Cortical interneurons that specialize in disinhibitory control

Abstract

In the mammalian cerebral cortex the diversity of interneuronal subtypes underlies a division of labour subserving distinct modes of inhibitory control. A unique mode of inhibitory control may be provided by inhibitory neurons that specifically suppress the firing of other inhibitory neurons. Such disinhibition could lead to the selective amplification of local processing and serve the important computational functions of gating and gain modulation. Although several interneuron populations are known to target other interneurons to varying degrees, little is known about interneurons specializing in disinhibition and their in vivo function. Here we show that a class of interneurons that express vasoactive intestinal polypeptide (VIP) mediates disinhibitory control in multiple areas of neocortex and is recruited by reinforcement signals. By combining optogenetic activation with single-cell recordings, we examined the functional role of VIP interneurons in awake mice, and investigated the underlying circuit mechanisms in vitro in auditory and medial prefrontal cortices. We identified a basic disinhibitory circuit module in which activation of VIP interneurons transiently suppresses primarily somatostatin- and a fraction of parvalbumin-expressing inhibitory interneurons that specialize in the control of the input and output of principal cells, respectively. During the performance of an auditory discrimination task, reinforcement signals (reward and punishment) strongly and uniformly activated VIP neurons in auditory cortex, and in turn VIP recruitment increased the gain of a functional subpopulation of principal neurons. These results reveal a specific cell type and microcircuit underlying disinhibitory control in cortex and demonstrate that it is activated under specific behavioural conditions.

Figure 1. VIP interneurons generate disinhibition in ACx and mPFC of awake mice

a, Expression of ChR2-YFP in a VIP-Cre mouse. Scale bar, 50μm. b, (left) VIP neurons were identified by optical stimulation in vivo. (right) Light-evoked spike waveforms (blue) were similar to spontaneous ones (black) (see Methods). c, d, e, Raster plot (top) and peri-stimulus time histogram (PSTH) (bottom) of representative neurons for directly activated (VIP neurons), inhibited, and delayed activated groups in ACx. f, Relative light-induced firing rate change (log scale) vs. latency of the maximal effect (peak/trough of PSTH). Three clearly separated groups of significantly modulated neurons are apparent: short-latency activated (VIP, green), inhibited (purple) and delayed activated (light brown). Solid lines indicate probability density functions of peak times (normalized separately to improve visibility). g, h, Average PSTH of the VIP, inhibited, and delayed activated neuron groups in ACx (g) and mPFC (h). The temporal differences between ACx and mPFC were due to the different ChR2 expression systems (Ai32 vs. viral expression, see Extended Data Fig. 9).

Figure 2. VIP interneurons inhibit Som and Pv interneurons in ACx and mPFC in vitro

a, b, c, (first column) Schematic of the in vitro experiments. A subpopulation of Som- (a) or Pv-expressing (b) neurons were identified under epifluorescence. Pyramidal (c) cells were identified by soma shape and were sampled from non-fluorescent neurons. (second column) Representative firing patterns of Som, Pv and pyramidal neurons during the injection of a depolarizing current (ACx). Scale bar, 40mV, 50pA, 200ms. (third and fourth columns) Photostimulation-induced (blue bars) IPSCs from Som neurons (a), Pv neurons (b) and pyramidal neurons (c) at 1Hz and 40Hz repetition rates. Scale bars, 1Hz stimulation: 50pA, 30ms (a), 40pA, 25ms (b), and 20pA, 25ms (c); 40Hz stimulation: 16pA, 125ms (a), 75pA, 50ms (b). d, Fraction of neurons responding to photostimulation in ACx and mPFC. e, Mean ± SEM of IPSCs for the significantly responsive neurons (paired t-test, p<0.01). Average IPSC amplitudes in ACx and mPFC were not significantly different (t-test, Som, p=0.125; Pv, p=0.83; Pyr, p=0.256; note the low sample sizes for pyramidal cells because of the low prevalence of evoked IPSCs. f, Short-term depression of IPSCs at 40Hz (ACx and mPFC combined).

Figure 3. Auditory responses of a functional subpopulation of principal neurons are modulated by disinhibition

a, Venn diagram showing the number of single neurons in ACx classified based on tone and light-responsiveness. b, (left) PSTHs aligned to stimulus onset show that tone-response of delayed activated cells is stronger than that of those unaffected by light (only cells firing >1Hz included in the unaffected population). (middle, right) Light-response is stronger in tone-responsive than -unresponsive neurons. c, d, e, f Auditory tuning of tone-responsive inhibited (c, d) and delayed activated (e, f) neurons in awake mice. c, e (top) Raster plots of representative neurons sorted by the frequency of the auditory stimulus. Gray shade, tone delivery; blue shade, tone+light stimulation. (bottom) Frequency tuning curves of the same cells. d, f Average tuning curves. Mustard solid/dashed lines are predictions from additive/multiplicative models, respectively.

Figure 4. VIP neurons are recruited by reinforcement signals

a, Schematic of behavioral setup and auditory discrimination task. b, Average performance and reaction time (mean ± SEM across sessions) c. Raster plots and peri-event time histograms (PETH) of example VIP neuron. Neural activity was aligned to reinforcement (reward/punishment; red). Blue, tone onset. d, Raster plots and average PETHs of VIP neurons (n=10). All VIP neurons showed strong increase of firing rate after punishment (VIP #1–4: foot shock; asterisk, #5–10: air puff). e, Cumulative fraction of tone-selectivity (left; Kolmogorov-Smirnov test, p=0.43) and firing rate change (right; Hit, p<0.01; False alarm, p<0.01; ΔResponse, see Methods). Response to reinforcement signals distinguishes VIP neurons from unidentified population. f, Normalized average PETH aligned to reinforcement of VIP and concurrently recorded unidentified neurons responsive to both positive and negative feedback (17/130; grey). (left) VIP neurons showed sustained activation after reward delivery. (right) VIP neurons increased firing rate abruptly after punishment, in contrast with unidentified cells. g, Schematic model of disinhibitory circuit. Feedback information (e.g. reinforcement signals) to VIP neurons disinhibits a functional subpopulation of pyramidal neurons.

Extended Data Figure 1. Specificity and efficiency of ChR2 expression, recording locations, and layer 1 VIP neurons

a, VIP-Cre:Ai32 (ACx). b, VIP-Cre::AAV-DIO-ChR2 (mPFC). Note that some green puncta were not somata and only somata were used for quantification. c, Quantification of the overlap of ChR2-YFP (green) with anti-VIP (red) in ACx. Overlap=88±6.6% (49/54 neurons), 6 slices from 4 mice (see Methods for a note on caveats). d, Quantification of the overlap of ChR2-YFP (green) with anti-VIP (red) in mPFC. Overlap=97±3.7% (35/36 neurons), 5 slices from 4 mice. Scale bar, 50 μm. e, f, Recording location in mPFC and ACx. Microdrives accommodating 6 tetrodes and 1 optical fiber were implanted in the ACx (e) or the mPFC (f). Recording sites were confirmed by histology using DiI (red) that was applied to the optical fiber before implantation. Histology results showed that the electrode locations were biased towards the middle layers. Green, VIP neurons expressing ChR2-YFP; red, DiI. Scale bar, 200μm. g, h, Most VIP neurons were located in layer 2/3, with a smaller fraction in layer 1. VIP comprised about 10% of layer 1 neurons. g, VIP neuron in layer 1 of the ACx (arrow; 6 VIP/60 layer 1 ACx neurons, n=6 slices from 2 mice). h, VIP neuron in layer 1 of the mPFC (5 VIP/56 layer 1 mPFC neurons, n=6 slices from 2 mice). Green, VIP; red, NeuN (neuronal marker) staining. Scale bar, 100 μm.

Extended Data Figure 2. Three distinct populations responsive to photostimulation in mPFC and spike width vs. firing rate

a, b, c Raster plots and PSTHs aligned to photostimulation for three distinct populations in mPFC. Examples of a directly activated (VIP, a), an inhibited (b), and a delayed activated neuron (c). Stimulation frequency, 10–20Hz. d, Photostimulation-evoked spike probability of a VIP interneuron. (left) Raster plot. (right) Firing probability as a function of photostimulation frequency. When all light pulses were considered, spike probability decreased with stimulation frequency (blue). However, the first 5 light pulses reliably evoke action potentials up to 100Hz (spike probability=0.78 at 100Hz). e, Relative light-induced firing rate change (log scale) vs. latency of the maximal effect (peak/trough of PSTH). Three separate groups are apparent in mPFC: short-latency activated (VIP, green), inhibited (purple) and delayed activated (light brown). f, g, (top left) Baseline firing rate vs. spike width in ACx (f) and mPFC (g). (top right) Cumulative fraction of firing rate. (bottom) Cumulative fraction of spike width. Green, directly activated (VIP); purple, inhibited; light brown, delayed activated group; dark grey, unidentified neurons. Light grey depicts neurons for which inhibition could not reliably be assessed because of very low baseline firing rates (see Methods).

Extended Data Figure 3. Inhibited and activated (Inh-act) population is a subgroup of the inhibited neurons

a, Average PSTH aligned to photostimulation (1ms pulses) for Inh-act cells (a subgroup of the inhibited neurons, Fig. 1 and 3). The color code of Fig. 1g, h applies. Inh-act neurons (purple) show initial inhibition followed by delayed activation after 1ms pulses. b, (top) Example raster plot aligned to auditory stimuli of a tone-responsive Inh-act neuron. Shading indicates the stimulation windows (grey, tone only; blue, tone- and photostimulation). Dashed boxes indicate time windows for frequency tuning analysis (early, 0–25ms; late, 75–100ms). (bottom) Frequency tuning curves of tone-responsive Inh-act neurons (population average, n=14). (bottom left) Tuning curve for the early time window (0–25ms). (bottom right) Tuning curve for the late time window (75–100ms). Simultaneous photostimulation (100ms) decreased the tone-evoked firing rates of Inh-act neurons in the early time window, whereas it increased the firing rates in the late time window. This pattern resembled the inhibition-activation sequence elicited by the 1ms light pulses (a).

Extended Data Figure 4. Light intensity-dependent changes in spike probability, delay and jitter in VIP interneuronsin vitro

a, Example traces of action potentials evoked by different light intensities. Blue bar, light stimulation. Scale bar, 10ms, 10mV. b, c, d Quantification. Spike probability (b) increased while delay (c) and jitter (d) decreased with increasing light intensities. The highest two intensities were used in the in vitro experiments.

Extended Data Figure 5. Photostimulation of VIP increases c-Fos in pyramidal neurons in mPFC

a, Schematic of c-Fos experiment. Animals were anesthetized for 2 hours to reduce the background c-Fos levels and photostimulation was applied. The expression level of c-Fos was captured 1 hour after photostimulation. b, Representative images of different experimental conditions. CTRL1, no ChR2 expression with photostimulation; CTRL2, ChR2 expression without photostimulation; EXP, ChR2 expression with photostimulation. (left column) Green, expression of ChR2.YFP; (middle) white, c-Fos staining; (right) merged images. Scale bar, 200 μm. c. Quantification of c-Fos levels. CTRL1: n=64 c-Fos immunopositive neurons, n=4 mice, CTRL2: n=58 neurons, n=4 mice, EXP: n=252 neurons, n=4 mice. d, e, f Representative images from different experimental conditions. White, c-Fos staining; green, ChR2-expression; red, CaMKIIα-staining. The arrow indicates a c-Fos-immunopositive VIP neuron. Scale bar, 50 μm. Note that some overlapping signals (c-Fos and CaMKIIα) are hard to appreciate in this image due to low resolution and uneven immunostaining. Additionally, due to the different signal strength, CaMKIIα immunopositivity is hard to appreciate for neurons that are slightly above or below the focal plane, while the strong c-Fos immunoreactivity is still detectable. For this reason, additional high power images were presented in (i). g, Colocalization of markers with c-Fos. h, Fraction of c-Fos positive cells. Among CaMKIIα positive neurons, the proportion of c-Fos-immunopositive cells was significantly higher in the experimental group as compared with the controls. CTRL1: fraction=11/208 (c-Fos/CaMKIIα), 4 mice; CTRL2: fraction=3/107, 3 mice; EXP: fraction=66/257, 4 mice. Approximately 64% (7/11) of the ChR2-expressing neurons were c-Fos-immunopositive. i, High resolution images of the co-localization between c-Fos and CaMKIIα in (f). Due to low resolution and uneven staining of CaMKIIα, some c-Fos signals seemingly do not co-localized with CaMKIIα+ neurons. However, in high resolution images, the co-localization is clearer. (top) Example of a weakly stained CaMKIIα+ neuron (arrow). In the high resolution image, CaMKIIα-staining is apparent. (bottom) Attributed to differences in immunofluorescence strength between c-Fos and CaMKIIα, neurons slightly out of focus may appear c-Fos-positive and CaMKIIα-negative. However, when the focal plane was adjusted, the co-localization became apparent. Blue, c-Fos; green, ChR2-YFP; red, CaMKIIα.

Extended Data Figure 6. Responses of VIP neurons in ACx during auditory stimulation, alone or combined with photostimulation

a, Raster plot (left) and PSTH (right) aligned to the onset of combined tone and photostimulation. Although all VIP neurons were responsive to photostimulation, individual VIP neurons showed heterogeneous response profiles. One VIP neuron (top) showed accommodation during the 200ms stimulation; two neurons (second and fourth) showed transient response; one neuron (third) fired persistently throughout the stimulation. Shaded boxes (left) or colored lines (right) indicate the stimulation duration. b, Average frequency tuning curve of VIP neurons (n=4). All 4 VIP neurons in the ACx for which tuning curves were recorded were responsive to pure tones; however, their tuning properties showed considerable heterogeneity.

Extended Data Figure 7. Auditory response profiles of different neuronal groups

a, b Single cell examples (top, raster plot; middle, PSTH) and population average (bottom, PSTH) of responses evoked by a brief 1ms light pulse. a, Delayed activated and tone-responsive neurons. b, Delayed activated and tone-unresponsive neurons. c, d Single cell examples (top, raster plot; middle, PSTH) and population average (bottom, PSTH) of responses evoked by combined auditory and light stimulation (100ms). c, Delayed activated and tone-responsive neurons. d, Delayed activated and tone-unresponsive neurons. Gray shaded box, tone stimulation; blue shaded box, tone+light stimulation. e, PSTH of tone-responsive (red) and tone-unresponsive (brown) delayed activated neurons for 100ms light pulses (without auditory stimulation). This experiment was performed in a subset of the frequency tuning experiments. f, Frequency tuning curve of tone-responsive neurons not modulated by 1ms light pulses. g, Fitting of one-parameter gain control models on tuning curve modulation of inhibited and delayed activated neurons. Tuning curves recorded during photostimulation were fitted with one-parameter models representing the scaled (multiplicative model) or shifted (additive model) versions of the baseline tuning curves (i.e., without photostimulation). The ratio of the least squared errors of the two model fits are plotted as a function of relative firing rate change after 1 ms light pulses on a logarithmic scale, for inhibited (purple) and delayed activated (brown) neurons (minus infinity corresponds to complete abolishment of firing). An error ratio >1 corresponds to a better fit of the additive model, whereas <1 means better fit of the multiplicative gain model. See also Methods.

Extended Data Figure 8. VIP neurons strongly respond to punishment in ACx

a, Raster plots (top) and PETHs (bottom) aligned to reinforcement (reward, green or punishment, red) for all VIP neurons recorded in the auditory go-nogo task. All VIP neurons were strongly recruited by punishment (foot shock: 1–4, marked by asterisks; air puff: 5–10), whereas water reward induced somewhat weaker and more sustained activation (9/10 cells showed significant firing rate increase for reward, see Results and Methods). b, Raster plots and PETHs of example unidentified neurons. Type 1 neurons (left) tended to be activated by tone onset. Type 2 neurons (right) tended to be inhibited by tone onset. c, (top) Normalized average PETH of VIP (green) and concurrently recorded non-VIP neurons aligned to feedback (left) and tone onset (right) for hit (top) and false alarm trials (bottom). VIP neurons showed an abrupt increase of firing rate after punishment (bottom left). The oscillatory pattern of VIP activation around reward delivery is a consequence of rhythmic firing rate modulations following the highly stereotypic pattern of licking in 4/10 VIP neurons (1/130 in non-VIP neurons). Gray, feedback-activated unidentified neurons (type 1). Pink, feedback-inhibited unidentified neurons (type 2). Insets, zoomed-in plots of PETHs. Arrows indicate the difference in activation pattern between VIP and unidentified type 1 neurons.

Extended Data Figure 9. Accounting for the temporal difference between VIP-Cre::Ai32 (ACx) and VIP-Cre::AAV.ChR2 (mPFC)

We observed a temporal difference in the firing pattern of VIP neurons between the ChR2 reporter line (Ai32) and the virus injected (AAV.ChR2) mice. We speculated that this difference could stem from the mutation in ChR2. The mutation in ChR2(H134R) of Ai32 mice produces larger currents and slower kinetics than ChR2(ChETA; AAV.ChR2). As a consequence, VIP neurons in VIP-Cre::Ai32 can fire bursts in response to single 1ms pulses and the activation can last more than 20ms (a). This sustained activity of VIP neurons prolonged the temporal dynamics of downstream neurons. a, Examples of VIP neurons that burst to 1ms photostimulation in the ACx of VIP-Cre::Ai32 mice. b, Model explaining the temporal difference between VIP-Cre::Ai32 and VIP-Cre::AAV-DIO-ChR2(ChETA). In VIP-Cre::Ai32 mice, almost all VIP neurons express ChR2 and exert stronger inhibition on the inhibited neuron group. Because the duration of VIP (green) activation varies, individual inhibited neurons (Inh, purple) receive different degrees of inhibition (strength and duration) from VIP neurons, therefore their firing rates recover to baseline at different time points. This variation propagates to the delayed activated group (dAct, orange), of which activation can start at different time instances.