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

Characterization 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 specific and sensitive mouse models¹⁸,¹⁹. Within the GIN line, green fluorescence protein (GFP)-expressing cells in cortical LII/LIII and LV were described as being almost exclusively MC²⁰,²¹,²². In agreement with previous literature, we showed that, also in triple transgenic mice, these cells often display a multipolar or bitufted somato-dendritic configuration and, as their most prominent feature, dense axonal clustering in LI (Fig. 1b). Furthermore, MC show an adapting firing 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 specificity 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-specific firing patterns and morphology (Supplementary Fig. 3a–c,e–g). Additionally, we controlled for adequate ChR2 expression levels. Indeed, optogenetic stimulation of these cells caused depolarizations sufficient to fire action potentials (APs) (Supplementary Fig. 3d,h–k).

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 P38–P60; VIP: 27 cells, 14 mice, age P35–P53) (Fig. 2a–c). 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: 76.7±15.0 μW; VIP: 124.0±27.1 μW; mean±s.e.m.) (Fig. 2d). PV cells evoked multicomponent IPSC with apparently larger amplitudes (PV: 456.55±43.22 pA; VIP: 275.70±46.59 pA; mean±s.e.m.) (Fig. 2a,b,e).

By means of optogenetic stimulation, we could define 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 (∼45%) of inhibitory fields was found in LII/LIII of the home column (n=10, 10 mice).

PV and VIP cell inhibition differs in synaptic properties

These findings 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: P21–P36; VIP MC: P21–P32; Fig. 3a). All PV and VIP cells matched their group-specific firing patterns and morphological characteristics, with PV cells being fast spiking multipolar cells and VIP cells being bipolar/bitufted cells (or partly modified 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 (∼58%, 12/21) than for VIP cells (∼35%, 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: ∼90%; VIP to MC: ∼80%) 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 significantly larger amplitude (PV to MC: 49.74±12.97 pA; VIP to MC: 12.13±3.57 pA; mean±s.e.m.), shorter latency (PV to MC: 0.60±0.07 ms; VIP to MC: 1.39±0.12 ms; mean±s.e.m.), shorter 10–90% rise time (PV to MC: 1.62±0.17 ms; VIP to MC: 4.59±0.64 ms; mean±s.e.m.) and higher normalized slope (PV to MC: 0.30±0.05 fraction of amplitude ms⁻¹; VIP to MC: 0.12±0.02 fraction of amplitude ms⁻¹; mean±s.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 transmission²⁴,²⁵,²⁶,²⁷. 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 significant 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 (19.20±4.07%–42.75±4.11%; mean±s.e.m.) already in the second response in every case (Fig. 4f; Supplementary Table 3a). By contrast, repetitive firing 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: P27–P49; Supplementary Fig. 6a and Supplementary Table 1b). PV cells target MC with a connection probability of ∼35% (6/17) (Supplementary Fig. 6b). Similar to S1, PV cells reliably (∼97%) caused IPSCs in postsynaptic MC (Supplementary Fig. 6b). On average (mean±s.e.m.), the IPSCs had an amplitude of 50.68±13.70 pA, a latency of 0.68±0.08 ms, a 10–90% rise time of 1.77±0.20 ms and a normalized slope of 0.24±0.03 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.

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 (Pvalb^tm1(cre)Arbr/J) or VIP-cre mice (VIP^tm1(cre)Zjh) were crossbred with homozygous Ai9 mice (B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J) to obtain PV-cre/VIP-cre::tdTomato mice. These mice were further crossed with 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 identified by their specific fluorescent label (PV/VIP cells: tdTomato fluorescence, GIN cells: GFP fluorescence). For optogenetic experiments, PV-cre (Pvalb^tm1(cre)Arbr/J) or VIP-cre mice (VIP^tm1(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 21–49) were deeply anaesthetized with isoflurane 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 μm thickness from the mouse barrel cortex were prepared according to Porter et al.⁴⁵ Coronal slices of 300 μm thickness were prepared from the primary visual cortex. Slices were incubated in oxygenated artificial cerebrospinal fluid (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 flow 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 μm liquid fibre to the epifluorescence 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 (5–8 MΩ) 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: 25–200 μm) PV and VIP cells. Intracellular solutions contained 0.3–0.5% biocytin for subsequent morphological visualization. Depolarizing current pulses above rheobase were used to characterize firing 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 (E_AMPA: ∼0 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 amplifier (npi electronics, Tamm, Germany) in discontinuous mode with a switching frequency of 50 kHz. The signals were filtered at 3 kHz and digitized at 10–25 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 P24–P34, either sex) were achieved (V_hold=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 a 50 × 50 μm² 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 final concentration of 330 μM. To reduce detection errors of IPSCs, laser stimulus (6 ms duration) was repeated three times per field 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 ∼120 μJ triggered spikes in ∼86% of all types of inhibitory cells but in only ∼25% 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 μm per step, 10 s intervals) controlled by the Morgentau M1 software (Morgentau Solutions GmbH, Munich, Germany). Thus up to 364 different fields 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 fields were assignable to specific columns and layers, maps were created representing the average IPSC amplitude in fields containing sources of inhibitory input (inhibitory fields). These maps were then converted into binary ones by assigning a grey-scale value of 255 to each inhibitory field and the value 0 to the remaining fields (Supplementary Fig. 3f). In addition, the number of inhibitory fields 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 confidence level for the position of inhibitory fields 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 floxed-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 18–27, either sex) were anaesthetized with 1–2% isoflurane (analgesia: Lidocaine s.c., metamizole p.o.) and positioned in a stereotactic frame. A small craniotomy (1–2 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 (anterior–posterior range: Bregma minus 1–2 mm, medial–lateral range: 2.5–3.5 mm) via a glass micropipette (25 μm inner diameter) connected to a Toohey Spritzer Pressure System IIe (Toohey Company, Fairfield, NJ, USA). The micropipette was positioned at 2–4 different locations, guided by surface blood vessel patterns and at 3 different depths (800, 500, 250 μm 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 10–15 min after pressure application. Finally, the animals were sutured and injected with carprofen (2.5 mg per 10 g body weight, Rymadyl, Pfizer, 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: ∼100 μm) 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: 3–1,000 μW) 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 (V_rest) in current clamp. Postsynaptic GIN cells were kept at V_hold=0 mV in voltage clamp. Consecutive brief current injections (5 ms per pulse, 20–650 pA, 10–20 sweeps, 10 s sweep interval) to presynaptic inhibitory neurons caused single spikes or a train of five 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), 10–90% 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 fit, 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 first response (nth response/first 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 fitted. This fit was extrapolated to baseline level. Response amplitude was then calculated as the difference between the peak of the response and the fit value at that point in time.

Staining

To visualize biocytin-filled 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 48–72 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 fluorescent images were taken using a Leica SP2 confocal microscope ( × 40 immersion objective; voxel size: 0.18 × 0.18 × 0.80 μm³), controlled by the Vision4d software (Arivis AG, Unterschleißheim, Germany) in order to acquire and stitch multiple, predefined tiles.

Reconstruction of biocytin-filled neurons

Fluorescently labelled neurons with 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, fine 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 file using defined layer borders (according to Prönneke et al.¹⁸) as a reference, somata aligned at the same vertical level and all fibers plotted as one binary image. This was filtered using a Gaussian filter 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, Mann–Whitney rank-sum tests were used. Results were given as P values. P<0.05 was interpreted as significantly different. Mean±s.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 files. Image brightness and contrast images were adjusted using the Photoshop software (Adobe, Dublin, Ireland).

Data availability

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