Corey D. Acker

Synchronization of strongly coupled excitatory neurons: relating network behavior to biophysics.

Behavior of a network of neurons is closely tied to the properties of the individual neurons. We study this relationship in models of layer II stellate cells (SCs) of the medial entorhinal cortex. SCs are thought to contribute to the mammalian theta rhythm (4–12 Hz), and are notable for the slow ionic conductances that constrain them to fire at rates within this frequency range. We apply “spike time response” (STR) methods, in which the effects of synaptic perturbations on the timing of subsequent spikes are used to predict how these neurons may synchronize at theta frequencies. Predictions from STR methods are verified using network simulations. Slow conductances often make small inputs “effectively large”; we suggest that this is due to reduced attractiveness or stability of the spiking limit cycle. When inputs are (effectively) large, changes in firing times depend nonlinearly on synaptic strength. One consequence of nonlinearityis to make a periodically firing model skip one or more beats, often leading to the elimination of the anti-synchronous state in bistable models. Biologically realistic membrane noise makes such “cycle skipping” more prevalent, and thus can eradicate bistability. Membrane noise also supports “sparse synchrony,” a phenomenon in which subthreshold behavior is uncorrelated, but there are brief periods of synchronous spiking.

Palette of fluorinated voltage-sensitive hemicyanine dyes

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440–670 nm; the two-photon excitation range is 900–1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Beyond Two-Cell Networks: Experimental Measurement of Neuronal Responses to Multiple Synaptic Inputs

Oscillations of large populations of neurons are thought to be important in the normal functioning of the brain. We have used phase response curve (PRC) methods to characterize the dynamics of single neurons and predict population dynamics. Our past experimental work was limited to special circumstances (e.g., 2-cell networks of periodically firing neurons). Here, we explore the feasibility of extending our methods to predict the synchronization properties of stellate cells (SCs) in the rat entorhinal cortex under broader conditions. In particular, we test the hypothesis that PRCs in SCs scale linearly with changes in synaptic amplitude, and measure how well responses to Poisson process-driven inputs can be predicted in terms of PRCs. Although we see nonlinear responses to excitatory and inhibitory inputs, we find that models based on weak coupling account for scaling and Poisson process-driven inputs reasonably accurately.

Single-Voxel Recording of Voltage Transients in Dendritic Spines

We report sensitive recording of membrane potential in single dendritic spines in cortical neurons within a brain slice using two-photon excitation and a new, fluorinated, intracellularly loaded organic dye, di-2-AN(F)EPPTEA. With a two-photon excitation wavelength of 1060 nm, we achieve voltage sensitivity of >16% change in fluorescence per 100 mV. By targeting single spines in single-voxel recordings, we attain excellent single/noise quality, with back-propagating action potentials (bAPs) visible in single sweeps while recording at 10 kHz. This recording rate allows us to reliably assess fast bAP dynamics on single sweeps including bAP rise times of 0.5 ms. The amplitude and propagation delays of the bAPs are similar among different spines located within the same dendritic region, and this is true despite large differences in spine size. The interregion differences in bAP waveforms in spines vary in relation to their distance from the soma and the caliber of their parent dendrites.

Quantitative Assessment of the Distributions of Membrane Conductances Involved in Action Potential Backpropagation Along Basal Dendrites

Basal dendrites of prefrontal cortical neurons receive strong synaptic drive from recurrent excitatory synaptic inputs. Synaptic integration within basal dendrites is therefore likely to play an important role in cortical information processing. Both synaptic integration and synaptic plasticity depend crucially on dendritic membrane excitability and the backpropagation of action potentials. We carried out multisite voltage-sensitive dye imaging of membrane potential transients from thin basal branches of prefrontal cortical pyramidal neurons before and after application of channel blockers. We found that backpropagating action potentials (bAPs) are predominantly controlled by voltage-gated sodium and A-type potassium channels. In contrast, pharmacologically blocking the delayed rectifier potassium, voltage-gated calcium, or I(h) conductance had little effect on dendritic AP propagation. Optically recorded bAP waveforms were quantified and multicompartmental modeling was used to link the observed behavior with the underlying biophysical properties. The best-fit model included a nonuniform sodium channel distribution with decreasing conductance with distance from the soma, together with a nonuniform (increasing) A-type potassium conductance. AP amplitudes decline with distance in this model, but to a lesser extent than previously thought. We used this model to explore the mechanisms underlying two sets of published data involving high-frequency trains of APs and the local generation of sodium spikelets. We also explored the conditions under which I(A) down-regulation would produce branch strength potentiation in the proposed model. Finally, we discuss the hypothesis that a fraction of basal branches may have different membrane properties compared with sister branches in the same dendritic tree.

EPSPs Measured in Proximal Dendritic Spines of Cortical Pyramidal Neurons.

Abstract EPSPs occur when the neurotransmitter glutamate binds to postsynaptic receptors located on small pleomorphic membrane protrusions called dendritic spines. To transmit the synaptic signal, these potentials must travel through the spine neck and the dendritic tree to reach the soma. Due to their small size, the electrical behavior of spines and their ability to compartmentalize electrical signals has been very difficult to assess experimentally. In this study, we developed a method to perform simultaneous two-photon voltage-sensitive dye recording with two-photon glutamate uncaging in order to measure the characteristics (amplitude and duration) of uncaging-evoked EPSPs in single spines on the basal dendrites of L5 pyramidal neurons in acute brain slices from CD1 control mice. We were able to record uncaging-evoked spine potentials that resembled miniature EPSPs at the soma from a wide range of spine morphologies. In proximal spines, these potentials averaged 13.0 mV (range, 6.5–30.8 mV; N = 20) for an average somatic EPSP of 0.59 mV, whereas the mean attenuation ratio (spine/soma) was found to be 25.3. Durations of spine EPSP waveforms were found to be 11.7 ms on average. Modeling studies demonstrate the important role that spine neck resistance (Rneck) plays in spine EPSP amplitudes. Simulations used to estimate Rneck by fits to voltage-sensitive dye measurements produced a mean of 179 MΩ (range, 23–420 MΩ; N = 19). Independent measurements based on fluorescence recovery after photobleaching of a cytosolic dye from spines of the same population of neurons produced a mean Rneck estimate of 204 MΩ (range, 52–521 MΩ; N = 34).

Roles of IA and morphology in action potential propagation in CA1 pyramidal cell dendrites

Dendrites of CA1 pyramidal cells of the hippocampus, along with those of a wide range of other cell types, support active backpropagation of axonal action potentials. Consistent with previous work, recent experiments demonstrating that properties of synaptic plasticity are different for distal synapses, suggest an important functional role of bAPs, which are known to be prone to failure in distal locations. Using conductance-based models of CA1 pyramidal cells, we show that underlying “traveling wave attractors” control action potential propagation in the apical dendrites. By computing these attractors, we dissect and quantify the effects of IA channels and dendritic morphology on bAP amplitudes. We find that non-uniform activation properties of IA can lead to backpropagation failure similar to that observed experimentally in these cells. Amplitude of forward propagation of dendritic spikes also depends strongly on the activation dynamics of IA. IA channel properties also influence transients at dendritic branch points and whether or not propagation failure results. The branching pattern in the distal apical dendrites, combined with IA channel properties in this region, ensure propagation failure in the apical tuft for a large range of IA conductance densities. At the same time, these same properties ensure failure of forward propagating dendritic spikes initiated in the distal tuft in the absence of some form of cooperativity of synaptic activation.

Characterization of Voltage-Sensitive Dyes in Living Cells Using Two-Photon Excitation

In this protocol, we describe the procedures we have developed to optimize the performance of voltage-sensitive dyes for recording changes in neuronal electrical activity. We emphasize our experience in finding the best dye conditions for recording backpropagating action potentials from individual dendritic spines in a neuron within a brain slice. We fully describe procedures for loading the dye through a patch pipette and for finding excitation and emission wavelengths for the best sensitivity of the fluorescence signal to membrane voltage. Many of these approaches can be adapted to in vivo preparations and to experiments on mapping brain activity via optical recording.

Nonlinear AMPA Sensitivity to Glutamate and Recruitment of NMDA Activity in Dendritic Spines

Our lab has been developing and improving the optical and molecular tools and techniques to record EPSPs (excitatory postsynaptic potentials) in dendritic spines. With a dual laser 2-photon microscope we are able to record from voltage-sensitive dye (VSD) labeled spines from cortical pyramidal neurons while evoking unitary EPSPs using glutamate uncaging. By varying the uncaging laser intensity we observe a nonlinear response to glutamate when the resulting EPSPs amplitudes are observed at the soma. By optically recording EPSPs in spines we can determine that the observed nonlinearity is often present even when spine EPSPs are relatively small, i.e. < 10 mV. Pharmacology is used to block NMDA receptors and determine the recruitment of these receptors and their contribution to both voltage and calcium influx. Calcium imaging reveals that NMDA receptors are recruited in a very linear fashion according to the spine depolarization. This calcium influx however, does not appear to play a significant role in the spine depolarization. Finally, modeling is used to reconcile the observed phenomena with detailed models of AMPA and NMDA receptor gating, and glutamate binding.

Membrane Potential Imaging in Neurons using Fluorinated Voltage-Sensitive Dyes and a Custom Multiphoton Brain Slice Microscope

In order to fully understand the physiology of fundamental neurophysiological processes such as synaptic integration and synaptic plasticity, direct recording of changes in membrane potential neuronal dendrites and spines is essential. In an effort to improve voltage-sensitive dye measurements of synaptic potentials and backpropagating action potentials, our group has developed new fluorinated dyes with enhanced photostability. We have also made performance improvements on our custom, non-linear optical microscope for greater sensitivity. By modifying a commercial Zeiss microscope we have added two “up front” epifluorescence detection channels and one transfluorescence detection channel. Optics for these new light paths were optimized using numerical ray tracing. Here we show that we are able to fill individual neurons with these dyes via somatic patch pipettes and record membrane potential changes in the soma and dendrites of Purkinje neurons in cerebellar brain slices. Using voltage clamp protocols, membrane potential was changed in a stepwise fashion, resulting in changes in membrane fluorescence. When excited with 1060 nm light, the new dyes typically produced changes in fluorescence (dF/F) between 3 and 7 % for 50 mV changes in membrane potential. Feasibility of using second harmonic generation to record membrane potential with these dyes was investigated in a cultured cell line by measuring dSHG/SHG, kinetics, and intensity as a function of dye concentration.Work funded by the following NIH grants: R01-EB001963 and P41-RR013186.