Fig 2: Supportive evidence for anatomical and functional segregation of the two MD cell types.a-c, Left: Starter neurons (arrowheads) in the PL of VIP-cre (a), PV-cre (b) and SST-cre (c) mice for monosynaptic retrograde tracing using rabies viruses. Right: Starter neurons identified by co-expression of TVA fused to GFP (top) and blue fluorescent protein from rabies viruses (bottom) in VIP, PV, and SST neurons, respectively. Scale bars in µm: 200 µm(left), 30 µm (right). d, Left: Representative images of MD neurons that monosynaptically target PL SST+ interneurons. Scale Bar in µm: 200. (Note a lack of preferential localization within the MDl) Right: 3D plot of the anatomical location of SST-projecting MDl neurons (n = 86 SST projecting neurons from 3 mice). e, Anatomical separation between SST-projecting MD neurons and VIP-/PV- projecting MD neurons quantified as high misclassification using KNN clustering. f, MDD2 and MDGRIK4 neuronal locations show low misclassification compared to VIP- and PV-projecting MD neurons respectively. g, Example of a PL neuron showing amplification of evoked responses through concurrent intra-PL and MDD2 optical stimulation (left), but not when intra-PL stimulation is combined with MDGRIK4 stimulation (right). h, Examples of excitatory (RS) and inhibitory (FS) PL neurons showing, respectively, suppression and increase in spike rates with optical activation of MDGRIK4 neurons but not with activation of MDD2 neurons. i, Parametric activation of MDGRIK4, but not MDD2, neurons increase spike rates of PL inhibitory neurons (n = 68 and n = 78 PL-FS neurons from 3 animals each of MDD2 and MDGRIK4 respectively; p = 0.874, For MDGRIK4 p = 0.556, *p = 0.0387, ***p = 9.36 x 10-6, *p = 0.0387 respectively for laser powers 0.65, 1.3, 3.5 and 7.0 mW/mm2; Mann-Whitney U test compared to baseline). j, left: D2 specific promoter (D2SP) driven expression of mCherry + (CreON) and co-expression of EYFP (CreOFF) in Cre-negative neurons using a Cre - Out intersectional strategy labels two populations similar to D2-cre and Grik4-cre, but in WT animal. Right: Magnified images showing mCherry (D2SP+) and eYFP (Cre negative) neurons. Scale bar = 200 µm k, Consistent anatomical similarity between MDD2SP and MDD2 populations and a corresponding segregation between MDD2SP and MDGRIK4 neurons, quantified using representational similarity analysis (n = 95 cells from 2 animals for MDD2SP). l, A comparable similarity and segregation as shown in (k) is found when comparing MDD2SP neurons to VIP-projecting and PV projecting neurons. m, top row: MDD2 Cre-expressing neurons (MDD2+) labelled with GFP have extremely sparse Grik4 protein expression (IHC) compared to MDD2 Cre-negative (MDD2- neurons (middle row) or MDGRIK4 expressing neurons (bottom row). Scale bar = 3 µm n, Quantification of data (n = 116 MDD2+, 106 MDD2- and 124 MDGRIK4 neurons from 2 animals, demonstrating substantial Grik4 immunolabelling overlap between D2- and Grik4+ neural populations (not significantly different), but both being different from the D2+ population. ***p = 0.0001 for both comparisons, Kruskal Wallis test). o, Direct comparison of Grik4 immunolabelling across D2+ and D2- neurons (thresholded by the lowest 10th percentile of this analysis puts an upper bound estimate of 15% overlap between the D2+ and Grik4+ population. ‘positive control’ Grik4+ neurons). All statistical tests are two-tailed. For box plot n boundaries, 25–75th percentiles; midline, median; whiskers, minimum–maximum. Data are presented as mean ± SEM for iSource data.

Fig 3: Two MD circuits for amplification and suppression of PFC activity.a, Prefrontal PV+ and VIP+ input mapping. b, MD neurons targeting VIP+ (left) and PV+ (right) interneurons occupy distinct MDl domains. Scale bars, 200 µm. c, Group summary for location of VIP- and PV-projecting MDl neurons (n = 73 VIP-projecting (7 mice) and n = 117 PV-projecting (4 mice)). d, KNN clustering and representational similarity analysis show robust separation. e, Labelling MDD2 and MDGRIK4 neurons using the corresponding Cre lines. f, MDD2 and MDGRIK4 neurons also occupy distinct anatomical locations. Scale bars, 200 μm. g, Group summary of MDD2 and MDGRIK4 neurons in MDl (n = 177 MDD2 neurons and 194 MDGRIK4 neurons from 3 mice each). h, MDD2 and MDGRIK4 locations show additional high representational similarity to VIP- and PV-projecting neurons, respectively. i, j, mGRASP labelling shows higher innervation of VIP+ neurons by MDD2 (i; n = 21 neurons from 3 D2-cre, n = 25 neurons from 3 GRIK4-cre mice, respectively; Kolmogorov–Smirnov) and higher innervation of PV+ neurons by MDGRIK4 (j; n = 27 neurons from 3 D2-cre, n = 32 neurons from 3 GRIK4-cre mice, respectively; Kolmogorov–Smirnov). Scale bars, 3 µm. k, Hypothesized circuit. l, Selective MD cell-type activation set-up. m, MDD2 but not MDGRIK4 amplify functional PL connectivity (n = 100 and n = 68 PL neural responses from 3 mice each for MDD2 and MDGRIK4, respectively; left to right: MDD2 P = 1.0 × 10−5 for all; MDGRIK4 P = 0.0599, 0.0789, 0.0575, 0.1311 (NS) for laser powers displayed; Mann-Whitney U, compared to baseline). n, MDGRIK4 but not MDD2 suppress PL neural spike rates (n = 1,257 and n = 697 putative excitatory PL neurons from 3 mice each; MDD2 P = 0.184, 0.605, 0.579, 0.739 (NS); MDGRIK4 P = 0.298, P = 0.067, *P = 0.033, ***P = 1.61 × 10−5, respectively, for laser powers displayed; Mann-Whitney U compared to baseline). All statistical tests are two-tailed. Box plot parameters as in Fig. 1. Data are mean ± s.e.m. for m, n.Source data.

Fig 4: mGRASP and synaptophysin labelling provide evidence for output segregation of the two MD cell types.a, Cartoon depicting strategy to label cell type specific MD→PL thalamocortical synapses using mGRASP. The pre mGRASP component is virally expressed in MDD2 or MDGRIK4 neurons in the respective Cre lines while the post mGRASP component is ubiquitously expressed in the PL. MDD2 or MDGRIK4 specific mGRASP synapses onto VIP vs PV neurons in the PL are identified by immunohistochemistry guided detection of PV and VIP neurons expressing post mGRASP in the PL. b–c, Left: Representative images of MDD2 (b) and MDGRIK4 (c) neurons expressing pre mGRASP in the MD of D2-cre and GRIK4-cre mice respectively. Right: Ubiquitous expression of post mGRASP+ neurons detected by TdTomato fluorescence in the PL of D2-cre (b) and GRIK4-cre (c) mice. Scale bar in µm: 200. d, Left to right: Examples of PL VIP+ neurons showing post mGRASP expression (magenta), VIP expression detected via immunohistochemistry (yellow) and mGRASP+ synapses from MDD2 (cyan dots, top row) or MDGRIK4 (cyan, dots, bottom row) neurons. e, Same as in d, for PL PV+ neurons. Scale bars in µm: 3 µm. f, Representative images showing layer-wise termination of synapses from MDD2 (left) and MDGRIK4 (right) neurons in the PL, labelled with virally expressed GFP fused to synaptic protein (synaptophysin). Scale bar in µm: 100. g, MDD2 neurons terminate in L1 of the PL with a higher frequency compared to MDGRIK4 neurons (n = 12 sections each from 3 D2-cre and 3 GRIK4-cre mice, *p = 0.0253, *p = 0.039, two-tailed Mann-Whitney U test comparing 50 µm bins from the pial surface across groups). All statistical tests are two-tailed. Data are presented as mean ± SEM for gSource data.

Fig 5: Untagged neurons in the tagging experiments are no different than generic recordings, and optical inhibition of terminals of the two cell types replicates cell body inactivation.a, (Top Left) Schematic of optogenetic tagging and identification of MDD2 and MDGRIK4 neurons. MDD2 or MDGRIK4 neurons are tagged with NpHR3.0 and identified via light activated spike rate suppression. (Bottom) Example tagged neuronal response to NpHR3.0 activation. (Right) Tagged neurons from one mouse (red) are identified using k-means clustering (features: change in firing rate, proportion of trials suppressed, and half-time to recover from suppression (n = 262 total number of neurons). b, Relative fraction of all MD neurons from GRIK4-cre mice that are conflict-preferring vs. non-preferring are comparable to that of wild-type animals (Fig. 3g) (n = 91 neurons from 3 mice; p = 0.429 (NS), chi-squared test). Note that tagged MDGRIK4 neurons are significantly more conflict-preferring compared to the whole population (Fig. 4b) (p = 0.0175; chi-squared test). c, Relative fraction of all MD neurons from D2-cre mice that are conflict-preferring vs. non-preferring, are also comparable to that of wild-type animals (Fig. 3g) (n = 95 neurons from 3 mice; p = 0.166 (NS), chi-squared test). Note, that tagged MDD2 neurons are significantly more conflict-non-preferring (Fig. 4d) (p=1.34 x 10-4; chi-squared test). d, Optical inhibition of MDGRIK4 terminals in the PL recapitulates the loss in task accuracy across low and high conflict trials as seen with optical MDGRIK4 inactivation (Fig. 4e; n = 20 sessions over 4 GRIK4-cre mice, *p = 0.0199, ***p = 0.0002; Mann-Whitney U test). e, Optical inhibition of MDD2 terminals in the PL enhances performance accuracy on trials with high cueing conflict, similar to the effect of optical MDD2 inactivation (Fig. 4f; n = 20 sessions over 4 D2-cre mice, p = 0.3941 (NS), **p = 0.0023; Mann-Whitney U test. f, Schematic of micro-drive bottom piece and the 3x3 grid organization of the tetrode array for MD recordings. g, Summary of the density of tagged neurons on the medial-lateral axis separated by animal. We show the result for 2 Grik4-cre (top and bottom) and 2 D2-cre animals ((top and bottom rows) that have enough numbers of tagged neurons. All statistical tests are two-tailed. For box plots b – e boundaries, 25–75th percentiles; midline, median; whiskers, minimum–maximumSource data.