The Enigmatic Function of Chandelier Cells

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provided by PubMed Central FOCUSED REVIEW published: 08 December 2010 doi: 10.3389/fnins.2010.00201

The enigmatic function of chandelier cells

Alan R. Woodruff 1*, Stewart A. Anderson 2 and Rafael Yuste1

1 Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, NY, USA 2 Department of Psychiatry, Weill Cornell Medical College, Cornell University, New York, NY, USA

Chandelier (or axo-axonic) cells are one of the most distinctive GABAergic interneurons in the brain. Their exquisite target specificity for the axon initial segment of pyramidal neurons, together with their GABAergic nature, long suggested the possibility that they provide the ultimate inhibitory control of pyramidal neuron output. Recent findings indicate that their function may be more complicated, and perhaps more interesting, than initially believed. Here we review these recent developments and their implications. We focus in particular on whether chandelier cells may provide a depolarizing, excitatory effect on pyramidal neuron output, in addition to a powerful inhibition.

Keywords: GABAergic depolarization, excitation, cortex, axon initial segment

Introduction arborizations of different interneurons reflects The mammalian brain is an organ of seemingly their subcellular target specificity, which in turn impossible complexity. There are billions of neu- has important implications for their function. rons, trillions of synaptic connections, and prob- Indeed, a common delineation exists between ably hundreds of distinct cell types, each of which interneurons targeting dendritic domains, such as is presumably specialized to perform distinct neocortical Martinotti and double bouquet cells, Edited by: tasks. A crucial step in addressing the underly- and those targeting axo-somatic domains, such David Linden, Johns Hopkins University, USA ing logic in the design of the brain is therefore as chandelier and basket cells (Markram et al., identifying the function, or functions, of a given 2004). Whereas dendrite-targeting neurons may Reviewed by: Stephen R. Williams, University of neuronal class. be more suited to modify and gate incoming exci- Cambridge, UK The most obvious separation in neuron func- tatory input (Murayama et al., 2009), axo-somatic Miles A. Whittington, Newcastle tion within cortical structures is between exci- interneurons are likely to exhibit a greater impact University, UK tatory, glutamatergic pyramidal (or principal) on the direct output of the postsynaptic neuron *Correspondence: neurons and inhibitory GABAergic interneurons. (Marr, 1970; Miles et al., 1996). Information is presumably carried and processed Here we focus on attempting to understand by pyramidal (Pyr) neurons, whose connections the chandelier cell (ChCs), an interneuron type traverse cortical layers and regions. Interneurons, that exemplifies the target specificity of cortical by contrast, are typically considered to project interneurons and thus potentially illustrates the only locally, and provide a means of controlling purposeful design of neuronal circuits. ChCs Alan R. Woodruff received his Ph.D. the excitation provided by pyramidal neurons. It were “missed” by Cajal and Lorente and were first from the University of Queensland under is, of course, not that simple. It turns out that a identified by Szentagothai and, independently, by the mentorship of Prof. Pankaj Sah, where he studied the local circuit vast heterogeneity exists amongst the GABAergic Jones (Jones, 1975; Szentagothai, 1975). The defin- properties of GABAergic neurons in the interneurons, which constitute ∼20% of all cor- ing morphological characteristic of ChCs is the basolateral amygdala. His current tical neurons. This heterogeneity has long been array of short, vertically oriented rows of terminal research, at Columbia University with recognized (Ramón y Cajal, 1899; Lorente de Nó, boutons, which resemble candlesticks. Originally Prof. Rafael Yuste, is focused on elucidating the function of cortical 1922), and one of the first aspects of this heteroge- believed to contact the apical dendrite of pyrami- chandelier cells. neity to be noticed, their “short axons,” is likely to dal cells (Szentagothai, 1975), these axonal car- [email protected] be one of the most important – the distinct axonal tridges were later shown via electron microscopic

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Chandelier cell reconstructions of Golgi-stained specimens to nomenclature that is used interchangeably. This GABAergic interneuron whose exclusive exclusively contact the axon initial segment (AIS) highly stereotyped and visually striking appear- postsynaptic target is the axon initial of pyramidal neurons (Figure 1C; Somogyi, 1977; ance of ChCs facilitated early studies regarding segment of pyramidal neurons. Fairen and Valverde, 1980). Because of this, chan- their location, abundance, and neurochemical delier neurons were renamed “axo-axonic cells,” a features. ChCs are present in all cortical layers,

L1 L2/3

100 µm

C D 20 mV 400 pA 100 ms 10 µm ChC

E F ChC BC

-58 mV -59 mV 0.5 mV Record ChC Record BC 50 mV 20 ms ChC 10 ms -66 mV -67 mV BC NBQX -73 mV -72 mV Pyr 5 pA -81 mV -85 mV (rest) (rest) 3.5 ms -91 mV -90 mV

Figure 1 | Characteristic features of chandelier cells. (A) Neurolucida reconstruction of a layer 2/3 chandelier cell. Cell body and dendrites are in blue, axon in red. Several vertically oriented axonal segments are visible, and are the characteristic morphological feature of ChCs. The long descending axon (arrowhead) reached and arborized in layer 6, but has been digitally truncated. (B) Parvalbumin immunoreactivity of a ChC. A GFP-labeled ChC (right panel, arrow) co-expresses PV (left panel, arrow). (C) Biocytin-filled ChC forms a row of cartridge synapses (arrowheads) on the AIS of a biocytin-filled cortical pyramidal neuron (axon marked by white arrow).(D) Chandelier and basket cells have characteristic responses to threshold current injection. Both cells exhibit the fast-spiking phenotype at higher current intensities (right

panel, 2× threshold illustrated). (E) In cortical layer 2/3 pyramids, EGABA differs at ChC–Pyr and BC–Pyr synapses. (F) ChCs can initiate polysynaptic events. Activation of a GABAergic ChC evokes a response in a simultaneously recorded basket cell. The response (left panel) is sensitive to the AMPA receptor antagonist NBQX, has a disynaptic latency, and is observed in a cell type (BC) that receives no direct synapses from ChCs. Schematic of disynaptic circuit is shown in right panel. (B, D and E) modified, fromWoodruff et al. (2009).

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most abundantly in layer 2/3 (DeFelipe et al., and resulted in ChCs being less receptive to 1985; Inda et al., 2007). Although originally ascending sensory input. However, ChCs mark- described in neocortex (Szentagothai, 1975), edly increased their output during conditions of ChCs have also been found in the CA3 (Sik et al., high network activity, implying that ChCs may 1993), CA1 (Somogyi et al., 1983) and dentate be recruited to dampen excessive excitation. gyrus (Soriano and Frotscher, 1989) regions of the Until recently, therefore, all evidence seemed to hippocampus, and in the amygdala (McDonald, suggest that ChCs were inhibitory GABAergic 1982). The expression of GABA (Somogyi et al., neurons, unique in their high degree of spatial 1985), parvalbumin (PV; DeFelipe et al., 1989), target selectivity, but otherwise not particularly and corticotropin-releasing factor (Lewis et al., remarkable in comparison to other GABAergic 1989) provides a neurochemical identity to these cell types. neurons (Figure 1B). Despite this knowledge, it has been signifi- Are chandelier cells depolarizing? cantly more difficult to ascertain the physiologi- In 2006, a controversial paper appeared demon- cal properties of ChCs, and more particularly strating an excitatory effect of cortical layer 2/3 their putative functions. This is in part due to chandelier cell activation (Szabadics et al., 2006). the rarity of ChCs and the fact that common Three pieces of evidence were presented in sup- markers label both chandelier and the much port of this. Firstly, disynaptic, glutamatergic exci- more abundant basket cells. To date there is cur- tatory postsynaptic potentials (EPSPs) could be rently no known unique ChC marker. Although elicited following a single spike in the chandelier a variety of transgenic mouse lines now exist in cell – an effect attributed to direct recruitment which various populations of interneurons are of pyramidal neurons by ChCs (Figure 1F). But fluorescently labeled, including PV-positive neu- for direct recruitment of a pyramidal neuron to rons (Meyer et al., 2002; Chattopadhyaya et al., occur with such a short latency, the ChC synapse 2004), ChCs have been recorded from much less at the AIS had to be depolarizing, a condition not frequently than have the more numerous basket previously considered for this particular synapse. cells. Physiologically, both ChCs and BCs fire Secondly, the authors confirmed that the ChC

high frequency, minimally adapting trains of synapse was indeed depolarizing, with the GABAA

narrow action potentials – a fast-spiking pheno- reversal potential (EGABA) significantly elevated

type. In the cortex, BCs and ChCs were believed above the neuron’s resting potential (Vrest), and indistinguishable on physiological grounds above that of basket cell synapses (Figure 1E).

(Kawaguchi, 1995; Gonzalez-Burgos et al., 2005), Importantly, EGABA in this study was measured while in the hippocampus, ChCs were reported using the antibiotic gramicidin in the postsynap- to exhibit a greater degree of spike frequency tic pipette of a paired recording. Gramicidin forms adaptation than BCs (Han, 1994). Thus in most membrane pores that allow exclusive exchange of cases, unequivocal identification of a ChC was monovalent cations and small uncharged mol- only possible following post-recording recov- ecules (Kyrozis and Reichling, 1995). Thus there ery of the neuron’s morphology (Figure 1A). is no dialysis of intracellular chloride, as occurs Whilst this is not a difficult procedure, it is not with whole-cell patching, and more physiological

routinely performed, and the number of physi- EGABA responses are recorded through the perfo- ological and functional studies on ChCs has been rated neuronal membrane. Thirdly, immunogold somewhat limited. labeling and electron microscopic reconstruction The studies hinting at a functional role for demonstrated an apparent absence of the potas- ChCs are therefore correlative rather than causa- sium-chloride cotransporter KCC2 from the AIS tive. In vivo recordings have revealed the firing – an effect that would theoretically lead to less pattern of hippocampal ChCs during various extrusion of intracellular chloride and lead to the

network states (Klausberger et al., 2003; Tukker depolarized EGABA reported. et al., 2007), showing that ChCs fire antiphase Thus, a powerful form of excitation had been to pyramidal neurons during theta activity, and demonstrated for a neuron previously believed – fire immediately prior to pyramidal neuron based on the strategic location of its GABAergic activation during sharp-wave-associated ripples synapses – to exert a powerful form of inhibition. Gramicidin (Klausberger et al., 2003). Perhaps a more direct The Szabadics data did not exclude an inhibitory Antibiotic that forms membrane functional role was suggested by whole-cell action of ChCs, but rather added an excitatory channels impermeable to chloride. recordings from ChCs in rat somatosensory cor- role to their function. Instead of being purely Gramicidin is useful for measuring GABA receptor-mediated events when tex during whisker deflection. Whisker-evoked inhibitory, ChCs now had the potential to be A stimulation revealed different sequences of affer- either inhibitory or excitatory, depending on the maintaining an accurate GABAA reversal potential is desired. ent input compared to BCs (Zhu et al., 2004), membrane potential of the postsynaptic neuron.

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By extension, the overall activity state of the net- Both the Szabadics and Khirug data were work would determine whether ChCs behaved as obtained primarily using the gramicidin per- excitatory or inhibitory neurons. forated patch technique. Although avoiding Further support for a depolarizing effect fol- direct chloride exchange between pipette and

lowing AIS GABAA receptor activation came when cell, gramicidin recordings may still be prone to

it was shown, again using gramicidin-based patch some error in calculating EGABA. This is because the recordings, that dentate granule cells exhibited an chloride equilibrium potential is set by the com-

axo-somato-dendritic gradient in EGABA (Khirug bined activity of the cation chloride cotransport- et al., 2008). The relatively depolarized axonal ers KCC2 and NKCC1 (Figure 2A). Because both

EGABA was shown to persist, to some degree, even sodium and potassium permeate the membrane in whole-cell recordings, an effect attributed to pores created by gramicidin, pipette concentra- efficient import of chloride by the Na-K-2Cl tions of these ions that don’t precisely match the cotransporter NKCC1 (Khirug et al., 2008), rather physiological intracellular concentrations can than, or perhaps in addition to, the absence of alter the activity of the transporters, and conse-

KCC2 suggested by Szabadics et al. (2006). A simi- quently shift EGABA, potentially giving a spurious + lar hyperpolarizing–depolarizing EGABA gradient reading. For example, resting [Na ]i is low relative + from dendrite to axon was shown for cortical to resting [K ]i, so slight inaccuracies in pipette pyramidal cells of layer 2/3. [Na+] in particular may produce significant effects

A B depolarizing

Cl– Cl– ChC

VThr

EGABA

hyperpolarizing K+ Cl– AXON SOMA Vrest

2 Cl– K+/Na+ K+ 2 Cl– Cl– + + VThr K /Na NKCC1 Nav1.6

EGABA / VNa KCC2 GABAA

Vrest Na+

small Gsyn/∆V

VA/S + Gsyn

p(AP) conductance – large Gsyn/∆V closed + p(AP) –

Figure 2 | GABAergic excitation. (A) The soma and axon of a pyramidal neuron are represented. Somatic ECl is

hyperpolarized due to the high expression of KCC2 and low expression of NKCC1. GABAA receptor activation leads to hyperpolarization, or no net flux of ions (shunting). At the AIS, the expression of cation chloride transporters is reversed,

favoring chloride efflux and depolarization upon GABAA channel opening. Low-threshold NaV1.6 sodium channels may be activated by the depolarization, enhancing the excitatory effect. (B) Although depolarizing, a GABAergic event may be inhibitory, due to the conductance effect of channel opening. However, the inhibitory conductance effect (red) decays more quickly than the excitatory membrane potential change (blue), providing at least some window of excitation (right panel, bottom). The strength and duration of excitation (blue) and inhibition (red), denoted here as spike probability (p(AP)), will depend on the magnitude of the conductance effect relative to the change in membrane potential. (C) The ChC synapse is expected to be hyperpolarizing during high activity periods (red) and depolarizing under resting conditions. + Because AIS EGABA is in the range of Na channel activation, some depolarizations may be enhanced by sodium currents (bottom panel).

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on transporter activity. For this reason, regions of of the synapse may be expected to dissipate if the neuron where sodium exchange via NKCC1 the recording pipette is moved hundreds of is used to set the transmembrane chloride gradi- microns away, along the somatodendritic axis. ent, such as the axon (Khirug et al., 2008), may Consistent with this, when the recording pipette be more prone to allowing inaccurate values of was moved to distal dendritic sites, the authors

EGABA to be recorded. Additionally, slight changes still observed unitary fields of a polarity consist- + + in [Na ]i and [K ]i introduced by the recording ent with a hyperpolarizing synapse at the AIS. pipette may alter the neuron’s resting membrane Thus even when not subjected to the possibil- potential, causing a compensatory redistribution ity of chloride leak from the recording pipette, of chloride ions across the membrane through the synapse was hyperpolarizing. Additionally, passive diffusion. the concern of Cl− leaking from the electrode These concerns were addressed using a novel, was addressed by replacing the 3 M NaCl in non-invasive approach to recording the polarity the recording pipette with artificial cerebrospi- of unitary GABAergic responses in hippocampal nal fluid (ACSF). This manipulation generated CA1 (Glickfeld et al., 2009). The authors recorded similar results in the observed amplitude of the “unitary fields” – i.e., extracellular field poten- unitary field evoked by basket synapses (Glickfeld tials produced by activation of a single, mor- et al., 2009), which would not be expected if chlo- phologically verified interneuron – at various ride leak was responsible for a hyperpolarizing

somatodendritic locations of the CA1 pyramidal shift in EGABA. cell population. These recordings rely on small These data, therefore, obtained using a non- changes in the flow of ions around the extracel- invasive technique (unitary field recordings), are lular recording electrode. As membrane channels at odds with the data of Szabadics et al. (2006) and open, transmembrane ion flux results in accumu- Khirug et al. (2008), both of which were obtained lations and depletions of the permeant ions in the using the slightly more invasive gramicidin perfo- regions proximal to the recording electrode. The rated patch technique. The polarity of effect of a direction of current flow is indicated by the polar- GABAergic synapse is likely a major indicator and ity of the extracellular response. Importantly, this determinant of that synapse’s functional role. The technique does not interfere in any way with the conflicting data described above, and the differing intracellular milieu of the postsynaptic neurons, techniques used to obtain the different results, and in this respect represents an improvement on prompted us to investigate whether the depolar- whole-cell and gramicidin recordings. For each izing ChC effect may be an artifact introduced by class of interneuron recorded, targeting either gramicidin recordings. the dendritic, somatic, or axonal compartment, the authors found only a hyperpolarizing effect. Depolarizing chandeliers, revisited Thus the non-invasive approach taken provided The goal of our experiments (Woodruff et al., no evidence for a depolarizing effect of ChCs. 2009) was to re-examine the depolarizing effect A potential caveat with the technique arises, of cortical ChCs. For this, we first found a method however, because Glickfeld et al. (2009) used very to routinely record from ChCs, something that to high (molar) concentrations of chloride ions in our knowledge had not previously been achieved. their extracellular recording electrodes, which We used Nkx2.1Cre MADM mice, a strain of may have leaked into the surrounding neuropil. genetically engineered animals that express GFP Typically, extracellular chloride concentrations in a subset of neocortical interneurons arising − are ∼130 mM. But by using 3 M [Cl ]o in their from the medial ganglionic eminence. This subset extracellular pipette, the authors could poten- included ChCs, which importantly were labeled tially have produced a ∼20-fold increase in brightly, allowing the distinctive axonal cartridges local chloride concentrations, should any chlo- of ChCs to be identified prior to recordings. ChCs ride leakage occur from the pipette solution. could therefore be easily targeted, particularly in Increased extracellular chloride levels would upper layer 2/3, near the layer 1 border. We rou-

shift EGABA to hyperpolarized potentials, favor- tinely recorded from ChCs in layer 2/3 and were

ing chloride influx upon GABAA channel open- able to distinguish them from basket cells based ing, and thus a hyperpolarizing response at the on their firing pattern and passive membrane synapse. Could the hyperpolarizing response properties (Figure 1D; Woodruff et al., 2009). of CA1 ChCs, which contrasts with the grami- This distinction was extremely reliable, as con- cidin recordings in cortex and in hippocampal firmed by the subsequent anatomical identifica- Extracellular field potentials The electrical potential produced by dentate granule cells, therefore be an artifact of tion of recorded cells, and obviated the need for neurons due to transmembrane ion the recording technique? If significant chloride morphological verification in the vast majority − flux, measured extracellularly. leak did occur, any changes in [Cl ]o at the site of subsequent cases.

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We first performed paired recordings from Can cortical chandelier cells be presynaptic ChCs and gramicidin-patched excitatory? postsynaptic pyramidal cells. Our data, taken But does the axonal depolarization caused by corti- from mouse neocortex, closely matched that of cal ChCs promote or inhibit action potential firing? Szabadics et al. (2006) from rat neocortex, with To date, the only demonstration that ChCs are exci-

EGABA of the ChC synapse lying ∼20 mV above tatory – that they promote firing – is the suprath- resting membrane potential, and ∼10 mV below reshold activation of pyramidal neurons reported action potential threshold. Similar experiments by several groups (Szabadics et al., 2006; Woodruff performed on basket cell synapses showed a pre- et al., 2006; Molnar et al., 2008; Glickfeld et al.,

dominantly shunting synapse, in which EGABA was 2009). But even this phenomenon, while clearly typically at the neuron’s resting membrane poten- excitatory, should be interpreted cautiously. tial. While this was consistent with the previous Firstly, nerve injury is known to result in an gramicidin-based recordings from Szabadics upregulation of NKCC1 and a downregulation and Khirug, the possibility remained that the of KCC2 (Hasbargen et al., 2010), both of which

depolarized axonal EGABA was simply the result would favor a depolarized EGABA. Thus the frequent of changes in the activity of NKCC1 that may axotomies that occur during the preparation of occur via alterations in intracellular Na+ concen- brain slices could create artificially high axonal

trations, as outlined above. We therefore com- EGABA values, leading to hyperexcitability. plemented these recordings with a completely Secondly, chandelier-triggered activation non-invasive technique, tight seal cell-attached of a pyramidal neuron has never been directly recordings. Cell-attached recordings have only observed, instead being inferred by the presence rarely been used to record synaptic potentials of a disynaptic, glutamatergic response that is (but see Kantrowitz et al., 2005; Perkins, 2006), time-locked to the ChC spike (Figure 1F) and

and to our knowledge, only to record large- which is sensitive to the GABAA receptor antago- amplitude, network driven events (Kantrowitz nist bicuculline. The reason for this may be merely et al., 2005). We first showed that detecting sub- statistical, in that the chances of recording from millivolt voltage deflections would be possible the one or two responsive pyramidal neurons are with tight seal cell-attached recordings. Our low. Alternatively, it may be because the active paired recordings then consistently revealed a pyramidal neurons are unhealthy, with a conse- depolarization of the postsynaptic pyramidal cell quent increase in NKCC1 expression responsible after ChC activation. All recordings, regardless of for the phenomenon. These neurons would not be whether they displayed evidence of a connection targeted for patching. Bulk loading of membrane- in the cell-attached configuration, were subse- permeable acetoxymethyl (AM) Ca2+ indicators, quently tested in whole-cell mode for the pres- which allows action potentials to be monitored ence of a synaptic response. We were thus able in hundreds of neurons (Yuste and Katz, 1991; to distinguish purely shunting synapses, which Smetters et al., 1999), should facilitate the detec- would not be detectable in cell-attached mode tion of active neurons, although we remain una- but would be revealed upon entering whole- ware of success with this technique. This failure cell mode, from unconnected ChC–Pyr pairs. could be due to low signal-to-noise, to unhealthy Importantly, we never observed purely shunt- neurons not incorporating the AM Ca2+ indica- ing or hyperpolarizing ChC–Pyr synapses. These tors, or to saturating concentrations of indicator data supported our gramicidin recordings, and that prevent the detection of action potential- furthermore demonstrated that the ChC-evoked induced fluorescence changes. Alternatively, the depolarization at the AIS observed using grami- action potential produced by chandelier activa- cidin recordings could not be disregarded as an tion may, depending on its initiation point rela- experimental artifact. tive to the ChC synapse, fail to backpropagate to These data demonstrated that at least for layer the soma. This would prevent its detection both 2/3 cortical ChCs, and seemingly also for dentate by somatic patch recording and somatic action- Cell-attached recordings granule cells, GABA at the AIS is depolarizing. potential derived fluorescence changes. Recordings in which the recording However, ChCs in CA1 produce a hyperpolariza- It is certainly not clear that the suprathreshold pipette is placed on the exterior of the cell membrane. Loose-seal cell-attached tion. Because the reversal potential of a GABAergic activation described by us and others – which is recordings are commonly used for synapse is likely an important determinant of that the sole excitatory ChC action reported to date – is recording currents due to action synapse’s function, an interesting dichotomy may a pathological response. However, until such acti- potentials. Tight-seal cell-attached exist in the roles played by ChCs in different parts vations are directly observed by recording from a recordings can be used to measure synaptic potentials. In the absence of of the brain, an intriguing possibility for a cell healthy pyramidal neuron, or until it is observed injected current, cell-attached type previously hypothesized to be a fine example in the intact animal, the possibility that they are recordings are completely non-invasive. of functional specialization. artifactual should be kept in mind.

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Disregarding the suprathreshold activations the driving force (EGABA − Vrest) is high enough,

induced by ChCs, we are left with the fact that in and EGABA is not too distant from VThr, GABA is some parts of the brain, but perhaps not in others, proven to be excitatory in these neurons. The val- ChCs are depolarizing. This subthreshold depo- ues reported for the three determining param- larization is, in fact, likely to be the effect felt by eters in this study are, in fact, in line with those the vast majority of recipient pyramidal neurons at the cortical ChC synapse, and an important upon ChC activation. A highly pertinent question, requirement for GABAergic excitation appears then, is whether this subthreshold depolarization to be the activation of voltage-gated sodium is excitatory. A few factors merit some thought. channels (VGSCs), which allow the neuron to

The opening of any membrane channel decreases continue depolarizing despite being above EGABA the neuronal input resistance, causing shunting (Figures 2A,B; Rheims et al., 2008; Valeeva et al., inhibition – a given current then produces a 2010). Since VGSCs at the AIS have a relatively smaller voltage deflection. This shunting effect hyperpolarized activation threshold (Colbert and is therefore by nature inhibitory, and antagonistic Pan, 2002; Astman et al., 2006; Hu et al., 2009)

to any depolarization (Figure 2C). Whether the due to the presence of NaV1.6 channels, in the

magnitude of the conductance shunt outweighs range of axonal EGABA, it certainly seems possible the magnitude of the depolarization is therefore that ChCs can be excitatory in a manner similar an important consideration, though it should to that seen in immature neurons. be noted that once the conductance closes, any It should be noted that there is considerable

remaining depolarization is likely to be excitatory neuron to neuron variability in both EGABA and

(Figure 2C). VThr. Both of these parameters may be plastic,

Secondly, EGABA for the ChC synapse lies below changing according to the neuron’s activity his- threshold. Although a ChC PSP may initially be tory (Henze and Buzsaki, 2001; Woodin et al., depolarizing, with the pyramidal neuron below 2003; Fiumelli et al., 2005), and small changes in

EGABA, continued depolarization of the pyra- these values are likely to be critical in determining

mid above EGABA, perhaps through additional whether GABA can be excitatory (Rheims et al., glutamatergic inputs, will result in the ChC PSP 2008). Thus, the precise interplay of the neuron’s

switching to hyperpolarizing and inhibitory, pro- membrane potential, EGABA and VThr, their modi- vided the conductance is still open. Whether the fication by prior activity, and the activation of net effect of ChC activation under these condi- axonal VGSCs seem to be the critical parameters tions will help or hinder firing is therefore not that will determine whether ChCs can provide straightforward. excitatory, as well as inhibitory, input. Another point worth considering is that con- The discussion so far has focused on experi- ductance effects aside, there still exists the theo- ments performed in vitro. However, neurons in retical possibility for a depolarization-induced vivo are constantly bombarded with synaptic inhibition due to inactivation of the Na+ chan- input, producing a depolarized and fluctuating nels responsible for spike generation. However, we membrane potential that promotes the opening believe this is unlikely to be a significant factor in and closure of a vast array of voltage depend- the case of ChCs, given that depolarization also ent channels (Destexhe et al., 2003). That in vivo greatly enhances Na+ channel open probability. membrane potentials are considerably more Indeed, if this possibility is to be considered, one depolarized than in the quiescent in vitro slice must also consider the possibility that similar may be particularly relevant for ChCs. Axonal

mechanisms can result in glutamatergic inputs EGABA in cortical layer 2/3 neurons is in the range being inhibitory due to the depolarization they of −65 to −55 mV, values similar to mean mem- produce – the situation is analogous. In addition, brane potentials recorded in awake animals the AIS contains not only fast-inactivating Na+ (Brecht et al., 2004; Poulet and Petersen, 2008; channels responsible for spike generation, but Gentet et al., 2010). Predicting the effect of ChCs also slowly inactivating channels with a negatively in vivo therefore becomes quite a complicated shifted activation threshold (Astman et al., 2006), exercise. If the in vivo membrane potential fluctu- which underlie the “persistent” sodium current, ates either side of E , the ChC has the potential Shunting inhibition GABA A form of inhibition that results from INAP (Fleidervish et al., 2010). This persistent cur- to be excitatory as well as inhibitory. This may the opening of a membrane rent may be important in aiding spike generation promote the homogenization of pyramidal neu- conductance and the decrease in following a ChC input. ron firing rates, inhibiting strongly active neu- excitability this causes. Typically used to A situation analogous to that in cortical ChCs, in rons but exciting those that are less active (Vida describe the effect of a GABAergic which EGABA lies significantly aboveV rest but below et al., 2006). In this scenario, ChCs may act as interneuron when EGABA of the synapse is equal to the membrane potential of VThr, exists in immature neocortical (Rheims et al., activity sensors, passively adjusting their function the neuron. 2008) and hippocampal pyramidal cells. Provided according to what is required by the surrounding

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network. The effect will be dictated, of course, by 2010; Varga et al., 2010). Whether there is spe- the pyramidal neuron membrane potential, and cificity in which particular local and interlaminar by the complex interplay of that neuron’s syn- neurons ChCs target is unknown, although there aptic inputs. Whether the membrane potential is some evidence that they may preferentially

ever stays below EGABA for a sufficient period of contact pyramidal neurons with predominantly time to allow depolarization, and whether the intracortical projections (De Carlos et al., 1985; presumably small amount of depolarization (so Farinas and DeFelipe, 1991). The presynaptic

close to EGABA) provided by the ChC will out- partners of ChCs are less well documented. Laser weigh the shunting effect, are critical questions. scanning photostimulation (LSPS) was recently Alternatively, perhaps the ChC synapse in vivo applied to L2/3 ChCs in mouse primary somato- should be considered predominantly shunting, sensory cortex, demonstrating excitatory input and thus purely inhibitory. Indeed, during active predominantly from layers 2/3 and 5a, and inhibi- states, the shunting effect often ascribed to basket tory input from L2/3 and L1 (Xu and Callaway,

cells, based on EGABA approximating resting poten- 2009). While this laminar input information is tial in a slice, may be more appropriate for ChCs, certainly useful, a higher resolution technique while BCs would instead provide hyperpolarizing such as two-photon photostimulation (Nikolenko inhibition. In this way, ChCs and BCs could have et al., 2007) could be useful in determining more quite distinct inhibitory, perisomatic functions. In precise input specificity. In addition, because these CA1, where the ChC and BC synapses presumably mapping experiments are performed in slices, a have more similar reversal potentials, any differ- great number of longer-range, inter-areal connec- ences in function must depend more critically on tions are likely to be severed. We commonly find the location of the synapse (AIS vs. soma/proxi- ChCs at the border between layer 1 and layer 2/3, mal dendrites), or on differences in their pre and a site from which their dendrites have easy access postsynaptic partners. to long-range “feedback” projections from higher cortical areas. A promising method for determin- Future Directions ing inter-areal connectivity is monosynaptic, ret- While it has recently become more feasible to rograde tracing using a deletion-mutant rabies record from ChCs, significant barriers to under- virus (Wickersham et al., 2007). These projec- standing their function remain. Firstly, because of tions are unlikely to be maintained in vitro. In their sparseness, recording from and manipulat- theory however, in vivo single-cell electroporation ing more than one or two at a time is still difficult. or whole-cell patching of a ChC at the layer 1 Although some studies report robust network- border is possible and should allow the introduc- level effects after manipulation of a single neuron tion of the virus, enabling both its local and long- (Brecht et al., 2004; Houweling and Brecht, 2008; range presynaptic connections to be elucidated Bonifazi et al., 2009), it would certainly be ben- (Marshel et al., 2010). It would be interesting, for eficial to selectively control several ChCs at once. example, to determine the similarity in inter-areal This sort of control has been demonstrated for input received by the apical tuft of L2/3 pyramidal PV-expressing fast-spiking cells using neurons neurons and the layer 1 dendrites of ChCs located transfected with channelrhodopsin-2 (ChR2; in upper layer 2/3, whose major targets are those Cardin et al., 2009; Sohal et al., 2009), a light- same pyramidal neurons. activated proton pump capable of depolarizing While dissecting the synaptic and network and activating neurons with high temporal reso- effects of chandelier neurons on cortical activ- lution (Boyden et al., 2005). As of now, however, ity remains a major challenge, considering how no unique genetic marker exists for ChCs, so that these effects may change during later postnatal interrogating their function in this manner is not development adds another important twist to yet possible. We expect this to happen in the near this story. Like other PV-expressing interneurons, future, however, and their currently murky role ChCs in the mouse originate in the medial gan- should become considerably clearer. glionic eminence from progenitors that express Another important direction will be to the fate-determining transcription factor, Nkx2.1 determine the synaptic partners of ChCs. Their (Xu et al., 2008; Fazzari et al., 2010). Factors that postsynaptic targets are quite well established, specify chandelier interneuron fate from other predominantly being local pyramidal neurons, Nkx2.1+ lineages, including other PV-expressing although some interlaminar connections also interneurons as well as those that express soma- exist (Somogyi et al., 1982). There are increasing tostatin, are at this point unknown. During post- numbers of reports demonstrating target specifi- natal development, the chandelier axon cartridges city in neuronal connections (Yoshimura et al., begin to be identifiable in mouse cortex at about 2005; Brown and Hestrin, 2009; Anderson et al., postnatal day 14, based on analysis of GFP+ cells

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in Nkx2.1Cre MADM mice. In the cat visual cor- tant to determine whether the quantity as well as tex, chandelier axon terminals undergo major the postsynaptic and network effects of chande- refinements between the early postnatal period lier neuron activity change during the pubertal and adulthood, although they appear to target age range of cortical development. only the AIS (Somogyi et al., 1982). Clearly, many questions remain regarding the Interestingly, this postnatal refinement may function of ChCs. Even the quite basic question be quite protracted. In the macaque neocortex, of whether their synapses are depolarizing or where chandelier cartridges are identifiable by hyperpolarizing is still somewhat open. While immunohistochemistry for PV (DeFelipe et al., they may exert different effects in different parts 1989), the detectability of these cartridges is of the brain, this implies a different function for highly dynamic (Anderson et al., 1995). A low what is otherwise considered a single neuronal density is present by 3 months, but they increase subtype, an intriguing possibility. Secondly, if greatly to reach a peak at about 15 months. the synapse can be depolarizing in vitro, that However, their numbers diminish over the does not necessarily hold for conditions in vivo pubertal age range, falling back to the level seen (at least in awake animals), and thus any func- in 3-month-old brains by about 3 years of age. tions ascribed to ChCs recorded in the slice must Since neither the density of GABA terminals be confirmed in the intact brain. Nor has it been (Erickson and Lewis, 2002) nor the density of established experimentally that the depolariza-

GABAA receptors on the AIS (in mice; Katagiri tion cortical ChCs can provide is excitatory. The et al., 2007) changes over this period, and since role that ChCs play in controlling or modulating PV expression is known to be dependent on neuronal communication remains an intrigu- extrinsic factors (Vogt Weisenhorn et al., 1998), ing mystery, hopefully to be solved in the near the reduction of PV detectability in chandelier future. axon terminals may reflect alterations of chandelier neuron activity over the pubertal age range. Acknowledgments Given the major role of GABA maturation on We thank laboratory members for comments, late-developing aspects of cortical plasticity Yeonsook Shin and Laura McGarry for help with (Hensch, 2005), evidence that chandelier axon anatomical reconstructions and histological pro- marker expression changes dramatically during cedures, and the National Eye Institute and the postnatal development suggests that it is impor- NIMH for support.

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cells in the mouse telencephalon. J. Yuste, R., and Katz, L. C. (1991). Control Conflict of Interest Statement: The chandelier cells. Front. Neurosci. 4:201. doi: Comp. Neurol. 506, 16–29. of postsynaptic Ca2+ influx in devel- authors declare that the research was 10.3389/fnins.2010.00201 Xu, X., and Callaway, E. M. (2009). oping neocortex by excitatory and conducted in the absence of any com- Copyright © 2010 Woodruff, Anderson Laminar specificity of functional input inhibitory neurotransmitters. Neuron mercial or financial relationships that and Yuste. This is an open-access arti- to distinct types of inhibitory cortical 6, 333–344. could be construed as a potential conflict cle subject to an exclusive license agree- neurons. J. Neurosci. 29, 70–85. Zhu, Y., Stornetta, R. L., and Zhu, J. J. of interest. ment between the authors and the Yoshimura, Y., Dantzker, J. L., and (2004). Chandelier cells control exces- Frontiers Research Foundation, which Callaway, E. M. (2005). Excitatory sive cortical excitation: characteristics of Received: 20 August 2010; accepted: 19 November permits unrestricted use, distribution, cortical neurons form fine-scale whisker-evoked synaptic responses of 2010; published online: 08 December 2010. and reproduction in any medium, pro- functional networks. Nature 433, layer 2/3 nonpyramidal and pyramidal Citation: Woodruff AR, Anderson SA and vided the original authors and source 868–873. neurons. J. Neurosci. 24, 5101–5108. Yuste R (2010) The enigmatic function of are credited.

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