Jian-young Wu

Spiral Waves in Disinhibited Mammalian Neocortex

Spiral waves are a basic feature of excitable systems. Although such waves have been observed in a variety of biological systems, they have not been observed in the mammalian cortex during neuronal activity. Here, we report stable rotating spiral waves in rat neocortical slices visualized by voltage-sensitive dye imaging. Tissue from the occipital cortex (visual) was sectioned parallel to cortical lamina to preserve horizontal connections in layers III-V (500-μm-thick, ∼4 × 6 mm2). In such tangential slices, excitation waves propagated in two dimensions during cholinergic oscillations. Spiral waves occurred spontaneously and alternated with plane, ring, and irregular waves. The rotation rate of the spirals was ∼10 turns per second, and the rotation was linked to the oscillations in a one-cycle- one-rotation manner. A small (<128 μm) phase singularity occurred at the center of the spirals, about which were observed oscillations of widely distributed phases. The phase singularity drifted slowly across the tissue (∼1 mm/10 turns). We introduced a computational model of a cortical layer that predicted and replicated many of the features of our experimental findings. We speculate that rotating spiral waves may provide a spatial framework to organize cortical oscillations.

Voltage-sensitive dye imaging of population neuronal activity in cortical tissue.

Voltage-sensitive dyes (VSDs) and optical imaging are useful for studying spatiotemporal patterns of population neuronal activity in cortical tissue. Using a photodiode array and absorption dyes we were able to detect neuronal activity in single trials before it could be detected by local field potential (LFP) recordings. Simultaneous electrical and optical recordings from the same tissue also showed that VSD and LFP signals have different waveforms during different activities, suggesting that they are sensitive to different aspects of the synchronization across the population. Noise, dye bleaching, phototoxicity and optical filter selection are important to the quality of the VSD signal and are discussed in this report. With optimized signal-to-noise ratio (S/N) and total recording time, we can optically monitor approximately 500 locations in an area of 1 mm(2) of cortical tissue with a sensitivity comparable to that of LFP electrodes. The total recording time and S/N of fluorescence and absorption dyes are also compared. At S/N of 8-10, absorption dye NK3630 allows a total recording time of 15-30 min, which can be divided into hundreds of 4-8 s recording trials over several hours, long enough for many kinds of experiments. In conclusion, the VSD method provides a reliable way for examining neuronal activity and pharmacological properties of synapses in brain slices.

TrkB signaling in parvalbumin-positive interneurons is critical for gamma-band network synchronization in hippocampus

Although brain-derived neurotrophic factor (BDNF) is known to regulate circuit development and synaptic plasticity, its exact role in neuronal network activity remains elusive. Using mutant mice (TrkB-PV−/−) in which the gene for the BDNF receptor, tyrosine kinase B receptor (trkB), has been specifically deleted in parvalbumin-expressing, fast-spiking GABAergic (PV+) interneurons, we show that TrkB is structurally and functionally important for the integrity of the hippocampal network. The amplitude of glutamatergic inputs to PV+ interneurons and the frequency of GABAergic inputs to excitatory pyramidal cells were reduced in the TrkB-PV−/− mice. Functionally, rhythmic network activity in the gamma-frequency band (30–80 Hz) was significantly decreased in hippocampal area CA1. This decrease was caused by a desynchronization and overall reduction in frequency of action potentials generated in PV+ interneurons of TrkB-PV−/− mice. Our results show that the integration of PV+ interneurons into the hippocampal microcircuit is impaired in TrkB-PV−/− mice, resulting in decreased rhythmic network activity in the gamma-frequency band.

INTERACTIONS BETWEEN TWO PROPAGATING WAVES IN RAT VISUAL CORTEX

Sensory-evoked propagating waves are frequently observed in sensory cortex. However, it is largely unknown how an evoked propagating wave affects the activity evoked by subsequent sensory inputs, or how two propagating waves interact when evoked by simultaneous sensory inputs. Using voltage-sensitive dye imaging, we investigated the interactions between two evoked waves in rat visual cortex, and the spatiotemporal patterns of depolarization in the neuronal population due to wave-to-wave interactions. We have found that visually-evoked propagating waves have a refractory period of about 300 ms, within which the response to a subsequent visual stimulus is suppressed. Simultaneous presentation of two visual stimuli at different locations can evoke two waves propagating toward each other, and these two waves fuse. Fusion significantly shortens the latency and half-width of the response, leading to changes in the spatial profile of evoked population activity. The visually-evoked propagating wave may also be suppressed by a preceding spontaneous wave. The refractory period following a propagating wave and the fusion between two waves may contribute to visual sensory processing by modifying the spatiotemporal profile of population neuronal activity evoked by sensory events.

Priming with real motion biases visual cortical response to bistable apparent motion

Apparent motion quartet is an ambiguous stimulus that elicits bistable perception, with the perceived motion alternating between two orthogonal paths. In human psychophysical experiments, the probability of perceiving motion in each path is greatly enhanced by a brief exposure to real motion along that path. To examine the neural mechanism underlying this priming effect, we used voltage-sensitive dye (VSD) imaging to measure the spatiotemporal activity in the primary visual cortex (V1) of awake mice. We found that a brief real motion stimulus transiently biased the cortical response to subsequent apparent motion toward the spatiotemporal pattern representing the real motion. Furthermore, intracellular recording from V1 neurons in anesthetized mice showed a similar increase in subthreshold depolarization in the neurons representing the path of real motion. Such short-term plasticity in early visual circuits may contribute to the priming effect in bistable visual perception.

5-HT3a Receptors Modulate Hippocampal Gamma Oscillations by Regulating Synchrony of Parvalbumin-Positive Interneurons

Gamma-frequency oscillatory activity plays an important role in information integration across brain areas. Disruption in gamma oscillations is implicated in cognitive impairments in psychiatric disorders, and 5-HT3 receptors (5-HT3Rs) are suggested as therapeutic targets for cognitive dysfunction in psychiatric disorders. Using a 5-HT3aR-EGFP transgenic mouse line and inducing gamma oscillations by carbachol in hippocampal slices, we show that activation of 5-HT3aRs, which are exclusively expressed in cholecystokinin (CCK)-containing interneurons, selectively suppressed and desynchronized firings in these interneurons by enhancing spike-frequency accommodation in a small conductance potassium (SK)-channel-dependent manner. Parvalbumin-positive interneurons therefore received diminished inhibitory input leading to increased but desynchronized firings of PV cells. As a consequence, the firing of pyramidal neurons was desynchronized and gamma oscillations were impaired. These effects were independent of 5-HT3aR-mediated CCK release. Our results therefore revealed an important role of 5-HT3aRs in gamma oscillations and identified a novel crosstalk among different types of interneurons for regulation of network oscillations. The functional link between 5-HT3aR and gamma oscillations may have implications for understanding the cognitive impairments in psychiatric disorders.

The role of inhibition in oscillatory wave dynamics in the cortex

Cortical oscillations arise during behavioral and mental tasks, and all temporal oscillations have particular spatial patterns. Studying the mechanisms that generate and modulate the spatiotemporal characteristics of oscillations is important for understanding neural information processing and the signs and symptoms of dynamical diseases of the brain. Nevertheless, it remains unclear how GABAergic inhibition modulates these oscillation dynamics. Using voltage‐sensitive dye imaging, pharmacological methods, and tangentially cut occipital neocortical brain slices (including layers 3–5) of Sprague‐Dawley rat, we found that GABAa disinhibition with bicuculline can progressively simplify oscillation dynamics in the presence of carbachol in a concentration‐dependent manner. Additionally, GABAb disinhibition can further simplify oscillation dynamics after GABAa receptors are blocked. Both GABAa and GABAb disinhibition increase the synchronization of the neural network. Theta frequency (5–15‐Hz) oscillations are reliably generated by using a combination of GABAa and GABAb antagonists alone. These theta oscillations have basic spatiotemporal patterns similar to those generated by carbachol/bicuculline. These results are illustrative of how GABAergic inhibition increases the complexity of patterns of activity and contributes to the regulation of the cortex.

Dynamical evolution of spatiotemporal patterns in mammalian middle cortex

The spatiotemporal structure of brain oscillations are important in understanding neural function. We analyze oscillatory episodes from isotropic preparations from the middle layers of a mammalian cortex which display irregular and chaotic spatiotemporal wave activity, within which spontaneously emerge spiral and plane waves. The dimensionality of these dynamics shows a consistent decrease during the middle of these episodes, regardless of the presence of simple spiral or plane waves. It is important to define the relevant biological order parameters which govern these dynamical bifurcations.

Pacing Hippocampal Sharp-Wave Ripples With Weak Electric Stimulation

Sharp-wave ripples (SWRs) are spontaneous neuronal population events that occur in the hippocampus during sleep and quiet restfulness, and are thought to play a critical role in the consolidation of episodic memory. SWRs occur at a rate of 30–200 events per minute. Their overall abundance may, however, be reduced with aging and neurodegenerative disease. Here we report that the abundance of SWR within murine hippocampal slices can be increased by paced administration of a weak electrical stimulus, especially when the spontaneously occurring rate is low or compromised. Resultant SWRs have large variations in amplitude and ripple patterns, which are morphologically indistinguishable from those of spontaneous SWRs, despite identical stimulus parameters which presumably activate the same CA3 neurons surrounding the electrode. The stimulus intensity for reliably pacing SWRs is weaker than that required for inducing detectable evoked field potentials in CA1. Moreover, repetitive ~1 Hz stimuli with low intensity can reliably evoke thousands of SWRs without detectable LTD or “habituation.” Our results suggest that weak stimuli may facilitate the spontaneous emergence of SWRs without significantly altering their characteristics. Pacing SWRs with weak electric stimuli could potentially be useful for restoring their abundance in the damaged hippocampus.

Now single spines: monitoring neuronal membrane potential with submicron and submillisecond resolution.

Since the spectacular and mysterious morphology of neurons was visualized by Cajal, scientists have wondered what happens when an action potential winds through the trajectory of the dendritic tree and reaches fine branches and synaptic spines. However, until recently we have not had a way to measure the time course, size and shape of a back-propagating action potential through a neurons fine branches. Such capability is important to our understanding of how information is processed in the dendritic tree and stored at the synapses. Pioneered by Antic & Zecevic (1995), the ‘inside dye’ technique allows us to monitor membrane potential from hundred of locations on a single neuron. The method is to infuse a voltage-sensitive dye (VSD) into a neuron with a patch pipette, allowing the dye to bind to the cellular membrane from inside, and to measure the dye signals that reflect the transmembrane potentials at different locations on the neuron. Voltage-sensitive dyes work differently from calcium dyes; they have to bind to the cellular membrane in order to measure the voltage across the membrane. Any unbound intracellular dye only adds noise. Thus the signal-to-noise ratio increases in finer branches, because the ratio of membrane area to intracellular volume increases. This method works better in fine branches than in the soma, which is the opposite of the patch-electrode techniques, which work better on larger structures. In a recent issue of The Journal of Physiology, Holthoff et al. (2010) have demonstrated improved methods with greatly increased signal-to-noise ratio. Terminal dendritic branches and individual synaptic spines can be monitored with a sensitivity that was not imagined by the original founders of voltage imaging. As described in Holthoff et al. (2010), the magic is achieved with the help of lasers. At fine neuronal branches the low density of photons limits the signal-to-noise ratio (S/N). The illumination intensity from an arc lamp is limited mainly by factors related to focusability of the emitted light. Solid state lasers, on the other hand, are, in biology, a practically limitless source of intensity. A second advantage of a laser source is the ability to use the wavelength where the dye has the largest fractional fluorescence change. As the report shows, the overall effect of using a near optimal wavelength at high intensity resulted in a S/N improvement by a factor of 10–40. This improvement resulted in a method with sub-micrometer and sub-millisecond resolution (Fig. 1). Figure 1 Locations now accessible for membrane potential measurements Whats next? Clearly, this new tool will enable neuroscientists to monitor electrical signals from axons, axon collaterals and axon terminals, as well as from terminal dendrites and individual dendritic spines, all important but tiny parts of neurons that are difficult to probe for electrical signals with any other measurement technique. There are a number of unresolved questions related to the physiology and pathology of these structures that can now be explored by direct measurement. One prominent example is the unresolved electrical role of dendritic spines in neuronal signal processing. Another area of research that will be greatly facilitated by high-sensitivity voltage imaging is the characterization of the currently unknown natural input to a dendritic tree (the spatio-temporal pattern of synaptic inputs). This information is critical for understanding the input–output function of any neuron. More ‘natural’ experiments should also be possible. Thus far, the action potentials in imaging experiments were evoked by some form of artificial stimulation of a neuron. It would be far more interesting to monitor action potentials generated naturally by the network. In a brain slice there are a variety of network events such as up–down states, theta oscillations and gamma oscillations. In these events it would be important to see if the shape of action potentials changes as well as where each action potential is initiated. In addition, most of the inside dye studies label and measure one neuron at a time. Can one inject a number of electrophysiologically identified pyramidal and fast-spiking neurons and watch their interactions during gamma oscillations? It would be possible to directly test the hypothesis that when oscillation frequency increases, the activations of pyramidal and GABAergic interneurons become more in phase (Mann & Mody, 2010). Finally, when the method is used in vivo, with the help of genetically encoded sensors and two-photon microscopy, one should be able to watch spike timing-dependent plasticity (Markram et al. 1997) at fine branches in which the temporal relationships can be largely different from location to location in the same neuronal pair. Several strategies for improving the fractional fluorescence change are under investigation. For example, the signal would be increased if unbound dye is quenched. Or a FRET mechanism or protein sensors may generate larger signals (Bradley et al. 2009). Thus the remarkable achievement of Holthoff et al. may not be the end of the line.