Extended Data Fig. 3: 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, MD_D2 and MD_GRIK4 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 MD_D2 optical stimulation (left), but not when intra-PL stimulation is combined with MD_GRIK4 stimulation (right). h, Examples of excitatory (RS) and inhibitory (FS) PL neurons showing, respectively, suppression and increase in spike rates with optical activation of MD_GRIK4 neurons but not with activation of MD_D2 neurons. i, Parametric activation of MD_GRIK4, but not MD_D2, neurons increase spike rates of PL inhibitory neurons (n = 68 and n = 78 PL-FS neurons from 3 animals each of MD_D2 and MD_GRIK4 respectively; p = 0.874, For MD_GRIK4 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 MD_D2SP and MD_D2 populations and a corresponding segregation between MD_D2SP and MD_GRIK4 neurons, quantified using representational similarity analysis (n = 95 cells from 2 animals for MD_D2SP). l, A comparable similarity and segregation as shown in (k) is found when comparing MD_D2SP neurons to VIP-projecting and PV projecting neurons. m, top row: MD_D2 Cre-expressing neurons (MD_D2+) labelled with GFP have extremely sparse Grik4 protein expression (IHC) compared to MD_D2 Cre-negative (MD_D2- neurons (middle row) or MD_GRIK4 expressing neurons (bottom row). Scale bar = 3 µm n, Quantification of data (n = 116 MD_D2+, 106 MD_D2- and 124 MD_GRIK4 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 i

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