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Translaminar recurrence from layer 5 suppresses superficial cortical layers

Information flow in the sensory cortex has been described as a predominantly feedforward sequence with deep layers as the output structure. Although recurrent excitatory projections from layer 5 (L5) to superficial L2/3 have been identified by anatomical and physiological studies, their functional impact on sensory processing remains unclear. Here, we use layer-selective optogenetic manipulations in the primary auditory cortex to demonstrate that feedback inputs from L5 suppress the activity of superficial layers regardless of the arousal level, contrary to the prediction from their excitatory connectivity. This suppressive effect is predominantly mediated by translaminar circuitry through intratelencephalic neurons, with an additional contribution of subcortical projections by pyramidal tract neurons. Furthermore, L5 activation sharpened tone-evoked responses of superficial layers in both frequency and time domains, indicating its impact on cortical spectro-temporal integration. Together, our findings establish a translaminar inhibitory recurrence from deep layers that sharpens feature selectivity in superficial cortical layers.


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Information flow in the sensory cortex has been described as a predominantly feedforward 23 sequence with deep layers as the output structure. Although recurrent excitatory projections from 24 layer 5 (L5) to superficial L2/3 have been identified by anatomical and physiological studies, 25 their functional impact on sensory processing remains unclear. Here, we use layer-selective 26 optogenetic manipulations in the primary auditory cortex to demonstrate that feedback inputs 27 from L5 suppress the activity of superficial layers, contrary to the prediction from their 28 excitatory connectivity. This suppressive effect is predominantly mediated by translaminar Revealing the principles governing interactions between the six layers of the cortex is 37 fundamental for understanding how these intricately woven circuits work together to process 38 sensory information. In the canonical cortical circuit model, information flow in the cortex has 39 been considered as a predominantly feedforward sequence along the L4→L2/3→L5/6 axis, from 40 which information is sent out to subcortical structures 1 . However, the cortex is not simply a 41 feedforward circuit but is full of recurrence 2,3 . In recurrent circuits, the activity of even a single 42 neuron can be transmitted back to itself, resulting in non-linear changes in the entire network. including the thalamus. Importantly, IT neurons, and a subset of PT neurons, send their axons to 55 superficial layers within the cortical column in addition to their distal targets 19 , providing a 56 potential substrate for translaminar recurrence between L2/3 and L5. 57 Indeed, this excitatory feedback projection from L5 pyramidal cells to L2/3 neurons has 58 6 variable (91%, 89%, and 63% of units showed suppression in L2/3, L4, and L6; L2/3: p = 105 2.4×10 −11 ; L4: p = 5.4×10 −10 ; L6: p = 6.3×10 −5 , Wilcoxon signed rank test; Fig. 1h,i). We 106 observed only a small fraction of activated units, which mostly resided in L6 (6%, 5%, 27% of 107 units in L2/3, L4, and L6). Notably, the suppression was stronger in L2/3 than L4 (p = 0.016, 108 Wilcoxon rank sum test), suggesting that L2/3 does not simply inherit its suppression from L4. 109 Thus, these data show that stimulation of L5 excitatory neurons suppresses the activity in the 110 superficial layers of A1.

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A potential concern of photoactivation results is that the artificial L5 activity patterns  Fig. 1l,m). Again, the enhancement of activity was more pronounced in L2/3 than L4 (p = 0.018,

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Wilcoxon rank sum test), suggesting a site of suppression within L2/3. Collectively, our 123 bidirectional manipulations demonstrate that both photoevoked and endogenous L5 activities 124 have suppressive effects on superficial cortical layers in A1 of awake mice.

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Our finding of L5-mediated negative regulation onto superficial layers contrasts with a 126 previous study that reported L5 activation in anesthetized mice triggered a transition to cortical 127 7 up state and increased L2/3 activity 31 . We wondered if this discrepancy could be due to different 128 cortical circuit operations across brain states in the recorded animals. To test the robustness of 129 our results across brain states, we examined if the arousal level of awake mice influenced the 130 suppressive effects of L5 onto other layers. Throughout the A1 unit recordings and L5 131 optogenetic manipulations, we monitored pupil diameter, a reliable readout for the animal's 132 arousal level 36,37 (Fig. 2a). We used moderate LED intensities to prevent activity from reaching 133 the floor or the ceiling. As reported previously 36 , L2/3 RS multi-unit spontaneous firing 134 exhibited a U-shaped relationship with pupil size in the control condition (Fig. 2b) 140 We next directly compared the effects of L5 optogenetic manipulations before and after 141 inducing urethane anesthesia within the same mice (Fig. 2f). Similar to our results in the awake 142 state, calculation of MI showed that L5 activation under anesthesia strongly suppressed 143 spontaneous firing in the superficial layers (96%, 80%, and 58% of single-units showed  the awake and anesthetized states (L2/3-L4: p = 0.91; L5: p = 0.55; Fig. 2l). We also confirmed 151 that the use of constant-intensity LED without a ramp, similar to the one used in the previous 152 study, or the use of weaker LED intensities did not affect our conclusions ( Supplementary Fig.   153 1). Taken together, L5 activity suppresses superficial cortical layers regardless of brain states, 154 indicating the continuous sparsening of L2/3 neural activity by negative feedback from L5. To address this question, we determined whether L5 activation alters the frequency tuning of A1 159 neurons across the depth of the cortical column. We generated tuning curves of RS single-units 160 by presenting pure tones across a range of frequencies (4-64 kHz, 70 dB SPL, 0.2 s), alternating 161 control trials with L5 photostimulation trials (Fig. 3a). On top of the suppression of spontaneous 162 firing, L5 activation attenuated tone-evoked activity in the superficial layers (Fig. 3a,b). 163 Calculation of MI for tone-evoked activity showed that most RS single-units in L2/3 and L4 164 decreased their response amplitudes, while the effect on L6 was variable (78%, 67%, and 46% of 165 unit-frequency pairs showed suppression in L2/3, L4, and L6; L2/3: p = 3.4×10 −9 ; L4: p = 166 4.9×10 −6 ; L6: p = 0.12, Wilcoxon signed rank test; Fig. 3c). Similar to the impact on spontaneous 167 activity, the suppression of tone responses was more robust in L2/3 than L4 (p = 0.035, 168 Wilcoxon rank sum test). Comparison of the best frequency (BF) between control and LED trials 169 showed that L5 photostimulation did not affect preferred frequency in other layers (Fig. 3d). 170 However, the overall suppression of tone-evoked activity narrowed the frequency tuning    Intracortical and subcortical mechanisms of L5-dependent inhibitory recurrence should 242 suppress the superficial layers with distinct kinetics. To test this prediction, we combined multi-243 unit spikes during L5 optogenetic activation across mice and compared the suppression kinetics 244 between A1 L2/3, L4, and MGv. Note that we excluded L2/3-L4 units with increased spikes to 245 clarify the suppression onset; however, the incomplete removal of activated units likely caused 246 an overestimation of suppression latency in these layers. Despite this overestimation, we found 247 that the suppression onset for L2/3 was earlier than L4 and comparable to MGv (9.0, 18.5, and 248 9.1 ms latency in L2/3, L4, and MGv; Fig. 5h), indicating that MGv suppression was not fast 249 enough to account for the short-latency suppression in L2/3. Indeed, the L2/3 spike rate was fit 250 the best by the sum of two exponential curves, which likely corresponds to fast intracortical and 251 slow subcortical mechanisms, while L4 and MGv data were fit better by a single exponential.  and IT neuron activation alone ( Fig. 3 and Supplementary Fig. 6). Therefore, intracortical 286 recurrence from IT neurons plays a crucial role in regulating both spontaneous and tone-evoked 287 14 activity of superficial layers. In contrast, subcortical projections from PT neurons suppress only 288 spontaneous firing and do not affect tone-evoked activity. 289 We next expressed eNpHR3.0 selectively in IT or PT neurons and conducted subtype-290 specific loss-of-function experiments (Fig. 6h,i). Surprisingly, although the magnitudes of L5  (Fig. 7a,b). Therefore, the suppressive effects of L5 cannot be simply explained by 310 increased activity of inhibitory neurons. However, focusing on spike changes at the LED onset, 311 we found many L2/3-L4 FS units that transiently increased their firing before switching to 312 suppression (Fig. 7c,d). Combining transient and sustained activation, we found a three times  prolongs sustained activity within the cortical column, which is contrary to our data. Although 389 the most obvious difference is their use of anesthesia, we found that the same anesthesia 390 condition (2 mg/kg urethane) did not affect our results and confirmed brain state-independent 391 suppression of superficial layers by L5 activation. One explanation is that, since the majority of 392 their data are LFP or multi-unit recordings with electrodes placed 450-700 μm deep, they may 393 reflect the measurement of optogenetically modulated L5 activity rather than global recurrent 394 activity (see Fig. 1b for distribution of Rbp4+ neurons across A1 cortical depth). Alternatively, 395 since the previous work was conducted in unspecified neocortical areas, potential differences in 396 the circuit wiring between cortical areas could account for the discrepancy. Indeed, previous 397 studies also found opposing roles for L6 corticothalamic neurons between A1 and V1;      The muscle overlying the right auditory cortex was removed, and a head-bar was secured on the 477 skull using dental cement. The brain surface was imaged through the skull kept transparent by across the cortical column, images were Z-projected with the maximum intensity, averaged 507 across the tangential axis, and normalized by the sum of intensity across depths. The cortical 508 depth was standardized with 0 for the brain surface and 1 for the white matter. Brains used for 509 unit recordings were also processed and sectioned in the same way, except they did not go  day 1-3).

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After the mice reached six weeks old, CAV-2.Cre (100 nL/site) was injected into three sites 533 within the external cortex of the right inferior colliculus, and recordings were conducted four 534 weeks after virus injections. In all experiments, black cement was used during head cap 535 implantation, and silicone was placed over the exposed skull to prevent light exposure of the 536 auditory cortex between virus injection and recording.

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On the day of recording, following a small craniotomy and durotomy in the right A1 538 identified by intrinsic signal imaging, mice were head-fixed in the awake state, and a 64-channel 539 silicon probe (ASSY-77-H3, sharpened, Cambridge Neurotech) was slowly (approximately 1 µm 540 per second) inserted perpendicular to the brain surface. Spikes were monitored during probe 541 insertion, and the probe was advanced until its tip reached the white matter, where no spikes 542 were observed. The reference electrode was placed at the dura above the visual cortex. The probe 543 was allowed to settle for at least 1 hour before collecting data. Unit activity was amplified,  Fig. 2b,c, 2 units in Fig. 2h,i, and 2 units in Fig. 5d,e) were 598 included in single-unit analyses but were excluded from multi-unit analyses to avoid their 599 dominance of the total spike count. For visualization of the laminar profile of L5 manipulation 600 effects, cortical depth was morphed into a normalized cortical column in each mouse such that 601 L1-3, L4, L5, and L6 are represented by 4, 4, 6, and 6 bins, respectively. For FS units, the bin 602 numbers were 2, 2, 3, and 3, respectively, to account for their sparsity. PSTHs were generated at 603 25 ms-bin, except for Fig. 5h, where 0.5 ms-bin was used, and Fig. 5d,f, Fig. 7c,g,   All data are presented as mean ± SEM or median with 25th and 75th percentiles, as stated in the 671 figure legend. Statistically significant differences between conditions were determined using 672 standard two-sided parametric or nonparametric tests in Matlab. Wilcoxon signed-rank test was 673 used for one-sample nonparametric tests. Paired t-test was used for paired data, and Wilcoxon 674 rank-sum test was used for independent group comparisons. Bonferroni correction was used for 675 multiple comparisons, and corrected p values are reported. In cases where parametric statistics 676 are reported, data distribution was assumed to be normal, but this was not formally tested.

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Randomization is not relevant for this study because there were no animal treatment groups. All

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The data that support the findings of this study are available from the corresponding authors 685 upon reasonable request. Source data are provided with this paper.