Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells

Abstract

Disinhibition of cortical excitatory cell gate information flow through and between cortical columns. The major contribution of Martinotti cells (MC) is providing dendritic inhibition to excitatory neurons and therefore they are a main component of disinhibitory connections. Here we show by means of optogenetics that MC in layers II/III of the mouse primary somatosensory cortex are inhibited by both parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing cells. Paired recordings revealed stronger synaptic input onto MC from PV cells than from VIP cells. Moreover, PV cell input showed frequency-independent depression, whereas VIP cell input facilitated at high frequencies. These differences in the properties of the two unitary connections enable disinhibition with distinct temporal features.

ARTICLE

Received 27 Oct 2015 | Accepted 21 Oct 2016 | Published 29 Nov 2016

DOI: 10.1038/ncomms13664 OPEN

Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells

F. Walker1, M. Mck1, M. Feyerabend1, J. Guy1, R.J. Wagener1,w, D. Schubert2, J.F. Staiger1,* & M. Witte1,*

Disinhibition of cortical excitatory cell gate information ow through and between cortical columns. The major contribution of Martinotti cells (MC) is providing dendritic inhibition to excitatory neurons and therefore they are a main component of disinhibitory connections. Here we show by means of optogenetics that MC in layers II/III of the mouse primary somatosensory cortex are inhibited by both parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing cells. Paired recordings revealed stronger synaptic input onto MC from PV cells than from VIP cells. Moreover, PV cell input showed frequency-independent depression, whereas VIP cell input facilitated at high frequencies. These differences in the properties of the two unitary connections enable disinhibition with distinct temporal features.

1 University Medical Center Gttingen, Institute for Neuroanatomy, Gttingen 37075, Germany. 2 Radboud University Medical Centre Nijmegen, Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, 6525 EN Nijmegen, Netherlands. w Present address: Department of Basic

Neurosciences, Faculty of Medicine, University of Geneva, CH-1211 GENEVE 4, Switzerland. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.W. (email: mailto:[email protected]

Web End [email protected] ).

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a

Cortical inhibitory interneurons (IN) are grouped into three major subpopulations dened by the expression of molecular markers, namely parvalbumin (PV),

somatostatin (SST) and the ionotropic serotonin receptor (5HT3aR)1,2. The main, but not exclusive, cell types within these subpopulations, largely dened by morphological features, are: PV-expressing basket cells, SST-expressing Martinotti cells (MC) and cells co-expressing 5HT3aR and vasoactive intestinal polypeptide (VIP)1,3,4. The functions of these IN are manifold. In general, they keep excitation in check, perform gain modulation and induce synchronization and oscillations, whereas more specically they open or close temporal or spatial windows for input control or output generation5. Their functional impact is not restricted to their interaction with excitatory neurons, but direct interactions between IN seem to be essential for sensory information processing as well69.

In the rodent primary somatosensory (barrel) cortex (S1), GABAergic MC in layer (L) II/III10 mediate disynaptic inhibition between neighbouring pyramidal cells (PCs) and thereby have a major impact on the ow of information within and across cortical columns2,1113. MC in turn are inhibited by other IN, leading to disinhibition of PCs68,14. Several recent in vivo studies have shown that such disinhibition via MC (or other types of IN) contributes to sensory-guided behaviour and learning7,14,15. Morphological and physiological differences of presynaptic IN subtypes16,17 may result in distinct versions of disinhibition in the spatial and/or temporal domain. If so, the regulation of cortical processing by disinhibition might be much more exible than simply being an on/off switch. Therefore, we aimed to dene the molecular identity of IN targeting LII/LIII MC by optogenetics, localize these cells by using glutamate uncaging and study the unitary properties and short-term plasticity of their synaptic transmission by paired recordings. We found that MC in mouse primary somatosensory cortex commonly receive inhibitory inputs from local PV- and VIP-expressing interneurons. Further, these inputs differ substantially in unitary properties and short-term plasticity.

ResultsCharacterization of GIN neurons. To investigate inhibitory inputs to MC, we used two triple transgenic mouse lines, namely PV-cre::tdTomato::GIN and VIP-cre::tdTomato::GIN (Fig. 1a). The cre-knock in lines have recently been reported to be highly specic and sensitive mouse models18,19. Within the GIN line, green uorescence protein (GFP)-expressing cells in cortical LII/LIII and LV were described as being almost exclusively MC2022. In agreement with previous literature, we showed that, also in triple transgenic mice, these cells often display a multipolar or bitufted somato-dendritic conguration and, as their most prominent feature, dense axonal clustering in LI (Fig. 1b). Furthermore, MC show an adapting ring pattern during strong current injections (Fig. 1c). Our experimental data set contained 100 biocytin-labelled GIN cells, of which 82 could be morphologically recovered. Of these recovered neurons, 79 possessed extensive axonal arborizations in LI. In three cases, the ascending axon was cut off before reaching LI. Eight well-preserved GIN cells were fully reconstructed (Supplementary Figs 1 and 2). The dendrites were primarily located in LII/LIII (Supplementary Fig. 1b). Axonal density, however, peaked in LI as well as in LII/LIII (Supplementary Fig. 1b). Therefore, we will use the term MC in the following text to refer to GFP-expressing cells.

MC are inhibited by local PV- and VIP-expressing cells. As mentioned above, PV and VIP cells form the main IN

subpopulations besides MC, which are not considered to interact with each other1,6. Accordingly, they are likely candidates for providing inhibitory inputs to MC. To test this hypothesis, we expressed channelrhodopsin 2 (ChR2) in PV and VIP cells throughout the entire cortical depth of S1 by viral transfection. In order to control for the specicity of the input population, we recorded from ChR2-transduced PV and VIP IN (PV: 11 cells, 5 mice; VIP: 8 cells, 4 mice). They show their subgroup-specic ring patterns and morphology (Supplementary Fig. 3ac,eg). Additionally, we controlled for adequate ChR2 expression levels. Indeed, optogenetic stimulation of these cells caused

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Figure 1 | LII/LIII GIN cells show typical characteristics of Martinotti cells. (a) Fluorescent staining of triple transgenic mice (left: PV-cre::tdTomato::GIN, right: VIP-cre::tdTomato::GIN) used for the present experiments. In all, 50-mm-thick frontal sections from the barrel cortex are shown. PV or VIP cells, respectively, are labelled red and GIN cells green.

Layers are indicated as IIV. Scale bar, 100 mm. (b) Neurolucida reconstructions of LII/LIII biocytin-lled GIN cells. Somatodendritic compartments are shown in orange and axonal arborizations in green. Note the dense axonal branching in LI, which is characteristic for MC. Layers are indicated as IVI. Scale bar, 100 mm. (c) Whole-cell current-clamp recordings of GIN cells shown in b. Depolarizing current injections caused an adapting ring pattern in these cells, as it is typical for MC.

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PV and VIP cell inhibition differs in synaptic properties. These ndings indicated the presence of two discrete inhibitory inputs onto MC, namely, from PV and VIP cells, which are restricted to LII/LIII. Hence, we performed simultaneous recordings of putative presynaptic PV or VIP cells and postsynaptic MCs in this compartment. In accordance with previous results, we indeed found connected pairs of both types (age: PV MC: P21P36; VIP MC: P21P32; Fig. 3a). All PV and VIP cells matched their group-specic ring patterns and morphological characteristics, with PV cells being fast spiking multipolar cells and VIP cells being bipolar/bitufted cells (or partly modied variations thereof) of the adapting or irregular spiking type (Fig. 3b,c; Supplementary Fig. 2). We further analysed the elementary synaptic properties of connected pairs on a unitary level (Fig. 4; Supplementary Table 1a). The connection probability was higher for PV cells (B58%, 12/21) than for VIP cells (B35%, 11/31) (Fig. 4b). Based on the observation that each MC receives input from both of the two other types, as shown by optogenetic stimulation above, the differences in connection probability might be due to a higher degree of divergence of PV cell axonal projections onto MC, as proposed for PC targets as well23. Single presynaptic spikes reliably (PV to MC: B90%; VIP to MC: B80%) elicited unitary

IPSCs in MC regardless of the type of presynaptic IN (Figs 3a and 4b). However, the average IPSC evoked by PV cells (n 12, 12

mice) showed signicantly larger amplitude (PV to MC:49.7412.97 pA; VIP to MC: 12.133.57 pA; means.e.m.), shorter latency (PV to MC: 0.600.07 ms; VIP to MC:1.390.12 ms; means.e.m.), shorter 1090% rise time (PV to MC: 1.620.17 ms; VIP to MC: 4.590.64 ms; means.e.m.) and higher normalized slope (PV to MC: 0.300.05 fraction of amplitude ms 1; VIP to MC: 0.120.02 fraction of amplitude ms 1; means.e.m.) in comparison to VIP cell-evoked IPSCs (n 11, 9 mice) (Fig. 4c,d). Finally, in most of the

cases we also probed for reciprocal connections. In six of the nine cases, PV cells were reciprocally connected with MC, whereas reciprocal connections between VIP cells and MC occurred only in one of the eight trials (Supplementary Fig. 5). We did not quantify these responses owing to differences in the intracellular solutions (see Methods section).

Information processing is subject to short-term dynamic changes in synaptic transmission2427. Therefore, we triggered trains of presynaptic spikes at different frequencies (1, 8, 40 Hz) to investigate short-term plasticity for both types of pairs (Fig. 4e; for original data, see Supplementary Table 2a). The PV-to-MC connection showed signicant depression in IPSC amplitude at all frequencies (1 Hz: n 11; 8 Hz: n 10; 40 Hz: n 10) (Fig. 4f;

Supplementary Table 3a). Although the amount of depression differed from frequency to frequency, it was apparent and substantial (19.204.07%42.754.11%; means.e.m.) already in the second response in every case (Fig. 4f; Supplementary Table 3a). By contrast, repetitive ring in VIP cells neither caused synaptic depression nor obvious facilitation of inhibitory inputs to MC at lower frequencies (1 Hz: n 11; 8 Hz: n 11; 40 Hz:

n 10). However, the amplitudes of IPSCs evoked by VIP cells

consistently facilitated at 40 Hz beginning from the third response onwards (Fig. 4f; Supplementary Table 3a).

PV-to-MC connections also exist in V1. The VIP-to-MC connection seems to be present in S1, as shown here and by Lee et al.8, and in the primary visual cortex (V1)14,28. However, it has been discussed recently whether or not the PV-to-MC connection does exist in V1 as well6,29,30. Accordingly, we performed paired recordings of PV cells and MC in LII/LIII of V1 (age: P27P49; Supplementary Fig. 6a and Supplementary Table 1b). PV cells target MC with a connection probability of B35% (6/17)

depolarizations sufcient to re action potentials (APs) (Supplementary Fig. 3d,hk).

Light-induced activation of ChR2 in either PV or VIP IN reliably evoked inhibitory postsynaptic currents (IPSCs) in every MC tested (PV: 23 cells, 12 mice, age P38P60; VIP: 27 cells, 14 mice, age P35P53) (Fig. 2ac). This indicates that each MC receives input from PV and VIP cells. The minimal laser energy required to elicit IPSCs was comparable for PV and VIP inputs (PV: 76.715.0 mW; VIP: 124.027.1 mW; means.e.m.)

(Fig. 2d). PV cells evoked multicomponent IPSC with apparently larger amplitudes (PV: 456.5543.22 pA; VIP: 275.7046.59 pA; means.e.m.) (Fig. 2a,b,e).

By means of optogenetic stimulation, we could dene presynaptic IN populations; however, their precise laminar location remained unclear. Thus we localized sources of monosynaptic inhibitory input to LII/LIII MC by focal photolysis of caged glutamate (Supplementary Fig. 4). The highest proportion (B45%) of inhibitory elds was found in LII/LIII of the home column (n 10, 10 mice).

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Figure 2 | PV and VIP cells reliably target MC in LII/LIII of S1.(a,b) Left: schematic of recording conguration for photostimulation of ChR2-expressing PV or VIP cells while recording from LII/LIII MC. Right: Examples of photostimulation-induced inhibitory postsynaptic currents (IPSC). Arrowheads indicate photostimulation (473 nm laser, 1 ms) of PV interneurons (a) or VIP interneurons (b) at three different intensities (subthreshold, threshold and 10 threshold). (c) Proportion of MC

responding to photostimulation of PV and VIP cells. In both experimental designs, the success rate was 100%. This indicates that each MC receives inhibitory input from PV and VIP cells. (d) Threshold laser energy to elicit IPSC in MC by photostimulation of PV and VIP cells. Note the same range (means.e.m) for both groups. (e) Means.e.m. of IPSC amplitudes(at 10 threshold level) in MC for each group (PV: n 16, age P38P60,

VIP: n 18, age P3553). Optical stimulation of the PV cell population tends

to result in larger multicomponent IPSC amplitudes.

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(Supplementary Fig. 6b). Similar to S1, PV cells reliably (B97%) caused IPSCs in postsynaptic MC (Supplementary Fig. 6b). On average (means.e.m.), the IPSCs had an amplitude of50.6813.70 pA, a latency of 0.680.08 ms, a 1090% rise time of 1.770.20 ms and a normalized slope of 0.240.03 fraction of amplitude ms 1 (n 6 and 4 mice; Supplementary

Fig. 6c). Concerning short-term plasticity, we observed synaptic depression already at 1-Hz stimulations (Supplementary Fig. 6d,e; for original data, see Supplementary Table 2b). This effect increased at higher frequencies of 8 and 40 Hz (Supplementary Fig. 6d,e; Supplementary Table 3b). Our data are thus in line with recent publications demonstrating that PV-to-MC connections are a frequent motif in V129,30. As MC are only a subpopulation of SST-expressing IN20, this might explain previous discrepant results on PV-to-SST connections6.

DiscussionThe present study demonstrates that both VIP and PV cells target MC in LII/LIII of S1 and differ strongly in unitary synaptic properties as well as short-term plasticity. Although the VIP-to-MC connection has already been shown to exist in LII/LIII of different sensory cortical areas7,8,14,29,30, the PV-to-MC connection has largely been explored in LII/LIII of V129,30. Here we show by combining optogenetics, glutamate uncaging, paired recordings and full morphological reconstruction that in S1 these two circuit motifs (VIP to MC and PV to MC) are fundamentally different.

An important nding is that these two inhibitory inputs onto MC differ substantially in IPSC amplitude, latency and kinetics. One mechanism possibly giving rise to such differences is divergent subcellular targeting. Owing to electrotonic spread, inputs located more distantly will be attenuated and slowed31. In addition, spread along the dendrite might account for the increase in latency. As VIP inputs in our sample are smaller in amplitude, slower in rise and relatively delayed, one might assume that VIP inputs onto MC are substantially more distal as compared with PV inputs. Indeed, such a separation of inhibitory inputs has been described for cortical PC3235. Although dendrite-targeting inhibition is in a position to selectively control excitatory inputs, perisomatic inhibition exerts a global control of spike output. To our knowledge, however, there is no direct ultrastructural evidence for this targeting pattern on MC or any other kind of cortical IN as of yet. Alternatively, differences in the subunit composition of GABAA-receptors in MC could also account for the differences in unitary properties mentioned above36. Interestingly, these two alternatives are not mutually exclusive but might occur in parallel37.

Besides differences in size and kinetics, these two unitary connections also differ in short-term plasticity. Frequency-independent depression of PV cell input onto MC in LII/LIII in S1, as shown in the present report, is in line with previous observations that PV cell input shows short-term depression regardless of the type of postsynaptic cell in sensory cortical areas38. By contrast, reports on short-term plasticity of unitary VIP cell inputs in these areas are lacking. Although the frequency range tested here is in line with many related studies, it must be

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Figure 3 | Electrophysiology and morphology of LII/LIII PVMC and VIPMC pairs. (a) Examples of connected pairs of presynaptic PV or VIP cells and postsynaptic MC in LII/LIII. The average of 10 individual IPSCs (grey traces, evoked by repetitive stimulation) is shown in colour (PV to MC: red, age P23; VIP to MC: blue, age P27). Presynaptic spikes reliably evoke IPSCs in both cases. (b) Whole-cell recordings of a presynaptic PV (left) and a VIP cell (right). During depolarizing current injections, the PV cell shows a fast spiking pattern, whereas the VIP cell shows an adapting ring pattern. (c) Staining of acute brain slices containing morphologically recovered and synaptically connected pairs as well as the corresponding Neurolucida reconstructions (left: PV to MC, right: VIP to MC). The connected cells are shown in white (pseudo-coloured). Asterisks mark MC somata, arrowheads somata of presynaptic cells. GIN cells are labelled green and the corresponding presynaptic population (PV or VIP) is labelled red (tdTomato-uorescence). For clarity, connected cells are shown separately as grey-scale images in the middle. The reconstructed pairs are shown at the bottom. Soma and dendrites of GIN cells are labelled black and the corresponding axon grey. The recorded PV cell exhibits a multipolar dendritic morphology (light red) and a locally dense axon (red), as described for basket cells. The VIP cell shows an atypical tripolar dendritic conguration (light blue) and an axon (blue) descending towards the white matter. Complete reconstructions are displayed in Supplementary Fig. 4. Scale bars, 100 mm.

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Figure 4 | Unitary connections of PV and VIP cells onto MC differ in their elementary synaptic properties and short-term plasticity. (a) Grand average of unitary IPSCs (red: PV to MC, n 12, age P21P36; blue: VIP to MC, n 11, age P21P32) in MC in response to single spikes, repeatedly evoked in

presynaptic IN. Averages of individual pairs are shown in grey. (b) Connection (left) and success rate of synaptic transmission (right) of the two different kinds of unitary connections. Note that the connection probability of PV cells (B58%, 12/21) is substantially larger than the one of VIP cells (B35%, 11/31). In connected pairs, synaptic transmission is highly reliable. (c) Overlay of grand averages (from a) aligned with respect to presynaptic spike peaks. IPSCs evoked by PV and VIP cells differ substantially in size and kinetics. For clarity, the boxed initial phase of both responses is shown at higher resolution as an inset. (d) Quantication of unitary IPSCs. Amplitude, latency, 1090% rise time and normalized slope as fraction of amplitude per ms were analysed based on averages of each individual connected pair (PV to MC: red; VIP to MC: blue). Means.e.m. was then calculated for each group separately. Asterisks indicate signicant differences (Po0.05) for all those parameters. (e) Individual examples of averaged IPSCs in MC in response to trains of ve spikes (1, 8 and 40 Hz) in a presynaptic IN (PV to MC: red trace; VIP to MC: blue trace). Individual traces are shown in grey. Quantication is shown in f. (f) Quantitative analysis of short-term plasticity at different frequencies (1 Hz: PV to MC, n 11; VIP to MC, n 11; 8 Hz: PV to MC, n 10; VIP

to MC, n 11; 40 Hz: PV to MC, n 10; VIP to MC, n 10). Amplitude ratio (nth response/rst response) of consecutive IPSCs plotted versus successive

IPSCs. At the population level, PV to MC responses show synaptic depression under all stimulus conditions, whereas VIP to MC responses show no signicant changes in amplitude at low frequencies but facilitate at 40 Hz. Values represent means.e.m.

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pointed out that both types of presynaptic neurons can re at higher instantaneous frequencies. To our knowledge, the only study using presynaptic spike trains with very short interspike intervals reported substantial synaptic depression in fast spiking as well as non-fast spiking interneurons in adult rats over a wide range of interspike intervals except for 1020 ms39. Short-term plasticity of PV inputs on MC might be described as phasic in comparison to the more tonic properties of VIP inputs. Nevertheless, even depressed PV inputs still exert a stronger inuence at the soma than the corresponding VIP inputs. Considering that VIP cells might target distal dendrites, their true impact on dendritic input control would be much stronger and facilitation at high frequencies could be a dominant factor in controlling activity in the postsynaptic cell. By this means, VIP and PV cells may provide different spatial and temporal windows of opportunities38.

In the present study, we have shown that each MC in LII/LIII of the barrel cortex receives two different inhibitory inputs with divergent properties. Two separate input channels may, on the one hand, allow different sources of excitatory drive to control PC inhibition by MC but it may as well allow one single excitatory input to utilize different kinds of MC inhibition. Both types of MC-inhibiting IN share much of their excitatory input, namely, local LII/LIII PC and excitatory cells in LIV of the same column4042. Recently, however, long-range connections from primary motor cortex have been described to preferentially target VIP cells in S1 (ref. 8). Accordingly, VIP cell-mediated inhibition of MC may integrate somatosensory and motor information, whereas PV cells are purely driven by somatosensory inputs. Besides glutamatergic input, VIP and PV cells also receive modulatory afferents. State-dependent cholinergic modulation may thereby selectively enhance and/or suppress activity in IN inhibiting MC43,44.

In summary, disinhibition of LII/LIII PC via MC is mediated by two distinct IN populations. As these inputs differ in strength, kinetics, short-term plasticity and possibly subcellular targeting, PV cells may induce a transient release from MC inhibition, whereas VIP cells may result in tonic disinhibition. Future studies will clarify whether tonic and phasic inhibition can be recruited by the same or different sources of inputs.

Methods

Animals. All experiments were performed in accordance with the German Law on the Protection of Animals. All animals used for breeding were obtained from Jackson Laboratory (Bar Harbor, USA) and kept under standard housing conditions. PV-cre (Pvalbtm1(cre)Arbr/J) or VIP-cre mice (VIPtm1(cre)Zjh) were crossbred with homozygous Ai9 mice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J)to obtain PV-cre/VIP-cre::tdTomato mice. These mice were further crossedwith homozygous GIN mice (FVB-Tg(GadGFP)45704Swn) to create the triple transgenic mouse lines PV-cre::tdTomato::GIN and VIP-cre::tdTomato::GIN. Using these lines, PV-expressing (PV cell), VIP-expressing (VIP cell) and GIN cells in LII/LIII could be identied by their specic uorescent label (PV/VIP cells: tdTomato uorescence, GIN cells: GFP uorescence). For optogenetic experiments, PV-cre (Pvalbtm1(cre)Arbr/J) or VIP-cre mice (VIPtm1(cre)Zjh) were crossbred with homozygous GIN mice (FVB-Tg(GadGFP)45704Swn) to create PV-cre::GIN and VIP-cre::GIN mouse lines.

Slice preparation and chemicals. To obtain acute brain slices, juvenile mice of either sex (postnatal days 2149) were deeply anaesthetized with isourane and decapitated. The brain was removed, the hemispheres separated and kept in cold (4 C), oxygenated (Carbogen: 95% O2/5% CO2) preparation solution used for cutting (in mmol: 75 sucrose, 87 NaCl, 2.5 KCL, 0.5 CaCl2, 7.0 MgCl2, 26 NaHCO3,1.25 NaH2PO4 and 10 glucose; pH: 7.4). Thalamo-cortical slices of 300 mm thickness from the mouse barrel cortex were prepared according to Porter et al.45

Coronal slices of 300 mm thickness were prepared from the primary visual cortex. Slices were incubated in oxygenated articial cerebrospinal uid (ACSF) (in mmol: 125 NaCl, 2.5 KCL, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4 and 25 glucose;

pH: 7.4) at 32 C for 30 min and later kept at room temperature until further processing.

Electrophysiology and data acquisition. Slices were transferred to a submerged recording chamber (ACSF ow rate of 2 ml min 1 at 32 C) in an upright microscope (Axio Examiner, Zeiss, Germany). For photostimulation, a 405 nm (DL-405, Rapp OptoElectronic, Wedel, Germany) or a 473 nm laser light (DL-473, Rapp OptoElectronic) was coupled via a 200 mm liquid bre to the epiuorescence path of the microscope and guided into the 40 objective. Whole-cell recordings

from MC in LII/LIII of the barrel cortex and from PV or VIP cells were performed in current clamp as well as voltage clamp. Borosilicate patch pipettes (58 MO)

contained a cesium-based intracellular solution (in mmol: 135 CsMeSO4, 5 CsCl,2 MgCl2, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine phosphate; pH: 7.4) for GIN cell recordings during optogenetics, uncaging experiments and paired recordings. We used a potassium-based solution (in mmol: 135 K-gluconate, 5 KCl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 Na-GTP,10 Na-phosphocreatine phosphate; pH: 7.4) for PV or VIP cells in all experiments and GIN cells during experiments performed in current clamp. In case of paired recordings, we recorded from nearby (distance: 25200 mm) PV and VIP cells.

Intracellular solutions contained 0.30.5% biocytin for subsequent morphological visualization. Depolarizing current pulses above rheobase were used to characterize ring patterns of PV, VIP and GIN cells during initial current clamp recordings. To investigate IPSC, in all subsequent experiments, GIN cells were kept close to AMPA-receptor equilibrium potential in voltage clamp (EAMPA: B0 mV). This was

carried out to increase the driving force for chloride, hence the amplitude of inhibitory postsynaptic currents, and to minimize contamination by excitatory postsynaptic currents. Data were acquired using a SEC-05 L amplier (npi electronics, Tamm, Germany) in discontinuous mode with a switching frequency of 50 kHz. The signals were ltered at 3 kHz and digitized at 1025 kHz using a CED Power 1401 interface (CED Limited, Cambridge, England). Data were collected, stored and analysed with Signal 5 (CED Limited, Cambridge, England).

Focal photolysis of caged glutamate. As soon as stable whole-cell voltage clamp recordings of LII/LIII GIN cells (age P24P34, either sex) were achieved(Vhold 0 mV), focal photolysis of caged glutamate with a 405 nm laser light

was carried out to activate presynaptic IN. The laser beam was focused on a50 50 mm2 area on the plane of the brain slice. Caged glutamate (CNB-caged-

L-

glutamate, Molecular Probes, Carlsbad, USA) was added to the ACSF perfusion with a nal concentration of 330 mM. To reduce detection errors of IPSCs, laser stimulus (6 ms duration) was repeated three times per eld at an interval of 3 s. In principle, glutamate release could activate all types of neurons, which could lead to disynaptically evoked inhibitory inputs. We set up a series of calibration experiments to determine an energy level at which the laser, with its beam centred on the soma, generated spikes. Therefore, we performed sequential whole-cell patch clamp recordings, with potassium-based intracellular solution, of cortical neurons(PV, VIP, SST, GIN and excitatory cells) and induced spiking by glutamate uncaging. A laser energy level of B120 mJ triggered spikes in B86% of all types of inhibitory cells but in only B25% of excitatory cells located throughout all cortical layers (Supplementary Fig. 3e).

In subsequent mapping experiments, IPSCs were only accepted as stimulus evoked if their amplitude exceeded the mean baseline 3 s.d. of the baseline, they

were detected in at least two out of three stimulus repetitions and they appeared within a 10 ms time window after stimulus offset. The laser was moved over an area covering three adjacent barrel-related columns (the middle one containing the recorded GIN cell) and the entire cortical depth, either from pia to white matter or vice versa. Scanning was carried out systematically along rows with alternating directions (50 mm per step, 10 s intervals) controlled by the Morgentau M1 software (Morgentau Solutions GmbH, Munich, Germany). Thus up to 364 different elds were stimulated without any intermittent gaps. In every slice containing a recorded GIN cell, layer and column borders were estimated from DAPI (4,6-diamidino-2-phenylindole) stainings and aligned with the scanned cortical area. Once individual elds were assignable to specic columns and layers, maps were created representing the average IPSC amplitude in elds containing sources of inhibitory input (inhibitory elds). These maps were then converted into binary ones by assigning a grey-scale value of 255 to each inhibitory eld and the value 0 to the remaining elds (Supplementary Fig. 3f). In addition, the number of inhibitory elds was counted per layer and column. Individual binary maps were then aligned in relation to the barrel-like structure in LIV of the home column and converted into an average map depicting the condence level for the position of inhibitory elds by means of a customized Matlab script (The MathWorks GmbH, Ismaning, Germany).

Viral injection and optogenetics. To test whether PV and/or VIP cells target LII/LIII MC in the barrel cortex, we used adeno-associated viral vectors (AAV) for cre-dependent ChR2 expression in Cre-expressing interneurons. The AAV (pAAV-EF1a-double oxed-hChR2(H134R)-mCherry-WPRE-HGHpA) were custom manufactured by the Viral Vectors Platform of the DFG research unit and cluster of excellence CNMPB Goettingen and packaged in AAV-6 capsids. PV-cre::GIN or VIP-cre::GIN mice (postnatal days 1827, either sex) were anaesthetized with 12% isourane (analgesia: Lidocaine s.c., metamizole p.o.) and positioned in a stereotactic frame. A small craniotomy (12 mm in diameter) was made with a dental drill (Osada Success 40, Osada, Tokyo, Japan) to expose the cortical surface. AAV were injected into the barrel cortex (anteriorposterior range:

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13664 ARTICLE

Bregma minus 12 mm, mediallateral range: 2.53.5 mm) via a glass micropipette (25 mm inner diameter) connected to a Toohey Spritzer Pressure System IIe (Toohey Company, Faireld, NJ, USA). The micropipette was positioned at 24 different locations, guided by surface blood vessel patterns and at 3 different depths (800, 500, 250 mm below the pial surface). Small amounts of virus in sterile phosphate-buffered saline (PBS; up to 150 nl) were injected by pressure application (3 psi, 250 ms pulse duration). The micropipette was withdrawn 1015 min after pressure application. Finally, the animals were sutured and injected with carprofen(2.5 mg per 10 g body weight, Rymadyl, Pzer, New York City, NY, USA). For postoperative care, animals were provided with wet food and metamizol(1.33 mg ml 1, Novaminsulfon-ratiopharm, Ratiopharm, Ulm, Germany) dissolved in drinking water. In accordance with animal care guidelines, metamizol was also applied a day before surgery.

Two-to-three weeks after viral transduction, acute brain slices were prepared and whole-cell patch clamp recordings of LII/LIII MC were performed as described above. Photostimulation (diameter: B100 mm) of ChR2-expressing cells was performed with a 473 nm laser light source (see above). The photostimulation (duration: 1 ms) was executed three times for three illumination intensities (subthreshold, threshold and 10 threshold for IPSC occurrence in MC; range:

31,000 mW) at a minimum inter-stimulus interval of 5 s.

Paired recordings. During paired recordings, presynaptic neurons (PV or VIP cells) remained at resting membrane potential (Vrest) in current clamp.

Postsynaptic GIN cells were kept at Vhold 0 mV in voltage clamp. Consecutive

brief current injections (5 ms per pulse, 20650 pA, 1020 sweeps, 10 s sweep interval) to presynaptic inhibitory neurons caused single spikes or a train of ve spikes with frequencies of 1, 8 and 40 Hz (short-term plasticity experiments), which led to IPSCs in GIN cells.

All measurements were carried out on averages of individual sweeps. Prior to averaging, all individual IPSCs of a connected pair were aligned with respect to the spike peak of the presynaptic action potential. This was carried out to prevent disturbance of the average IPSC waveform owing to spike jitter. For responses from single spike stimulations, we analysed the following parameters: latency (time from presynaptic spike peak to IPSC onset), 1090% rise time (time between 10% and 90% of IPSC peak amplitude), amplitude (difference from baseline to peak) and normalized slope of the ascending phase (average slope, determined by means of a least-square best t, divided by the maximum amplitude) of the IPSC.

Short-term plasticity was tested by applying trains of presynaptic spikes. Here we only measured the peak amplitudes of the average IPSCs and calculated the response ratio for each IPSC relative to the amplitude of the rst response(nth response/rst response). Consecutive IPSCs overlapped only during 40 Hz stimulations. To measure the amplitude of single responses in this case, the decay phase of the preceding IPSC was exponentially tted. This t was extrapolated to baseline level. Response amplitude was then calculated as the difference between the peak of the response and the t value at that point in time.

Staining. To visualize biocytin-lled neurons as well as GFP-, mCherry- and tdTomato-expressing cells, slices were processed as follows. Slices were rinsed with PBS and incubated with primary antibodies (rabbit anti-RFP, 1:500, Rockland, Limerick, PA, USA; goat anti-GFP, 1:2,000, Abcam, Cambridge, UK) in blocking solution (0.25% bovine serum albumin, 10% normal donkey serum and 0.5% Triton X-100, pH 7.6, in PBS) for 4872 h at 4 C.Then they were rinsed in PBS (5 ), followed by 4 h of secondary antibody incubation at room temperature,

rinsed in PBS (6 ) and stained by DAPI (1:1,000, Molecular Probes, Carlsbad,

CA, USA). Secondary antibodies used were donkey anti-goat AF488 (1:500, Invitrogen, Carlsbad, CA, USA), donkey anti-rabbit AF546 (1:500) and streptavidin-conjugated AF633 (1:500). Slices were mounted in AquaPolyMount and uorescent images were taken using a Leica SP2 confocal microscope( 40 immersion objective; voxel size: 0.18 0.18 0.80 mm3), controlled by the

Vision4d software (Arivis AG, Unterschleiheim, Germany) in order to acquire and stitch multiple, predened tiles.

Reconstruction of biocytin-lled neurons. Fluorescently labelled neuronswith consistently intense staining of neurites and no obvious truncation of main processes were reconstructed by acquiring large XYZ-stacks with a confocal microscope (Leica SP2; equipped with Arivis software) and subsequently loading these stacks into Neurolucida Workstation (MBF Bioscience, Colchester, VT, USA). Dendritic processes were distinguished from axonal structures by their diameter, ne structure and branching pattern.

To superimpose multiple reconstructions and show the distribution of neuronal processes as a population average, reconstructed neurons were registered into one le using dened layer borders (according to Prnneke et al.18) as a reference, somata aligned at the same vertical level and all bers plotted as one binary image. This was ltered using a Gaussian lter with a comparable radial sigma (25 at 300 dpi) for all structures and a colour look-up-table ranging from cold (blue and green for white to light grey) to warm colours (yellow and red for dark grey to black) was applied to the resulting grey-scale image. This was then merged with the original black and white image and resulted in heat maps visualizing areas of the highest density of dendritic and axonal trees.

Statistics. For statistical comparisons, MannWhitney rank-sum tests were used. Results were given as P values. Po0.05 was interpreted as signicantly different.

Means.e.m. are given for all other values, if not stated otherwise.

Digital illustrations. Confocal image stacks were exported as maximum intensity projections and stored as TIFF les. Image brightness and contrast images were adjusted using the Photoshop software (Adobe, Dublin, Ireland).

Data availability. The data that support the ndings of this study are available from the corresponding author on request. Neurolucida reconstructions will be made available through Neuromorpho.org.

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Acknowledgements

We cordially thank Patricia Sprysch for technical support. Thanks are due to Dirk Feldmeyer and Guanxiao Xi for initial help with paired recordings as well as Swen Hlsmann and Michal Fortuna for supplying viral vectors. We thank Anna Dudek for proofreading this manuscript. This work was supported by a University Medical Center start-up grant to Mirko Witte and by the Deutsche Forschungsgemeinschaft (Sta 431/8-1 and SFB 889, TP C7 to Jochen F. Staiger).

Author contributions

F.W., M.W., M.F., J.G. and R.J.W. carried out experiments. F.W., M.M., J.F.S. and M.W. were involved in study design and wrote the paper. M.W. and J.F.S. supervised all experiments. F.W., M.W., M.M. and D.S. carried out data analysis.

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How to cite this article: Walker, F. et al. Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells. Nat. Commun. 7:13664 doi: 10.1038/ncomms13664 (2016).

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