Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Channelrhodopsin-2 Localised to the Axon Initial Segment

  • Matthew S. Grubb ,

    matthew.grubb@kcl.ac.uk

    Affiliation Medical Research Council (MRC) Centre for Developmental Neurobiology, King's College London, London, United Kingdom

  • Juan Burrone

    Affiliation Medical Research Council (MRC) Centre for Developmental Neurobiology, King's College London, London, United Kingdom

Channelrhodopsin-2 Localised to the Axon Initial Segment

  • Matthew S. Grubb, 
  • Juan Burrone
PLOS
x

Abstract

The light-gated cation channel Channelrhodopsin-2 (ChR2) is a powerful and versatile tool for controlling neuronal activity. Currently available versions of ChR2 either distribute uniformly throughout the plasma membrane or are localised specifically to somatodendritic or synaptic domains. Localising ChR2 instead to the axon initial segment (AIS) could prove an extremely useful addition to the optogenetic repertoire, targeting the channel directly to the site of action potential initiation, and limiting depolarisation and associated calcium entry elsewhere in the neuron. Here, we describe a ChR2 construct that we localised specifically to the AIS by adding the ankyrinG-binding loop of voltage-gated sodium channels (NavII-III) to its intracellular terminus. Expression of ChR2-YFP-NavII-III did not significantly affect the passive or active electrical properties of cultured rat hippocampal neurons. However, the tiny ChR2 currents and small membrane depolarisations resulting from AIS targeting meant that optogenetic control of action potential firing with ChR2-YFP-NavII-III was unsuccessful in baseline conditions. We did succeed in stimulating action potentials with light in some ChR2-YFP-NavII-III-expressing neurons, but only when blocking KCNQ voltage-gated potassium channels. We discuss possible alternative approaches to obtaining precise control of neuronal spiking with AIS-targeted optogenetic constructs and propose potential uses for our ChR2-YFP-NavII-III probe where subthreshold modulation of action potential initiation is desirable.

Introduction

Gaining precise control over neuronal activity in space and time is a major goal in experimental and translational neuroscience. In various model systems, and with increasingly sophisticated technology, attempts to activate endogenous neural tissue have employed electrical [e.g. [1],[2],[3],[4],[5]], magnetic [6] and ultrasound [7], [8] stimulation, with varying degrees of success. Recent years have also seen major advances in means of controlling neuronal activity using light [9]. Photocaged compounds such as neurotransmitters or divalent ions can be used in combination with focal photostimulation to mimic fast, local neuronal signals [10], while synthetically created photoswitch molecules can turn native or genetically altered proteins, including potassium channels [e.g. [11]] and glutamate receptors [e.g.[12]], into light-responsive proteins. Both of these approaches, however, require application of exogenous compounds to neuronal tissue. An alternative strategy is to genetically modify neurons so that they produce inherently light-responsive molecules, an approach known as optogenetics [13] (although optogenetic proteins do require additional chromophores such as all-trans retinal to become light-sensitive, these are usually already present in sufficient concentrations in in vitro neuronal preparations, and in the mammalian brain). The available range of optogenetic tools is becoming impressively large. We now have means of hyperpolarising neurons by activating chloride pumps with yellow light (halorhodopsin, or (e)NpHR) [14], [15], [16], [17], or by activating proton pumps with yellow or blue-green light (Arch and Mac, respectively) [18]. There are also increasingly sophisticated ways to depolarise neurons, including activation of a cation channel with yellow light (Volvox channelrhodopsin-1, or VChR1) [19], bi-stable excitation that can be switched on with blue light and off with green light (step-function opsins or SFOs) [20], [21], channelrhodopsin variants with improved kinetics (e.g. ChEF) [22], and ultra-fast light-sensitive channels for high-frequency neuronal stimulation (ChETA) [23]. The most intensely studied, best understood and most widely used optogenetic tool, however, is channelrhodopsin-2 (ChR2). ChR2 is a cation channel, isolated from the eyespot of Chlamydomonas algae, which is directly opened by illumination with a sufficient intensity of blue light (peak excitation ∼460 nm) [24]. When expressed in vertebrate neurons, it mobilises efficiently to the plasma membrane, and upon light stimulation can produce rapid, large currents carried predominantly by sodium, potassium and calcium ions [24], [25], [26]. Activating these conductances under resting physiological conditions depolarises the neuronal membrane, so if the light stimulus is sufficiently strong, photostimulation combined with ChR2 expression can be used to trigger action potentials with millisecond resolution [e.g. [25],[26]]. Amongst a plethora of current applications, ChR2-induced firing has now been employed in vitro and in vivo to map neuronal connectivity [27], [28], [29], [30], [31], [32], [33], [34], to induce synaptic plasticity [35], [36], to describe the activity patterns needed for homeostatic plasticity [37], to demonstrate the formation of functional outputs [38], to alter behaviour [39], [40], [41], [42], [43], [44], [45], [46], and even to restore function after spinal cord injury [47] or retinal degeneration [48,49,50, but see 51].

A vital component in determining spiking behaviour in neurons, whether ChR2-driven or not, is a highly-specialised structure known as the axon initial segment, or AIS. As its name suggests, the AIS is located near the start of the axon, where it comprises a complex arrangement of specialised scaffolding proteins, signalling molecules and ion channels [52], [53], [54], [55]. This dense meshwork of interconnected proteins provides a barrier for molecular movement both within the cell membrane [56] and within the cytoplasm [57], making the AIS crucial in regulating axonal transport, and a key player in determining the boundary between axonal and somatodendritic neuronal compartments [55], [58], [59], [60]. In terms of electrical activity in the nervous system, however, the AIS plays a different, vital role. Electrophysiological recordings [e.g. [61],[62],[63],[64],[65],[66],[67],[68],[69],[70]], mathematical modelling approaches [e.g. [68],[69],[71],[72],[73]] and experiments using voltage [70], [74], [75] or sodium [68] indicator dyes all point to the AIS as the subcellular location where action potentials initiate. This ‘hotspot’ for neuronal excitability exists by virtue of the AIS's high concentration of voltage-gated sodium channels (VGSCs) [68], [76], and because of the low voltage activation threshold of certain VGSC subtypes found preferentially in its distal region [73], [77]. It may also be a surprisingly plastic functional compartment, with recent data showing not only that the AIS can disassemble rapidly following cellular injury [60], but also that the structure can relocate along the axon [37], or change its length [78] in response to long-term changes in electrical activity.

Basic fluorophore-bound versions of ChR2 distribute indiscriminately throughout the cell membrane, including the AIS [e.g. [26],[79]]. ChR2 can be targeted to specific neuronal subcompartments, however, by creating fusion protein constructs that bind preferentially to molecules found in particular cellular regions. For example, adding the myosin-Va binding domain (MBD) of melanophilin to the C-terminus of ChR2-YFP produces somatodendritic localisation, with ChR2-YFP-MBD largely excluded from the axon [79]. Fusing the PDZ binding motif ETQV to ChR2-YFP, on the other hand, targets the ion channel preferentially to the postsynaptic side of glutamatergic synapses [80]. So far, though, there are no versions of ChR2 that localise to the AIS, despite the obvious theoretical benefits of an AIS-targeted optogenetic tool. Since action potentials initiate at the AIS, being able to control neuronal depolarisation (or hyperpolarisation) specifically at that initiation site should allow precise temporal control over firing behaviour. Action potentials are triggered at the AIS because of its high concentration of low-threshold voltage-gated sodium channels [68], [73], [76], so targeting ChR2 to this most excitable portion of the cell might also allow for more efficient control over spiking, using lower light stimulus intensities. Finally, for applications where accurate spike triggering is the only goal, localising ChR2 to the AIS avoids any complications associated with depolarisation in the soma and dendrites. This is particularly relevant because ChR2 is permeable to calcium [24], meaning that strong whole-cell photostimulation could lead to significant elevations in somatodendritic [Ca2]i, and subsequently trigger a range of unwanted intracellular signalling cascades [81].

Localising ChR2 to the AIS might therefore prove extremely useful. But how could it be achieved? An obvious approach is to co-opt strategies already used by native AIS-localised proteins. For example, cell adhesion molecules such as neurofascin (NF)-186 localise to the AIS by binding to the scaffolding protein ankyrin-G, an interaction which depends upon a C-terminus FIGQY domain [82], [83], [84]. Better understood, however, are the AIS-targeting mechanisms of ion channels, particularly VGSCs. Sodium channel subunits Nav1.1, 1.2, 1.3 and 1.6 share an ankyrinG binding motif, critically dependent upon a 9 amino acid sequence, which is located in the intracellular loop between their second and third transmembrane domains (II-III loop) [84], [85], [86], [87]. A homologous motif is also found in KCNQ2/3-type potassium channel subunits, which, along with VGSCs, bind to ankyrinG and localise to the AIS [88], [89]. The ankyrinG binding domain alone, however, is not sufficient for AIS targeting of VGSC subunits [87], which also use a removal sequence in their II-III intracellular loop to ensure that they are not stably inserted in the membrane elsewhere in the neuron [87], and several phosphorylation target sites for protein kinase CK2, also in the II-III loop, to regulate ankyrinG binding [90], [91]. Since all of these necessary components for AIS targeting are found in the II-III intracellular loop of all Nav1.1, 1.2, 1.3 and 1.6 VGSC subunits [91], it would be no surprise if the NavII-III loop sequence from any of these subunits were sufficient to target novel proteins to the AIS. This is indeed the case: fusing the II-III loop from Nav1.2 to a CD4 extracellular domain [86], or the extracellular domain of neurofascin [84], GFP [86] or YFP [37] produces reliable AIS localisation in dissociated hippocampal cultures.

Here we undertook a similar strategy for ChR2, successfully localising ChR2-YFP to the AIS by fusing it to the II-III loop sequence of Nav1.2. This construct was expressed in dissociated hippocampal neurons without significantly altering their electrical properties. However, the extremely low magnitude light-evoked depolarisations resulting from its restriction to the AIS meant that we were unable to induce light-evoked spikes in cells expressing ChR2-YFP-NavII-III, except under conditions of artificially increased neuronal excitability.

Methods

Molecular Biology and Transfections

The ChR2-YFP construct was pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE, a gift from Karl Deisseroth (www.optogenetics.org). We made ChR2-YFP-NavII-III by cloning the II-III intracellular loop sequence of Nav1.2 [86] (SSFSSDNLAATDDDNEMNNLQIAVGRMQKGIDFVKRKIREFIQKAFVRKQKALDEIKPLEDLNNKKDSCISNHTTIEIGKDLNYLKDGNGTTSGIGSSVEKYVVDESDYMSFINNPSLTVTVPIALGESDFENLNTEEFSSESDMEESKEKLNATSSSEGSTVDIGAPAEGEQPEAEPEESLEPEACFTEDCVRKFKCCQISIEEGKGKLWWNLRKTCYKX; MWG) into the BsrgI site found at the C-terminal end of YFP in the ChR2-YFP plasmid. The appropriate base pairs were added to complete the YFP sequence before the NavII-III sequence began. The construct was verified with sequencing, and has been submitted for general distribution with Addgene (www.addgene.org).

Dissociated Hippocampal Cultures

Humane killing for tissue collection conformed to local King's College London ethical approval under the UK Supplementary Code of Practice, The Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986. We prepared dissociated mixed neuronal and glial cultures from E 18 Sprague-Dawley rat hippocampi according to standard protocols, as described previously [37]. Briefly, hippocampi were dissected in HBSS, digested in trypsin (Worthington; 0.5 mg/ml; 15 min 37°C), triturated through Pasteur pipettes of increasingly narrow diameter, and plated at ∼240 cells/mm2 on glass coverslips pre-coated with PDL (50 µg/ml) and laminin (40 µg/ml). Cultures were incubated (37°C, 6% CO2) in a 1∶1 mixture of neurobasal medium plus B27 supplement (2%) and neurobasal medium plus foetal calf serum (2%), with additional glutamax (500 µM) and penicillin-streptomycin (100 µg/ml). At 7 days in vitro (DIV) half of this medium was replaced with neurobasal medium plus B27 supplement. Cells were transfected at 7 DIV using lipofectamine2000, and were studied between 9 DIV and 12 DIV. Unless otherwise specified, all culture reagents were from Invitrogen, UK.

Immunocytochemistry

We fixed cells with 1% paraformaldehyde (TAAB Laboratories, UK; in 3% sucrose, 60 mM PIPES, 25 mM HEPES, 5 mM EGTA, 1 mM MgCl2; pH 7.4; 20 min RT), washed in PBS, then blocked and permeabilised in 3% bovine serum albumin (BSA, Sigma) with 0.4% saponin (Sigma, UK; 1 h RT). We labelled AISs with a chicken polyclonal anti-βIV-spectrin antibody (a gift from M. Komada, Tokyo Institute of Technology, Japan; 1∶500 in PBS with 3% BSA, 1 h RT), followed by PBS washes, and then an Alexa Fluor goat anti-chicken 568 secondary antibody (Invitrogen, UK; 1∶1000; 1 h RT). Coverslips were then given a final PBS wash, and mounted in Mowiol (Calbiochem, UK).

Imaging and image analysis

Immunostained coverslips were imaged using an Olympus FluoView 1000 laser scanning confocal microscope coupled to FluoView software. We used an Olympus 40x oil immersion objective (0.8 NA). YFP and Alexa Fluor 568 fluorophores were imaged sequentially using 488 nm and 543 nm lasers, respectively, along with appropriate excitation and emission filters. Z-axis image stacks were acquired (1024×1024 pixels at 2x digital zoom, 0.5 µm z-axis steps), exported as raw 16-bit TIFF files, converted into single maximum intensity z-axis projections, and imported into Matlab (Mathworks, UK) for analysis using a custom-written function [37] that is freely available at www.grubblab.org/Matlab-scripts.php and www.mathworks.com/matlabcentral/fileexchange/28181-ais-quantification. We drew a line profile starting at the soma that extended down the axon, through and past the AIS. For our first measure of AIS localisation, we simply took raw fluorescence values along this line profile for both YFP and βIV-spectrin and calculated the Spearman correlation coefficient r between the two samples. For our second measure of AIS localisation, we used measures of the start and end positions of the AIS, as described previously [37]. At each pixel (1 pixel  = 0.155 µm) along the axon line profile, βIV-spectrin fluorescence intensity values were averaged over a 3×3 pixel square centred on the pixel of interest. Averaged profiles were then smoothed using a 40-point (∼6 µm) sliding mean, and normalised between 1 (maximum smoothed fluorescence) and 0 (minimum smoothed fluorescence). AIS start and end positions were obtained at the proximal and distal axonal positions, respectively, where the normalised and smoothed profile declined to 0.33. The AIS region defined by these start and end positions was then used to calculate an AIS localisation index (AISi) for the ChR2 constructs based on normalised, smoothed YFP fluorescence intensities within and outside the AIS: where meanAIS is the mean YFP fluorescence within the AIS region, and meannon-AIS is the mean YFP fluorescence over all points outside the AIS region.

Electrophysiology

As previously described [37], we made visually targeted whole-cell patch-clamp recordings from cultures maintained in a basic HEPES-buffered saline external solution (pH 7.4, ∼280 mOsm, ∼23°C) that contained, in mM: 136 NaCl, 2.5 KCl, 10 HEPES, 10 d-glucose, 2 CaCl2, 1.3 MgCl2. Pipettes were pulled from borosilicate glass (OD 1.5 mm, ID 1.17 mm, Harvard Apparatus, Edenbridge, UK), with a resistance of 3–7 MΩ, and were filled with an internal solution containing, in mM: 130 K-gluconate, 10 NaCl, 1 EGTA, 0.133 CaCl2, 2 MgCl2, 10 HEPES, 3.5 NaATP, 1 NaGTP. Recordings were obtained with a Heka EPC10/2 amplifier coupled to Pulse acquisition software. Signals were Bessel filtered at 10 kHz (filter 1), and 2.9 kHz (filter 2), digitised, and sampled at 25–50 kHz (20–40 µs sample interval). Fast capacitance was compensated in the On-cell configuration.

Electrophysiological parameters were analysed using custom routines written in Matlab. The resting membrane potential (Vrest) was estimated immediately following membrane patch rupture in current clamp mode with zero holding current (I = 0) and, like all voltages in this paper, was adjusted for an estimated liquid junction potential of ∼+15 mV. With slow capacitance compensation inactive in voltage-clamp mode, we used responses to a 10 mV hyperpolarisation step to estimate the neuron's membrane resistance (Rm; from the steady holding current at the new voltage) and membrane capacitance (Cm; from the area under the exponentially-decaying current from peak to holding).

Voltage-gated sodium currents were measured in basic HBS external medium (see above) with the addition of 10 mM tetraethylammonium (TEA; Alfa Aesar, UK) to block voltage-gated potassium currents, 100 µM CdCl2 (Sigma, UK) to block voltage-gated calcium currents, 10 µM SR-95531 (gabazine; Sigma, UK) to block GABAA receptors, 20 µM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; Sigma, UK) to block AMPA receptors, and 25 µM dl-2-amino-5-phosphonovaleric acid (APV, Sigma, UK) to block NMDA receptors. Potassium conductances were additionally blocked with a Cs-gluconate-based internal solution which contained, in mM: 130 Cs-gluconate, 10 NaCl, 1 EGTA, 0.133 CaCl2, 2 MgCl2, 10 HEPES, 3.5 NaATP, 1 NaGTP. For these recordings, series resistance was compensated at ≥50% (10 µs). Peak transient sodium currents were measured after subtraction of scaled passive current responses to the appropriate voltage steps. Persistent sodium currents were estimated from the mean whole-cell current over the final 50 ms of prolonged 500 ms membrane potential steps. To compensate for passive current contributions the initial portion of the resulting IV curve was fitted with a regression line. Subtracting this line from the raw data points then gave an estimate of persistent sodium current magnitude.

Spiking behaviour in response to 500 ms somatic current injections was recorded in current-clamp mode with a holding voltage of −75 mV, using the basic HBS external and K-gluconate-based internal solutions described above. Spikes were counted as transient depolarisations passing 0 mV, current threshold (Ithresh) was measured from the lowest current input that reliably evoked at least one spike, and voltage threshold (Vthresh) was measured as the voltage at which the first derivative of the recording (dV/dt) exceeded 10x the standard deviation of its baseline.

Responses of ChR2-expressing neurons to photostimulation in on-cell or current-clamp modes were recorded using the basic HBS external and K-gluconate-based internal solutions described above, with the addition of 10 µM gabazine, 10 µM CNQX and 25 µM APV to block synaptic conductances extracellularly. In some recordings, 100 µM Xe 991 and 100 µM linopirdine (Tocris, UK) were added to the external medium to block KCNQ channels. Whole-cell ChR2 currents were measured in voltage-clamp (−75 mV) using the Cs-gluconate-based internal solution and basic HBS external solution supplemented with 10 mM TEA, 1 µM tetrodotoxin (TTX; Alomone Labs, Israel), and 100 µM CdCl2 to block all main voltage-gated channels, and 10 µM gabazine, 10 µM CNQX and 25 µM APV to block all main synaptic conductances. ∼488 nm photostimulation at ∼170 mW/mm2 was provided by a shutter-controlled Xenon-arc lamp (Lambda-LS, Sutter Instruments, UK) with appropriate excitation filters (Chroma Tech. Corp., USA) but no neutral density filtering. More powerful ∼1 mW photostimulation was provided by a near-violet diode 405 nm laser controlled by an Olympus FluoView 1000 confocal microscope.

Statistics

Statistical analysis was carried out in Prism (GraphPad, USA). All tests were two-tailed, with the level of significance (α) set at 0.05.

Results

We targeted ChR2-YFP to the AIS by adding the Nav1.2 ankyrinG-binding loop NavII-III [84], [86] to its intracellular terminus. When expressed in cultured hippocampal neurons, the resulting fusion protein ChR2-YFP-NavII-III localised extremely well to the AIS (Figure 1A, top), unlike ChR2-YFP (Figure 1A, bottom). We quantified the degree of AIS localisation of these fusion protein constructs in two ways, both using parallel fluorescent immunocytochemical staining for the AIS scaffolding protein βIV-spectrin. In the first, we obtained normalised fluorescence profiles for βIV-spectrin and YFP along the axon (Figure 1A, right), and simply calculated the Spearman correlation coefficient r between these distributions as a measure of YFP AIS localisation. Mean r was far higher in neurons expressing ChR2-YFP- NavII-III (mean ± SEM, 0.54±0.04, n = 15) than in neurons expressing ChR2-YFP (0.09±0.06, 11; t-test, p<0.0001; Figure 1B). In the second approach, we used the βIV-spectrin fluorescence distribution to define the extent of the AIS (see Methods) [37], and obtained an AIS index (AISi) from the ratio of YFP fluorescence within the AIS region to YFP fluorescence outside the AIS region (see Methods). Mean AISi values were also much greater in ChR2-YFP- NavII-III -expressing neurons (mean ± SEM, 0.56±0.04; n = 15 cells) versus cells expressing ChR2-YFP (0.06±0.09, 11; t-test with Welch's correction for unequal variances, p = 0.0002; Figure 1C). Addition of the ankyrinG-binding loop NavII-III therefore successfully targets ChR2-YFP to the AIS.

thumbnail
Figure 1. The sodium channel loop NavII-III targets ChR2 to the AIS.

A) Example images (left) of neurons expressing ChR2-YFP-NavII-III (top, note background fluorescence revealing the soma of a non-transfected neuron in the YFP channel, with this cell's AIS also clear in the βIV-spectrin channel), or ChR2-YFP (bottom), co-stained for βIV-spectrin. Scalebar, 25 µm. Plots (right) show normalised YFP and βIV-spectrin fluorescence along the axon, with associated AIS localisation indices. Grey box shows AIS location. B, C) Mean correlation r (B) between YFP and βIV-spectrin fluorescence, and mean AISi (C; see text) showing AIS localisation of ChR2-YFP-NavII-III. Numbers in bars show n for each group; error bars show SEM; p values report results of t-tests.

https://doi.org/10.1371/journal.pone.0013761.g001

We used whole-cell patch-clamp recordings to verify that expressing AIS-targeted ChR2-YFP-NavII-III in neurons did not affect their fundamental electrophysiological properties. Basic functional parameters including the resting membrane potential (Vm), whole-cell capacitance (Cm), and passive membrane resistance (Rm) were not significantly different between neurons expressing ChR2-YFP-NavII-III (Vm: mean ± SEM, −61.8±2.6 mV; Cm: 44.2±8.5 pF; Rm: 403±74 MΩ; n = 8), those expressing ChR2-YFP (Vm: −61.2±1.8 mV; Cm: 39.4±6.1 pF; Rm: 478±67 MΩ; n = 9), and non-transfected wild-type (wt) cells (Vm: −57.9±2.2 mV; Cm: 43.4±3.9 pF; Rm: 354±30 MΩ; n = 11; Figure 2A–C; one-way ANOVA, F2,27<1.3, p>0.29).

thumbnail
Figure 2. Passive electrical properties of neurons expressing ChR2-YFP-NavII-III.

A–C) Mean resting membrane potential (A), cell capacitance (B) and membrane resistance (C) do not differ significantly between ChR2-YFP-NavII-III-positive, ChR2-YFP-positive, or wild-type neurons (1-way ANOVA, p>0.29). Numbers in bars show n for each group; error bars show SEM.

https://doi.org/10.1371/journal.pone.0013761.g002

Because ChR2-YFP-NavII-III uses the same mechanism as native voltage-gated sodium channels to localise to the AIS, we were concerned that ChR2-YFP-NavII-III expression might result in competition for ankyrinG binding sites and a consequent reduction in sodium current magnitude [86]. However, although there was a trend for ChR2-YFP-NavII-III-expressing neurons to have lower voltage-gated transient sodium current densities than wt cells, they displayed a trend towards larger transient sodium current densities than ChR2-YFP-expressing neurons, and neither of these trends was statistically significant (Figure 3A,B; repeated-measures 2-way ANOVA: effect of construct, F2,462 = 1.39, p = 0.27; effect of voltage, F22,462 = 66.75, p<0.0001; effect of interaction, F44,462 = 0.94, p = 0.58. Note that a small but significant difference in series resistance (Rs) was also observed in these recordings; ChR2-YFP-NavII-III mean ± SEM, 10.7±0.8 MΩ, n = 8; wt, 14.2±1.2 MΩ, n = 8; Dunn post-test following Kruskal-Wallis non-parametric one-way ANOVA, p<0.05). Because whole-cell transient sodium current measurements are particularly sensitive to space-clamp errors, we also assessed persistent sodium current (INaP) magnitude in cells expressing ChR2-YFP-NavII-III. This current can be clamped well in dendritic cells, and is known to emanate from the AIS [76], [92]. We estimated INaP in conditions of synaptic, calcium and potassium channel blockade by measuring whole-cell currents observed at the end of prolonged 500 ms voltage steps, after subtraction of passive current contributions (see Methods; Figure 3C). The resulting INaP current was entirely blocked by 5 µM TTX (n = 3 wild-type neurons, data not shown). We found no significant differences in INaP magnitude between ChR2-YFP-NavII-III-positive, ChR2-YFP-positive, and wild-type neurons (Figure 3D,E; repeated-measures 2-way ANOVA on INaP current density: effect of construct, F2,361 = 0.52, p = 0.60; effect of voltage, F19,361 = 26.95, p<0.0001; effect of interaction, F38,361 = 1.05, p = 0.40. One-way ANOVA on maximum INaP current density, F2,21 = 0.22, p = 0.81).

thumbnail
Figure 3. Whole-cell sodium currents in cells expressing ChR2-YFP-NavII-III.

A) Example whole-cell transient sodium currents in response to depolarisation steps from −105 mV in ChR2-YFP-NavII-III-expressing, ChR2-YFP-expressing, and wild-type neurons. B) No significant difference in mean whole-cell transient sodium current INa density in the three cell types (effect of cell type in 2-way repeated-measures ANOVA, p = 0.27). Error bars show SEM. C) Example IV relationships for whole-cell persistent currents following 500 ms membrane potential steps in the three cell types. The linear fit used to subtract passive currents is shown in magenta. D) No significant difference in mean whole-cell persistent sodium current INaP density across the three cell types (effect of cell type in 2-way repeated-measures ANOVA, p = 0.60). Error bars show SEM. E) No significant difference in maximum persistent sodium current INaP density in the three cell types (one-way ANOVA, p = 0.58). Error bars show SEM.

https://doi.org/10.1371/journal.pone.0013761.g003

These results were mirrored in measures of action potential firing. Cells transfected with ChR2-YFP-NavII-III could fire spikes in current-clamp mode perfectly well (Figure 4A), and although they showed trends towards depolarised voltage thresholds (Figure 4B; ChR2-YFP-NavII-III mean ± SEM, −33.8±2.4 mV, n = 8; ChR2-YFP, −38.0±2.5 mV, n = 8; wt, −40.5±1.3 mV, n = 11; one-way ANOVA, F2,26 = 2.96, p = 0.07), higher current thresholds (Figure 4C; ChR2-YFP-NavII-III mean ± SEM, 2.44±0.31 pA/pF, n = 8; ChR2-YFP, 2.15±0.58 pA/pF, n = 8; wt, 1.84±0.31 pA/pF, n = 11; Kruskal-Wallis non-parametric one-way ANOVA, p = 0.38), and lower maximum spike number (across all current injection magnitudes for a given cell; Figure 4D; ChR2-YFP-NavII-III mean ± SEM, 8.75±1.41, n = 8; ChR2-YFP, 8.00±1.98, n = 8; wt, 12.27±2.08, n = 11; one-way ANOVA, F2,26 = 1.51, p = 0.24), none of these trends was statistically significant.

thumbnail
Figure 4. Spiking properties of cells expressing ChR2-YFP-NavII-III.

A) Example current-clamp traces in response to 500 ms positive somatic current injection in the ChR2-YFP-NavII-III-expressing, ChR2-YFP-expressing, and wild-type neurons. Values beneath each trace show current injection magnitude in pA/pF. B–D) Mean voltage threshold (B; 1-way ANOVA, p = 0.07), current threshold (C; p = 0.38), and maximum spike number (D; p = 0.24) do not differ significantly across the three cell types. Numbers in bars show n for each group; error bars show SEM.

https://doi.org/10.1371/journal.pone.0013761.g004

ChR2-YFP-NavII-III therefore localises to the AIS without affecting the basic health or action potential-generating properties of its host cells. But can the construct be used to provide optogenetic control over spiking behaviour? We initially attempted to evoke action potentials in ChR2-YFP-NavII-III-expressing cells using blue light from a Xenon arc fluorescent lamp at maximum intensity (∼170 mW/mm2). This light source is routinely used to evoke spikes in ChR2-YFP-positive cells in our laboratory, but even long, 500 ms flashes never produced an action potential in ChR2-YFP-NavII-III-expressing cells (Figure 5A; on-cell and current-clamp I = 0 recording configurations, n = 5). As shown above, this was not because ChR2-YFP-NavII-III-positive neurons were unable to fire action potentials per se (Figure 5A, grey trace). Instead, the problem was one of ChR2 response magnitude, with the maximum depolarisation produced by light stimulation measured at the soma (mean ± SEM, 6.72±0.99 mV) being ∼20 mV smaller than the depolarisation needed to reach spike threshold from rest (25.3±3.15 mV; Figure 5B). We tried to augment light-evoked ChR2-YFP-NavII-III responses by increasing the intensity of our light stimulus, employing focal AIS stimulation with a near-violet diode 405 nm laser (∼1 mW at the coverslip surface). However, although this strong stimulus reliably evoked spikes in ChR2-YFP-positive cells, even when directed to the soma (Figure 5C, left; 6/6 cells), it never evoked an action potential in neurons expressing ChR2-YFP-NavII-III (Figure 5C, right; 0/4 cells). The magnitude of light-evoked responses in ChR2-YFP-NavII-III-positive cells therefore appears limited by the low total number of ChR2 molecules present in the cell membrane. This was evidenced by voltage-clamp recordings of whole-cell ChR2 currents (Figure 5D), which showed that peak light-evoked responses were ∼28 –fold smaller in neurons expressing ChR2-YFP-NavII-III versus those expressing ChR2-YFP (Figure 5E; Xenon lamp stimulation at ∼170 mW/mm2; ChR2-YFP-NavII-III mean ± SEM, 42.1±8.4 pA, n = 8; ChR2-YFP, 1188.5±235.3 pA, n = 8). So, whilst it localises extremely well to the AIS, the ChR2-YFP-NavII-III construct simply provides too little light-evoked current to allow optogenetic control of spiking under baseline conditions.

thumbnail
Figure 5. Attempting to control neuronal activity with ChR2-YFP-NavII-III.

A) Example on-cell (top) and current-clamp I = 0 (bottom) recordings in control HBS solution, showing lack of spiking in a ChR2-YFP-NavII-III-expressing neuron in response to 500 ms blue light stimulation. Grey trace shows spiking in the same cell in response to somatic current injection. B) Mean depolarisation produced by light stimulation in ChR2-YFP-NavII-III-positive cells (current-clamp I = 0) is much smaller than the mean depolarisation required to fire an action potential from rest. Numbers in bars show n for each group; asterisks show t-test result; ***, p<0.001. Error bars show SEM. C) ChR2-YFP-NavII-III-positive cells fail to fire even to high intensity laser photostimulation. Left, brief ∼1 mW 405 nm laser illumination targeted to the soma of ChR2-positive cells always produced an action potential. Right, the same stimulus at the AIS of ChR2-YFP-NavII-III-positive cells never produced an action potential, even though these cells were capable of firing spontaneous spikes. D) Example whole-cell ChR2 currents in a ChR2-YFP-NavII-III-positive neuron (left) and scaled in comparison to a typical ChR2-YFP-expressing cell (right), recorded at −75 mV in response to 500 ms blue light stimulation. E) Mean peak whole-cell ChR2 current is significantly smaller in ChR2-YFP-NavII-III-positive neurons. Numbers in bars show n for each group; asterisks show t-test result; ***, p<0.001. F) Blocking K+ channels at the AIS can allow optogenetic control of neuronal activity with ChR2-YFP-NavII-III. Example on-cell (top) and current-clamp I = 0 (bottom) traces in HBS supplemented with 100 µM Xe-991 (xe) and 100 µM linopirdine (lino), showing broad spikes in a ChR2-YFP-NavII-III-expressing neuron in response to 500 ms blue light stimulation.

https://doi.org/10.1371/journal.pone.0013761.g005

We did, however, succeed in producing light-evoked action potentials in some ChR2-YFP-NavII-III-expressing cells under rather specialised conditions. As well as clustering the voltage-gated sodium channels that are vital for spike initiation, the AIS also contains high densities of voltage-gated potassium channels that limit and control action potential production [e.g. [53]]. Blocking KCNQ potassium channels, which cluster preferentially at the AIS [88], with the antagonists Xe-991 (100 µM) and linopirdine (100 µM) produced broad spikes that were initiated at much less depolarised membrane potentials, making cells significantly more excitable. In a subset of neurons expressing ChR2-YFP-NavII-III (on-cell, 10/17 cells = 59%; current-clamp I = 0, 9/17 cells = 53%), this allowed the production of single action potentials using long-duration high-intensity blue light stimulation (Figure 5F). The temporal precision of spike initiation under these conditions was poor, however: mean time-to-spike following light stimulus onset in on-cell mode was 70 ms, with a standard deviation of 37 ms across all ChR2-YFP-NavII-III-expressing cells.

Discussion

We have shown that the sodium channel NavII-III loop sequence is sufficient to accurately target ChR2 to the AIS in cultured hippocampal neurons. Expressing this construct does not significantly alter the basic functional properties of its host neurons. However, controlling action potential initiation with ChR2-YFP-NavII-III proved impossible, even with strong AIS-directed laser photostimulation. ChR2 currents localised to the AIS were simply too small to take a neuron past spike threshold, except under artificial conditions of increased excitability.

As predicted by previous success with simple fluorophore [37], [86] and membrane-bound [84], [86] molecules, adding the entire sodium channel II-III loop to the intracellular terminus of ChR2-YFP was sufficient to target the protein specifically to the AIS. Attempts at localising ChR2 to the AIS using only the 9 aa [84] or 27 aa [86] ankyrinG binding domains were less successful (data not shown), suggesting that ChR2, like VGSCs, requires the II-III loop membrane removal sequence [87] and/or CK2 phosphorylation sites [90] for complete AIS targeting. These multiple mechanisms of subcellular localisation, however, make it unlikely that the number of ChR2-YFP-NavII-III molecules could be increased at the AIS by simply increasing expression of the protein with, say, stronger promoters or lentiviral infection [e.g. [16],[93]]. Although producing more foreign protein is an obvious approach to the problem of low ChR2 currents and lack of light-induced spiking, the need for ChR2-YFP-NavII-III to be constitutively removed from non-AIS locations [87] means that cells expressing very high levels of the construct would probably display impaired AIS targeting – they would be simply swamped with excess ChR2. Even more pertinent is the fact that ChR2-YFP-NavII-III uses the same mechanisms as native sodium [84], [86], [87], [90] and potassium [88] channels to localise to the AIS. We saw no significant decrease in sodium current density in our ChR2-YFP-NavII-III-expressing neurons, but we did see a trend towards lower transient INa amplitude (Figure 3), while others have reported large decreases in transient sodium currents with strong expression of GFP-NavII-III [86]. Even if hugely-increased densities of ChR2-YFP-NavII-III could be precisely localised to the AIS, then, they might out-compete native VGSCs to such an extent that action potential initiation is impaired and light-evoked spiking remains impossible.

Are there other means by which ChR2 can be localised to the AIS, without competing with native VGSCs? Cell adhesion molecules such as neurofascin-186 bind ankyrinG and become AIS-targeted via an intracellular FIGQY motif [82], [83], [84], so in theory adding this sequence to ChR2-YFP could localise it to the AIS without interfering with sodium channel binding sites. However, although the FIGQY motif is necessary for cell adhesion molecule binding to ankyrinG, it is not known whether it is sufficient for AIS targeting. Similarly, another AIS scaffolding molecule βIV-spectrin requires spectrin repeat 15 for ankyrinG binding and AIS localisation, but a combination of spectrin repeats 14+15 proved insufficient for full AIS targeting of a myc construct [94]. Finally, the clustering of Kv1 channels at the AIS is dependent upon the scaffolding molecule PSD-93 [95], but the molecular mechanisms governing this interaction are presently unclear. There are many alternative potential means of targeting ChR2 to the AIS, therefore, but none currently understood sufficiently to be able to be put into practice.

Instead, perhaps the answer to controlling spikes with ChR2 at the AIS lies away from the AIS targeting mechanism, and more towards ChR2 itself. Simple cell compartment trafficking sequences added to the ChR2-YFP-NavII-III construct, for example, might increase the density of ChR2 molecules at the AIS and improve optogenetic efficiency [e.g. [17]], but with all of the caveats associated with overexpression outlined above. Alternatively, variants of ChR2 could be utilised that are more sensitive to light, or have improved single-channel conductance, producing larger light-evoked currents with unchanged AIS channel densities. Such variants are a major future target of optogenetic molecular engineering. In the meantime there are slow-kinetic ChR2 mutants which, over time, produce hugely increased integral currents for a given light stimulus [20], [21], and which could prove useful in evoking spikes via AIS-targeted ChR2 expression, so long as low frequency light-evoked firing is the goal.

Finally, rather than attempting full control over spike initiation with AIS-localised ChR2, maybe the most attainable target is to influence action potential firing with optogenetic tools localised to the AIS. Rather than initiating spikes with AIS-based optogenetics, action potentials could be specifically inhibited or shaped by light-driven chloride [14], [15], [16], [17] or proton [18] pumps fused to the NavII-III sequence. In its native state, the AIS contains multiple mechanisms for altering the initiation and subsequent properties of action potentials [53], including voltage-gated potassium channels [e.g. [96],[97]], voltage-gated calcium channels [98], [99], sodium channel modulators [100], [101] and synaptic inputs [102], [103], [104], [105]. Returning to the construct described here, perhaps the small photocurrents produced by ChR2-YFP-NavII-III could be used in various experimental settings to mimic, trigger, or influence these mechanisms of action potential modulation.

Acknowledgments

We thank K. Deisseroth for the ChR2-YFP construct, M. Komada for the anti-βIV-spectrin antibody, N. Ben Fredj, A. Chand and M. Evans for assistance with cell culture, and S. Poopalasundaram for help with molecular biology.

Author Contributions

Conceived and designed the experiments: MSG JB. Performed the experiments: MSG JB. Analyzed the data: MSG. Wrote the paper: MSG JB.

References

  1. 1. Torebjork HE, Vallbo AB, Ochoa JL (1987) Intraneural microstimulation in man: Its relation to specificity of tactile sensations. Brain 110: 1509–1529.HE TorebjorkAB VallboJL Ochoa1987Intraneural microstimulation in man: Its relation to specificity of tactile sensations.Brain11015091529
  2. 2. Tehovnik EJ, Tolias AS, Sultan F, Slocum WM, Logothetis NK (2006) Direct and indirect activation of cortical neurons by electrical microstimulation. J Neurophysiol 96: 512–521.EJ TehovnikAS ToliasF. SultanWM SlocumNK Logothetis2006Direct and indirect activation of cortical neurons by electrical microstimulation.J Neurophysiol96512521
  3. 3. Wilson BS, Dorman MF (2008) Cochlear implants: a remarkable past and a brilliant future. Hear Res 242: 3–21.BS WilsonMF Dorman2008Cochlear implants: a remarkable past and a brilliant future.Hear Res242321
  4. 4. Benabid AL, Chabardes S, Mitrofanis J, Pollak P (2009) Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson's disease. Lancet Neurol 8: 67–81.AL BenabidS. ChabardesJ. MitrofanisP. Pollak2009Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson's disease.Lancet Neurol86781
  5. 5. Hales CM, Rolston JD, Potter SM (2010) How to culture, record and stimulate neuronal networks on micro-electrode arrays (MEAs). J Vis Exp. CM HalesJD RolstonSM Potter2010How to culture, record and stimulate neuronal networks on micro-electrode arrays (MEAs).J Vis Exp
  6. 6. Barker AT (1999) The history and basic principles of magnetic nerve stimulation. Electroencephalogr Clin Neurophysiol Suppl 513–21.AT Barker1999The history and basic principles of magnetic nerve stimulation.Electroencephalogr Clin NeurophysiolSuppl 51321
  7. 7. Tyler WJ, Tufail Y, Finsterwald M, Tauchmann ML, Olson EJ, et al. (2008) Remote excitation of neuronal circuits using low-intensity, low-frequency ultrasound. PLoS One 3: e3511.WJ TylerY. TufailM. FinsterwaldML TauchmannEJ Olson2008Remote excitation of neuronal circuits using low-intensity, low-frequency ultrasound.PLoS One3e3511
  8. 8. Tufail Y, Matyushov A, Baldwin N, Tauchmann ML, Georges J, et al. (2010) Transcranial pulsed ultrasound stimulates intact brain circuits. Neuron 66: 681–694.Y. TufailA. MatyushovN. BaldwinML TauchmannJ. Georges2010Transcranial pulsed ultrasound stimulates intact brain circuits.Neuron66681694
  9. 9. Kramer RH, Fortin DL, Trauner D (2009) New photochemical tools for controlling neuronal activity. Curr Opin Neurobiol 19: 544–552.RH KramerDL FortinD. Trauner2009New photochemical tools for controlling neuronal activity.Curr Opin Neurobiol19544552
  10. 10. Ellis-Davies GC (2007) Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods 4: 619–628.GC Ellis-Davies2007Caged compounds: photorelease technology for control of cellular chemistry and physiology.Nat Methods4619628
  11. 11. Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7: 1381–1386.M. BanghartK. BorgesE. IsacoffD. TraunerRH Kramer2004Light-activated ion channels for remote control of neuronal firing.Nat Neurosci713811386
  12. 12. Volgraf M, Gorostiza P, Numano R, Kramer RH, Isacoff EY, et al. (2006) Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat Chem Biol 2: 47–52.M. VolgrafP. GorostizaR. NumanoRH KramerEY Isacoff2006Allosteric control of an ionotropic glutamate receptor with an optical switch.Nat Chem Biol24752
  13. 13. Deisseroth K, Feng G, Majewska AK, Miesenbock G, Ting A, et al. (2006) Next-generation optical technologies for illuminating genetically targeted brain circuits. J Neurosci 26: 10380–10386.K. DeisserothG. FengAK MajewskaG. MiesenbockA. Ting2006Next-generation optical technologies for illuminating genetically targeted brain circuits.J Neurosci261038010386
  14. 14. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, et al. (2007) Multimodal fast optical interrogation of neural circuitry. Nature 446: 633–639.F. ZhangLP WangM. BraunerJF LiewaldK. Kay2007Multimodal fast optical interrogation of neural circuitry.Nature446633639
  15. 15. Han X, Boyden ES (2007) Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution. PLoS One 2: e299.X. HanES Boyden2007Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution.PLoS One2e299
  16. 16. Gradinaru V, Thompson KR, Deisseroth K (2008) eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol 36: 129–139.V. GradinaruKR ThompsonK. Deisseroth2008eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications.Brain Cell Biol36129139
  17. 17. Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, et al. (2010) Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141: 154–165.V. GradinaruF. ZhangC. RamakrishnanJ. MattisR. Prakash2010Molecular and cellular approaches for diversifying and extending optogenetics.Cell141154165
  18. 18. Chow BY, Han X, Dobry AS, Qian X, Chuong AS, et al. (2010) High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463: 98–102.BY ChowX. HanAS DobryX. QianAS Chuong2010High-performance genetically targetable optical neural silencing by light-driven proton pumps.Nature46398102
  19. 19. Zhang F, Prigge M, Beyriere F, Tsunoda SP, Mattis J, et al. (2008) Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat Neurosci 11: 631–633.F. ZhangM. PriggeF. BeyriereSP TsunodaJ. Mattis2008Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri.Nat Neurosci11631633
  20. 20. Berndt A, Prigge M, Gradmann D, Hegemann P (2008) Two open states with progressive proton selectivities in the branched channelrhodopsin-2 photocycle. Biophys J 98: 753–761.A. BerndtM. PriggeD. GradmannP. Hegemann2008Two open states with progressive proton selectivities in the branched channelrhodopsin-2 photocycle.Biophys J98753761
  21. 21. Bamann C, Gueta R, Kleinlogel S, Nagel G, Bamberg E (2010) Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond. Biochemistry 49: 267–278.C. BamannR. GuetaS. KleinlogelG. NagelE. Bamberg2010Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond.Biochemistry49267278
  22. 22. Lin JY, Lin MZ, Steinbach P, Tsien RY (2009) Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96: 1803–1814.JY LinMZ LinP. SteinbachRY Tsien2009Characterization of engineered channelrhodopsin variants with improved properties and kinetics.Biophys J9618031814
  23. 23. Gunaydin LA, Yizhar O, Berndt A, Sohal VS, Deisseroth K, et al. (2010) Ultrafast optogenetic control. Nat Neurosci 13: 387–392.LA GunaydinO. YizharA. BerndtVS SohalK. Deisseroth2010Ultrafast optogenetic control.Nat Neurosci13387392
  24. 24. Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, et al. (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A 100: 13940–13945.G. NagelT. SzellasW. HuhnS. KateriyaN. Adeishvili2003Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.Proc Natl Acad Sci U S A1001394013945
  25. 25. Li X, Gutierrez DV, Hanson MG, Han J, Mark MD, et al. (2005) Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc Natl Acad Sci U S A 102: 17816–17821.X. LiDV GutierrezMG HansonJ. HanMD Mark2005Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.Proc Natl Acad Sci U S A1021781617821
  26. 26. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8: 1263–1268.ES BoydenF. ZhangE. BambergG. NagelK. Deisseroth2005Millisecond-timescale, genetically targeted optical control of neural activity.Nat Neurosci812631268
  27. 27. Arenkiel BR, Peca J, Davison IG, Feliciano C, Deisseroth K, et al. (2007) In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54: 205–218.BR ArenkielJ. PecaIG DavisonC. FelicianoK. Deisseroth2007In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2.Neuron54205218
  28. 28. Wang H, Peca J, Matsuzaki M, Matsuzaki K, Noguchi J, et al. (2007) High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc Natl Acad Sci U S A 104: 8143–8148.H. WangJ. PecaM. MatsuzakiK. MatsuzakiJ. Noguchi2007High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice.Proc Natl Acad Sci U S A10481438148
  29. 29. Petreanu L, Huber D, Sobczyk A, Svoboda K (2007) Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat Neurosci 10: 663–668.L. PetreanuD. HuberA. SobczykK. Svoboda2007Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.Nat Neurosci10663668
  30. 30. Petreanu L, Mao T, Sternson SM, Svoboda K (2009) The subcellular organization of neocortical excitatory connections. Nature 457: 1142–1145.L. PetreanuT. MaoSM SternsonK. Svoboda2009The subcellular organization of neocortical excitatory connections.Nature45711421145
  31. 31. Cruikshank SJ, Urabe H, Nurmikko AV, Connors BW (2009) Pathway-specific feedforward circuits between thalamus and neocortex revealed by selective optical stimulation of axons. Neuron 65: 230–245.SJ CruikshankH. UrabeAV NurmikkoBW Connors2009Pathway-specific feedforward circuits between thalamus and neocortex revealed by selective optical stimulation of axons.Neuron65230245
  32. 32. Wang H, Zylka MJ (2009) Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons. J Neurosci 29: 13202–13209.H. WangMJ Zylka2009Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.J Neurosci291320213209
  33. 33. Stuber GD, Hnasko TS, Britt JP, Edwards RH, Bonci A (2010) Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamate. J Neurosci 30: 8229–8233.GD StuberTS HnaskoJP BrittRH EdwardsA. Bonci2010Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamate.J Neurosci3082298233
  34. 34. Adesnik H, Scanziani M (2010) Lateral competition for cortical space by layer-specific horizontal circuits. Nature 464: 1155–1160.H. AdesnikM. Scanziani2010Lateral competition for cortical space by layer-specific horizontal circuits.Nature46411551160
  35. 35. Zhang YP, Oertner TG (2007) Optical induction of synaptic plasticity using a light-sensitive channel. Nat Methods 4: 139–141.YP ZhangTG Oertner2007Optical induction of synaptic plasticity using a light-sensitive channel.Nat Methods4139141
  36. 36. Zhang YP, Holbro N, Oertner TG (2008) Optical induction of plasticity at single synapses reveals input-specific accumulation of alphaCaMKII. Proc Natl Acad Sci U S A 105: 12039–12044.YP ZhangN. HolbroTG Oertner2008Optical induction of plasticity at single synapses reveals input-specific accumulation of alphaCaMKII.Proc Natl Acad Sci U S A1051203912044
  37. 37. Grubb MS, Burrone J (2010) Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability. Nature 465: 1070–1074.MS GrubbJ. Burrone2010Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability.Nature46510701074
  38. 38. Toni N, Laplagne DA, Zhao C, Lombardi G, Ribak CE, et al. (2008) Neurons born in the adult dentate gyrus form functional synapses with target cells. Nat Neurosci 11: 901–907.N. ToniDA LaplagneC. ZhaoG. LombardiCE Ribak2008Neurons born in the adult dentate gyrus form functional synapses with target cells.Nat Neurosci11901907
  39. 39. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, et al. (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol 15: 2279–2284.G. NagelM. BraunerJF LiewaldN. AdeishviliE. Bamberg2005Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.Curr Biol1522792284
  40. 40. Douglass AD, Kraves S, Deisseroth K, Schier AF, Engert F (2008) Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons. Curr Biol 18: 1133–1137.AD DouglassS. KravesK. DeisserothAF SchierF. Engert2008Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons.Curr Biol1811331137
  41. 41. Huber D, Petreanu L, Ghitani N, Ranade S, Hromadka T, et al. (2008) Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451: 61–64.D. HuberL. PetreanuN. GhitaniS. RanadeT. Hromadka2008Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice.Nature4516164
  42. 42. Gradinaru V, Mogri M, Thompson KR, Henderson JM, Deisseroth K (2009) Optical deconstruction of parkinsonian neural circuitry. Science 324: 354–359.V. GradinaruM. MogriKR ThompsonJM HendersonK. Deisseroth2009Optical deconstruction of parkinsonian neural circuitry.Science324354359
  43. 43. Tsai HC, Zhang F, Adamantidis A, Stuber GD, Bonci A, et al. (2009) Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324: 1080–1084.HC TsaiF. ZhangA. AdamantidisGD StuberA. Bonci2009Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning.Science32410801084
  44. 44. Bellmann D, Richardt A, Freyberger R, Nuwal N, Schwarzel M, et al. (2010) Optogenetically Induced Olfactory Stimulation in Drosophila Larvae Reveals the Neuronal Basis of Odor-Aversion behavior. Front Behav Neurosci 4: 27.D. BellmannA. RichardtR. FreybergerN. NuwalM. Schwarzel2010Optogenetically Induced Olfactory Stimulation in Drosophila Larvae Reveals the Neuronal Basis of Odor-Aversion behavior.Front Behav Neurosci427
  45. 45. Hagglund M, Borgius L, Dougherty KJ, Kiehn O (2010) Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion. Nat Neurosci 13: 246–252.M. HagglundL. BorgiusKJ DoughertyO. Kiehn2010Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion.Nat Neurosci13246252
  46. 46. Schoonheim PJ, Arrenberg AB, Del Bene F, Baier H (2010) Optogenetic localization and genetic perturbation of saccade-generating neurons in zebrafish. J Neurosci 30: 7111–7120.PJ SchoonheimAB ArrenbergF. Del BeneH. Baier2010Optogenetic localization and genetic perturbation of saccade-generating neurons in zebrafish.J Neurosci3071117120
  47. 47. Alilain WJ, Li X, Horn KP, Dhingra R, Dick TE, et al. (2008) Light-induced rescue of breathing after spinal cord injury. J Neurosci 28: 11862–11870.WJ AlilainX. LiKP HornR. DhingraTE Dick2008Light-induced rescue of breathing after spinal cord injury.J Neurosci281186211870
  48. 48. Bi A, Cui J, Ma YP, Olshevskaya E, Pu M, et al. (2006) Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron 50: 23–33.A. BiJ. CuiYP MaE. OlshevskayaM. Pu2006Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration.Neuron502333
  49. 49. Lagali PS, Balya D, Awatramani GB, Munch TA, Kim DS, et al. (2008) Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat Neurosci 11: 667–675.PS LagaliD. BalyaGB AwatramaniTA MunchDS Kim2008Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.Nat Neurosci11667675
  50. 50. Tomita H, Sugano E, Fukazawa Y, Isago H, Sugiyama Y, et al. (2009) Visual properties of transgenic rats harboring the channelrhodopsin-2 gene regulated by the thy-1.2 promoter. PLoS One 4: e7679.H. TomitaE. SuganoY. FukazawaH. IsagoY. Sugiyama2009Visual properties of transgenic rats harboring the channelrhodopsin-2 gene regulated by the thy-1.2 promoter.PLoS One4e7679
  51. 51. Thyagarajan S, van Wyk M, Lehmann K, Lowel S, Feng G, et al. (2010) Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells. J Neurosci 30: 8745–8758.S. ThyagarajanM. van WykK. LehmannS. LowelG. Feng2010Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells.J Neurosci3087458758
  52. 52. Ogawa Y, Rasband MN (2008) The functional organization and assembly of the axon initial segment. Curr Opin Neurobiol 18: 307–313.Y. OgawaMN Rasband2008The functional organization and assembly of the axon initial segment.Curr Opin Neurobiol18307313
  53. 53. Clark BD, Goldberg EM, Rudy B (2009) Electrogenic tuning of the axon initial segment. Neuroscientist 15: 651–668.BD ClarkEM GoldbergB. Rudy2009Electrogenic tuning of the axon initial segment.Neuroscientist15651668
  54. 54. Grubb MS, Burrone J (2010) Building and maintaining the axon initial segment. Curr Opin Neurobiol 20: 481–488.MS GrubbJ. Burrone2010Building and maintaining the axon initial segment.Curr Opin Neurobiol20481488
  55. 55. Rasband MN (2010) The axon initial segment and the maintenance of neuronal polarity. Nat Rev Neurosci 11: 552–562.MN Rasband2010The axon initial segment and the maintenance of neuronal polarity.Nat Rev Neurosci11552562
  56. 56. Winckler B, Forscher P, Mellman I (1999) A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature 397: 698–701.B. WincklerP. ForscherI. Mellman1999A diffusion barrier maintains distribution of membrane proteins in polarized neurons.Nature397698701
  57. 57. Song AH, Wang D, Chen G, Li Y, Luo J, et al. (2009) A selective filter for cytoplasmic transport at the axon initial segment. Cell 136: 1148–1160.AH SongD. WangG. ChenY. LiJ. Luo2009A selective filter for cytoplasmic transport at the axon initial segment.Cell13611481160
  58. 58. Hedstrom KL, Ogawa Y, Rasband MN (2008) AnkyrinG is required for maintenance of the axon initial segment and neuronal polarity. J Cell Biol 183: 635–640.KL HedstromY. OgawaMN Rasband2008AnkyrinG is required for maintenance of the axon initial segment and neuronal polarity.J Cell Biol183635640
  59. 59. Sobotzik JM, Sie JM, Politi C, Del Turco D, Bennett V, et al. (2009) AnkyrinG is required to maintain axo-dendritic polarity in vivo. Proc Natl Acad Sci U S A 106: 17564–17569.JM SobotzikJM SieC. PolitiD. Del TurcoV. Bennett2009AnkyrinG is required to maintain axo-dendritic polarity in vivo.Proc Natl Acad Sci U S A1061756417569
  60. 60. Schafer DP, Jha S, Liu F, Akella T, McCullough LD, et al. (2009) Disruption of the axon initial segment cytoskeleton is a new mechanism for neuronal injury. J Neurosci 29: 13242–13254.DP SchaferS. JhaF. LiuT. AkellaLD McCullough2009Disruption of the axon initial segment cytoskeleton is a new mechanism for neuronal injury.J Neurosci291324213254
  61. 61. Coombs JS, Curtis DR, Eccles JC (1957) The generation of impulses in motoneurones. J Physiol 139: 232–249.JS CoombsDR CurtisJC Eccles1957The generation of impulses in motoneurones.J Physiol139232249
  62. 62. Fatt P (1957) Sequence of events in synaptic activation of a motoneurone. J Neurophysiol 20: 61–80.P. Fatt1957Sequence of events in synaptic activation of a motoneurone.J Neurophysiol206180
  63. 63. Fuortes MG, Frank K, Becker MC (1957) Steps in the production of motoneuron spikes. J Gen Physiol 40: 735–752.MG FuortesK. FrankMC Becker1957Steps in the production of motoneuron spikes.J Gen Physiol40735752
  64. 64. Stuart GJ, Sakmann B (1994) Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367: 69–72.GJ StuartB. Sakmann1994Active propagation of somatic action potentials into neocortical pyramidal cell dendrites.Nature3676972
  65. 65. Stuart G, Hausser M (1994) Initiation and spread of sodium action potentials in cerebellar Purkinje cells. Neuron 13: 703–712.G. StuartM. Hausser1994Initiation and spread of sodium action potentials in cerebellar Purkinje cells.Neuron13703712
  66. 66. Colbert CM, Johnston D (1996) Axonal action-potential initiation and Na+ channel densities in the soma and axon initial segment of subicular pyramidal neurons. J Neurosci 16: 6676–6686.CM ColbertD. Johnston1996Axonal action-potential initiation and Na+ channel densities in the soma and axon initial segment of subicular pyramidal neurons.J Neurosci1666766686
  67. 67. Shu Y, Duque A, Yu Y, Haider B, McCormick DA (2007) Properties of action-potential initiation in neocortical pyramidal cells: evidence from whole cell axon recordings. J Neurophysiol 97: 746–760.Y. ShuA. DuqueY. YuB. HaiderDA McCormick2007Properties of action-potential initiation in neocortical pyramidal cells: evidence from whole cell axon recordings.J Neurophysiol97746760
  68. 68. Kole MH, Ilschner SU, Kampa BM, Williams SR, Ruben PC, et al. (2008) Action potential generation requires a high sodium channel density in the axon initial segment. Nat Neurosci 11: 178–186.MH KoleSU IlschnerBM KampaSR WilliamsPC Ruben2008Action potential generation requires a high sodium channel density in the axon initial segment.Nat Neurosci11178186
  69. 69. Yu Y, Shu Y, McCormick DA (2008) Cortical action potential backpropagation explains spike threshold variability and rapid-onset kinetics. J Neurosci 28: 7260–7272.Y. YuY. ShuDA McCormick2008Cortical action potential backpropagation explains spike threshold variability and rapid-onset kinetics.J Neurosci2872607272
  70. 70. Palmer LM, Clark BA, Grundemann J, Roth A, Stuart GJ, et al. (2010) Initiation of simple and complex spikes in cerebellar Purkinje cells. J Physiol 588: 1709–1717.LM PalmerBA ClarkJ. GrundemannA. RothGJ Stuart2010Initiation of simple and complex spikes in cerebellar Purkinje cells.J Physiol58817091717
  71. 71. Mainen ZF, Joerges J, Huguenard JR, Sejnowski TJ (1995) A model of spike initiation in neocortical pyramidal neurons. Neuron 15: 1427–1439.ZF MainenJ. JoergesJR HuguenardTJ Sejnowski1995A model of spike initiation in neocortical pyramidal neurons.Neuron1514271439
  72. 72. Meeks JP, Mennerick S (2007) Action potential initiation and propagation in CA3 pyramidal axons. J Neurophysiol 97: 3460–3472.JP MeeksS. Mennerick2007Action potential initiation and propagation in CA3 pyramidal axons.J Neurophysiol9734603472
  73. 73. Hu W, Tian C, Li T, Yang M, Hou H, et al. (2009) Distinct contributions of Na(v)1.6 and Na(v)1.2 in action potential initiation and backpropagation. Nat Neurosci 12: 996–1002.W. HuC. TianT. LiM. YangH. Hou2009Distinct contributions of Na(v)1.6 and Na(v)1.2 in action potential initiation and backpropagation.Nat Neurosci129961002
  74. 74. Palmer LM, Stuart GJ (2006) Site of action potential initiation in layer 5 pyramidal neurons. J Neurosci 26: 1854–1863.LM PalmerGJ Stuart2006Site of action potential initiation in layer 5 pyramidal neurons.J Neurosci2618541863
  75. 75. Foust A, Popovic M, Zecevic D, McCormick DA (2010) Action potentials initiate in the axon initial segment and propagate through axon collaterals reliably in cerebellar Purkinje neurons. J Neurosci 30: 6891–6902.A. FoustM. PopovicD. ZecevicDA McCormick2010Action potentials initiate in the axon initial segment and propagate through axon collaterals reliably in cerebellar Purkinje neurons.J Neurosci3068916902
  76. 76. Fleidervish IA, Lasser-Ross N, Gutnick MJ, Ross WN (2010) Na+ imaging reveals little difference in action potential-evoked Na+ influx between axon and soma. Nat Neurosci 13: 852–860.IA FleidervishN. Lasser-RossMJ GutnickWN Ross2010Na+ imaging reveals little difference in action potential-evoked Na+ influx between axon and soma.Nat Neurosci13852860
  77. 77. Rush AM, Dib-Hajj SD, Waxman SG (2005) Electrophysiological properties of two axonal sodium channels, Nav1.2 and Nav1.6, expressed in mouse spinal sensory neurones. J Physiol 564: 803–815.AM RushSD Dib-HajjSG Waxman2005Electrophysiological properties of two axonal sodium channels, Nav1.2 and Nav1.6, expressed in mouse spinal sensory neurones.J Physiol564803815
  78. 78. Kuba H, Oichi Y, Ohmori H (2010) Presynaptic activity regulates Na(+) channel distribution at the axon initial segment. Nature 465: 1075–1078.H. KubaY. OichiH. Ohmori2010Presynaptic activity regulates Na(+) channel distribution at the axon initial segment.Nature46510751078
  79. 79. Lewis TL Jr, Mao T, Svoboda K, Arnold DB (2009) Myosin-dependent targeting of transmembrane proteins to neuronal dendrites. Nat Neurosci 12: 568–576.TL Lewis JrT. MaoK. SvobodaDB Arnold2009Myosin-dependent targeting of transmembrane proteins to neuronal dendrites.Nat Neurosci12568576
  80. 80. Gradinaru V, Thompson KR, Zhang F, Mogri M, Kay K, et al. (2007) Targeting and readout strategies for fast optical neural control in vitro and in vivo. J Neurosci 27: 14231–14238.V. GradinaruKR ThompsonF. ZhangM. MogriK. Kay2007Targeting and readout strategies for fast optical neural control in vitro and in vivo.J Neurosci271423114238
  81. 81. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21.MJ BerridgeP. LippMD Bootman2000The versatility and universality of calcium signalling.Nat Rev Mol Cell Biol11121
  82. 82. Davis JQ, McLaughlin T, Bennett V (1993) Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain. J Cell Biol 121: 121–133.JQ DavisT. McLaughlinV. Bennett1993Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.J Cell Biol121121133
  83. 83. Garver TD, Ren Q, Tuvia S, Bennett V (1997) Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin. J Cell Biol 137: 703–714.TD GarverQ. RenS. TuviaV. Bennett1997Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.J Cell Biol137703714
  84. 84. Lemaillet G, Walker B, Lambert S (2003) Identification of a conserved ankyrin-binding motif in the family of sodium channel alpha subunits. J Biol Chem 278: 27333–27339.G. LemailletB. WalkerS. Lambert2003Identification of a conserved ankyrin-binding motif in the family of sodium channel alpha subunits.J Biol Chem2782733327339
  85. 85. Zhou D, Lambert S, Malen PL, Carpenter S, Boland LM, et al. (1998) AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing. J Cell Biol 143: 1295–1304.D. ZhouS. LambertPL MalenS. CarpenterLM Boland1998AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing.J Cell Biol14312951304
  86. 86. Garrido JJ, Giraud P, Carlier E, Fernandes F, Moussif A, et al. (2003) A targeting motif involved in sodium channel clustering at the axon initial segment. Science 300: 2091–2094.JJ GarridoP. GiraudE. CarlierF. FernandesA. Moussif2003A targeting motif involved in sodium channel clustering at the axon initial segment.Science30020912094
  87. 87. Fache MP, Moussif A, Fernandes F, Giraud P, Garrido JJ, et al. (2004) Endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment. J Cell Biol 166: 571–578.MP FacheA. MoussifF. FernandesP. GiraudJJ Garrido2004Endocytotic elimination and domain-selective tethering constitute a potential mechanism of protein segregation at the axonal initial segment.J Cell Biol166571578
  88. 88. Pan Z, Kao T, Horvath Z, Lemos J, Sul JY, et al. (2006) A common ankyrin-G-based mechanism retains KCNQ and NaV channels at electrically active domains of the axon. J Neurosci 26: 2599–2613.Z. PanT. KaoZ. HorvathJ. LemosJY Sul2006A common ankyrin-G-based mechanism retains KCNQ and NaV channels at electrically active domains of the axon.J Neurosci2625992613
  89. 89. Rasmussen HB, Frokjaer-Jensen C, Jensen CS, Jensen HS, Jorgensen NK, et al. (2007) Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment. J Cell Sci 120: 953–963.HB RasmussenC. Frokjaer-JensenCS JensenHS JensenNK Jorgensen2007Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment.J Cell Sci120953963
  90. 90. Brechet A, Fache MP, Brachet A, Ferracci G, Baude A, et al. (2008) Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G. J Cell Biol 183: 1101–1114.A. BrechetMP FacheA. BrachetG. FerracciA. Baude2008Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G.J Cell Biol18311011114
  91. 91. Leterrier C, Brachet A, Fache MP, Dargent B (2010) Voltage-gated sodium channel organization in neurons: protein interactions and trafficking pathways. Neurosci Lett, epub ahead of print. C. LeterrierA. BrachetMP FacheB. Dargent2010Voltage-gated sodium channel organization in neurons: protein interactions and trafficking pathways.Neurosci Lett, epub ahead of print
  92. 92. Taddese A, Bean BP (2002) Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons. Neuron 33: 587–600.A. TaddeseBP Bean2002Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons.Neuron33587600
  93. 93. Zhang F, Gradinaru V, Adamantidis AR, Durand R, Airan RD, et al. (2010) Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat Protoc 5: 439–456.F. ZhangV. GradinaruAR AdamantidisR. DurandRD Airan2010Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures.Nat Protoc5439456
  94. 94. Yang Y, Ogawa Y, Hedstrom KL, Rasband MN (2007) betaIV spectrin is recruited to axon initial segments and nodes of Ranvier by ankyrinG. J Cell Biol 176: 509–519.Y. YangY. OgawaKL HedstromMN Rasband2007betaIV spectrin is recruited to axon initial segments and nodes of Ranvier by ankyrinG.J Cell Biol176509519
  95. 95. Ogawa Y, Horresh I, Trimmer JS, Bredt DS, Peles E, et al. (2008) Postsynaptic density-93 clusters Kv1 channels at axon initial segments independently of Caspr2. J Neurosci 28: 5731–5739.Y. OgawaI. HorreshJS TrimmerDS BredtE. Peles2008Postsynaptic density-93 clusters Kv1 channels at axon initial segments independently of Caspr2.J Neurosci2857315739
  96. 96. Kole MH, Letzkus JJ, Stuart GJ (2007) Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy. Neuron 55: 633–647.MH KoleJJ LetzkusGJ Stuart2007Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.Neuron55633647
  97. 97. Shah MM, Migliore M, Valencia I, Cooper EC, Brown DA (2008) Functional significance of axonal Kv7 channels in hippocampal pyramidal neurons. Proc Natl Acad Sci U S A 105: 7869–7874.MM ShahM. MiglioreI. ValenciaEC CooperDA Brown2008Functional significance of axonal Kv7 channels in hippocampal pyramidal neurons.Proc Natl Acad Sci U S A10578697874
  98. 98. Bender KJ, Trussell LO (2009) Axon initial segment Ca2+ channels influence action potential generation and timing. Neuron 61: 259–271.KJ BenderLO Trussell2009Axon initial segment Ca2+ channels influence action potential generation and timing.Neuron61259271
  99. 99. Yu Y, Maureira C, Liu X, McCormick D (2010) P/Q and N channels control baseline and spike-triggered calcium levels in neocortical axons and synaptic boutons. J Neurosci 30: 11858–11869.Y. YuC. MaureiraX. LiuD. McCormick2010P/Q and N channels control baseline and spike-triggered calcium levels in neocortical axons and synaptic boutons.J Neurosci301185811869
  100. 100. Laezza F, Gerber BR, Lou JY, Kozel MA, Hartman H, et al. (2007) The FGF14(F145S) mutation disrupts the interaction of FGF14 with voltage-gated Na+ channels and impairs neuronal excitability. J Neurosci 27: 12033–12044.F. LaezzaBR GerberJY LouMA KozelH. Hartman2007The FGF14(F145S) mutation disrupts the interaction of FGF14 with voltage-gated Na+ channels and impairs neuronal excitability.J Neurosci271203312044
  101. 101. Brackenbury WJ, Calhoun JD, Chen C, Miyazaki H, Nukina N, et al. (2010) Functional reciprocity between Na+ channel Nav1.6 and beta1 subunits in the coordinated regulation of excitability and neurite outgrowth. Proc Natl Acad Sci U S A 107: 2283–2288.WJ BrackenburyJD CalhounC. ChenH. MiyazakiN. Nukina2010Functional reciprocity between Na+ channel Nav1.6 and beta1 subunits in the coordinated regulation of excitability and neurite outgrowth.Proc Natl Acad Sci U S A10722832288
  102. 102. Somogyi P, Nunzi MG, Gorio A, Smith AD (1983) A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells. Brain Res 259: 137–142.P. SomogyiMG NunziA. GorioAD Smith1983A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells.Brain Res259137142
  103. 103. Szabadics J, Varga C, Molnar G, Olah S, Barzo P, et al. (2006) Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311: 233–235.J. SzabadicsC. VargaG. MolnarS. OlahP. Barzo2006Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits.Science311233235
  104. 104. Khirug S, Yamada J, Afzalov R, Voipio J, Khiroug L, et al. (2008) GABAergic depolarization of the axon initial segment in cortical principal neurons is caused by the Na-K-2Cl cotransporter NKCC1. J Neurosci 28: 4635–4639.S. KhirugJ. YamadaR. AfzalovJ. VoipioL. Khiroug2008GABAergic depolarization of the axon initial segment in cortical principal neurons is caused by the Na-K-2Cl cotransporter NKCC1.J Neurosci2846354639
  105. 105. Glickfeld LL, Roberts JD, Somogyi P, Scanziani M (2009) Interneurons hyperpolarize pyramidal cells along their entire somatodendritic axis. Nat Neurosci 12: 21–23.LL GlickfeldJD RobertsP. SomogyiM. Scanziani2009Interneurons hyperpolarize pyramidal cells along their entire somatodendritic axis.Nat Neurosci122123