Specificity of Synaptic Connectivity between Layer 1 Inhibitory Interneurons and Layer 2/3 Pyramidal Neurons in the Rat Neocortex.

0.74 MB·English

by  Wozny, Christian

CerebralCortexAugust2011;21:1818--1826
doi:10.1093/cercor/bhq257
AdvanceAccesspublicationJanuary10,2011
Specificity of Synaptic Connectivity between Layer 1 Inhibitory Interneurons and
Layer 2/3 Pyramidal Neurons in the Rat Neocortex
Christian Wozny1and StephenR. Williams1,2
1MRCLaboratoryofMolecularBiology,CambridgeCB20QH,UKand2TheQueenslandBrainInstitute,StLucia,QLD4072,Australia
AddresscorrespondencetoStephenR.WilliamsandChristianWozny.Email:[email protected]@mrc-lmb.cam.ac.uk.
Understanding the structure and function of the neocortical to L1 neurons (Zhu Y and Zhu JJ 2004), which, in turn, act
microcircuit requires a description of the synaptic connectivity to powerfully inhibit sensory-evoked responses in L2/3
between identified neuronal populations. Here, we investigate the (Shlosbergetal.2006).Surprisingly,theinterneuronalcompo-
electrophysiological properties of layer 1 (L1) neurons of the rat sition and synaptic connectivity of L1 neurons have not been
somatosensoryneocortex(postnatalday24--36)andtheirsynaptic explored in detail (Hestrin and Armstrong 1996; Zhou and
connectivitywithsupragranularpyramidalneurons.Theactiveand Hablitz1996a,1996b;Chuetal.2003;ZhuYandZhuJJ2004).
passive properties of visually identified L1 neurons (n 5 266) Layer 1 constitutes an important site of integration in the
suggested division into 4 groups according to the Petilla neocortical column as it contains a dense plexus of the apical
classification scheme with characteristics of neurogliaform cells dendritesoflayer2/3andlayer5pyramidalneuronsandaxons
(NGFCs)(n572),classical-accommodating(n5137),fast-spiking originatingfromavarietyofcorticalareas(Caulleretal.1998;
(n 5 23), and burst-spiking neurons (n 5 34). Anatomical Mitchell and Cauller 2001; Petreanu et al. 2009) and the
reconstructions of L1 neurons supported the existence of 4 major thalamus (Rubio-Garrido et al. 2009). Long-range intracortical
neuronal groups. Multiparameter unsupervised cluster analysis excitatoryaxons,conveyedinL1,synapsewithboththeapical
confirmed the existence of 4 groups, revealing a high degree of dendritesofpyramidalneurons(Petreanuetal.2009)andlocal
similarity with the Petilla scheme. Simultaneous recordings GABAergicinterneurons(AndersonandMartin2006).Thus,L1
between synaptically connected L1 neurons and L2/3 pyramidal inhibitory interneurons are ideally placed to control dendritic
neurons(n5384)demonstratedneuronalclassspecificityinboth synapticintegrationinpyramidalneurons(WilliamsandStuart
excitatoryandinhibitoryconnectivityandthepropertiesofsynaptic 2002; Waterset al. 2003;Williams2004; Larkum et al.2009).
potentials.Notably,allgroupsofL1neuronsreceivedmonosynaptic Inthisstudy,wehavecharacterizedtheelectrophysiological
excitatory input from L2/3 pyramidal neurons (n 5 33), with the propertiesofL1neuronsoftheratneocortexandinvestigated
exception of NGFCs (n 5 68 pairs tested). In contrast, NGFCs the synaptic connectivity between L1 inhibitory interneurons
strongly inhibited L2/3 pyramidal neurons (n 5 12 out 27 pairs and L2/3 pyramidal neurons. Our results provide new insights
tested). These data reveal a high specificity of excitatory and into the wiring diagram of the superficial layers of the
inhibitory connections inthesuperficial layersofthe neocortex. neocortical column.
Keywords: corticalcolumn, GABAergic interneuron, layer 1, layer2/3,
Materials and Methods
neurogliaform cell,synapticconnectivity, target-cellspecificity
SlicesandRecordings
Wistar rats (P24 to P36) were decapitated under deep isoflurane
anesthesiafollowingUKHomeofficeandinstitutionalguidelines.Brains
Introduction were quickly removed and placed into ice-cold solution containing
(mM):NaCl125,NaHCO 25,KCl3,NaH PO41.25,CaCl 1,MgCl 6,
3 2 2 2
The neocortex is a 6-layered structure which is functionally sodiumpyruvate3,andglucose25,oxygenatedat95%O and5%CO .
2 2
organizedintocolumns(Mountcastle1997).Understandingthe After cutting coronal sections (300--350 lm) of the somatosensory
wiringdiagramofacorticalcolumnhasreceivedmuchattention cortex (slices were collected corresponding to the coordinates of
in recent years (Douglas and Martin 2004). The excitatory Bregmaca.--3.8mmtoBregmaca.--1.8mmPaxinosandWatson1998),
slices were incubated at 34 (cid:1)C for 30 min and then stored at room
synapticcircuitryofthecorticalcolumnhasbeenwelldescribed
temperature. For recordings, a single brain slice was placed in
(Lubke and Feldmeyer 2007; Schubert et al. 2007). The
a chamber perfused with a solution of composition (mM): NaCl 125,
neocortex,however,isnotonlycomposedofexcitatoryneurons NaHCO 25,KCl3,NaH PO41.25,CaCl 2,MgCl 1,sodiumpyruvate3,
3 2 2 2
but also c-aminobutyric acid (GABA)ergic inhibitory interneur- and glucose 25 at 34--36 (cid:1)C. Double and triple whole-cell recordings
ons, which make up ~20% of the neocortical neuronal from the somata of layer 1 (L1) and layer 2/3 (L2/3) somatosensory
population. Inhibitory interneurons can be classified by mor- neocorticalneuronsweremadewithidenticalcurrentclampamplifiers
(BVC 700A; Dagan). The L1--L2 border was defined by the abrupt
phological,electrophysiological,neurochemicalfeatures(Ascoli
changeinthedensityofneuronsvisualizedunderinfrareddifferential
et al. 2008), and their transcriptional regulation during de-
interference contrast video microscopy. Pipettes were filled with
velopment(Buttetal.2007;Batista-BritoandFishell2009).The
a solution containing (mM): potassium gluconate 135, NaCl 7, Hepes
distribution of interneuron classes, however, differs across 10,Na -ATP2,Na-GTP0.3,MgCl 2,andoccasionallywithAlexaFluor
2 2
neocorticallayers(Markrametal.2004;Ascolietal.2008). 5680.01--0.04(MolecularProbes)forvisualizationafterrecording;pH
Layer 1 (L1) of the neocortex is a cell-sparse synaptic and wasadjustedto7.2--7.3withKOH.
axon dense zone. In contrast to other neocortical layers, L1 is
composedofmorethan90%ofGABAergicneurons(Winerand
IntrinsicPropertiesofL1andL2/3Neurons
Larue 1989; Prieto et al. 1994). In the primary somatosensory Theactionpotential(AP)firingpatternofneuronswasinvestigatedby
cortex, whisker-evoked sensory information is rapidly relayed theinjectionofaseriesof600mspositivecurrentstepsthroughthe
(cid:2)TheAuthors2011.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/2.5),whichpermits
unrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
recording pipette (100 pA steps up to a maximum of 1 nA). Input resistance and prominent rectification of the voltage--current
resistance (Rin) was calculated from the steady-state voltage change relationship, see Supplementary Fig. 1). We first classified the
evoked by the injection of a 100 pA negative current step. Resting remaining 266 L1 neurons according to their repetitive AP
membrane potential (RMP) was measured at the beginning of each
firing patterns in response to suprathreshold injection of
recordingandwasnotcorrectedfortheliquidjunctionpotential.The
positive current steps (Fig. 1). We observed key differences
accommodation of a neuron’s AP firing frequency was assessed by
calculatingan accommodationindex,whichisdefinedastheratioof between groups of neurons in the initial AP firing frequency,
thefirstandlastinterspikeintervalatthresholdforrepetitiveAPfiring. thehalf-widthofthefirstAP,andaccommodationindex(Fig.1;
We did not estimate the rheobase of neurons as we injected current seeMaterialsandMethods). AccordingtothePetillaclassifica-
through the patch pipette in 100 pA steps and therefore the tionscheme(Ascolietal.2008),weobservedregular-(n=209),
accommodationindexwascalculatedatnear-thresholdvoltages.
fast-(n=23),andburst-spiking(BS)neurons(n=34).Regular-
spiking behavior was observed in a group of neurons termed
ClusterAnalysis classical-accommodatingneurons(c-AC,n=137)andinagroup
To cluster L1 neurons into groups, unsupervised clustering was resembling neurogliaform cells (NGFCs, n = 72) (Tamas et al.
performed using Ward’s method (WinSTAT) (for details, see Ward 2003; Olah et al. 2007; Szabadics et al. 2007). Most of the fast-
1963; Cauli et al. 2000). The results of the cluster analysis were
spiking (FS) neurons were observed at the L1-L2 boarder (see
compared with the classification of neurons according to the Petilla
anatomical features below). BS neurons showed high initial
terminology(seeResults;Ascolietal.2008).
(burst) frequency (Fig. 1A,B). We divided neuronal classes
according to their accommodation index (see Materials and
SynapticConnectivity
Methods),whichseparatedc-ACfromNGFCcells(Fig.1C)and
Excitatoryandinhibitorysynaptictransmissionwasexaminedbetween
the half-width of the first AP, which was narrowest in FS cells
simultaneously recorded L2/3 pyramidal neurons and L1 neurons.
Synaptic connectivity was tested for by the repeated generation of (Fig.1D,Table1).Thesequantitativemeasuresallowedtheclear
apairofAPs(evokedby2mstestpulses;0.5--5nA;separatedby50ms; divisionofL1neuronsinto4groups.
deliveredat0.33Hz).Ifneuronswerenotsynapticallyconnected,one When we examined the passive properties of neurons
oftherecordingelectrodeswaswithdrawnandeitheranewpyramidal grouped by their AP properties, we found that the input
neuronorinterneuronrecorded.Voltageandcurrentsignalswerelow-
resistance(R )andRMPofeachgrouppossessedsurprisingly
pass filtered at 10 kHz and acquired at 25--50 kHz using an ITC-18 in
narrow boundaries. c-AC cells had an average input resistance
interface(HEKA)controlledbyanApplecomputer.Datawereanalyzed
of166±5MX(n=137),NGFCcells107±3MX(n=72),FS
and curve fitting performed using Axograph X. Unitary postsynaptic
potential (uPSP) onset time, rise time, and half-width was measured cells 87 ± 7 MX (n = 23), and BS cells 201 ± 15 MX (n = 34)
from either single episodes or from averages (usually 50 responses). (ANOVA on ranks, Kruskal--Wallis followed by a Dunn’s test:
Onsetwasdeterminedat5%ofpeakresponse,risetimewasmeasured
asthetimetoreachbetween10and90%ofthemaximalamplitudeof
theuPSP,andthedecaytimeconstantsweredeterminedbyfittingthe
decay with a single exponential function. To accurately measure the
amplitude of the second uPSP following a paired-pulse stimulus, the
decayoffirstuPSPwasfittedwithanexponentialfunction,whichwas
subsequentlysubtractedfromthetrace.Theamplitudeofthesecond
uPSP was measured over a window centered around peak amplitude
relativetothesubtractedtrace.Statisticalanalysiswasperformedwith
Student’st-test(Excel,Microsoft),chi-squaredtest,Fisher’sExacttest,
orwithananalysisofvariance(ANOVA)test(allSigma-Plot).
Reconstructions
Neuronswerefilledwithbiocytin(0.5%,addedtothepipetterecording
solution)andslicesfixedovernightin4%paraformaldehydedissolved
in0.1Msodiumphosphate-bufferedsaline(PBS,pH7.4).Subsequently,
slices were incubated for 12--18 h in PBS supplemented with 0.5--2%
Triton X-100 and 0.2% streptavidin, Alexa Fluor 488 conjugate
(Invitrogen) at 4--6 (cid:1)C. After washing with PBS, slices were mounted
on slides, covered with Vectashield and coverslipped (Vector
Laboratories). Confocal images were taken with Zeiss 510 or Zeiss
710 confocal microscope, respectively. Neuronal morpholology was
reconstructedfromconfocalimagestacksusingNeurolucidasoftware
(MBFBioscience).
Results
Electrophysiological PropertiesofLayer 1 Neurons
Werecordedfrom271layer1(L1)neuronslocatedatleast20 Figure1. ElectrophysiologicalcharacterizationofL1neurons.(A)RepresentativeAP
lm from the L1--L2 border, selected irrespective of their firingpatternsofL1neurons(uppertraces),generatedinresponsetotheinjectionof
morphology under infrared differential interference contrast positivecurrentsteps(lowertraces).L1neuronswereclassifiedaccordingtotheir
firingpropertieswithrespecttothePetillaclassificationscheme:c-AC,NGFCs,FS,
videomicroscopy, using whole-cellcurrent clamptechniques.
and BS neurons. (B--D) The cumulative probability distributions of initial firing
Despite this selection criteria, 5 of 271 neurons had electro-
frequency,accommodationindex,andhalf-widthofthefirstAP areshownforeach
1
physiological properties similar to those of simultaneously typeofL1neurons(colorcode:c-AC:black,n5137;NGFC:green,n572;FS:
recorded L2/3 pyramidal neurons (n = 384; low input orange,n523;BS:blue,n534).
CerebralCortexAugust2011,V21N8 1819
Table1
IntrinsicpropertiesofL1neurons
RMP(mV) Rin(MX) FiringFreqinitial.(Hz) AP1half-width(ms) Accommodationindex
(t(AP2(cid:1)AP1)/t(APn(cid:1)1(cid:1)APn))
Petilla
NGFC(72) (cid:1)73.4±0.5 107±3 23.0±1.6 0.48±0.01 1.12±0.04
FS(23) (cid:1)72.0±1.1 87±7 93.0±12.5 0.28±0.01 0.93±0.1
c-AC(137) 67.3±0.4 166±5 44.5±4.2 0.5±0.01 0.50±0.02
BS(34) (cid:1)69.2±0.9 201±15 285.9±11.3 0.43±0.01 0.09±0.01
Clustering
Cluster1(81)(81%NGFC) (cid:1)74.0±0.4((cid:1)74.1±0.5) 108±3(106±3) 23.8±2.3(21.7±1.5) 0.49±0.1(0.48±0.01) 1.06±0.04(1.14±0.04)
Cluster2(24)(96%FS) (cid:1)72.0±1.1((cid:1)72.0±1.1) 88±7(87±8) 92.8±12.0(93.0±12.5) 0.28±0.01(0.28±0.01) 0.91±0.1(0.93±0.1)
Cluster3(125)(94%c-AC) (cid:1)66.2±0.4((cid:1)66.4±0.4) 167±4(169±4) 41.2±3.2(39.7±3.2) 0.50±0.01(0.50±0.01) 0.51±0.02(0.49±0.02)
Cluster4(36)(89%BS) (cid:1)69.7±0.7((cid:1)69.8±0.8) 209±17(202±16) 286.3±11.3(295.2±9.9) 0.43±0.01(0.43±0.01) 0.09±0.01(0.08±0.01)
Petilla:TheactiveandpassivepropertiesofL1neuronsgroupedaccordingtothePetillaclassificationscheme(c-AC,NGFC,FS,andBS).Numberofneuronsaregiveninbrackets.Clustering:unsupervised
clustering(hierarchicalclusteringusingWard’smethod)wasperformedandacutoffcriteriawasdeterminedtoreveal4clusters.Numberofneuronsineachclusteraregiveninbrackets.Theprimary
compositionofneuronsineachclusterwhenidentifiedbythePetillaclassificationschemeareindicated:81%ofneuronsincluster1wereNGFCs,96%ofneuronsincluster2FSneurons,94%ofneurons
incluster3c-ACneurons,and89%ofneuronsincluster4BSneurons.Theaveragevaluesfortheactiveandpassivepropertiesfortheprimarycompositionofneuronsineachcluster.Allvaluesaregiven
asmean±standarderrorofthemean.
P <0.05forallpairwisemultiplecomparisons,exceptforc-AC
vs.BSandNGFCvs.FS).Seventy-ninepercentofNGFCcellsand
83% of FS cells had R of less than 130 MX, whereas 80% of
in
eitherc-ACcellsand82%BScellshadR ofmorethan130MX
in
(Fig.2A,B).TheRMPof81%ofNGFCcellsand78%ofFScells
hadanRMPdepolarizedto–70mV,whereastheRMPof23%of
c-AC cells were hyperpolarized to –70 mV (Fig. 2C). BS cells
showed a heterogeneous RMP distribution (Fig. 2C). These
results are summarized in Table 1 (RMP: ANOVA on ranks,
Kruskal--Wallis followed by a Dunn’s test: P < 0.05 for all
pairwise multiple comparisons, except for c-AC vs. BS and
NGFCvs. FS).
Anatomical FeaturesofL1Neurons
Neuronswereregularlyfilledwithbiocytinandsubsequently
processedformorphologicalreconstruction(n=71).In40of
71 neurons, the axonal arborization was sufficiently stained
forfurtheranalysis.Typicalreconstructionsforeachneuronal
type are shown in Figure 3. The neurites of 13 of 21 c-AC
neurons were confined to L1 (62%, Fig. 3A), 5 had 1 or 2
axonalbranchesdescendingtodeeperlayers(Supplementary Figure 2. Passive properties of c-AC, NGFCs, FS, and BS neurons. (A) Example
Fig.2B),andafurther2neuronshadmorethan2descending tracesofvoltageresponses(uppertraces)evokedtheinjectionofa100pAnegative
currentstep(lowertrace)ineachcelltype.TheRMPisindicated.(B)Distributionof
axonal branches. One c-AC neuron showed a divergent
apparent input resistance (R , measured at steady state, indicated as DV by the
morphology. All successfully reconstructed NGFCs (n = 12) arrow in A) and (C) RMP oinf each cell type. Data are presented as cumulative
hadcharacteristicanatomicalfeatures,typifiedbya largeand probabilityplots(colorcode:c-AC:black,n5137;NGFC:green,n572;FS:orange,
dense axonal field and short dendrites (Fig. 4B) (Tamas et al. n523;BS:blue,n534).
2003; Olah et al. 2007; Szabadics et al. 2007). The axonal
arborization of 5 NGFCs was confined to layer 1, while 7 havemultipolardendritesandaxonalbranchesendinginlayer
neurons had multiple descending axons (Fig. 3B). Located 1 and layer 2/3 (Fig. 3D).
closetotheL1-L2/3boarder5FScellswerereconstructed;1
had multipolar dendrites and an axonal arbor in deep L1 and
Unsupervised Clustering
superficial part of L2 (Fig. 3C) and 4 were found to be
chandelier cells, which had the well-described feature of We used unsupervised cluster analysis (using Ward’s method)
several hundreds of vertically oriented axonal segments toindependentlyclassifyL1neurons.Fiveelectrophysiological
targeting the axon initial segments of pyramidal neurons parameters were used in this analysis: initial firing rate,
(Supplementary Fig. 3) (Somogyi 1977; Howard et al. 2005). accommodation index, half-width of the first AP, Rin, and
The active properties of FS neurons that were anatomically RMP. Figure 4A shows the tree diagram of clusters based on
identified as chandelier cells had unique electrophysiological theseparameters;whenathresholdof4clusterswasselected,
properties, exhibiting a left-shifted relationship between we found that neuronal grouping was similar to the Petilla
injected current and AP firing frequency (Supplementary classification.Thefirstclusterwascomposedof66NGFCsand
Fig. 4A) and transient stuttering AP firing upon threshold 15 c-AC neurons. The second cluster contained 96% FS cells.
current injection (Supplementary Fig. 4C1,4C2), which was ThemajorityofL1neuronsweregroupedinthethirdcluster:
not be observed in other FS cells (Supplementary Fig. 4B) 117 c-AC neurons, 6 NGFCs, and 2 BS neurons. The fourth
(Woodruff et al. 2009). Two BS neurons were also found to cluster was composed of 89% BS cells (Fig. 4). Thus,
1820 Layer1--Layer2/3SynapticConnectivity d WoznyandWilliams
Figure3. AnatomicalreconstructionsofL1neurons.(A1)Typicalexampleofac-ACneuronwithneuritesconfinedtoL1,dendritesareshowninblack,andaxoninred.(B1)
RepresentativeexampleofanNGFCwithanatomicalcharacteristicsofalargeanddenseaxonalfieldandshortdendrites.(C1)ReconstructionofanFSneuronlocatedatthe
boarderofL1andL2.(D1)ExampleofaBSneuronwithmultipolardendriticandaxonalarborinL1andL2/3.Laminarbordersareindicatedwithdashedlines.(A2--D2)APfiring
patternofthereconstructedL1neurons(uppertraces)inresponsetocurrentinjection(lowertraces).
horizontal distance less than 100 lm between L2/3 Pyr--L1
pairs). The properties of unitary excitatory postsynaptic
potentials (uEPSPs) were found to be dependent on the
identity of the postsynaptic neuron. We analyzed the latency,
the rise time, the amplitude, and the half-width of the uEPSPs
(Fig.5).L2/3-FSuEPSPshadalatencyof1.4±0.2ms,arisetime
of1.1±0.1ms,amplitudeof1.1±0.3mV,ahalf-widthof5.7±
0.7 ms, and a decay time constant of 7.4 ± 1.1 ms (n = 7,
SupplementaryFig.5).L2/3-c-ACuEPSPshadalatencyof1.8±
0.1 ms (P = 0.06), a slower mean rise time of 2.2 ± 0.2 ms
(P <0.001),asimilaraveragemeanamplitudeof1.1±0.2mV,
alongerhalf-widthof17.2±1.2ms(P <0.001),andagreater
decay time constant of 25.0 ± 4.2 (P < 0.001, n = 21,
Supplementary Fig. 5). L2/3-BS connections had a latency
similartotheL2/3-c-ACandL2/3-FSconnections(1.4±0.3ms,
P >0.05),arisetimeof2.4±0.1ms,anamplitudeof1.1±0.4
mV,ahalf-widthof14.5±2.0ms,andadecaytimeconstantof
21.5 ± 3.5 ms (n = 5, Supplementary Fig. 5). The latter
propertieswerenotsignificantlydifferentfromtheproperties
of L2/3-c-AC connections but distinct from L2/3-FS connec-
Figure4. ClusteranalysisoftheelectrophysiologicalpropertiesofL1neurons.(A) tions (rise time, P < 0.001; half-width, P < 0.05; decay time
TreediagramobtainedusingWard’smethod.Fiveparameterswereusedtocluster constant, P < 0.05). The connectivity probability between
neurons:initialfiringrate,accommodationindex,half-widthofthefirstAP,R ,and
in identified L2/3 pyramidal neurons and L1 interneurons was
RMP.Thedashedlineindicatesthechosencutoff.Clustersarenumberedfrom1to
4. (B) Pie chart illustrating the composition of each cluster with reference to the calculated as the ratio of the number of synaptic connected
Petillaclassificationscheme.Thefirstclustercomprises81neurons,66ofwhichhad neurons to the number of connections tested (Fig. 5).
beenclassifiedasNGFCsand15asc-ACs.Thesecondclusteriscomposedof24
Surprisingly, none of the NGFCs tested received excitatory
neurons,23FSand1c-ACneurons,thethirdclusterof117c-AC,6NGFCs,2BS
input from L2/3 pyramidal neurons (68 connections tested),
neurons,andthefourthclusterof32BSand4c-ACneurons(colorcode:c-AC:black,
NGFC:green,FS:orange,BS:blue). whereas L2/3 pyramidal neurons had a high probability of
connectivity with FS cells (0.35, 7/20 connections tested,
significantly different from L2/3 Pyr-NGFC ratio, P < 0.001,
unsupervised cluster analysis confirmed the presence of 4
Fisher’s Exact test, and from L2/3 Pyr-c-AC and L2/3 Pyr-BS
groups: NGFCs, FS, c-AC, and BS neurons. For all subsequent
ratio, P < 0.05, chi-squared test). c-AC and BS neurons were
analysis, we excluded all neurons that did not unequivocally
foundtoreceiveexcitatoryinputfromL2/3pyramidalneurons
belongtothesamegroupusingbothclassificationschemes.We
therefore excluded 8% of NGFC, 15% of c-AC, and 6% of BS with a probability of 0.16 (21/134) and 0.12 (5/42), re-
cells from our initial groups based on the Petilla scheme (Fig. spectively (both significantly different from L2/3-NGFC ratio,
4B). The electrophysiological properties of neurons grouped Fisher’s Exact test, P < 0.05). These data indicate that L2/3
according to the Petilla classification scheme and following pyramidal neurons target L1 interneurons in a class-specific
refinementbasedonclusteranalysisaresummarizedinTable1. manner.
We next investigated if the use-dependent properties (use-
dependent depression orfacilitation)of theexcitatory synaptic
Synaptic Interaction betweenL2/3Pyramidal Neurons
input to classesof L1 interneuronswere target cell dependent.
andL1Interneurons
To test this, we evoked 2 APs in L2/3 pyramidal neurons at an
Next, we investigated how efficiently L2/3 pyramidal neurons intervalof50ms.Notably,use-dependentplasticityofL2/3Pyr-
excited L1 interneurons (vertical distance up to ~250 lm, FS and L2/3 Pyr-c-AC uEPSPs was distinct, L2/3 Pyr-FS uEPSPs
CerebralCortexAugust2011,V21N8 1821
uIPSPs with a significantly shorter latency (1.8 ± 0.3 ms, P <
0.05),smalleramplitude(0.28±0.04mV,P <0.001),andfaster
kinetics(risetime6.5±0.5ms,P <0.001;half-width29±8ms,P
< 0.01;n= 4;SupplementaryFig.5;decaytimeconstant39.6±
6.9ms[datanotshown]).Notably,theprobabilityofconnectivity
betweencACandL2/3pyramidalneuronswaslow(0.06,4out
of67connectionstested).Furthermore,c-ACneuronsinhibited
L2/3 pyramidal neurons through the activation of GABA
A
receptors alone as uIPSPs were completely blocked by SR
95531(2lM,Fig.7B,7D,c-AC:n=3).
FSneuronsareknowntoactasfastsignalingdevicesinother
neuronalcircuits(Jonasetal.2004).Consistentwiththisrolein
the superficial cortical layers, FS cells evoked uIPSPs in L2/3
pyramidal neurons with a shorter latency than NGFCs and
a faster rise time than other classes of L1 interneurons (uIPSP
latency0.9±0.1ms,FSvs.NGFCP <0.01,FSvs.c-ACP=0.08;
risetime4±0.4ms,FSvs.NGFCP <0.001,FSvs.c-ACP <0.05;
amplitude 0.27 ± 0.04 mV, FS vs. NGFC P < 0.001, FS vs. c-AC
P=0.77;half-width27±1ms,FSvs.NGFCP <0.001,FSvs.c-AC
P = 0.85; n = 3; Supplementary Fig. 5; and decay time constant
31.7 ± 9.5 ms [data not shown]). Moreover, a high connection
probability was found between FS and L2/3 pyramidal neurons
(0.33,3/9connectionstested).Onechandeliercellwasfoundto
besynapticallyconnectedwithalayer2/3pyramidalneuronand
followingreconstruction,putativesynapticcontactswerefound
decorating theaxon initial segmentof thepostsynaptic neuron
(Supplementary Fig. 7). In line with recent studies (Szabadics
etal.2006;Woodruffetal.2006,2009);butsee(Glickfeldetal.
2009),weobservedthatthechandeliercellevokeddepolarizing
synaptic responses at a membrane potential of –60 mV
Figure5. PropertiesofunitaryEPSPsrecordedindifferenttypesofL1interneurons (Supplementary Fig. 7), in contrast to hyperpolarizing uIPSPs
evokedbysingleAPfiringinL2/3pyramidalneurons.(A)OverlaintracesofuEPSPs
evoked in L2/3 pyramidal neurons by all other classes of L1
recordedfromac-AC(black),FS(orange),andBS(green)neuron.AverageuEPSPs
are shown as thick black traces. Pre, presynaptic L2/3 pyramidal neuron, post, interneuronsatthismembranepotential(seeFig.7).Incommon
postsynapticL1neuron.InsetshowsprobabilityofconnectivityforeachtypeofL1 withhyperpolarizinguIPSPsevokedbyFSneurons,depolarizing
neuron. (B--E) Distributions of the peak amplitude (B), half-width (C), 10--90% rise Chandlier-L2/3PyruIPSPswereblockedbytheGABA receptor
A
time(D),andlatency(E)ofuEPSPsrecordedfrom21c-AC(n5792uEPSPs,black), antagonist SR 95531 (2 lM, FS: n = 2; Supplementary Fig. 6).
7FS(n5182uEPSPs,orange),and5BSneurons(n5111uEPSPs,blue).Dataare
Interestingly, none of the BS neurons we recorded were
presentedascumulativeprobabilityplots.
synaptically connected with L2/3 pyramidal neurons (20
connectionstested);however,oneBS-BSconnectionconfirmed
exhibitedpaired-pulsefacilitation(1.39±0.09;n=7),whileL2/3 theGABAergicnatureofthisconnection(SupplementaryFig.8).
Pyr-c-AC connections displayed, on average, paired-pulse de-
pression (0.92 ± 0.07; n = 21; P < 0.01; Fig. 6). L2/3 Pyr-BS
Discussion
connections, however, showed both facilitation and depression
uponpaired-pulsestimulation(1.1±0.2,n=5,datanotshown). In this study, we have shown that neocortical L1 neurons can
be divided into classes according to the Petilla classification
Inhibitory Synaptic Connectivity betweenL1 scheme (Ascoli et al. 2008). Hierarchical unsupervised cluster
Interneurons andL2/3Pyramidal Neurons analysis confirmed thedivisionofneurons into4groups.L2/3
The properties of unitary inhibitory postsynaptic potentials pyramidal neurons were found to excite L1 neurons in a cell
(uIPSPs) recorded from L2/3 pyramidal neurons were found to class--specific manner. Surprisingly, we found no excitatory
be dependent on the identity of the presynaptic L1 neuron. In connection between L2/3 pyramidal neurons and NGFCs. In
contrast to excitatory connectivity, NGFCs were the most contrast, NGFCs provided strong inhibition of L2/3 pyramidal
prominent source of inhibition to L2/3 pyramidal neurons, neurons.
with a connectivity probability of 0.44 (12 connected out 27
connectionstested).NGFC-L2/3PyruIPSPswereoflonglatency, How Many ClassesofInterneuron Exist inL1ofthe
slowlyrising,andlargeamplitude(latency4.1±0.9ms,risetime Neocortex?
53.2±10.8ms,amplitude0.58±0.1mV,andhalf-width100±19 Previous studies have distinguished, at least, 2 classes of L1
ms, n = 12). In support of recent observations (Tamas et al. neurons based on electrophysiological and anatomical criteria
2003), we observed that a single AP in NGFCs evoked uIPSPs in brain slices prepared from early postnatal (P7--24) rats
mediatedbytheactivationofbothGABA andGABA receptors (Hestrin and Armstrong 1996; Zhou and Hablitz 1996a; Chu
A B
(GABA receptorantagonistSR95531,2lMandGABA receptor et al. 2003). Local circuit neurons were found to have axonal
A B
antagonistCGP52432,10lM,n=10,Fig.7E,F).Incontrast,the and dendritic arbors restricted to L1, while deeper layer--
other class of regular-spiking L1 neuron, c-AC cells, generated projecting neurons possessed descending axons that
1822 Layer1--Layer2/3SynapticConnectivity d WoznyandWilliams
Figure6. Target-specificshort-termplasticityofL2/3toL1unitaryEPSPs.(A1,B1)APfiringpatternofac-AC(left)andanFSneuron(right,uppertraces)inresponsetocurrent
injection(lowertraces).(A2,B2)Paired-pulsestimulationinsynapticallycoupledL2/3pyramidal--L1neuronpairs(sameneuronsasshowninA1,B1).Toptrace(pre,presynaptic
L2/3pyramidalneuron)and10consecutiveuEPSPs(post,postsynapticL1neuron)thatwereonaverage(lowertrace)depressinginac-ACandfacilitatinginanFSneuron.(C)
Barplotofthepaired-pulseratio(EPSP2/EPSP1)forL2/3Pyr-c-AC(n521)andL2/3Pyr-FS(n57)connections(Student’st-test,**P\0.01).
Figure7. PropertiesofunitaryIPSPsrecordedfromL2/3pyramidalneuronsevokedbysingleAPfiringinL1neurons.(A)Typicalexampleofthefiringpatternofac-ACneuron
(uppertrace)inresponsetocurrentinjection(lowertraces).(B)AverageuIPSPsrecordedfromanL2/3pyramidalneuron(averageof10uIPSPs,Post)evokedbyAPfiringinac-
ACL1neuron(Pre,sameneuronasinA).uIPSPswereblockedbytheGABA receptorantagonistSR95531(2lM).(C)TypicalexampleofthefiringpatternofanFSneuron.(D)
A
AverageuIPSPsinthisFS-L2/3pairwereblockedbySR95531(2lM,Pre,sameneuronasinC).(E)FiringpatternofanNGFC.(F)AverageuIPSPsinanNGFC-L2/3connection
(Pre,sameneuronasinE).Notethelongertimebase.TheslowcomponentoftheuIPSPswasblockedbytheGABA receptorantagonistCGP52432(10lM,shadedareain
B
lowerpanel)andthefastcomponentbySR95531(2lM).B,D,F,InsetsshowprobabilityofconnectivityforeachtypeofL1neuron.
innervated deeper neocortical layers (Hestrin and Armstrong arborizationsthatinvadedthedeeperlayeroftheneocortex.In
1996; Zhou and Hablitz 1996a; Chu et al. 2003). Chu et al. this study, we investigated the properties of L1 neurons
(2003) identified delayed AP firing neurons as NGFCs, that maintained in brain slices prepared from P24--36 rats. Over
were restricted to L1, while regular firing neurons had axonal thispostnatalagerange,theelectrophysiologicalpropertiesof
CerebralCortexAugust2011,V21N8 1823
pyramidal (Zhu 2000; Atkinson and Williams 2009) and Svoboda 2005). L2/3 pyramidal neurons target neighboring
interneurons (Doischer et al. 2008) as well as the use- L2/3 (Feldmeyer et al. 2006) and L5 pyramidal neurons
dependent dynamics of unitary excitatory and inhibitory (Thomson and Bannister 1998; Schubert et al. 2001; Williams
synaptic transmission reach adult levels (Reyes and Sakmann andAtkinson2007).However,L2/3andL5pyramidalneurons
1999; Angulo et al. 1999; Frick et al. 2007; Feldmeyer and also show extensive axonal arborization within L1 (Feldmeyer
Radnikow2009).Inthematureneocortex,wehaveidentified4 et al. 2006; Brown and Hestrin 2009). We have found that
classes of L1 neurons using unsupervised hierarchical cluster various interneuron types of L1 receive monosynaptic excit-
analysis of active and passive electrophysiological properties, atory input from L2/3 pyramidal neurons. Wefind the highest
a classification schemesupported by morphologicalanalysis. connectivityprobabilityinL2/3-FScellpairs,followedbyL2/3-
Theroleofinhibitoryinterneuronsinneuronalcomputation c-AC and L2/3-BS connections. As FS and c-AC interneurons
is largely determined by the subcellular sites at which werefoundtoinhibitL2/3pyramidalneurons,weproposethat
inhibitory synaptic contacts are made on pyramidal neurons these celltypes mayfunction toprovide feedback inhibition.
(Somogyi et al. 1998). For example, classes of cortical The use-dependent properties of excitatory synapses have
interneurons target the axon initial segment, perisomatic beensuggestedtodeterminetheirroleinnetworkfunction,by
regions,orthedistaldendritesofpyramidalneurons(Markram shaping the flow of information in neuronal circuits during
et al. 2004; Pouille and Scanziani 2004; Ascoli et al. 2008; repetitive AP firing (Abbott and Regehr 2004). Target-specific
KlausbergerandSomogyi2008).Here,wefindthattheaxonsof use-dependent modifications of uEPSPs have been reported in
60% of c-AC neurons and more than 40% of NGFCs were several excitatory synaptic connections in the neocortex
restricted to L1, suggesting that the majority of L1 neurons (Tsodyks and Markram 1997; Reyes et al. 1998; Markram
inhibit pyramidal neurons at the level of the apical dendritic et al. 1998; Gupta et al. 2000; Rozov et al. 2001; Koester and
arbor.ThereareonlyfewreportsonFSneuronsdescribedinL1 Johnston2005;Westetal.2006;WilliamsandAtkinson2007).
(Foehring et al. 2002). L1 is relatively void of parvalbumin- Indeed, the use-dependent properties of uEPSPs evoked from
positive cells, a calcium-binding protein expressed in FS a single presynaptic pyramidal neuron has been found to be
neurons of other neocortical layers (Xu et al. 2010). We have, dependent upon the identity of the postsynaptic partner
however, also identified a sparse population (23 of 266) of (Reyes et al. 1998; Markram et al. 1998; Koester and Johnston
neurons in L1 with electrophysiological properties of FS 2005). In line with this, we found that the use-dependent
neurons that possessed anatomical characteristics of axon or properties of excitatory synaptic transmission was dependent
perisomatic-targeting interneurons. We cannot rule out that upon the postsynaptic target in L1, with L2/3-FS uEPSPs
a small fraction of these neurons have been assigned to the showing paired-pulse facilitation, and L2/3-c-AC depression.
wrong cortical layer because of the proximity of their somata PairedrecordingsfromadultratbrainsliceshaveshownthatL5
with the L1-L2/3 border. However, based on the finding that and L6 pyramidal neurons generate facilitating uEPSPs in FS
only 5 of 271 neurons recorded from layer 1 had properties neurons (Angulo et al. 1999; West et al. 2006), whereas
reminiscent of layer 2/3 pyramidal neurons, we estimate this synapticdepressionhasbeenreportedforL2/3-FSsynapsesin
error to be less than 2%. In support of the view that FS L1 younger animals (P14) (Reyes et al. 1998) and for L4 regular-
neurons target proximal sites of L2/3 pyramidal neurons, we spiking neurons onto FS neurons (P14--21) (Beierlein et al.
observedthatuIPSPsrecordedbetweenFSL1neuronsandL2/ 2003).Adevelopmentalstudy,however,hasdemonstratedthat
3 pyramidal neurons had significantly faster rise times than inmotorcortices fromWistarratsexcitation ontoFSundergo
those generated by dendritic targeting c-AC neurons, pre- marked changes during maturation switching from use-de-
sumably because of the influence of dendritic filtering (Miles pendent depression at P14--P20 to facilitation at P27--P36
et al. 1996). As the rise time of uIPSPs also depends on the (Angulo et al. 1999).
subunit composition of postsynaptic GABA receptors, it is, L1 neurons have been shown to powerfully control the
A
however, possible that GABA receptor composition may excitabilityofL2/3pyramidalneuronsinvivo(Shlosbergetal.
A
influence these findings(Farrant andNusser 2005). 2006). However, the functional role of different classes of L1
TheminorityofL1neuronspossessedaxonalprojectionsto inhibitory neurons is unknown. Here, we have revealed the
deeperlayersoftheneocortexandwereelectrophysiologically existence of 2 inhibitory feedback circuits between L2/3
classified as BS neurons, NGFCs, and chandelier cells. As we pyramidalandL1neurons.Wesuggestthatthecontrastinguse-
failedtoobserveanyinhibitorysynapticinputfromBSneurons dependent properties observed in the excitatory arm of these
toL2/3pyramidalneurons,wespeculatethatsuchapathwayis circuits suggest that c-AC neurons, which target the apical
sparse and that BS neurons might belong to a group of dendriticarborofL2/3pyramidalneuronsandperisomatically
interneurons which have previously been described to targeting FS neurons will be differentially engaged in a fre-
preferentially target other interneurons (Supplementary Fig. quency-dependent manner. Both cell types provide feedback
8;(Gulyas etal. 1996;Caputi et al.2009). inhibition when L2/3 neurons fire APs at low frequencies,
however,use-dependentdepressionatL2/3-c-ACsynapseswill
ensure that dendritic inhibition is dampened during higher
Neuronal Microcircuits inSuperficial Layers ofthe frequencies of AP firing. This frequency-dependent routing of
Neocortex feed-backinhibitionisincontrasttothatfoundinareaCA1of
It is widely believed that a canonical microcircuit exists in the hippocampus, where pyramidal to interneuron synapses
various cortical areas. In sensory cortices, the flow of show use-dependent depression for interneurons that inner-
information in excitatory neurons of the microcircuit is well vate perisomatic sites but facilitation for interneuron that
described: Thalamocortical projection neurons mainly target innervate apical dendritic sites of pyramidal neurons (Pouille
spiny stellate cells in L4, which, in turn, innervate L2/3 andScanziani2004).IncontrasttootherclassesofL1neuron,
pyramidal neurons (Feldmeyer et al. 2002; Shepherd and wefailedtofindexcitatorysynapticdrivetoNGFCsfromL2/3
1824 Layer1--Layer2/3SynapticConnectivity d WoznyandWilliams
pyramidal neurons. We, however, found that NGFCs generate BrownSP,HestrinS.2009.Intracorticalcircuitsofpyramidalneurons
combinedGABA -andGABA -mediatedinhibitoryresponsesto reflecttheirlong-rangeaxonaltargets.Nature.457:1133--1136.
A B
powerfully inhibit L2/3 pyramidal neurons. Notably, in line Butt SJ, Cobos I, Golden J, Kessaris N, Pachnis V, Anderson S. 2007.
Transcriptional regulation of cortical interneuron development.
withthelonglatencyofNGFCsevokedIPSPs,NGFCshavebeen
JNeurosci.27:11847--11850.
shown to release GABA into the extracellular space providing
CaputiA,RozovA,BlatowM,MonyerH.2009.Twocalretinin-positive
widespreadinhibitoryinputtoneighboringneurons(Olahetal. GABAergiccelltypesinlayer2/3ofthemouseneocortexprovide
2009). Because of the large and dense axonal arborization of differentformsofinhibition.CerebCortex.19:1345--1359.
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columnarcircuittocontrolsynapticintegrationindistalapical AudinatE.2000.Classificationoffusiformneocorticalinterneurons
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Williams2004;Larkumetal.2007;Larkumetal.2009).Indeed,
CaullerLJ,ClancyB,ConnorsBW.1998.Backwardcorticalprojections
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DoischerD,HospJA,YanagawaY,ObataK,JonasP,VidaI,BartosM.
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2008. Postnatal differentiation of basket cells from slow to fast
the columnar circuitry but from long-range intracortical
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DouglasRJ,MartinKA.2004.Neuronalcircuitsoftheneocortex.Annu
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2009) and L1 interneurons (Anderson and Martin 2006). We Elfant D, Pal BZ, Emptage N, Capogna M. 2008. Specific inhibitory
speculate therefore that NGFCs function to provide powerful synapsesshiftthebalancefromfeedforwardtofeedbackinhibition
feed-forward inhibition of long-range intracortical excitatory ofhippocampalCA1pyramidalcells.EurJNeurosci.27:104--113.
Farrant M, Nusser Z. 2005. Variations on an inhibitory theme: phasic
input.
and tonic activation of GABA(A) receptors. Nat Rev Neurosci.
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Feldmeyer D, Lubke J, Sakmann B. 2006. Efficacy and connectivity of
Supplementary Material intracolumnarpairsoflayer2/3pyramidalcellsinthebarrelcortex
ofjuvenilerats.JPhysiol.575:583--602.
Supplementary material can be found at: http://www.cercor.
FeldmeyerD,LubkeJ,SilverRA,SakmannB.2002.Synapticconnections
oxfordjournals.org/.
between layer 4 spiny neurone-layer 2/3 pyramidal cell pairs in
juvenile rat barrel cortex: physiology and anatomy of interlaminar
signallingwithinacorticalcolumn.JPhysiol.538:803--822.
Funding Feldmeyer D, Radnikow G. 2009. Developmental alterations in the
functional properties of excitatory neocortical synapses. J Physiol.
Intramural funding from the Medical Research Council to
587:1889--1896.
S.R.W.; EUMarie-Curie Fellowship to C.W.
Foehring RC, van Brederode JF, Kinney GA, Spain WJ. 2002.
Serotonergic modulation of supragranular neurons in rat sensori-
Notes motorcortex.JNeurosci.22:8238--8250.
Frick A, Feldmeyer D, Sakmann B. 2007. Postnatal development of
WewouldliketothankthepresentandpreviousmembersofStephen
synaptictransmissioninlocalnetworksofL5Apyramidalneuronsin
Williams’ laboratory for helpful discussions. We are grateful to Rick
ratsomatosensorycortex.JPhysiol.585:103--116.
Livesey from the Gurdon Institute, Cambridge, UK and the members
Glickfeld LL, Roberts JD, Somogyi P, Scanziani M. 2009. Interneurons
of the Gurdon Imaging Facility for initial help with confocal images.
hyperpolarize pyramidal cells along their entire somatodendritic
ConflictofInterest:Nonedeclared.
axis.NatNeurosci.12:21--23.
GulyasAI,HajosN,FreundTF.1996.Interneuronscontainingcalretinin
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