Ca2+ imaging of mouse neocortical interneurone -

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Neurones in the mammalian CNS are characterized by an

exuberant diversity of dendritic morphologies (Ramón y

Cajal, 1904). Dendrites were thought for decades to be

passive cables, yet it has become clear than many

mammalian neurones have dendrites with active

conductances and rich intrinsic electrophysiological

properties (Johnston et al. 1996; Yuste and Tank, 1996;

Llinás, 1988; Stuart & Sakmann, 1994). In particular, in

pyramidal cells, electrophysiological and imaging studies

have demonstrated the existence of backpropagating

sodium-based action potentials (APs), which can quickly

propagate through large territories of the dendritic tree

and trigger essentially instantaneous calcium accumul-

ations in spines and dendritic shafts (Stuart & Sakmann,

1994; Yuste & Denk, 1995). In addition, local dendritic

spikes, mediated by sodium or calcium channels or by

regenerative activation of NMDA receptors (NMDARs),

can activate restricted regions of the dendritic tree and

trigger more localized calcium accumulations (Pockberger,

1991; Amitai et al. 1993; Yuste et al. 1994; Schiller et al.1997; Schiller et al. 2000). These different types of dendritic

spiking have been implicated in the implementation of

synaptic learning rules (Magee & Johnston, 1997;

Markram et al. 1997) and in the temporal firing patterns of

the cell (Larkum et al. 2001).

GABAergic cells are thought to play an essential role in

controlling the excitability and spike timing in cortical

networks (Somogyi et al. 1998; Pouille & Scanziani, 2001).

Although they have prominent dendritic trees with a large

diversity of morphologies, their dendritic physiology is

relatively unexplored. An indication that the dendrites of

GABAergic cells are endowed with spiking properties

came from modelling studies to explain the paradoxical

activation of interneurones by single release site EPSPs

(Gulyas et al. 1993; Traub & Miles, 1995). Two recent

studies have demonstrated that one class of hippocampal

interneurone and a potentially homologue neocortical cell

type also have active dendrites, although it is still unclear if

other classes of interneurone behave similarly. Specifically,

dendritic recordings from oriens-alveus interneurones in

the hippocampus have established that these cells exhibit

dendritic APs that are mediated by sodium channels and

can backpropagate to the dendritic tree (Martina et al.2000). In addition, bitufted, somatostatin-positive inter-

neurones in layer 2/3 from the rat neocortex also have

backpropagating dendritic APs, which cause EPSP

depression via dendritic calcium accumulations (Zilberter,

2000; Kaiser et al. 2001). These calcium accumulations

were reported to be smaller than those measured in

pyramidal neurones, perhaps due to the larger calcium-

Ca2+ imaging of mouse neocortical interneurone dendrites:Ia-type K+ channels control action potential backpropagationJesse H. Goldberg, Gabor Tamas* and Rafael Yuste

Department of Biological Sciences, Columbia University, New York, NY 10027, USA and *Department of Comparative Physiology, University ofSzeged, Szeged, Hungary H-6726

GABAergic interneurones are essential in cortical processing, yet the functional properties of their

dendrites are still poorly understood. In this first study, we combined two-photon calcium imaging

with whole-cell recording and anatomical reconstructions to examine the calcium dynamics during

action potential (AP) backpropagation in three types of V1 supragranular interneurones:

parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells

(AD). Somatically generated APs actively backpropagated into the dendritic tree and evoked

instantaneous calcium accumulations. Although voltage-gated calcium channels were expressed

throughout the dendritic arbor, calcium signals during backpropagation of both single APs and AP

trains were restricted to proximal dendrites. This spatial control of AP backpropagation was

mediated by Ia-type potassium currents and could be mitigated by by previous synaptic activity.

Further, we observed supralinear summation of calcium signals in synaptically activated dendritic

compartments. Together, these findings indicate that in interneurons, dendritic AP propagation is

synaptically regulated. We propose that interneurones have a perisomatic and a distal dendritic

functional compartment, with different integrative functions.

(Received 7 March 2003; accepted after revision 8 May 2003; first published online 4 July 2003)

Corresponding author J. H. Goldberg: Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, Box2435, New York, NY 10027, USA. Email: [email protected]

J Physiol (2003), 551.1, pp. 49–65 DOI: 10.1113/jphysiol.2003.042580

© The Physiological Society 2003 www.jphysiol.org

) at MASS INST OF TECHNOLOGY on October 4, 2011jp.physoc.orgDownloaded from J Physiol (

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buffering capacity of interneurones (Lee et al. 2000a;

Kaiser et al. 2001).

We have used two-photon calcium imaging to

systematically explore the phenomenology and mechanisms

underlying calcium accumulations in different types of

supragranular V1 neocortical interneurones. We focused

on two groups (multipolar, parvalbumin-positive fast

spikers (FS) and bipolar, calretinin-positive irregular spikers

(IS)) based on their morphology, intrinsic electro-

physiology, and immunocytochemistry. In addition, we

include data from a third, heterogeneous, group of

interneurones which we term adapting (AD), due to their

spike frequency adaptation during depolarizing current

injections (Dantzker & Callaway, 2000).

We found that APs required sodium channels to

backpropagate and produced calcium accumulations

mediated by voltage-gated calcium channels (VGCCs).

We observed that VGCCs were expressed throughout the

dendritic tree, and that calcium signals during back-

propagating APs were proximally restricted by potassium

currents. In addition, we found that calcium influx due to

dendritic AP invasion was enhanced specifically in

synaptically activated dendritic compartments.

METHODS Slice preparation and electrophysiologyExperiments were carried out in accordance with the NIH Guidefor the Care and Use of Laboratory Animals (NIH publication no.86–23, revised 1987) and with the Society for Neuroscience 1995Statement (http://www.jneurosci.org/misc/itoa.shtml). Coronalslices of primary visual cortex were made from P13–17 C57BL/6mice. Animals were anaesthetized with ketamine–xylazine (50and 10 mg kg_1). After decapitation, brains were rapidly removedand transferred into ice-cold cutting solution containing (mM):222 sucrose, 27 NaHCO3, 2.5 KCl, 1.5 NaH2PO4, bubbled with95 % O2–5 % CO2 to pH 7.4. Brains were cooled for at least 2 minand 300-mm-thick slices were prepared with a Vibratome(VT1000, Leitz, Germany). Slices were then transferred to aheated solution (35 °C) containing (mM): 126 NaCl, 3 KCl, 1.1NaH2PO4, 26 NaHCO3, 1 CaCl2, 3 MgSO4, bubbled with 95 %O2_5 % CO2 to pH 7.4, which cooled down in the next 30 min toroom temperature. Slices were transferred to the imagingchamber 1–7 h after cutting. Artificial cerebral spinal fluid(ACSF) during experiments contained (mM): 126 NaCl, 3 KCl, 1.1NaH2PO4, 26 NaHCO3, 3 CaCl2, 1 Mg2SO4, bubbled with 95 %O2–5 % CO2 to pH 7.4. All experiments were performed at 37 °C.Whole-cell recordings from non-pyramidal cells in layer 2/3 wereobtained with a patch-clamp amplifier (Axoclamp 2B, AxonInstruments, Foster City, CA, USA, or BVC-700, Dagan Corp.,Minneapolis, MN, USA). Mechanisms of backpropagation wereexplored with several drugs (Sigma), including CPA (50 mM),NiCl2 (1 mM), TTX (1 mM), TEA (24 mM), 4-AP (1 mM), andDl-APV (100–200mM). 6-Cyano-7-nitroquinoxaline-2,3-dione(CNQX) (100 mM) was washed in during some 4-AP experimentsto prevent background synaptic activity, and Trolox (100 mM,Aldrich) was used in some Fluo-4 experiments to reducephototoxicity. Neurones were stimulated synaptically using anextracellular pipette filled with 200 mM Alexa-488 dextran

(Molecular Probes, Eugene, OR, USA) in ACSF. Tips ofstimulation pipettes were bent by about 70 deg with a microforge(Narishige, Japan). This allowed positioning the stimulationpipette perpendicular to the slice surface. In order to achieve localsubthreshold stimulation it was necessary to place glass electrodesin the immediate vicinity (< 15 mm) of the dendrite of interest,and use small amplitude (5–20 mA or 1 V), and short duration(100 ms) single shocks.

Two-photon imagingCells were filled via patch pipette with 200 mM CaGreen-1 or400 mM Fluo-4 (Molecular Probes). Pipette solution contained(mM): 130 KMeO4 , 5 KCl, 5 NaCl, 10 Hepes, 2.5 Mg-ATP, 0.3GTP, 0.2 CaGreen-1 (or 0.4 Fluo-4), and 0.03 % biocytin and wastitrated to pH 7.3. Following break-in, we waited for 30 minbefore imaging to ensure that dendrites filled with indicator.Imaging was done using a custom-made two-photon laser scanningmicroscope, consisting of a modified Fluoview (Olympus,Melville, NY, USA) confocal microscope with a Ti:sapphire laserproviding 130 fs pulses at 75 MHz (Mira, Coherent, Santa Clara,CA, USA), and pumped by a solid-state source (Verdi, Coherent).A 60 w, 0.9 NA water immersion objective (IR1, Olympus) wasused. Fluorescence was detected with photo-multiplier tubes(HC125-02, Hamamatsu, Hamamatsu City, Japan) in externalwhole-area detection mode, and images were acquired andanalysed with Fluoview (Olympus) software. Images of dendriteswere acquired at 10 w digital zoom, resulting in a nominal spatialresolution of 30 pixels mm_1 and at a time resolution of 12.64 msper point (79 Hz) in line-scan mode.

AnalysisFluorescence levels of calcium measurements were analysed usingFluoview (Olympus) and ImageJ (NIH, Bethesda, MD, USA).Time courses were analysed using Igor (Wavemetrics, LakeOswego, OR, USA). Calcium signals during AP generation weredetected in line-scan mode and were corrected for backgroundfluorescence by measuring a non-fluorescent area close to thedendrite. The relative change of fluorescence of baseline (from400 ms prior to AP generation) (DF/F) was used as an indicatorfor the change in calcium. Between 5 and 15 line scans weretypically averaged to generate DF/F transients during APs. Decaykinetics were fitted using single exponential fitting algorithms ofIgor. Unless mentioned, two-sided Student t tests were used, anddata are presented as mean ± standard deviation (S.D.). Distancesfrom the soma were measured from the site of dendritic imagingto the location where the parent dendrite emerged from the soma.AP repolarization in 1 mM TEA experiments was measured as thetime from initial resting potential to return to resting potentialafter a single AP. Calcium transients in Fig. 4 were filtered with asliding Hanning kernel.

HistologyVisualization of biocytin was performed as described (Buhl et al.1994; Tamas et al. 1997). Three-dimensional light microscopicreconstructions were carried out using Neurolucida and NeuroExplorer (MicroBrightfield, Colchester, VT, USA) with a 100 w oilobjective. Monoclonal antibodies to parvalbumin (Swant,Bellinzona, Switzerland, diluted 1:2000) and calretinin (Swant,1:1000) were applied to characterize interneurones. Dualfluorescence labelling of cortical slices was carried out as described(Reyes et al. 1998; Tamas et al. 2000), using Alexa488-conjugatedstreptavidin (Molecular Probes) revealing biocytin and CY3-conjugated anti-mouse IgG (Jackson Labs, West Grove, PA, USA)for parvalbumin and calretinin.

J. H. Goldberg, G. Tamas and R. Yuste50 J Physiol 551.1



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RESULTSDifferent classes of interneurones in layer 2/3 frommouse primary visual cortexTo characterize the intrinsic calcium dynamics of dendrites

from neocortical interneurones we performed combined

imaging–electrophysiological–anatomical experiments with

100 layer 2/3 non-pyramidal neurones in slices from

mouse primary visual cortex. Somata without detectable

apical dendrites were targeted using differential

interference contrast (DIC) for whole-cell recordings,

filled with the high-affinity calcium indicator calcium

green (200 mM) or Fluo-4 (400 mM), imaged with a

custom-made laser scanning two-photon microscope and,

in some cases, also reconstructed anatomically.

We characterized two groups of interneurones (Fig. 1): fast

spiking cells (FS, n = 41) and irregularly spiking cells (IS,

n = 39). FS cells (Connors & Gutnick, 1990) were

characterized by high-frequency non-adapting spike

trains during sustained current injection, multipolar

dendritic morphologies with dense axonal arborizations

generally restricted to layer 2/3, and parvalbumin

immunoreactivity (n = 12/13) (Fig. 1A). IS cells (Cauli etal. 1997) fired an initial burst of APs often followed

by single spikes at an irregular frequency, had

characteristically bipolar dendritic morphologies with a

narrow columnar axonal arbor that could reach layer 6,

and were preferentially immunoreactive for calretinin

(n = 7/10) (Fig. 1B). Finally, we encountered a third,

heterogeneous group of interneurones with adapting

firing patterns, characteristically different from the FS

and IS neurones (AD, n = 20) (Fig. 1C). Some AD cells

had bitufted dendritic arbors (n = 8) and expressed

somatostatin (n = 1/2) (data not shown) (Reyes et al. 1998;

Kaiser et al. 2001), whereas other AD cells, of variable

morphology, had regular spiking electrophysiological

characteristics (n = 9) (Szabadics et al. 2001).

Action potentials triggered dendritic calciumaccumulations in interneuronesTo explore the dendritic expression of voltage-gated

channels and to characterize the intrinsic calcium

dynamics we used APs to trigger stereotyped and

reproducible calcium accumulations (Majewska et al.2000). In these experiments we stimulated neurones with

single APs or trains of 10 APs (40 Hz), evoked with

depolarizing current steps injected in the soma while we

imaged different regions of the dendritic tree using line

scans with 79 Hz resolution (Fig. 2).

We found similar dendritic calcium accumulations in all

three types of interneurones, which were significantly

different from those found in neighbouring pyramidal

neurones (Fig. 2D–F). With 200 mM calcium green as the

indicator, in all FS, IS and AD cells, single APs caused

barely detectable calcium accumulations even in proximal

dendrites, at distances of < 50 mm from the soma

(Fig. 2C–F, left panels). However, trains of APs at

20–100 Hz reliably caused a time-locked calcium

accumulation which depended on the number of APs

fired. To investigate calcium accumulations during back-

propagation systematically, we chose a physiologically

relevant stimulation protocol of 10 APs at 40 Hz (Csicsvari

et al. 1998; Swadlow et al. 1998). We compared peak

calcium signals during 10 APs (40 Hz) in proximal

dendrites (< 50 mm), and did not observe significant class-

specific differences (Fig. 2D–F, right panels; for FS:

48 ± 35 % DF/F, n = 31; for IS: 47 ± 39 % DF/F, n = 26;

for AD: 78 ± 43 % DF/F, n = 15; mean ± S.D. for all

measurements). However, we observed that across classes

interneurones with < 50 % DF/F peak amplitude had

significantly longer decay time constant (t) values than

those with > 100 % DF/F peak amplitude (1500 ± 850 and

860 ± 370 ms, respectively P < 0.01). We assume that

indicator completely washed into the cell in the 30 min we

waited prior to imaging; thus, these results were consistent

with a high interneurone-group non-specific variability in

endogenous buffer capacities (Lee et al. 2000a).

Calcium signals during AP backpropagation in inter-

neurones were significantly smaller and slower than in

pyramidal cells. Peak amplitudes of calcium signals

(%DF/F) in proximal ( < 50 mm) dendrites of inter-

neurones during 10 APs were comparable to those found

in proximal apical dendrites from pyramidal cells during a

single AP (Fig. 2C, 48 ± 27 % DF/F, n = 45 (K. Holthoff &

R. Yuste, unpublished observations). In addition, decay

time constants of the calcium accumulations in pyramidal

cells (430 ± 240 ms; n = 42) were faster than those of FS

(1050 ± 650 ms; n = 31, P < 0.001), IS (1490 ± 810 ms;

n = 26, P < 0.001) and AD cells (1170 ± 550 ms, n = 15,

P < 0.001). Since higher buffer capacities decrease the

amplitude and prolong the decays of fluorescent calcium

transients (Helmchen, 1999), these results were consistent

with studies demonstrating that interneurones have

higher endogenous buffer capacities than pyramidal

neurones (Lee et al. 2000a; Kaiser et al. 2001).

Distinct populations of cortical interneurones express a

diversity of calcium-binding proteins in a cell-type-

specific fashion (DeFelipe, 1993). FS cells expressed

parvalbumin (Fig. 1A) (Kawaguchi & Kubota, 1993),

bipolar IS cells expressed calretinin (Fig. 1B) (Cauli et al.1997), and AD cells were a heterogeneous class with

potentially different calcium buffers. Because kinetically

distinct buffers are predicted to differentially shape dendritic

calcium dynamics during AP backpropagation (Markram

et al. 1998), we wondered whether there were kinetic

differences in the FS, IS or AD calcium transients. At all

distances from the soma, FS cells had faster decay kinetics

than IS cells, although this trend was only significant at

intermediate distances (P = 0.007, Mann–Whitney U-test;

Backpropagation in interneuronesJ Physiol 551.1 51



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Figure 1. Morphology and intrinsic electrophysiology of different types of interneuronesAa, fast spiking (FS) firing pattern in response to 800 ms depolarizing (above) and hyperpolarizing (below)current injections. Ab, representative FS cell morphology with multipolar dendritic arbor (blue) and localaxonal collaterals (red). Ac, parvalbumin (PV) immunopositivity of an FS cell with firing pattern andmorphology as shown above. The red PV immunostained cell in the left panel and the green biocytin-filledcell in the right panel indicated by arrows are the same cell. Ba, IS firing pattern, same regime as in Aa.



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Fig. 3D). This may have been due to the presence of

parvalbumin in these cells, which has been shown to

accelerate the initial component of the decay phase due to

its high affinity for but slow binding to calcium (Chard etal. 1993; Lee et al. 2000b). It is important to note that the

intracellular environment during whole-cell recording is

highly dialysed, suggesting that under more physiological

conditions, the impact of mobile buffers such as

parvalbumin and calretinin on calcium kinetics could be

more profound.

Calcium accumulations induced by AP trains wererestricted from the distal dendritic treeThe spatial extent of calcium accumulations induced by a

train of APs was not uniform along the dendritic tree. In all

cell types, (FS, n = 30; IS, n = 23; AD, n = 13), the peak

amplitude of the accumulations was reduced at distal

dendritic sites (Fig. 3A). Across cell types, the average

amplitude (DF/F %) at < 50 mm from the soma was

61 ± 37 (n = 49), at 50–100 mm from the soma was

55 ± 40 (n = 30, P = 0.426; Fig. 3B), whereas at > 100 mm

from the soma it was 19 ± 21 (n = 20 all cells, P < 0.001

compared to proximal measurement; Fig. 3B). There were

no significant differences between FS and IS cells at either

proximal, intermediate or distal dendritic positions

(proximal: 56 ± 31, n = 25 FS; 60 ± 42, n = 16 IS, P = 0.71;

intermediate: 56 ± 41, n = 15 FS; 42 ± 36, n = 12 IS,

P = 0.27; distal: 19 ± 24, n = 10 FS; 19 ± 19, n = 8 IS,

P = 0.94), but at intermediate positions, AD cells had

higher calcium accumulations than the other two cell groups

(proximal: 85 ± 41, n = 7 P = 0.425 vs. FS, P = 0.224 vs. IS;

intermediate: 100 ± 20, n = 4 P = 0.044 vs. FS, P = 0.010

vs. IS; distal: 21 ± 20 n = 4, P = 0.89 vs. FS, P = 0.84 vs. IS,

n = 4).

Mechanisms of calcium influx and efflux duringaction potential backpropagationWhy was there a limited spatial spread of the AP-induced

calcium accumulations in interneurone dendrites?

Although multicompartamental models suggest that the

passive cable properties of interneurones are well suited

for efficient AP backpropagation (Vetter et al. 2001b), a

non-uniform distribution of dendritic conductances or

buffer capacity could greatly influence the extent of AP

backpropagation or subsequent AP-triggered calcium

accumulations, respectively. We therefore considered the

following hypotheses: (1) distal dendrites had a higher

endogenous buffer capacity, (2) the AP train did not

invade distal dendrites, or (3) the AP train faithfully

invaded the distal dendritic tree but no calcium


Bb, representative IS morphology, with bipolar dendritic organization (blue). Basal dendrites tended to bemore branched than apical, especially in lower layers, and axonal collaterals (red) were vertically distributed.Bc, calretinin immunopositivity of an IS cell with firing pattern and morphology as shown above. Filled(right) and labelled (left) cell indicated by arrows. Ca, firing pattern and Cb, light microscopic reconstructionof an adapting (AD) cell.

Figure 2. AP-induced calcium accumulations in interneurone dendrites were slower andsmaller than in pyramidal cellsA, projected two-photon z-scan of the basal dendritic tree of an FS cell, pia top. B, protocol used. Left: singleAP; right: train of ten APs (40 Hz). C, pyramidal cell dendritic calcium accumulations during both protocols.D–F, FS, IS and AD responses. Time constants (t) are from mono-exponential fits to decays.



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accumulations were produced, due to a lack of VGCCs

distally. For the rest of the study we focused exclusively on

interneurones of the FS and IS types, because they could be

immunohistochemically defined, and therefore represented

a more homogenous group, and because they could be

reliably targeted under DIC.

If distal dendritic domains were targeted with higher

endogenous buffer capacity, we would expect to see at

distal sites a prolonged decay as well as a reduction of peak

amplitude of the calcium transient. Decays of calcium

transients did not change significantly over distance from

soma (Fig. 3C), suggesting that the reduction of peak

signal was either due to absence of voltage-gated calcium

channels distally, or to failure of the AP train to

regeneratively propagate to distal sites.

One explanation for the poor calcium signals into the

distal dendrites could be that APs were passively

propagating along dendrites devoid of sodium channels.

We measured calcium accumulations in the presence of

the voltage-gated sodium channel blocker, TTX (1 mM)

using a train of brief (3 ms), large-amplitude (100 mV)

depolarizing currents to simulate APs (Fig. 4B). To avoid

underestimating the extent of passive AP propagation, we

simulated APs 3–6 times wider than the normal APs in

these cells. Still, calcium influx was reduced even in the

proximal 50 mm of the dendritic tree (27 ± 9 % from

control, n = 3 FS, P < 0.05; 41 ± 15 % from control, n = 3

IS, P = 0.11 Fig. 4B), and failed at distances greater than

50 mm (4 ± 5 % from control, n = 3 FS, P < 0.05; 13 ± 19 %

from control, n = 3 IS, P < 0.05). We concluded that, since

passive propagation alone could initiate calcium influx

only very proximally (< 50 mm) and at reduced amplitudes,

sodium channels were expressed on the dendrites of both

FS and IS cells.

To confirm that the AP-induced dendritic calcium influx

was due to the opening of voltage-gated calcium channels,

we applied nickel at a high concentration (1 mM) to block

both high- and low-voltage-activated calcium channels. In

both FS and IS cells, practically all calcium accumulations

were blocked by Ni2+ (Fig. 4C; 12 ± 2 %, n = 2 FS;

15 ± 4 %, n = 2 IS; P < 0.001, all cells) without any

significant effect on AP physiology (not shown). Calcium

influx through VGCCs can initiate further calcium release

from internal stores (Nakamura et al. 1999), and we tested

this possibility by depleting internal calcium stores with

the SERCA (smooth endoplasmic reticulum calcium

ATPase)-pump antagonist cyclopiazonic acid (CPA)

(Kovalchuk et al. 2000). CPA (30–50 mM) did not change

the amplitude of the calcium transients significantly

(Fig. 4D; 79 ± 21 %, n = 4 FS; 70 ± 14 %, n = 6 IS of

control), but prolonged the decay time constants of

calcium transients (172 ± 50 % n = 4 FS; 240 ± 140 % in

CPA, n = 6 IS), confirming wash-in of drug, and

indicating that SERCA pumps were involved in calcium

clearance.

We conclude that sodium-based APs actively back-

propagated into the dendritic tree and caused calcium

influxes via activation of voltage-gated calcium channels.

These calcium accumulations were then cleared in part by

SERCA pumps, into intracellular calcium stores.

Existence of VGCCs throughout the interneuronedendritic treeSince the calcium influxes we measured during AP

backpropagation were due to opening of VGCCs, it

remained possible that AP trains successfully invaded

distal dendrites but failed to elicit calcium accumulations

due to an absence of calcium channels distally. We thus

tested if VGCCs were expressed on distal dendrites by


Figure 3. Calcium influx during backpropagation of AP trains was proximally restrictedA, calcium transients during 10 APs (40 Hz) from IS (1), FS (•) and AD (8) cell types, plotted againstdistance from the soma. Each line represents signals from a single cell imaged at different distances from thesoma. B, data were pooled into three compartments: proximal, intermediate, and distal, and comparedbetween FS (4), IS (5) and AD (Æ). * P < 0.05 on two-tailed Student’s t test, distal signals versus proximalfor each cell type. C, time constants (t) of mono-exponential fits of calcium decays, plotted versus distancefrom soma as in B.



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somatically injecting sustained (250 ms) depolarizing

currents in the presence of high concentrations of the

potassium channel blocker TEA (24 mM) and the sodium

channel antagonist TTX (1 mM). Because calcium

diffusion is relatively slow (Allbritton et al. 1992; Neher &

Augustine, 1992), imaging calcium accumulations in

dendrites under TEA/TTX in response to somatic

depolarizations can reveal regions of the dendritic tree that

have functional VGCCs (Yuste et al. 1994).

In all tested cells (n = 4 FS and 6 IS), sustained somatic

current depolarizations in TEA/TTX gave rise to

immediate calcium transients throughout the dendritic

tree which were significantly larger than those produced

by trains of backpropagating APs (Fig. 5). Importantly,

sustained depolarizations caused large transients even at

distal dendrites where AP trains had previously failed to

produce detectable signals (Fig. 5). Although there were no

systematic dendritic distance-dependent trends in peak

signal amplitude during sustained depolarizations, peak

signals in the soma were significantly smaller. We attribute

this to the low surface-to-volume ratio in the soma.

Interestingly, plateau potentials were triggered by these

depolarizations and closely resembled those previously

observed in pyramidal neurones under TTX/TEA (arrow,

Fig. 5C; Reuveni et al. 1993; Yuste et al. 1994).

We conclude that our inability to detect calcium signals in

distal dendrites during AP trains was not due to an absence

of VGCCs in distal dendrites. Rather, these data suggested

that AP trains did not invade distal dendrites.

Potassium channels controlled calcium influxduring backpropagating AP trainsThe failure of AP trains to invade distal dendritic

compartments could be explained by several different

scenarios: (1) a lack of functional sodium channels in

distal dendrites, (2) a high density of potassium currents in

distal dendrites, or (3) slow sodium channel inactivation

developing during the train and disproportionately

affecting the distal compartment (Mickus et al. 1999).

To test if potassium currents controlled AP propagation in

interneurone dendrites, we measured AP-induced

calcium accumulations in FS and IS cells in the presence of

1 mM 4-AP, a concentration that is relatively specific for Ia

and Kv3-type potassium channels (Kirsch & Drewe, 1993).

4-AP had a powerful effect on calcium accumulations

during backpropagating AP trains and preferentially

enhanced signals at distal sites (Fig. 6). In proximal

(< 50 mm) and intermediate (50–100 mm) dendritic

regions, no significant increases were observed. However,

at distal sites, addition of 4-AP endowed unresponsive

distal dendritic segments with prominent AP-initiated

Ca2+ events (Fig. 6; control/4-AP peak response ratios (%)

were: proximal, 97 ± 21, P = 0.76, n = 7; intermediate,

77 ± 36, P = 0.23, n = 6; distal, 18 ± 18, P < 0.0005, n = 5).


Figure 4. Mechanism of backpropagation-initiatedcalcium transientsA, percentage of control (dashed line at 100 %) DF/F signal afteraddition of TTX (1 mM), nickel (1 mM) (55 mm from soma) or CPA(50 mM) (35 mm from soma). FS, filled bars; IS, open bars.* P < 0.05. B–D, effect of drug addition (light trace) on controlDF/F signal (dark trace) during 10 APs (40 Hz). Examples from FScells are shown on the left and IS cells on the right. B, light traces arecalcium response to ten 3-ms-wide simulated APs in the presenceof TTX. Left, FS cell at 25 mm from the soma, above, and 60 mmfrom the soma along the same dendrite, below. Right, IS cell 20 mmfrom the soma (upper) and 90 mm from the soma along the samedendrite, below. C, nickel (1 mM) blockade. E, CPA failed to blockthe signal, but prolonged decay kinetics.



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Figure 5. Voltage-gated calcium channels wereexpressed throughout the dendritic treeA, basal dendritic arbor of a bipolar IS cell. The top halfof the soma was clipped during imaging to sample distalbasal dendrites. Lines transecting the dendrites indicatesites that were selected for line-scan imaging at anadditional 10 w digital zoom (not shown). B, a train of10 APs at 40 Hz was generated by 10 separate 3 mscurrent injections in the soma (shown at top). Calciumtransients were imaged in line-scan mode at the soma,and at three positions along the basal dendrite. C, a250 ms somatic current injection in the presence of TEA(24 mM) and TTX (1 mM) caused a plateau potential(arrow). Calcium transients were imaged at identicalsites to those in B. D, data pooled from 6 IS and 4 FScells. In each experiment, peak signals were normalizedto the control AP train signal at the soma. * P < 0.05,** P < 0.01.



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Dendritic invasion of single action potentials wasalso controlled by K+ currentsAfter observing the prominent role of potassium channels

in limiting calcium influx during backpropagation of AP

trains, we wondered whether single APs were similarly

controlled. We were able to image the calcium influx

during a single backpropagating AP by switching to the

calcium indicator Fluo-4. Because Fluo-4 undergoes a

near 100-fold increase in fluorescence on binding calcium,

it is more responsive to small calcium influxes than

calcium green. As shown in Fig. 7A, calcium influx during

a single backpropagating AP was also reduced, and often

undetectable, at distal (> 100 mm) sites of FS and IS cells

(P << 0.001 n = 8 FS; P < 0.001, n = 12 IS). We again

observed a reduction in calcium accumulations with

increasing distance from the soma during AP trains (10

at 40 Hz) (P = 0.006, n = 8 FS; P = 0.006, n = 12 IS),

although calcium accumulations imaged with Fluo-4 were

often detectable even at terminal dendrites > 170 mm

from the soma (Fig. 7B). Thus, in a separate set of

experiments under different exogenous buffer conditions,

we confirmed that calcium signals due to AP back-

propagation were spatially restricted.

In addition, we again observed that application of 1 mM

4-AP preferentially increased the distal Fluo-4 signal of

both single APs (P = 0.021, n = 6 FS; P = 0.001, n = 7 IS)

and AP trains (P = 0.007, n = 6 FS; P = 0.049, n = 6 IS;

Fig. 8). In both cell types, 4-AP application also

significantly increased the DF/F signals at intermediate

(51–100 mm) dendritic segments during single APs

(P = 0.004, n = 6 FS; P = 0.001, n = 7 IS). Calcium signals


Figure 6. AP trains were proximally restricted bypotassium currentsA, AP train (10 APs at 40 Hz) was generated at the soma andimaged 65 mm and 120 mm from the soma of an IS cell, undercontrol conditions (thick line) and in the presence of 4-AP (thinline). B, same as in A for an FS cell. Tested sites were 70 and 140 mmfrom the soma. C, pooled data from 6 cells showing the % DF/Famplitude before (filled bars) and after (open bars) 4-AP onproximal, intermediate, and distal dendritic sites. Note that theeffect of 4-AP was only significant at distal sites, *** P < 0.0005.

Figure 7. Single APs and AP trains imaged with Fluo-4were also proximally restrictedIn a separate set of experiments using Fluo-4 as the calciumindicator, calcium signals due to single APs could be resolved.A and B, left, data presented as in Fig. 3. Each line represents signalsfrom a single cell imaged at different distances from the somaduring a single backpropagating AP (A) and during 10 APs at40 Hz (B). Right , data from the graph at the left were pooled intoproximal, intermediate and distal groups. * P < 0.005,** P < 0.001.



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evoked by single APs and AP trains were greatly reduced at

the distal compartment, and 1 mM 4-AP eliminated this

distance-dependent reduction. These results demonstrated

that K+ currents altered the propagation of both single APs

and AP trains into the dendritic tree and preferentially

affected the excitability and calcium dynamics of the distal

compartment.

4-AP also produced an increase in somatic AP width

(193 ± 49 % of control, n = 13 for FS; 196 ± 67 % of

control, n = 7 for IS; Fig. 8C). It is thus possible that the

effect that 4-AP had on dendritic AP-induced calcium

accumulations was due to an enhanced backpropagation

of wider spikes. However, the impact of 4-AP on dendritic

excitability appeared essential since the distal compart-

ments were primarily affected by the drug.

Ia-type potassium currents controlled APbackpropagationThe two targets of the 1 mM 4-AP, Ia- and Kv3-type

potassium currents, are both expressed in interneurones

(Zhang & McBain, 1995; Rudy & McBain, 2001). To

determine which of these potassium channel subtypes

controlled calcium influx during AP backpropagation, we

repeated experiments in the presence 1 mM TEA. This

concentration of TEA targets Kv3 channels while leaving

Ia-type channels intact (Erisiret al. 1999; Lien et al. 2002).

We found that blockade of Kv3 channels alone did not

affect calcium influx during AP backpropagation (Fig. 9).

Together with the 4-AP results, these data indicate that Ia

channels controlled AP backpropagation.

Interestingly, 1 mM TEA did not significantly increase the

half-width in either IS or FS cells (TEA/control: 99 ± 6 %,

n = 3 IS; 110 ± 11 %, n = 3 FS); however, specifically in FS

cells, 1 mM TEA slowed AP repolarization (TEA/control:

86 ± 14 %, n = 3 IS; 151 ± 9 %, n = 3 FS).

EPSP–AP coupling caused supralinear calciuminflux adjacent to activated synapsesOur results in FS and IS interneurones were reminiscent of

Ia-type potassium channel control of AP propagation in

pyramidal neurones (Hoffman et al. 1997). An important

characteristic of Ia currents is that they inactivate during

subthreshold depolarizations, such as during synaptic

activity (Migliore et al. 1999a). We wondered if in

interneurones, previous synaptic activation could affect

the invasion of APs into activated dendritic compart-

ments, and if calcium influx at synaptic sites was

modulated by backpropagating APs. To address these

issues, we somatically generated a single AP 10 ms after

evoking an EPSP with a stimulation electrode placed in the

immediate vicinity (< 15 mm) of the dendrite of interest

(see Methods and accompanying paper, Goldberg et al.2003). Importantly, we exclusively sought out dendritic

segments with orientations parallel to our line scan, and

used small stimulation intensities to activate restricted

domains along that segment (Fig. 10A). This allowed us to

quantitatively examine the interplay between EPSPs and

APs at three sites: (1) synaptic sites (where synaptic

activation alone caused calcium signals), (2) immediately

adjacent to synaptic sites but where synaptic activation

alone did not cause calcium influx (9.2 ± 2.3 mm, n = 10

dendrites in 2 FS, 4 IS and 4 AD cells) and (3) on dendrites

where no synaptic calcium signal was detected.

We reliably observed supralinear calcium signals during

EPSP–AP pairing only at sites adjacent to activated

synapses. In the experiment illustrated in Fig. 10, the apical

branch of a bipolar IS cell was activated at a local site in a

dendritic compartment. In interleaved trials, synaptic

stimulation or APs were evoked in isolation or coupled

with EPSPs preceding the AP by 10 ms. At the site of the

synaptically evoked calcium entry, this coupling did not

significantly alter the signal. However, 6 mm distal along

the branch, the pairing of the AP and EPSP resulted in a

large supralinear calcium influx (arrow, Fig. 10D). This

effect was observed in all three cell types (Fig. 10E, n = 2/2

FS, 4/4 IS, 3/4 AD).

As shown in Fig. 10E, we found that the degree of

supralinearity at sites where synaptic activation alone

caused calcium influx was highly variable in all three cell

types. While some cells exhibited modest supralinearity at

synaptic sites, suggestive of AP-mediated NMDA receptor

(NMDAR) recruitment (Yuste & Denk, 1995; Magee &

Johnston, 1997), others showed sublinearity, suggesting


Figure 8. Single action potentials were also proximally restricted by K+ currentsA, left, XYZ-projection of a basal dendritic branch of an FS cell. Lines transecting dendrites at 10, 50 and

120 mm from the soma, indicate regions of interest where line scans were conducted at an additional 10 wdigital zoom (not shown). B, traces were recorded while eliciting single APs (left, top) or trains of 10 APs at40 Hz (right, top). Note the different % DF/F scale bars for the two stimulation regimes. Relative to control(dark traces), addition of 4-AP (1 mM, light traces) specifically increased distal signals, eliminating thedistance-dependent reduction in calcium signal for both single APs (left) and AP trains (right). C, 4-APincreased AP half-width. Top, single AP in control (dark trace), and in the presence of 1 mM 4-AP (lighttrace), generated by a 5 ms current injection at the soma, bottom. D, pooled data from FS cell group (n = 6)demonstrate the effect of 4-AP (open bars) on single APs (black bars) and AP trains (grey bars) at proximal,intermediate and distal dendritic sites. Data are normalized to the control signal at the proximal site. E, datapresented as in D for IS cell group (n = 7). * P < 0.05, ** P < 0.01.



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that VGCCs activated synaptically were left unavailable to

the backpropagating AP. However most fell very close to

the linear range, as shown in Fig. 10D. Although addition

of APV (100 mM) to block NMDAR-mediated calcium

entry significantly reduced synaptic calcium signals (see

accompanying paper, Goldberg et al. 2003), it did not

significantly change the distribution of paired signals

about the computed linear sum, revealing that in at least

some cases, supralinear EPSP–AP interaction was not

NMDAR-dependent.

Our failure to observe supralinearity at synaptic sites could

be due to indicator saturation during the strong synaptic

calcium signal. However, the peak amplitude of the

synaptic signal in the sublinear cases (151 ± 105 %DF/F,

n = 13 dendritic branches) was not significantly different

from that in supralinear cases (128 ± 118 %DF/F, n = 11

branches). Further, we found that the saturating calcium

influx in many of these experiments was at a DF/F around

500 % (n = 15). Thus we think it was unlikely that our

inability to reliably observe supralinearity at synaptic sites

was due to indicator saturation. Moreover, in identical

recording and indicator conditions during stronger synaptic

stimulation of clustered synapses we routinely observed

peak influxes 2–3 times greater (298 ± 101 %DF/F, n = 7).

We did not consistently observe supralinearity on

dendritic segments where we did not observe synaptic

calcium signals (ratio observed/predicted linearity =

1.64 ± 1.4, P = 0.14, n = 12). Thus signals were reliably

enhanced specifically at synaptically activated branches.

This finding suggests that previous synaptic activity is

capable of controlling the spatial dynamics of dendritic AP

invasion, and is consistent with our finding that Ia-type

potassium channels controlled calcium influx during AP

backpropagation.


Figure 9. Ia-type potassium channels controlled AP backpropagationData are laid out as in Figure 8. A, XYZ-projection of an FS cell, pia at right, medial is at bottom. Open barsindicate regions of interest examined at an additional 10 w zoom for traces in B. B, traces were recorded whileeliciting single APs (left, top) or trains of 10 APs at 40 Hz (right, top). Note the different % DF/F scale bars forthe two stimulation regimes. Relative to control (dark traces), addition of TEA (1 mM, light traces) did notsignificantly alter calcium signals at any distance from the soma during both single APs (left) or AP trains(right). C, 1 mM TEA did not significantly change the AP half-width, but slowed repolarization in FS cells.Top, single AP in control (dark trace), and in the presence of 1 mM TEA (light trace), generated by a 4 mscurrent injection at the soma, bottom. D, pooled data from FS cell group (n = 3) demonstrate the effect of1 mM TEA (5) on single APs (4) and AP trains (Æ) at proximal, intermediate and distal dendritic sites. Dataare normalized to control signal at the proximal site. E, data presented as in D for IS cell group (n = 3).

Figure 10. EPSP–AP coupling caused supralinear calcium influxes adjacent to activatedsynapsesA, the position of the line scan, arrowheads, is indicated on an apical dendrite of an IS cell 40 mm from thesoma. The stimulation electrode, S, was placed approximately 8 mm beneath the dendrite. B–D, line scansand calcium transients (red at synaptic site, black 6 mm distal) during three stimulation protocols: synapticstimulation alone, Syn (B), single AP alone, 1 AP (C), and paired, Syn + 1 AP (D). Blue traces represent thecalculated sum of 1 AP and Syn signals. Physiology traces for each experimental protocol are at the bottom.Each line-scan image is an average of four interleaved trials. Arrows at 400 ms into the line scan, also beneatheach physiology trace, indicate the time of stimulation. Note different time scales for physiology and calciumtraces. E, summed responses during Syn + 1 AP at synaptic sites (syn), adjacent (adj), and under NMDAreceptor blockade (apv + mk-801) were normalized to the computed sum (blue line at y = 1). Data are from11 IS, 10 FS and 4 AD cells, shown individually at the left, and pooled at the right. Multiple dendriticpositions were tested on individual cells.



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DISCUSSION Using two-photon calcium imaging we have characterized

the AP-induced calcium dynamics in dendrites from

different classes of interneurones. Our goals were to

investigate their intrinsic calcium dynamics and explore

their electrical excitability using calcium accumulations to

monitor the activation of particular dendritic regions by

single APs and AP trains. The main findings of this study

were (1) dendrites of FS, IS and AD cells actively support

AP backpropagation due the expression of voltage-gated

sodium, potassium and calcium channels, (2) calcium

signals during backpropagation of both single APs and AP

trains were reduced at distal dendritic regions due to the

activation of Ia-type potassium currents, and (3) synaptic

activation was able to enhance AP-mediated calcium

signals specifically in activated compartments. Together,

these findings indicate that interneurone dendrites are

active structures which can dynamically control signal

propagation.

Mechanisms of AP-induced calcium accumulationsin neocortical interneuronesSomatically generated APs propagated actively to the

dendritic tree and by opening VGCCs produced calcium

influx in the proximal regions. The peak accumulations

were unaffected by blockers of internal release, although

SERCA pumps were involved in dendritic calcium

clearance. Therefore both calcium influx and efflux

pathways in interneurone dendrites were the same as has

been reported in pyramidal cells (Regehr & Tank, 1994;

Yuste et al. 1994; Markram et al. 1995; Yuste & Denk, 1995;

Helmchen, 1999).

We did not encounter any appreciable differences in these

mechanisms among different types of interneurones

examined. However, calcium signals during AP back-

propagation had smaller amplitudes and slower offset

kinetics compared with pyramidal cells (Fig. 2), consistent

with a larger interneurone calcium-buffering capacity (Lee

et al. 2000a; Kaiser et al. 2001; Rozov et al. 2001).

Ia-type K+ channel control of AP backpropagation inGABAergic cellsOur data reveal that the dendrites of multiple types of

neocortical interneurones actively express voltage-gated

sodium, potassium and calcium channels. We observed

that, although VGCCs were located throughout the

dendritic tree (Fig. 5), and passive cable properties appear

ideal for backpropagation in interneurones (Vetter et al.2001a), under normal conditions the AP-induced calcium

accumulations were reduced at distal (> 100 mm)

dendritic regions (Figs 3 and 7).

Based on the large effect that 4-AP had on the spatial

pattern of AP-induced calcium accumulations (Figs 6 and

8), we conclude that AP backpropagation was controlled

by potassium channels. In pyramidal neurones, Ia-type

potassium channels appear at high densities in apical

dendrites (Hoffman et al. 1997; Korngreen & Sakmann,

2000), where they play a prominent role in regulating

dendritic AP invasion (Hoffman et al. 1997), and in fast-

spiking interneurones Kv3-type potassium channels

facilitate high-frequency firing (Martina et al. 1998; Erisir

et al. 1999; Rudy & McBain, 2001). Both targets of 1 mM

4-AP, Ia and Kv3-type K+ currents, are reportedly

expressed in interneurones, and thus may have been

involved in controlling dendritic AP propagation (Zhang

& McBain, 1995; Rudy & McBain, 2001). In order to

distinguish between these two potassium channel subtypes

implicated in the 1 mM 4-AP experiments, we specifically

blocked Kv3 potassium channels in 1 mM TEA (Erisir et al.1999; Lien et al. 2002). Since blockade of Kv3 alone did not

significantly enhance calcium signals during AP

backpropagation (Fig. 9), we conclude that Ia-type

potassium channels controlled dendritic calcium

accumulations during AP backpropagation.

We found it particularly interesting that Ia potassium

currents were so important in controlling AP propagation,

in the light of two recent studies in interneurones which

have emphasized the importance of dendritic potassium

currents in regulating synaptic activation and spike

initiation (Fricker & Miles, 2000; Galarreta & Hestrin,

2001). High dendritic Ia-potassium channel expression on

the dendrites of interneurones could thus serve the dual

function of regulating both spike initiation and

propagation.

EPSP interaction with backpropagating actionpotentialsAlthough AP backpropagation has recently been

demonstrated in a variety of interneuronal classes

(Tombaugh, 1998; Martina et al. 2000; Kaiser et al. 2001),

there has, as yet, been no description of how bAPs interact

with EPSPs in interneurones. Because of the unique

subthreshold inactivation kinetics of Ia channels, we

wondered if synaptic activity could regulate AP

backpropagation. We found that when evoked EPSPs

preceded somatically generated APs by 10 ms, calcium

influx in the compartment of the activated synapse was

supralinearly increased in some cells (Fig. 9).

There are at least two possible mechanisms for the

enhancement of AP-mediated calcium signals by

preceding EPSPs. First, EPSPs could eliminate potassium

channel control of APs by inactivating Ia, as has been

shown in CA1 pyramidal neurones (Hoffman et al. 1997;

Migliore et al. 1999a). Alternatively, EPSPs could boost

dissipating backpropagating APs by providing the

necessary dendritic depolarization to reach sodium

channel threshold (Stuart & Hausser, 2001). Given the

importance of K+ channels in controlling AP propagation

under control conditions (Figs 6 and 8), we prefer the first

explanation. However, direct dendritic recordings of these




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narrow dendrites will be necessary to clarify the precise

mechanisms of EPSP-mediated enhancement of AP-

induced calcium influxes.

It is important to note that we observed no supralinearity

in dendritic compartments where we did not observe

synaptic calcium influx. Given that the propagation of

dendritic APs through gap junctions may facilitate the

synchronization of the interneuronal syncytium (Beierlein

et al. 2000), our finding that EPSPs can control AP

propagation specifically in activated compartments

suggests that the spatial component of synaptic activity

may affect synchronization of interneuronal ensembles.

Functional compartments in GABAergic dendritesOur experiments suggest that spatially limited calcium

signalling during active backpropagation of somatic APs

defines at least two functional compartments in the

somatodendritic domain of neocortical interneurones: a

perisomatic region of the dendritic tree, where back-

propagating APs can reach, in normal conditions, and a

distal region, which may only be affected by back-

propagating APs when dendritic potassium channels are

inactivated. While proximal dendrites may undergo

calcium-dependent processes that involve the timing of

the firing of the postsynaptic cell, synaptic mechanisms of

calcium influx may dominate distal compartments. Also,

selective invasion of APs to proximal or recently activated

dendrites favours the propagation of both electrical and

calcium signals through gap junctions, which are

expressed within the networks of FS and regular spiking

non-pyramidal cells (Galarreta & Hestrin, 1999; Gibson etal. 1999; Tamas et al. 2000; Szabadics et al. 2001). In

addition, Ia potassium currents are regulated by

neurotransmitters and second messenger pathways (Hille,

1992; Migliore et al. 1999b; Atzori et al. 2000) suggesting

that the multi-compartment picture of interneurone

dendritic physiology is a dynamic one.

Differential calcium dynamics in proximal and distal

dendritic regions of interneurones might selectively

interact with inputs targeting different somatodendritic

regions. The proximal dendritic domain of cortical

interneurones is selectively innervated by glutamatergic

afferents from the thalamus (Freund et al. 1985) and by

GABAergic inputs from subcortical sources (Freund &

Meskenaite, 1992) and from local basket cells (Tamas et al.1998). Distal dendritic branches of interneurones, on the

other hand, are innervated by other local GABAergic cell

classes (Tamas et al. 1998) and by predominantly cortical

glutamatergic afferents. These two dendritic regions

correspond nicely to those identified in our study as

having different backpropagation and calcium dynamics.

It is possible that functional pairing between AP back-

propagation, postsynaptic calcium dynamics and particular

afferent pathways could dramatically increase the

computational power of individual cortical interneurones.

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Acknowledgements We thank Misha Beierlein, Josh Brumberg and Jason MacLean forcomments. This study was funded by the NEI (EY11787 ), NINDS(NS40726), the New York STAR Center for High ResolutionImaging of Functional Neural Circuits and the John Merck Fund.




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