCE STATEMENT SNARE proteins function broadly in biological membrane fusion. Evidence from non-neuronal systems suggests that SNARE proteins initiate fusion by forming a fusion pore lined by transmembrane domains, but this model has not yet been tested in synapses. The present study addressed this question by testing mutations in the synaptic vesicle SNARE synaptobrevin for an influence on the rise rate of miniature synaptic currents. These results indicate that synaptobrevin's transmembrane domain interacts with glutamate as it passes through the fusion pore. The sites in synaptobrevin that influence this flux are identical to those shown previously to influence flux through endocrine fusion pores. Thus, SNARE transmembrane domains may function in the fusion pores of Ca2+-triggered exocytosis of both neurotransmitters and hormones.

Imaging Voltage in Genetically Defined Neuronal Subpopulations with a Cre Recombinase-Targeted Hybrid Voltage Sensor.

Genetically encoded voltage indicators create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of genetically encoded voltage indicators requires a generalized strategy for targeting the probe to genetically defined populations of cells. To this end, we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase-dependent expression. Crossing this mouse with Cre drivers generated double transgenics expressing hVOS probe in GABAergic, parvalbumin, and calretinin interneurons, as well as hilar mossy cells, new adult-born neurons, and recently active neurons. In each case, imaging in brain slices from male or female animals revealed electrically evoked optical signals from multiple individual neurons in single trials. These imaging experiments revealed action potentials, dynamic aspects of dendritic integration, and trial-to-trial fluctuations in response latency. The rapid time response of hVOS imaging revealed action potentials with high temporal fidelity, and enabled accurate measurements of spike half-widths characteristic of each cell type. Simultaneous recording of rapid voltage changes in multiple neurons with a common genetic signature offers a powerful approach to the study of neural circuit function and the investigation of how neural networks encode, process, and store information.SIGNIFICANCE STATEMENT Genetically encoded voltage indicators hold great promise in the study of neural circuitry, but realizing their full potential depends on targeting the sensor to distinct cell types. Here we present a new mouse line that expresses a hybrid optical voltage sensor under the control of Cre recombinase. Crossing this line with Cre drivers generated double-transgenic mice, which express this sensor in targeted cell types. In brain slices from these animals, single-trial hybrid optical voltage sensor recordings revealed voltage changes with submillisecond resolution in multiple neurons simultaneously. This imaging tool will allow for the study of the emergent properties of neural circuits and permit experimental tests of the roles of specific types of neurons in complex circuit activity.

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion.

SNARE proteins catalyze many forms of biological membrane fusion, including Ca(2+)-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca(2+)-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca(2+)-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.

A structural role for the synaptobrevin 2 transmembrane domain in dense-core vesicle fusion pores.

Ca(2+)-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two α-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD.

A sodium afterdepolarization in rat superior colliculus neurons and its contribution to population activity.

The mammalian superior colliculus (SC) is a midbrain structure that integrates multimodal sensory inputs and computes commands to initiate rapid eye movements. SC neurons burst with the sudden onset of a visual stimulus, followed by persistent activity that may underlie shifts of attention and decision making. Experiments in vitro suggest that circuit reverberations play a role in the burst activity in the SC, but the origin of persistent activity is unclear. In the present study we characterized an afterdepolarization (ADP) that follows action potentials in slices of rat SC. Population responses seen with voltage-sensitive dye imaging consisted of rapid spikes followed immediately by a second distinct depolarization of lower amplitude and longer duration. Patch-clamp recordings showed qualitatively similar behavior: in nearly all neurons throughout the SC, rapid spikes were followed by an ADP. Ionic and pharmacological manipulations along with experiments with current and voltage steps indicated that the ADP of SC neurons arises from Na(+) current that either persists or resurges following Na(+) channel inactivation at the end of an action potential. Comparisons of pharmacological properties and frequency dependence revealed a clear parallel between patch-clamp recordings and voltage imaging experiments, indicating a common underlying membrane mechanism for the ADP in both single neurons and populations. The ADP can initiate repetitive spiking at intervals consistent with the frequency of persistent activity in the SC. These results indicate that SC neurons have intrinsic membrane properties that can contribute to electrical activity that underlies shifts of attention and decision making.

Synaptobrevin transmembrane domain influences exocytosis by perturbing vesicle membrane curvature.

Membrane fusion requires that nearly flat lipid bilayers deform into shapes with very high curvature. This makes membrane bending a critical force in determining fusion mechanisms. A lipid bilayer will bend spontaneously when material is distributed asymmetrically between its two monolayers, and its spontaneous curvature (C0) will influence the stability of curved fusion intermediates. Prior work on Ca(2+)-triggered exocytosis revealed that fusion pore lifetime (τ) varies with vesicle content (Q), and showed that this relation reflects membrane bending energetics. Lipids that alter C0 change the dependence of τ on Q. These results suggested that the greater stability of an initial exocytotic fusion pore associated with larger vesicles reflects the need to bend more membrane during fusion pore dilation. In this study, we explored the possibility of manipulating C0 by mutating the transmembrane domain (TMD) of the vesicle membrane protein synaptobrevin 2 (syb2). Amperometric measurements of exocytosis in mouse chromaffin cells revealed that syb2 TMD mutations altered the relation between τ and Q. The effects of these mutations showed a striking periodicity, changing sign as the structural perturbation moved through the inner and outer leaflets. Some glycine and charge mutations also influenced the dependence of τ on Q in a manner consistent with expected changes in C0. These results suggest that side chains in the syb2 TMD influence the kinetics of exocytosis by perturbing the packing of the surrounding lipids. The present results support the view that membrane bending occurs during fusion pore expansion rather than during fusion pore formation. This supports the view of an initial fusion pore through two relatively flat membranes formed by protein.

Long-term potentiation in hilar circuitry modulates gating by the dentate gyrus.

The dentate gyrus serves as a gateway to the hippocampus, filtering and processing sensory inputs as an animal explores its environment. The hilus occupies a strategic position within the dentate gyrus from which it can play a pivotal role in these functions. Inputs from dentate granule cells converge on the hilus, and excitatory hilar mossy cells redistribute these signals back to granule cells to transform a pattern of cortical input into a new pattern of output to the hippocampal CA3 region. Using voltage-sensitive dye to image electrical activity in rat hippocampal slices, we explored how long-term potentiation (LTP) of different excitatory synapses modifies the flow of information. Theta burst stimulation of the perforant path potentiated responses throughout the molecular layer, but left responses in the CA3 region unchanged. By contrast, theta burst stimulation of the granule cell layer potentiated responses throughout the molecular layer, as well as in the CA3 region. Theta burst stimulation of the granule cell layer potentiated CA3 responses not only to granule cell layer stimulation but also to perforant path stimulation. Potentiation of responses in the CA3 region reflected NMDA receptor-dependent LTP of upstream synapses between granule cells and mossy cells, with no detectable contribution from NMDA receptor-independent LTP of local CA3 mossy fiber synapses. Potentiation of transmission to the CA3 region required LTP in both granule cell→mossy cell and mossy cell→granule cell synapses. This bidirectional plasticity enables hilar circuitry to regulate the flow of information through the dentate gyrus and on to the hippocampus.

Response normalization in the superficial layers of the superior colliculus as a possible mechanism for saccadic averaging.

How does the brain decide where to look? Neuronal networks within the superior colliculus (SC) encode locations of intended eye movements. When faced with multiple targets, the relative activities of neuronal populations compete for the selection of a saccade. However, the computational principles underlying saccadic choices remain poorly understood. We used voltage imaging of slices of rat SC to record circuit dynamics of population responses to single- and dual-site electrical stimulation to begin to reveal some of the principles of how populations of neurons interact. Stimulation of two distant sites simultaneously within the SC produced two distinct peaks of activity, whereas stimulation of two nearby sites simultaneously exhibited a single, merged peak centered between the two sites. The distances required to produce merged peaks of activity corresponded to target separations that evoked averaging saccades in humans performing a corresponding dual target task. The merged activity was well accounted for by a linear weighed summation and a divisive normalization of the responses evoked by the single-site stimulations. Interestingly, the merging of activity occurred within the superficial SC, suggesting a novel pathway for saccadic eye movement choice.

Excitatory synaptic feedback from the motor layer to the sensory layers of the superior colliculus.

Neural circuits that translate sensory information into motor commands are organized in a feedforward manner converting sensory information into motor output. The superior colliculus (SC) follows this pattern as it plays a role in converting visual information from the retina and visual cortex into motor commands for rapid eye movements (saccades). Feedback from movement to sensory regions is hypothesized to play critical roles in attention, visual image stability, and saccadic suppression, but in contrast to feedforward pathways, motor feedback to sensory regions has received much less attention. The present study used voltage imaging and patch-clamp recording in slices of rat SC to test the hypothesis of an excitatory synaptic pathway from the motor layers of the SC back to the sensory superficial layers. Voltage imaging revealed an extensive depolarization of the superficial layers evoked by electrical stimulation of the motor layers. A pharmacologically isolated excitatory synaptic potential in the superficial layers depended on stimulus strength in the motor layers in a manner consistent with orthodromic excitation. Patch-clamp recording from neurons in the sensory layers revealed excitatory synaptic potentials in response to glutamate application in the motor layers. The location, size, and morphology of responsive neurons indicated they were likely to be narrow-field vertical cells. This excitatory projection from motor to sensory layers adds an important element to the circuitry of the SC and reveals a novel feedback pathway that could play a role in enhancing sensory responses to attended targets as well as visual image stabilization.

Single-trial imaging of spikes and synaptic potentials in single neurons in brain slices with genetically encoded hybrid voltage sensor.

Genetically encoded voltage sensors expand the optogenetics toolkit into the important realm of electrical recording, enabling researchers to study the dynamic activity of complex neural circuits in real time. However, these probes have thus far performed poorly when tested in intact neural circuits. Hybrid voltage sensors (hVOS) enable the imaging of voltage by harnessing the resonant energy transfer that occurs between a genetically encoded component, a membrane-tethered fluorescent protein that serves as a donor, and a small charged molecule, dipicrylamine, which serves as an acceptor. hVOS generates optical signals as a result of voltage-induced changes in donor-acceptor distance. We expressed the hVOS probe in mouse brain by in utero electroporation and in transgenic mice with a neuronal promoter. Under conditions favoring sparse labeling we could visualize single-labeled neurons. hVOS imaging reported electrically evoked fluorescence changes from individual neurons in slices from entorhinal cortex, somatosensory cortex, and hippocampus. These fluorescence signals tracked action potentials in individual neurons in a single trial with excellent temporal fidelity, producing changes that exceeded background noise by as much as 16-fold. Subthreshold synaptic potentials were detected in single trials in multiple distinct cells simultaneously. We followed signal propagation between different cells within one field of view and between dendrites and somata of the same cell. hVOS imaging thus provides a tool for high-resolution recording of electrical activity from genetically targeted cells in intact neuronal circuits.

A hard-wired priority map in the superior colliculus shaped by asymmetric inhibitory circuitry.

The mammalian superior colliculus (SC) is a laminar midbrain structure that translates visual signals into commands to shift the focus of attention and gaze. The SC plays an integral role in selecting targets and ultimately generating rapid eye movements to those targets. In all mammals studied to date, neurons in the SC are arranged topographically such that the location of visual stimuli and the endpoints of orienting movements form organized maps in superficial and deeper layers, respectively. The organization of these maps is thought to underlie attentional priority by assessing which regions of the visual field contain behaviorally relevant information. Using voltage imaging and patch-clamp recordings in parasagittal SC slices from the rat, we found the synaptic circuitry of the visuosensory map in the SC imposes a strong bias. Voltage imaging of responses to electrical stimulation revealed more spread in the caudal direction than the rostral direction. Pharmacological experiments demonstrated that this asymmetry arises from GABAA receptor activation rostral to the site of stimulation. Patch-clamp recordings confirmed this rostrally directed inhibitory circuit and showed that it is contained within the visuosensory layers of the SC. Stimulation of two sites showed that initial stimulation of a caudal site can take priority over subsequent stimulation of a rostral site. Taken together, our data indicate that the circuitry of the visuosensory SC is hard-wired to give higher priority to more peripheral targets, and this property is conferred by a uniquely structured, dedicated inhibitory circuit.