, = 11 mice (DG/ChR2/light) versus n = 11 (DG/ChR2/no light) in f (**P = 0.0013, F 987 (20, 100) = 2.882), n = 11 (DG/eNpHR/light) versus n = 11 (DG/eNpHR/no light) in g (****P <, EYFP controls in Fig. 4b-e. Two-way ANOVA with Bonferroni post 986 hoc test

, Extended Data Figure 10 | Synaptic transmission from SuM terminals to DG granule cells 993 (GCs) and CA2 pyramidal neurons (PNs). a, Identification of direct glutamatergic 994 transmission in CA2 from SuM inputs. (left) Diagram illustrating the whole-cell recordings of 995 hippocampal pyramidal neurons (PNs) and SuM fiber stimulation in acute slice preparation

, Cesium-based intracellular solution was used in the recording pipette and all cells were recorded 997 in voltage-clamp mode with a membrane potential held at -70 mV in order to record excitatory 998 glutamatergic transmission. (right) Diagram mapping the location of all PNs recorded in the 999 hippocampus with closed circles indicating connected cells and open circles indicating 1000 unconnected cells with no detected transmission. Cells were classified as CA1 (n = 11

, unconnected) based on morphology (the presence of thorny excrecsences for CA3) and location

, Circle color indicates cell type with CA2 PNs black, CA3 PNs brown and CA1 PNs orange, p.1004

, Diagram 1005 illustrating the local circuitry involved in the feed-forward inhibition recruited by SuM inputs in 1006 the hippocampus. The same recording configuration was used as shown in a, except that cells 1007 were held at +10 mV in order to measure feed-forward inhibitory transmission, CA2 from SuM inputs. (left), p.1008

, No 1010 CA1 PNs received feed-forward inhibition (n = 11) while over half of CA2 PNs received 1011 detectable feed-forward inhibition (n = 30 connected, n = 18 unconnected) as well as a fraction 1012 of CA3 PNs (n = 20 connected, n = 18 unconnected). c, Identification of excitatory and 1013 inhibitory transmission to DG granule cells (GCs) from SuM inputs. (left) Diagram illustrating 1014 the experimental setup to measure local excitatory and inhibitory circuitry recruited by SuM 1015 inputs in the DG. The same recording configuration was used as shown in a and b. Cells were 1016 held at -70 mV to measure excitatory transmission and then at +10 mV. Inhibitory transmission 1017 was measured before and after the addition of 10 µM NBQX & 50 µM APV to distinguish feed-1018 forward and direct inhibitory transmission. (right) Diagram mapping GCs in the DG that receive 1019 excitatory and inhibitory (both feed-forward and direct) transmission from light-evoked SuM 1020 inputs. Blue filled circles mark the location of cells that received both excitatory and inhibitory 1021 transmission (n = 21). All cells received both direct and feed-forward inhibition. Yellow-filled 1022 circles mark cells that did not receive detectable excitatory transmission, but both direct and 1023 feed-forward inhibitory transmission (n = 3). Data for panels a.-c. were collected from 156 1024 slices from 92 mice. Abbreviations: so, stratum oriens; sp, stratum pyramidale; sr, stratum 1025 radiatum; mf, mossy fiber; ml, molecular layer; gcl, granule cell layer. d, Inverted epifluorescent 1026 image of a hippocampal slice with a DG GC and a CA2 PN, mapping PNs in the hippocampus that receive feed-forward transmission from light-evoked SuM 1009 inputs with unconnected cells shown as open circles and connected cells as filled circles

, Diagram illustrating the local circuitry and whole-cell recording configuration of DG GCs in 1030 acute brain slices prepared from SuM-Cre mice injected with AAV-DIO-ChR2-EYFP. SuM 1031 axonal terminals were illuminated by a 0.5 ms 488 nm light stimulation, p.1032

, Sample traces of light-evoked EPSCs recorded at ?70 mV (individual traces in grey, average 1033 trace in black) and IPSCs recorded at +10 mV, p.1034

, The blue line denotes when the light stimulus was 1035 applied. h, Normalized cumulative distribution of latencies for DG GC EPSCs (black) and IPSCs 1036 (red), both displaying response latencies consistent with direct monosynaptic transmission

, Kolmogorov-Smirnov test, P = 0.97). i, j, Sample traces (i) and time course of amplitudes (j, p.1039

, IPSCs only, n = 22 GCs from 8 mice) of light-evoked EPSCs (black) and IPSCs (red) recorded 1040 in DG GCs before and after application of 10 µM NBQX & 50 µM APV (grey), and further 1041 application of 1 µM SR95531 & 2 µM CGP55845A (green). AMPA and NMDA receptor 1042 blockers completely, p.1044

, indicating that there is feed-forward 1045 inhibition recruited by SuM inputs, accompanied by a larger amount of direct inhibitory 1046 transmission. The remaining light-evoked IPSCs were entirely blocked by the subsequent 1047 addition of GABAA and GABAB receptor blockers, suggesting that DG-projecting neurons in 1048 the SuM are capable of simultaneously releasing both glutamate and GABA. k, Diagram similar 1049 to f, showing the local circuitry and whole-cell recording configuration of CA2 PNs. l, Sample 1050 traces of EPSCs recorded at ?70 mV, p.1051

, IPSCs recorded at +10 mV (individual traces in light red, average trace in red) in a same CA2 1052 PN under voltage clamp. Note the increased latency of IPSCs onset compared to EPSCs, p.1053

, Normalized cumulative distribution of latencies for CA2 PN EPSCs (black) and IPSCs (red)

, IPSCs displayed significantly different response latencies from the EPSCs, with a longer IPSC 1055 response latency consistent with bi-synaptic feed-forward inhibition (2.9 ± 0.1 ms for EPSCs

. ****p-&lt;-0, , vol.0001

*. Kolmogorov-smirnov-test and . ***p-&lt;-0, 0001, data from 92 mice). n, o, Sample traces (n) and 1058 time course of amplitudes (o, IPSCs only, p.1059

, Both the EPSCs (16 ± 4.8 pA, n = 6 PNs from 4 mice) and IPSCs (167 ± 40 pA, IPSCs (red) recorded in CA2 PNs before and after application of 10 µM NBQX & 50 µM APV 1060 (grey)

, PNs from 4 mice) were completely blocked by the application of AMPA and NMDA receptor 1062 blockers, indicating that the synaptic transmission from the SuM is entirely glutamatergic in 1063

. Ca2 and . Notably, Confirmation of SuM-CA2 transmission by the addition of tetrodotoxin (TTX) and 1065 4-aminopyridine (4-AP) to isolate transmitter release resulting from direct optical terminal 1066 depolarization. Application of TTX & 4-AP abolishes IPSCs and spares EPSCs in CA2 1067 pyramidal neurons (PNs), consistent with mono-synaptic excitation and di-synaptic inhibition, both SuM-DG and SuM-CA2 transmissions recruit a robust feed-forward 1064 inhibition. p

, Time course of light-evoked EPSC and IPSC 1070 amplitudes upon application of 0.2 µM TTX & 100 µM 4-AP in which a longer light stimulus 1071 was used to directly depolarize the SuM axonal terminal. Initial amplitudes were 20.2 ± 6.1 pA 1072 for EPSCs and 110 ± 50 pA for IPSCs, Control sample traces are shown in black (EPSC) and red (IPSC), while traces following 1069 application of TTX & 4-AP are shown in grey. q, vol.9

, Extended Data Figure 11 | Retrograde tracing of upstream inputs to the DG-and CA2-1078 projecting SuM neurons. a, b, Strategies of projection-specific retrograde tracing to map the 1079 upstream inputs to the DG-(a) and CA2-projecting (b) neurons. Retrograde AAV helper viruses 1080 expressing the rabies G protein and the TVA receptor are injected into DG (a) or into CA2 (b), 1081 and three weeks later EnvA pseudotyped rabies virus expressing mCherry is, p.1082

. Sum, This allows for the retrograde labeling of upstream neurons that send efferent axons to 1083

, DG-or CA2-projecting SuM cells by the rabies virus. c, d, Coronal sections showing DG-(c), p.1084

, Cells in red (mCherry) are TVA+ cells that 1085 express rabies virus (one example of four mice for DG injections, one example of five mice for 1086 CA2 injections). Cells in green (GFP) express oG, and CA2-projecting (d) starter cells in the SuM

, mCherry+/GFP+ overlapping cells (yellow) are starter cells where the trans-complementation of 1088

, Scale bar, 1 mm. e, f, Coronal sections with trans-synaptically labeled input cells upstream 1090 to the DG-(e) and CA2-projecting (f) SuM neurons. Scale bars, 1 mm. g, h, Quantification of 1091 inputs from various brain regions to the DG-(g, n = 4 mice) and CA2-projecting, RVdG with oG results in the production of oG+RVdG that spreads trans-synaptically to input 1089 cells, p.1092

, Both populations received extensive inputs from subcortical regions including the 1093 hypothalamus, brainstem, septum and nucleus accumbens. However, the inputs to the DG-1094 projecting population were comparatively biased to brain regions in the reward and motor 1095 systems, such as the VTA, SI, AcbSh, LS and MS, while the CA2 projectors received 1096 proportionally greater inputs from neurons in socially engaged regions

, Mann-Whitney U test) and MPO. All error bars show mean ± s.e.m. LS, lateral septal 1098 nucleus

, MS, medial septal nucleus

S. Si and . Innominata,

, AcbSh, accumbens nucleus, shell

Z. Zi and . Incerta, LHA, lateral hypothalamic area

. Mpo,

, LPO, lateral 1100 preoptic area; PVH, paraventricular hypothalamic nucleus; PH, posterior hypothalamic nucleus

. Pag and . Mrn,

, Raphe: DR, dorsal raphe nucleus & MnR, median raphe nucleus