Louis Richevaux
email: louis.richevaux@parisdescartes.fr

Dongkyun Lim

Mérie Nassar

Léa Dias

Constanze Mauthe

Ivan Cohen

Nathalie Sol-Foulon

Desdemona Fricker
email: desdemona.fricker@parisdescartes.fr

Vh = -65 Mv

Projection-specific integration of convergent thalamic and retrosplenial signals in the presubicular head direction cortex Authors

Keywords: postsubiculum, presubiculum, head direc on, mul sensory, dendri c integra

come

Introduction

The head direc on (HD) system func ons as the brain's compass system. It is distributed across several interconnected brain structures, and hierarchically organized from the brainstem to the lateral mammillary nucleus (LMN; [START_REF] Stackman | Firing proper es of rat lateral mammillary single units: head direc on, head pitch, and angular head velocity[END_REF], the anterior thalamic nuclei (ATN; [START_REF] Taube | Head direc on cells recorded in the anterior thalamic nuclei of freely moving rats[END_REF], and the dorsal Presubiculum (PrS; [START_REF] Ranck | Head direc on cells in the deep layer of dorsal presubiculum in freely moving rats[END_REF]Taube et al., 1990a). Modeling work suggests the HD system func ons as a ring a ractor, where the popula on of HD neurons is arranged in a one dimensional manifold [START_REF] Blair | An cipatory head direc on signals in anterior thalamus: evidence for a thalamocor cal circuit that integrates angular head mo on to compute head direc on[END_REF][START_REF] Mcnaughton | Path integra on and the neural basis of the "cogni ve map[END_REF][START_REF] Skaggs | A model of the neural basis of the rat's sense of direc on[END_REF]. The ac vity of thalamic and PrS HD neurons is correlated across different brain states, even during sleep, independently of sensory cues [START_REF] Peyrache | Internally organized mechanisms of the head direc on sense[END_REF]. The bump-like ac vity dynamics of an a ractor network can maintain HD signals based on excitatory-excitatory or excitatory-inhibitory interac ons [START_REF] Knierim | A ractor dynamics of spa ally correlated neural ac vity in the limbic system[END_REF][START_REF] Simonnet | Ac vity dependent feedback inhibi on may maintain head direc on signals in mouse presubiculum[END_REF]. However, HD signals may dri in the dark (Taube et al., 1990b;[START_REF] Zugaro | mm Bregma -0.82mm Bregma -0.70 mm Bregma -0.58mm Bregma -0.46 mm Bregma -0.34mm Bregma 0.26mm Bregma 0.86mm Bregma 1.18mm Bregma 1[END_REF]. For precise naviga on, internally generated informa on on head direc on must be combined with awareness of loca on in an environment. Mechanisms underlying the combina on of egocentric head direc on signals with anchoring to allocentric landmarks remain to be clarified.

Head direc on and visual landmark signals may be integrated in the presubiculum [START_REF] Jeffery | Op mal cue combina on and landmark-stability learning in the head direc on system[END_REF][START_REF] Yoder | Bilateral postsubiculum lesions impair visual and nonvisual homing performance in rats[END_REF]. Head direc on cells are found in the superficial and deep layers of dorsal presubiculum, also termed postsubiculum [START_REF] Boccara | Grid cells in pre-and parasubiculum[END_REF], and most layer 3 neurons in the dorsal presubiculum are head direc on cells [START_REF] Tukker | Head-Direc onal Tuning and Theta Modula on of Anatomically Iden fied Neurons in the Presubiculum[END_REF][START_REF] Preston-Ferrer | Anatomical organiza on of presubicular head-direc on circuits[END_REF]. These cells receive monosynap c head direc on inputs from the anterior thalamus [START_REF] Nassar | Anterior Thalamic Excita on and Feedforward Inhibi on of Presubicular Neurons Projec ng to Medial Entorhinal Cortex[END_REF][START_REF] Peyrache | Internally organized mechanisms of the head direc on sense[END_REF][START_REF] Balsamo | Modular microcircuit organiza on of the presubicular head-direc on map[END_REF]. Lesions of the PrS impair the visual landmark control of a cell's preferred direc on in ATN and in LMN [START_REF] Goodridge | Interac on between the postsubiculum and anterior thalamus in the genera on of head direc on cell ac vity[END_REF][START_REF] Yoder | Visual landmark informa on gains control of the head direc on signal at the lateral mammillary nuclei[END_REF]. Presubicular lesions also induce place field instability in the hippocampus [START_REF] Calton | Hippocampal place cell instability a er lesions of the head direc on cell network[END_REF], sugges ng this region may be crucial to the anchoring of direc onally modulated neurons to environmental landmarks.

Landmark based naviga on depends on reliable visual cues. The PrS appears to receive direct projec ons from the primary visual cortex and indirect visual input via the retrosplenial cortex [START_REF] Van Groen | Connec ons of the retrosplenial granular b cortex in the rat[END_REF][START_REF] Vogt | Cor cal connec ons between rat cingulate cortex and visual, motor, and postsubicular cor ces[END_REF]. The retrosplenial cortex encodes angular head velocity [START_REF] Alexander | Retrosplenial cortex maps the conjunc on of internal and external spaces[END_REF][START_REF] Keshavarzi | Mul sensory coding of angular head velocity in the retrosplenial cortex[END_REF]) and visual landmark informa on [START_REF] Auger | Retrosplenial Cortex Codes for Permanent Landmarks[END_REF][START_REF] Clark | Impaired Head Direc on Cell Representa on in the Anterodorsal Thalamus a er Lesions of the Retrosplenial Cortex[END_REF][START_REF] Sit | Coregistra on of heading to visual cues in retrosplenial cortex[END_REF]. In par cular, neurons in the dysgranular retrosplenial cortex encode landmark-dominated head-direc on signals [START_REF] Jacob | An independent, landmark-dominated head-direc on signal in dysgranular retrosplenial cortex[END_REF]. Presubicular layer 3 cells receiving projec ons from both the anterior thalamus and the retrosplenial cortex [START_REF] Kononenko | Presubiculum layer III conveys retrosplenial input to the medial entorhinal cortex[END_REF] could update the compass and bind landmarks to head direc on signals. Presubicular neurons could then broadcast integrated HD-landmark signals directly to the MEC, via deep layers to the ADN and via layer 4 neurons to the LMN [START_REF] Huang | Laminar Localiza on and Projec on-Specific Proper es of Presubicular Neurons Targe ng the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex[END_REF][START_REF] Yoder | Visual landmark informa on gains control of the head direc on signal at the lateral mammillary nuclei[END_REF][START_REF] Yoder | Projec ons to the anterodorsal thalamus and lateral mammillary nuclei arise from different cell popula ons within the postsubiculum: implica ons for the control of head direc on cells[END_REF]. The different target-specific presubicular projec on neurons are well posi oned to integrate anterior thalamic and retrosplenial inputs, but technical constraints have limited understanding of how known anatomical inputs are transformed into func onal integrated output signals.

antero-posterior axis.

Projec ons of the ATN to the presubiculum were examined in horizontal sec ons (Figure 1G). ATN axons expressing the Chronos-GFP construct targeted layers 1 and 3 of the presubiculum, avoiding layer 2 [START_REF] Simonnet | Ac vity dependent feedback inhibi on may maintain head direc on signals in mouse presubiculum[END_REF][START_REF] Nassar | Anterior Thalamic Excita on and Feedforward Inhibi on of Presubicular Neurons Projec ng to Medial Entorhinal Cortex[END_REF]. Labeling was precisely limited to the presubiculum with a sharp reduc on of green fluorescence at the posterior border with the parasubiculum, the anterior border with the subiculum and the lateral border with deep layers. Axon terminals in layer 3 were not homogeneously distributed. Patches of higher density were evident in deep layer 3 (Figure 1G). Along the dorso-ventral axis, ATN axons projected from -2 to -4 mm ventral to Bregma. Fluorescence was not detected in adjacent regions including the Sub, PaS, and DG. ATN axons avoided layer 2 of the PrS, including anterior regions with patches of calbindin posi ve neurons that project to contralateral PrS [START_REF] Preston-Ferrer | Anatomical organiza on of presubicular head-direc on circuits[END_REF]; Figure 1K).

RSC projec ons to the presubiculum (Figure 1H), traced with the fluorescent Chronos-GFP construct, also projected to layers 1 and 3. Unlike ATN axons, they were restricted to dorsal regions of the presubiculum (from -2 to -3 mm ventrally to Bregma on the dorso-ventral axis; Figure 1H,I,J). RSC axon labeling tended to avoid patches of highdensity ATN projec ons (Figure 1G, K. Figure 3). Overall, these data show ATN and RSC afferents innervate overlapping dorsal regions of the PrS, in layer 1 and deep parts of layer 3. Different spa al pa erns of fiber termina ons suggest dis nct local connec vi es.

Layer 3 neurons are directly excited by both ATN and RSC afferents

We next examined physiological effects of ATN and RSC afferents on PrS pyramidal cells [START_REF] Rees | Weighing the Evidence in Peters' Rule: Does Neuronal Morphology Predict Connec vity?[END_REF]. Responses to photo-s mula on of the Chronos-GFP construct injected either in the thalamus or in the retrosplenial cortex (Figure 2A) were recorded in layer 3 neurons (Figure 2B). These cells had robust regular firing pa erns in response to depolarizing current injec ons (Figure 2C). Intrinsic proper es of layer 3 cells were rather uniform (Figure 2-figure supplement 1) and an unsupervised cluster analysis suggested they formed a single popula on (Figure 2D).

Blue light ac va on of either ATN or RSC axons expressing Chronos-GFP reliably evoked synap c responses in whole-cell patch-clamp records from layer 3 cells. High intensity s mula on (1 -3 mW) evoked EPSCs or EPSPs with a probability of 84% (70/83) for ATN fibers and 85% (104/123) for RSC fibers (Figure 2E). Excitatory postsynap c currents or poten als evoked by op cal s mula on of thalamic and retrosplenial inputs will be referred to as oEPSCs and oEPSPs. Mean oEPSC amplitudes (Figure 2F) were similar for ATN (-305.0 ± 61.0 pA, n = 24) and RSC fiber s muli (-305.8 ± 59.1 pA, n = 27). Response latency (Figure 2G) to ATN axon s mula on was 2.45 ± 0.19 ms (n = 24) and that for RSC axon s mula on was 2.93 ± 0.23 ms (n = 27). The variability in latency of oEPSCs (Figure 2-figure supplement 2A, B) induced by RSC afferent s muli was 0.35 ± 0.09 ms (n = 27) and that for oEPSCs induced by ATN s muli was 0.19 ± 0.06 ms (n = 24). These short oEPSC latencies suggest monosynap c transmission. We confirmed this point by showing that oEPSCs were maintained in presence of TTX (1µM) and 4AP (100µM) (Figure 2H, n = 2 and 12 for ATN and RSC respec vely). EPSCs induced by s mula on of ATN and RSC fibers were reduced in amplitude and decayed more quickly in the presence of the NMDA receptor antagonist APV (50 µM) and were completely abolished by further applica on of the AMPA receptor antagonist NBQX (1 mM) (Figure 2I). The shape of oEPSCs induced by light s mula on of ATN and RSC fibers was comparable. Rise mes were 1.38 ± 0.15 ms for ATN EPSCs (n = 15) and 1.38 ± 0.08 ms for RSC-mediated EPSCs (n = 15) and mean half-widths were 4.00 ± 1.02 ms for ATN-induced ESPCs (n = 15) and 4.34 ± 1.18 ms for RSC EPSCs (n = 15). The mean me constant (tau decay) for ATN-mediated EPSCs was 3.01 ± 0.21 (n = 15) and for RSC-induced EPSCs 3.17 ± 0.27 (n = 15) (Figure 2-figure supplement 2C-E.).

Op cal s mula on of ATN and RSC fibers at lower light intensi es elicited subthreshold synap c events in layer 3 cells. oEPSP amplitude for ATN mediated events was 4.09 ± 1.18 mV (n = 11) and for RSC events it was 4.80 ± 0.84 mV (n = 11). Maximal rising slopes were 2.93 ± 0.82 mV/ms for ATN-mediated events (n = 11) and 3.02 ± 0.53 mV/ms for RSC EPSPs (n = 11), and decay me constants were 81.4 ± 11.6 ms for ATN EPSPs (n = 11) and 86.9 ± 11.2 ms for RSC EPSPs (n = 11). Rise mes were shorter for subthreshold EPSPs induced by ATN afferents at 3.00 ± 0.24 ms (n = 11) while those for RSC ini ated EPSPs were 3.59 ± 0.34 (n = 11, p = 0.042, Wilcoxon test; Figure 2-figure supplement 2F-J). These data show that ATN and RSC axons make monosynap c, glutamatergic excitatory contacts on layer 3 PrS cells.

The dynamics of responses to repe ve s mula on of layer 3 PrS cells were tested using 20 Hz trains of light s muli to ac vate either ATN or RSC afferents. oEPSCs for both inputs followed depressing kine cs. Amplitude decreased for both inputs (Figure 2J, K), significantly a er the fourth pulse (Friedman's test and Dunn's mul ple comparison test). The 10/1 ra o was significantly lower than the paired-pulse ra o (PPR) in both cases (ATN 10/1 0.56 ± 0.05 vs PPR 0.86 ± 0.05, n = 15, p < 0.0001, Wilcoxon test, RSC 10/1 0.64 ± 0.05 vs PPR 0.83 ± 0.03, n = 15, Wilcoxon test) (Fig 2L). ATN fiber s mula on induced PrS cell firing in current-clamp with a high probability for ini al s muli and lower stable probabili es for later s muli in the train. Similarly, 20 Hz s mula on of RSC afferents evoked spikes with high probability for ini al s muli and decreasing probabili es for later s muli (on fourth and 7 th -10 th pulses, Friedman's and Dunn's mul ple comparison tests; Figure 2M,N). Responses to repeated subthreshold s muli had similar dynamics (Figure 2O). S mula on with lower light intensi es (< 1 mW) to ini ate sub-threshold oEPSPs elicited different dynamic responses. ATN afferent trains elicited responses with depressing dynamics: the amplitude of the fi h oEPSP was significantly less than the first one (3.18 ± 0.88 vs 5.11 ± 1.64 mV, p = 0.0185, Friedman's and Dunn's tests). In contrast, subthreshold 20 Hz s mula on of RSC afferents evoked li le or no oEPSP depression (Figure 2-figure supplement 2K-M). These data reveal dis nct pa erns of integra on of repe ve ac vity in glutamatergic ATN and RSC afferents termina ng on PrS layer 3 pyramidal cells.

Convergence of ATN and RSC inputs on single layer 3 neurons

Are single layer 3 pyramidal cells innervated by both ATN and RSC afferents? It seemed likely since the probability of connec on to a given cell was 84% for ATN fibers and 85% for RSC afferents, and cluster analysis (Figure 2D) provided no evidence for subpopula ons of layer 3 cells (but see [START_REF] Balsamo | Modular microcircuit organiza on of the presubicular head-direc on map[END_REF].

We tested the hypothesis using photos mula on at two different light frequencies to ac vate ATN and RSC afferents independently. However this approach may be compromised since there is an overlap in the excita on spectra of blue-light ac vated Chronos (400-600 nm) and red-light ac vated Chrimson (450-700 nm; [START_REF] Klapoetke | Independent op cal excita on of dis nct neural popula ons[END_REF]. Excita on of Chronos with high intensi es of blue light might also excite Chrimson. S mula ng with red light at 630 nm should elicit neurotransmi er release in Chrimson-containing but not Chronos-expressing fibers, which we found was indeed the case. Blue light s muli at 470 nm and intensi es higher than 0.01 mW elicited oEPSPs in layer 3 cells of animals with Chronosexpressing fibers. In contrast, blue light intensi es higher than 0.25 mW were needed to induce oEPSPs in different animals with Chrimson-expressing axons (Figure 3-figure supplement 1A-D). Moreover, the amplitude of these events was smaller in Chrimsoninjected than in Chronos-injected mice. Thus, in our experimental condi ons, blue light intensi es up to 0.25 mW could be used to excite Chronos-posi ve axons with confidence that Chrimson-expressing fibers would not be s mulated.

We also tested the inverse, red light component of the dual s mulus strategy. Intensi es were tested in mice injected either with AAV5-Chronos to label ATN fibers or AAV5-Chrimson to label RSC afferents. The strategy was validated by showing that red light induced oEPSPs in Chrimson-containing, but not Chronos-expressing fibers, while blue light evoked synap c events in Chronos-, but not Chrimson-expressing fibers (Figure 3-figure supplement 1E-K).

Injec on in the same animal of the Chronos construc on to the thalamus and that for Chrimson to the retrosplenial cortex let us visualize ATN and RSC afferents to the presubiculum (Figure 3A,B). Axon terminal fields of ATN and RSC projec ons were evident in superficial layers of dorsal PrS (cf. Figure 1). Layer specifici es, and labeling inhomogenei es were apparent as were high density ATN patches below layer 2 with no RSC axon innerva on (Figure 3B). Layer 3 pyramidal cells could be innervated by ATN and RSC contacts both with apical dendrites in layer 1 and basilar dendrites of layer 3 (Figure 3D). We found op cal s mula on of ATN and RSC axons, evoked synap c responses in 14 out of 17 layer 3 pyramidal cells tested (current-clamp and voltage-clamp; Figure 3E,F).

In order to quan fy substrates of these innerva ons, we counted numbers of labeled ATN or RSC terminals located at less than 1µm from dendrites of biocy n filled layer 3 pyramidal cells (n = 6; Figure 3G). The distribu on of these poten al contact sites on dendri c trees of layer 3 neurons was diverse. Some neurons exhibited highly segregated domains receiving either ATN or RSC inputs. For instance, cell 1 had a high propor on of clustered RSC puta ve synapses on its apical tu while basilar dendrites were mostly surrounded by poten al thalamic terminals. In cell 2, RSC terminals were clustered close to apical dendrites and also together with ATN terminals close to basilar dendrites. In contrast, many ATN terminals were located closer to apical dendrites of cell 3. Segrega on of cor cal RSC inputs to apical dendrites and thalamic ATN inputs to basal dendrites might favor supralinear EPSP summa on. However, afferent terminal distribu ons differed and we found no clear pa ern of sub-or supralinear summa on (cell 1, supralinear summa on (Dual/summed EPSP, 1.39); cell 2, roughly linear (0.91); cell 3, sublinear (0.69), Figure 3G,H). Furthermore, we noted certain poten al contacts were located with separa ons less than 20 µm on the same dendrite (Figure 3I). Such proximity might underly local dendri c computa ons and supra-lineari es such as NMDA-mediated dendri c spikes which are suggested to contribute to non-linear summa on of synap c events [START_REF] Larkum | A new cellular mechanism for coupling inputs arriving at different cor cal layers[END_REF][START_REF] Larkum | Dendri c spikes in apical dendrites of neocor cal layer 2/3 pyramidal neurons[END_REF][START_REF] Larkum | Synap c Integra on in Tu Dendrites of Layer 5 Pyramidal Neurons: A New Unifying Principle[END_REF].

Supralinear integra on and amplifica on of ATN and RSC excitatory postsynap c poten als

We explored interac ons between excitatory inputs to layer 3 neurons by s mula ng ATN and RSC axons separately with blue or red light. Excitatory synap c poten als evoked by ATN or RSC axon s mula on, were compared to responses elicited when both sets of axons were ac vated at short me intervals (dual; Figure 4A). The mean amplitude of dual oEPSPs (synchronous light onset, Figure 4A-C) was significantly larger than calculated summed amplitudes of single oEPSPs (n = 11 cells, dual/summed amplitude 2.06 ± 0.55 mV, p = 0.0068, Wilcoxon test). Charge transfer, quan fied as the surface under the dual oEPSP, was greater than the summed values for single oEPSPs (Dual/summed surface 2.21 ± 0.54 mV.s, n = 11, p = 0.0098, Wilcoxon test). These mean values cover variability between individual neurons. Summa on of responses to ATN and RSC axon s muli was supralinear in 7 out of 11 cells tested, linear for 3 cells and sublinear for 1 cell.

Dynamic responses to repe ve s mula ons were also transformed by dual photos mula on. Amplitudes of 5 dual oEPSPs elicited at 20 Hz were higher than for a linear summa on (Amplitudes in mV, Summed vs. Dual: S m 1, 7.7 ± 1.9 vs. 11.1 ± 1.9; S m 2, 6.7 ± 1.6 vs. 9.5 ± 1.5; S m 3, 6.5 ± 1.4 vs. 9.5 ± 1.5; S m 4, 6.1 ± 1.3 vs. 9.2 ± 1.5; S m 5, 5.9 ± 1.3 vs. 8.6 ± 1.4, p = 0.0117, Two-way ANOVA; Figure 4E) and charge transfer, quan fied as the surface under the 5 oEPSPs, also typically summed supralinearly (20 Hz, Surface in mV.s, Summed vs. Dual: S m 1, 136.9 ± 30.6 vs. 221.2 ± 41.5; S m 2, 143.3 ± 25.7 vs. 242 ± 43.9; S m 3, 147.3 ± 22.5 vs. 259.2 ± 44; S m 4, 145.1 ± 22 vs. 263.5 ± 49.2; S m 5, 145.6 ± 23.2 vs. 258.7 ± 45.2, p = 0.0128, Two-way ANOVA; Figure 4F). Trains of dual ATN and RSC fiber s mula ons also showed increased oEPSP integrals, compared to the s mula on of either one set of fibers alone, indica ng stronger excita on over me (Figure 4E,F). Taken together, these data show supralinear summa on of ATN and RSC inputs in most single layer 3 pyramidal cells, and also a facilita on of dynamic responses to repe ve inputs at 20 Hz.

We next asked how efficiently ac on poten als were induced by combined ATN and RSC inputs. Firing probability of layer 3 cells was higher for dual than for single input s mula on at a membrane poten al of -65 mV (ATN 0.008 vs Dual 0.268, n = 5, p = 0.0216, RSC 0.024 vs Dual 0.268, n = 5, p = 0.1195, Friedman's and Dunn's tests) and higher s ll at -55 mV (Figure 4G,H). Firing was induced over a narrow window of me delays between s mula on of the two inputs (Figure 4I,J). It occurred for delays of -2 to +5 ms (RSC preceding ATN s mulus) reaching a maximum at a delay of +1 ms.

Supralinear summed responses of excitatory synap c events may be mediated via the ac va on of NMDA receptors or voltage dependent intrinsic currents that amplify EPSPs [START_REF] Fricker | EPSP amplifica on and the precision of spike ming in hippocampal neurons[END_REF], especially if synap c inhibi on is reduced. We assessed these mechanisms in records made from layer 3 pyramidal neurons using a Cesium-based internal solu on to favor ca onic amplifying currents, and also containing QX314 to suppress ac on poten als (Figure 5). In 3 cells photos mula on of ATN fibers evoked oEPSPs of amplitude 10.9 ± 2.0 mV and RSC fiber s mula on induced events of 13.7 ± 2.5 mV. The response dynamics to 5 light pulses at 20 Hz were moderately facilita ng (2nd vs 1st amplitude ra o: ATN 1.39, RSC 1.17; 5th vs 1st: ATN 1.18, RSC 1.20; n = 3). Coincident ac va on of ATN and RSC afferents induced supralinear oEPSP summa on. Furthermore, repe ve s mula on generated large all-or-none depolariza ons on the second or third s mulus at 20 Hz (2nd vs 1st amplitude ra o: 1.52; 5th vs 1st: 2.90; n = 3 cells, Figure 5A). It may be mediated by VGCC or a QX-314 resistant Na+ inward current [START_REF] Fricker | Pyramidal cells of rodent presubiculum express a tetrodotoxin-insensi ve Na+ current[END_REF]. This component appeared with higher probability and shorter latency in the presence of GABA A receptor blocker gabazine (Figure 5B). The NMDA receptor antagonist APV largely abolished the dual EPSP amplifica on ini ally (Figure 5C, black trace; 2nd vs 1st integral ra o: 1.22; 5th vs 1st: 1.32), although amplifica on was par ally restored by increasing RSC s mulus intensity (pale pink trace). NMDA receptor ac va on thus assists depolariza on towards the threshold of a voltage-dependent process contribu ng to supra-linear EPSP summa on, but is not the only charge carrier involved.

Cholinergic modula on and recruitment of presubicular layer 4 neurons by ATN and RSC afferents

Presubicular layer 4 neurons are intrinsic burs ng pyramidal neurons that project to the lateral mammillary nucleus [START_REF] Huang | Laminar Localiza on and Projec on-Specific Proper es of Presubicular Neurons Targe ng the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex[END_REF]. This pathway is cri cal for the coordinated stable landmark control of HD cells in the thalamus and throughout the HD cell circuit [START_REF] Yoder | Visual landmark informa on gains control of the head direc on signal at the lateral mammillary nuclei[END_REF][START_REF] Yoder | Acetylcholine Contributes to the Integra on of Self-Movement Cues in Head Direc on Cells[END_REF] To inves gate the input connec vity of layer 4 principal cells, we recorded responses of these neurons to s mula on of ATN and RSC afferents.

Layer 4 neurons labeled by retrograde tracers injected in the lateral mammillary nucleus were located in the lamina dissecans of the presubiculum, below layer 3, where thalamic axons ramify (Figure 6A-D). The apical dendrites of layer 4 pyramidal neurons extended towards presubicular layer 1 as previously described [START_REF] Huang | Laminar Localiza on and Projec on-Specific Proper es of Presubicular Neurons Targe ng the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex[END_REF], but tended to circumvent layer 3 and avoid thalamic afferents, by swerving towards the subiculum. Apical dendrites of some neurons crossed layer 3 obliquely, while others avoided thalamic afferents in layer 3 (Figure 6C, D, Figure 6-figure supplement 1).

Layer 4 neurons had a more depolarized res ng membrane poten al, lower input resistance and me constant than layer 3 neurons (Figure 6-figure supplement 2 compares ac ve and passive proper es). A characteris c voltage sag in responses to hyperpolarizing steps, indicated the presence of an I h current. Layer 4 neurons discharged bursts of two or three ac on poten als at the onset of a depolarizing step current injec on and also a er the offset of hyperpolarizing steps (Figure 6E). These bursts were abolished by the T-type Ca 2+ channel blocker TTA-P2 (Figure 6F).

We next recorded responses of layer 4 neurons to op cal ac va on of ATN or RSC afferents together with effects of the same s muli on layer 3 pyramidal cells (Figure 6G). Overall, latencies of oEPSCs in layer 4 neurons were longer than for layer 3 (ATN layer 4, 6.2 ± 0.6 ms, n = 8, vs. layer 3, 2.4 ± 0.2 ms, n = 24; RSC layer 4, 5.6 ± 0.4 ms, n = 9, vs. layer 3, 2.9 ± 0.2 ms, n = 27; Figure 6H), indica ng possible polysynap c excita on of layer 4 neurons. Bath applica on of TTX-4AP did not abolish oEPSPs en rely, leaving a low amplitude component with short, poten ally monosynap c latencies (latency ATN 3.9 ± 0.6 ms, RSC 2.9 ± 0.2 ms, n = 5; Figure 6I).

Comparison of the ming of synap c events and firing (Fig 7A -C) showed that oEPSP onset in a layer 4 neuron occurred a er firing in the layer 3 neuron, for either ATN (4/5) or RSC (1/1) afferent s muli. Depolariza on of the layer 3 cell via the patch pipe e, ini ated firing, but EPSPs were not elicited in any simultaneously recorded layer 4 neuron (0 out of 6 cell pairs tested). These data suggest that excita on of layer 3 cells by ATN and RSC afferents is transmi ed to layer 4 neurons, even in the absence of direct evidence for mono-synap c coupling between cell pairs.

In records from pairs of layer 3 and 4 neurons (n = 3; Figure 7D,E) layer 3 cells responded with precisely med ac on poten als to low intensity s mula on of ATN afferents, while only very small oEPSPs were ini ated in layer 4 neurons. Increasing the excitatory drive by s mula ng RSC afferents elicited larger oEPSPs more reliably in layer 4 neurons. Higher intensity s mula on of both ATN and RSC axons, could evoke bursts of ac on poten als, with the ac va on of an underlying Ca 2+ current in layer 4 cells (Figure 7E). We hypothesize that increasing the ac vity of layer 3 neurons by strong (and non-specific) s mula on of ATN and RSC afferents is needed to induce discharges in layer 4 cells. We should also note that strong photos mula on of a single set of afferent fibers could ini ate discharges in layer 4 pyramidal cells, as was shown in records from mice where only one afferent brain area, ATN or RSC, expressed an opsin (Figure 7-figure supplement 1).

Overall, it seems probable that layer 3 neurons relay ac vity projected by ATN and RSC inputs onto layer 4 pyramidal neurons. However, evidence on this point could be improved. In dual records, we did not detect func onal synapses between layer 3 and layer 4 neurons (n = 9). Anatomically, puta ve synap c contacts between filled axons of layer 3 and dendrites of layer 4 cells, were not evident (n = 5 filled layer 4 cells; see also [START_REF] Peng | Layer-Specific Organiza on of Local Excitatory and Inhibitory Synap c Connec vity in the Rat Presubiculum[END_REF]. We did find a possible synap c connec on between two neighboring L4 neurons in one case (Figure 6-figure supplement 1D), with a close apposi on between axon and dendrite. Recurrent excita on may promote burs ng in a posi ve feedback loop in layer 4 neurons, and NMDA receptor related EPSP amplifica on favors burst firing (Figure 7F).

Neuromodulatory factors may help drive layer 4 cells to fire ac on poten als. As Presubiculum is rich in acetylcholinesterase [START_REF] Slomianka | Distribu on of acetylcholinesterase in the hippocampal region of the mouse: I. Entorhinal area, parasubiculum, retrosplenial area, and presubiculum[END_REF], we examined excitability changes in the presence of a broad cholinergic agonist. The applica on of Carbachol (10 µM; Figure 7G) led to a depolariza on of the recorded layer 4 neurons' membrane poten al by 13 ± 4 mV, with increased ac on poten al firing during a posi ve step current injec on, and from the baseline (Figure 7Gi,ii). The depolarizing effect of carbachol was mimicked by muscarine (10 µM; n = 3), and persisted in the presence of TTX (1 µM; n = 2), indica ng a direct effect on layer 4 neurons.

Discussion

Understanding the anchoring of HD signals with visual landmarks provides a useful framework to ask how external informa on is integrated to revise and improve brain representa ons. Here we show that thalamic ATN afferents signaling HD data and cor cal RSC projec ons that carry visual landmark informa on converge on layer 3 pyramidal cells of the dorsal presubiculum. Independent optogene c s mula on of these two glutamatergic afferents in slices was used to define mechanisms of their integra on in the presubiculum. Most layer 3 cells were innervated by both ATN and RSC fibers, some mes close on the same dendri c branches, some mes on different dendrites. Nearly coincident EPSPs (2-5 ms) induced independently by ATN and RSC afferents, evoked non-linear membrane responses to trigger layer 3 cell firing, transmi ed to the MEC. In a second processing step, layer 3 neurons also excite layer 4 pyramidal cells which receive li le direct ATN and RSC innerva on. These burst firing neurons, project a dis nct, di-synap cally mediated, visuallyupdated HD signal to the LMN. Our data thus suggest that the presubiculum integrates HD and landmark signals producing two dis nct output signals which are transmi ed to different regions. Cholinergic modula on may facilitate responses to salient s muli, for flexible anchoring to landmarks.

Anatomical convergence of ATN and RSC projec ons in the dorsal Presubiculum

Retrograde tracing in this study confirmed strong projec ons to the presubiculum from the ATN and the RSC to the presubiculum (Figure 1). Anterior thalamic fibers (Van Groen et Wyss 1990c;1990a;1990b;1992;[START_REF] Shibata | Thalamocor cal projec ons of the anterodorsal thalamic nucleus in the rabbit[END_REF][START_REF] Vogt | Cor cal connec ons between rat cingulate cortex and visual, motor, and postsubicular cor ces[END_REF] ramify in and delimit the anatomical borders of the presubiculum [START_REF] Liu | Molecular characteriza on of superficial layers of the presubiculum during development[END_REF][START_REF] Simonnet | Ac vity dependent feedback inhibi on may maintain head direc on signals in mouse presubiculum[END_REF]. HD signals from HD cells of the anterior thalamus project to the presubiculum [START_REF] Goodridge | Interac on between the postsubiculum and anterior thalamus in the genera on of head direc on cell ac vity[END_REF] where they form synapses directly with layer 3 pyramidal cells [START_REF] Nassar | Anterior Thalamic Excita on and Feedforward Inhibi on of Presubicular Neurons Projec ng to Medial Entorhinal Cortex[END_REF]. The retrosplenial cortex was the most strongly labeled cor cal region innerva ng the presubiculum. We detected retrogradely transported beads in cells of layers 2 and 5 of dysgranular RSC, and layer 5 of the granular RSC across its antero-posterior axis [START_REF] Sugar | Postnatal development of retrosplenial projec ons to the parahippocampal region of the rat[END_REF]. We examined the anatomy and physiology of these two afferent systems to ask how visual landmark signals from the RSC are combined with HD signals relayed via the ATN. Anterograde fiber tracing, with an AAV5 construct expressing Chronos-GFP, confirmed that axons from both regions project to superficial layers of the dorsal presubiculum. No RSC projec ons were found in ventral PrS for injec ons in rostral RSC, as previously noted [START_REF] Jones | Cingulate cortex projec ons to the parahippocampal region and hippocampal forma on in the rat[END_REF][START_REF] Kononenko | Presubiculum layer III conveys retrosplenial input to the medial entorhinal cortex[END_REF]. Both RSC and ATN innervated superficial presubicular layers 1 and 3, while sparing layer 2 containing somata of calbindin posi ve neurons (Figure 1K and cf. [START_REF] Balsamo | Modular microcircuit organiza on of the presubicular head-direc on map[END_REF]. We detected microzones containing a high density of ATN-posi ve fibers, but no RSC fibers, in upper layer 3.

Pathway specific func onal connec vity onto PrS layer 3 neurons.

Photos mula on of GFP-Chronos expressing axons let us compare synap c events ini ated in layer 3 pyramidal cells by fibers from the ATN or the RSC in dorsal presubiculum slices (Figure 2). Both ATN and RSC afferents formed mono-synap c glutamatergic connec ons with components mediated by NMDA and AMPA receptors. The amplitudes of synap c currents varied, with an overall similar amplitude distribu on for ATN and RSC s mula on. This may be due to variable expression levels [START_REF] Hooks | Dual-channel circuit mapping reveals sensorimotor convergence in the primary motor cortex[END_REF], or may imply varia ons in coupling weights across cells. ATN and RSC fiber mediated EPSCs depressed during repe ve ac va on at 20 Hz. EPSPs induced firing early during repe ve s mula on. RSC inputs tended to produce more sustained EPSP dynamics and slower rise mes than ATN afferents for low intensity s mula on. ATN synapses with presubicular pyramidal cells resemble those made by thalamic afferents in the somatosensory cortex -high release with depressing dynamics, [START_REF] Gil | Efficacy of thalamocor cal and intracor cal synap c connec ons: quanta, innerva on, and reliability[END_REF] possibly due to presynap c expression of VGLut2 [START_REF] Liu | Molecular characteriza on of superficial layers of the presubiculum during development[END_REF].

Dual wavelength optogene c s mula on was used for independent ac va on of intermingled afferents expressing blue-light sensi ve Chronos in ATN fibers and red-shi ed Chrimson in RSC fibers [START_REF] Klapoetke | Independent op cal excita on of dis nct neural popula ons[END_REF]. Precau ons were taken to avoid crosss mula on since all channelrhodopsin variants are somewhat sensi ve to blue light. With the fast, sensi ve opsin Chronos expressed in ATN fibers, synap c events were induced by very short (0.5 ms), low intensity, 0.25 mW, s muli. For RSC fibers expressing Chrimson, using light s muli of dura on 2 ms, adjus ng intensity up to 2 mW ini ated synap c events of comparable amplitude. Calibra on experiments (Figure 3-figure supplement 1) provided strong evidence for the independence of responses. While Chrimson has slower dynamics than Chronos [START_REF] Klapoetke | Independent op cal excita on of dis nct neural popula ons[END_REF], synap c events induced by s mula ng either opsin were similar, as we showed by swapping the two opsin variants injected in ATN and RSC fibers respec vely (Figure 4). This dual opsin approach permi ed independent s mula on with blue and red light pulses. Most (76%) recorded layer 3 neurons generated synap c events in response to s mula on of both ATN and RSC fibers providing a substrate for integra on of landmark informa on from the RSC with thalamic HD signals. Layer 3 cell firing was most effec vely triggered by nearly coincident inputs (-2 to +5 ms separa on, Figure 4I,J). This is of interest since HD representa ons in the AD and RSC are highly coherent [START_REF] Fallahnezhad | Cerebellar control of a unitary head direc on sense[END_REF], with short intervals between spikes in these two regions (< 5ms; van der Goes et al. 2022). Converging inputs from thalamic and retrosplenial axons can therefore excite common postsynap c presubicular layer 3 neurons with very short delays, such that coincidence detec on by these cells will tend to enhance HD signals transmi ed to the MEC. Response dynamics to combined s mula on of both inputs at 20 Hz were maintained or facilita ng, in contrast to the depressing dynamics of repe ve s mula on of one set of afferent fibers. Combined and temporally precise inputs from ATN and RSC may thus help maintain HD signaling during immobility.

Nonlinear signal integra on in layer 3 thalamo-recipient neurons

Convergence of ATN and RSC axons onto single layer 3 pyramidal cells provides the anatomical basis of synap c integra on. Puta ve synap c contacts from both afferent fiber systems were found on the basal dendrites of pyramidal cells (Figure 3), some mes on the same branch. Photos mula on centered on the soma of recorded neurons predominantly ac vated synapses on basal dendrites. Supralinear summa on could result from local spikegenera ng mechanisms if the ac vated synapses were located on a same dendri c branch [START_REF] Makarov | Dendrites and Efficiency: Op mizing Performance and Resource U liza on[END_REF][START_REF] Poirazi | Illumina ng dendri c func on with computa onal models[END_REF][START_REF] Polsky | Computa onal subunits in thin dendrites of pyramidal cells[END_REF]. Clustered synapses could also guide the forma on of new spines and synapses during integra on of landmark informa on in the HD signal. Learning might bind new inputs into func onal synap c clusters [START_REF] Hedrick | Learning binds new inputs into func onal synap c clusters via spinogenesis[END_REF]. Our small sample of layer 3 pyramidal cells suggest that both ATN and RSC axons target basal dendrites, and more RSC than ATN axons contact apical dendrites (Figure 3). Tests on the effects of precise, near coincident ac va on of basal and apical synapses could be revealing but were not technically possible in this study. Dis nct dendri c inputs to layer 3 cells may improve spa al percep on [START_REF] Takahashi | Ac ve cor cal dendrites modulate percep on[END_REF] enhancing HD signal quality by integra on with landmark informa on.

Our data show that EPSPs elicited by nearly coincident ATN and RSC inputs which exceed a threshold are amplified by NMDA receptor ac va on and voltage gated inward dendri c currents (Figure 5; [START_REF] Fricker | Pyramidal cells of rodent presubiculum express a tetrodotoxin-insensi ve Na+ current[END_REF]. EPSP amplifica on is facilitated by a reduc on in synap c inhibi on (Figure 5), indica ng that disinhibi on may be permissive for supralinearity and gate firing by dynamic modula on of the balance between inhibi on and excita on [START_REF] Milstein | Inhibitory Ga ng of Input Comparison in the CA1 Microcircuit[END_REF]. VIP expressing interneurons, which are excitated by cholinergic modula on could provide such disinhibi on of the presubicular microcircuit [START_REF] Porter | Selec ve excita on of subtypes of neocor cal interneurons by nico nic receptors[END_REF][START_REF] Slomianka | Distribu on of acetylcholinesterase in the hippocampal region of the mouse: I. Entorhinal area, parasubiculum, retrosplenial area, and presubiculum[END_REF]. A 'when-to-learn' signal for HD upda ng in the presubiculum might func on analogously to the promo on by dopamine of associa ons between sensory cues and head direc on cells in the fly compass neurons [START_REF] Fisher | Dopamine promotes head direc on plas city during orien ng movements[END_REF]. It is however unclear whether LTP type learning takes place in the mammalian head direc on circuit. Presubicular layer 3 cells do not express the GluR1 subunit of AMPA receptors (Mar n et al 1993; [START_REF] Ishihara | Immunohistochemical inves ga on of the internal structure of the mouse subiculum[END_REF] that is cri cal for LTP expression [START_REF] Boehm | Synap c incorpora on of AMPA receptors during LTP is controlled by a PKC phosphoryla on site on GluR1[END_REF]. The absence of GluR1 might indicate that the thalamopresubicular synapses in layer 3 func on without classical long-term plas city.

Layer 3 pyramidal cells may be described as mul -compartmental computa onal devices [START_REF] Häusser | Dendrites: bug or feature?[END_REF][START_REF] Mel | Synap c integra on in an excitable dendri c tree[END_REF][START_REF] Poirazi | Arithme c of subthreshold synap c summa on in a model CA1 pyramidal cell[END_REF][START_REF] Spruston | Pyramidal neurons: dendri c structure and synap c integra on[END_REF] which integrate HD and landmark informa on. ATN axons drive presubicular HD neurons [START_REF] Peyrache | Internally organized mechanisms of the head direc on sense[END_REF] and RSC mixed selec vity neurons contribute visual landmark informa on and allocentric spa al references [START_REF] Jacob | An independent, landmark-dominated head-direc on signal in dysgranular retrosplenial cortex[END_REF][START_REF] Mitchell | Retrosplenial cortex and its role in spa al cogni on[END_REF][START_REF] Vann | What does the retrosplenial cortex do?[END_REF]. NMDA mediated dendri c spikes enhance tuning selec vity in visual cortex [START_REF] Smith | Dendri c spikes enhance s mulus selec vity in cor cal neurons in vivo[END_REF][START_REF] Wilson | Orienta on selec vity and the func onal clustering of synap c inputs in primary visual cortex[END_REF] and barrel cortex [START_REF] Lavzin | Nonlinear dendri c processing determines angular tuning of barrel cortex neurons in vivo[END_REF]. Dendri c events in Layer 3 PrS cells may enable binding of visual landmarks with HD tuning. It is temp ng to speculate that nonlinear synap c integra on and inhibitory ga ng may be involved in flexibly upda ng the allocentric direc on of HD cells based on the integra on of visual landmark informa on to the current HD signal. In the primary sensory cortex, nonlineari es act to increase perceptual threshold of sensory informa on [START_REF] Takahashi | Ac ve cor cal dendrites modulate percep on[END_REF]2020). The a ractor network in the PrS could thus be either stabilized or flexibly reset to external spa al cues.

Func onal significance of two-layer processing preceding projec on to LMN

Burst-firing layer 4 PrS cells project a dis nct version of HD-landmark signals to the LMN. The dendrites of these LMN projec ng neurons overlap li le with direct ATN and RSC inputs gran ng par al isola on from direct excita on, and permi ng a second level of integra on of informa on from layer 3 neurons. Layer 3 axons may innervate layer 4 neurons basal dendrites, which may be driven to fire by sufficiently strong excitatory inputs from layer 3, especially if combined with cholinergic ac va on.

There are advantages to segrega ng the integra on of converging input signals from the upda ng signal across layers. This separa on permits both a fast transmission of an integrated signal to the medial entorhinal cortex, and the condi onal transmission of a upda ng signal mediated by burst firing to upstream HD circuit elements. The synap c threshold implicit in this two-stage system permits a gated upda ng. Func onally layer 4 neurons are uniquely posi oned to update the HD signal in the LMN with visual landmark informa on [START_REF] Yoder | Visual landmark informa on gains control of the head direc on signal at the lateral mammillary nuclei[END_REF]. In this way, cell-type specific cholinergic facilita on may help idiothe c cue based naviga on [START_REF] Yoder | Acetylcholine Contributes to the Integra on of Self-Movement Cues in Head Direc on Cells[END_REF]. These cellular and synap c circuit data support and expand the findings of [START_REF] Yoder | Visual landmark informa on gains control of the head direc on signal at the lateral mammillary nuclei[END_REF]2017). They clarify how the thalamic HD signal integrated with visual landmark informa on is relayed to the grid cell system in the medial entorhinal cortex and to the lateral mammillary nuclei.

Limita ons

Burst firing may play a role for learning in hierarchical circuits [START_REF] Naud | Silences, Spikes and Bursts: Three-Part Knot of the Neural Code[END_REF][START_REF] Payeur | Burst-dependent synap c 26 plas city can coordinate learning in hierarchical circuits[END_REF]. In the HD circuit, visual landmark informa on contained in bursts might reset or anchor the HD a ractor in the LMN and beyond. The effects of burst firing signals transmi ed to the LMN remain to be assessed. Poten ally HD signals may be updated with visual signals at several sites including the PrS, but this region uniquely provides a feed-back projec on to the lateral mammillary nucleus. Further work on layer 3 to layer 4 transmission is warranted, and the link to spa al percep on and behavioural upda ng needs to be strengthened.

Methods

Animals

Experiments were performed on wild-type and transgenic C57Bl6 mice, housed on a 12 hours light/dark cycle with food and water available ad libitum. Animal care and use conformed to the European Community Council Direc ve (2010/63/EU) and French law (87/848). Our study was approved by the local ethics commi ee (CEEA -34) and the French Ministry for Research 01025.02.

Viral vectors and beads

Projec ng neurons were labeled with retrograde fluorescent tracers (Retrobeads, Lumafluor, and pAAV-CAG-tdTomato, Addgene 59462P). Fluorescent beads were stored at 4°C before use. Channelrhodopsin expression was achieved by injec ng Adeno-associated viral construc ons. AAV5.Syn.Chronos-GFP.WPRE.bGH (AAV5-Chronos, Penn Vector Core, Addgene 59170P) was used to induce neuronal expression of the blue light induced channelrhodopsin Chronos fused to the GFP marker and under the control of the Synapsin promoter. AAV5.Syn.ChrimsonR-tdTomato.WPRE.bGH (AAV5-Chrimson, Penn Vector Core, Addgene 59171P) induced neuronal expression of the red light gated channelrhodopsin Chrimson fused to the tdTomato marker, under the control of the Synapsin promoter. Viral vectors were stored at -80°C before use.

Stereotaxic surgery

Mice at ages of 4-5 weeks were anesthe zed by intraperitoneal (i.p.) injec on of a mixture of ketamine hydrochloride and xylazine (100 and 15 mg/kg respec vely, in NaCl 0.9%). They were placed in a stereotaxic frame for injec ons. Fluorescent beads for retrograde tracing were injected (300-500 nl) into the PrS at the coordinates: -4.06 anteroposterior (AP), 2.00 medio-lateral (ML) and -2.15 mm dorso-ventral (DV) and into the LMN (-2.8 AP, 0.75 ML, -5.35 DV) with respect to the bregma. Viral injec ons were performed unilaterally [START_REF] Mathon | Increasing the effec veness of intracerebral injec ons in adult and neonatal mice: a neurosurgical point of view[END_REF][START_REF] Richevaux | In Vivo Intracerebral Stereotaxic Injec ons for Optogene c S mula on of Long-Range Inputs in Mouse Brain Slices[END_REF] at the coordinates -0.82 AP, 0.75 ML and -3.2 mm DV for the ADN, and at -2.1 to -2.15 AP, 0.65 ML and -0.65 mm DV for the RSC. Volumes of 200 to 250 nl were injected with a 10 µL Hamilton syringe equipped with 33ga needle over a me of 10 min. The needle was slowly removed a er a delay of 10 min to avoid leakage from the injec on site. Best expression of AAV5 serotypes was achieved a er 3 to 4 weeks.

Tissue fixa on and slicing for retrograde tracing

Brains were removed for anatomy at 4 days a er retrobead injec on. Mice were anesthe zed by i.p. injec on of the ketamine/xylazine mixture. An intracardiac perfusion with 0.1M PBS was followed by perfusion with 4% paraformaldehyde. Brains were stored overnight in paraformaldehyde at 4°C and then washed in PBS. Coronal or horizontal sec ons were cut at 100 µm with a vibratome and stored in sucrose at 4°C.

Prepara on of brain slices for physiology

Slices of the temporal lobe were prepared 3-4 weeks a er injec on of AAV5 viral construc ons. Mice were anesthe zed by i.p. injec on of the ketamine/xylazine mixture. They were then perfused intracardially with a cu ng solu on containing (in mM): 125 NaCl, 25 sucrose, 2.5 KCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 D-glucose, 0.1 CaCl 2 , 7 MgCl 2 , cooled to 4°C, and oxygenated with a 5% CO 2 / 95% O 2 . The brain was removed and a vibratome was used to cut horizontal slices at 300 µm in the same solu on. Slices were stored for 15 min at 34°C in an ACSF containing (in mM): 124 NaCl, 2.5 KCl, 26 NaHCO 3 , 1 NaH 2 PO 4 , 2 CaCl 2 , 2 MgCl 2 , and 11 D-glucose, bubbled with 5% CO 2 / 95% O 2 . They were then kept in the same solu on at room temperature un l recording.

Whole-cell patch-clamp recordings

Slices were transferred to a recording chamber perfused with oxygenated, warmed (~32 °C) ACSF mounted on an epifluorescence microscope. Patch-clamp records were made from neurons with borosilicate glass pipe es of external diameter 1.5 mm (Clark Capillary Glass, Harvard Apparatus) pulled with a Brown-Flaming electrode puller (Su er Instruments). Electrodes, filled with a potassium-gluconate based solu on containing (in mM): 135 K-gluconate, 1.2 KCl, 10 HEPES, 0.2 EGTA, 2 MgCl 2 , 4 MgATP, 0.4 Tris-GTP and 10 Na 2 -phosphocrea ne, had a resistance of 4-8 MΩ. An alterna ve, cesium-gluconate based solu on facilitated neuronal depolariza on to examine synap c inhibi on. It contained (in mM): 125 Cs-gluconate, 10 HEPES, 0.2 EGTA, 2 MgCl 2 , 4 MgATP, 0.4 Tris-GTP and 10 Na 2 -Phosphocrea ne, together with 5 mM QX-314 to block Na + channels. Pipe e solu ons also contained 3 mM biocy n to reveal morphology a er recording. They were adjusted to pH 7.3 and osmolarity 290 mOsm. Whole-cell, patch-clamp signals were filtered at 3kHz, amplified with a Mul Clamp 700B amplifier and acquired with pCLAMP so ware (Molecular Devices). Monosynap c excita on induced by op cal s mula on was tested in the presence of TTX (1 µM) and 4-AP (100 µM). NBQX (10 µM) and APV (100 µM) were used to block AMPA and NMDA receptors respec vely. Gabazine (10 µM) was used to block GABA A receptors. Carbachol (10 µM) was used to ac vate acetylcholine receptors. All drugs were bath applied.

Op cal s mula on

LED illumina on (Cairn Research, OptoLED) was used to visualize the fluorescent reporters GFP and tdTomato, and to s mulate opsin-expressing axons, using a 470 nm LED for Chronos and a 627 nm LED for Chrimson. Illuminated spots had a diameter of 200 µm with a 60x objec ve and were centered on the recorded cell soma. Photos mula on thus covered most of the basilar dendrites of layer 3 pyramidal neurons (typical distance from p of apical to p of basilar dendrite <400µm). S muli consisted of light pulses of 0.5 to 5 ms dura on, repeated 5-10 mes at 20 Hz.

A mul band filter allowed simultaneous s mula on by blue and red LEDs of axons containing Chronos and Chrimson [START_REF] Simonnet | Single or Double Patch-Clamp Recordings In Ex Vivo Slice Prepara on: Func onal Connec vity, Synapse Dynamics, and Optogene cs[END_REF]. S mulus power intensity (set in mV) was calibrated as light intensity. The response probability of layer 3 cells was calibrated to blue or red illumina on of ATN or RSC afferents expressing Chronos or Chrimson to avoid s mulus overlap (Figure 3-figure supplement 1). Chronos was targeted to the ATN and Chrimson to the RSC a er tes ng the reverse configura on. Projec ons from the thalamus are larger and Chronos is more sensi ve to blue light, so this configura on assured reliable ac va on of thalamic fibers at minimal blue-light intensi es. For experiments inves ga ng the integra on of ATN and RSC inputs, we aimed to give similar weights to both inputs: Chrimson-expressing fibers were s mulated with light of intensity adjusted to ini ate op cally evoked excitatory postsynap c poten als (oEPSPs) of amplitude similar to those induced by blue light Chronos-fiber s muli.

Data analysis

ATN and RSC projec ons to the PrS, were analyzed from Chronos and Chrimson expression, using the ImageJ Plot Profile plug-in to quan tate normalized plot profiles (2000 pixels) of horizontal presubicular sec ons. Dorso-ventral differences were derived by dividing differences in labeling intensity between the PrS and the dentate gyrus (DG) molecular layer by DG intensity in slices from 5 dorso-ventral PrS levels. Values from all animals were averaged and then normalized.

Cells were qualified as synap cally 'connected' when they responded to light s mula on of afferent axons with a delay < 8 ms. Slices with very low or absent expression of the fluorescent reporter were excluded. Cells were qualified as 'non-connected' synap cally, if they did not respond to light s mula on, but at least one neighboring cell in the same slice did.

Intrinsic neuronal proper es were analyzed with custom MATLAB rou nes to derive 15 electrophysiological parameters [START_REF] Huang | Laminar Localiza on and Projec on-Specific Proper es of Presubicular Neurons Targe ng the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex[END_REF]. Parameters were standardized and unsupervised cluster analysis performed with MATLAB was used to compare different neurons [START_REF] Huang | Laminar Localiza on and Projec on-Specific Proper es of Presubicular Neurons Targe ng the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex[END_REF][START_REF] Simonnet | Cellular neuroanatomy of rat presubiculum[END_REF].

Axograph was used to analyze responses to op cal s mula on of ATN and RSC fibers. Layer 3 neurons were recorded at poten als near -65 mV. Responses to light were averaged from 10 s mulus trains at 20 Hz. Amplitudes and latencies of ini al light evoked EPSCs, of latency shorter than 10ms, were quan fied from voltage-clamp records. Latency was measured from s mulus onset to 10% of the peak amplitude of the first op cally induced EPSC. Amplitude was measured with respect to the pre-s mulus baseline. Paired-pulse ra o (PPR) was defined as the ra o of the amplitude of the second to the first EPSC and 10/1 ra o as that between the 10th and the 1st EPSC, in responses to 20 Hz s mula ons. Spike probability was counted over 5 to 10 trials per cell and then averaged over all cells. EPSPs induced by dual wavelength s mula on were analyzed using Axograph and a custom-made so ware. Events evoked by light s muli were detected in a window of 1-10 ms a er s mula on. EPSP amplitude and integrated area were calculated over 50 ms a er s mula on. Baseline suppression was applied using an average of membrane poten al during 50 ms before s mula on. Summa on of ATN and RSC evoked EPSCs in layer 3 neurons was determined from the amplitude and integral of averaged events. Summa on was considered to be supralinear, if values were more than 10% higher than a linear addi on, linear for values within ± 10%, and sublinear for values more than 10% lower.

Biocy n revela on and morphology

Recorded neurons were filled with biocy n to visualize their morphology and loca on. Slices were fixed in 4% PFA in 0.1 M PBS at 4°C overnight, washed 3 mes in 0.1 PBS and cryoprotected in 30% sucrose. Cell membranes were permeabilized by three freezethaw cycles over dry ice and rinsed in PBS. Slices were agitated in a satura on buffer (2% milk, 1% Triton X-100 in 0.1 M PBS) for 2 hours at room temperature. They were then incubated with Streptavidin-Cy5 conjugate (1:500) and DAPI (1:1000) overnight at 4°C. Sec ons were washed 3 mes with PBS and mounted on coverslips with ProLong gold an fade mountant. For anatomical study they were mounted in Mowiol medium. Cell, bead and virus expression were visualized with an epifluorescence microscope. Higher resolu on of morphology was obtained with confocal microscopy. Cell morphologies were reconstructed using IMARIS.

Quan fica on and sta s cal analysis

Data was analyzed with AxoGraphX and custom-wri en so ware (MATLAB, The MathWorks). Results are given as mean ± SEM. Sta s cal analysis was performed with Prism (GraphPad So ware). The Mann-Whitney unpaired t-test was used to compare two groups. The Wilcoxon or Kruskal-Wallis test was used to compare paired groups. Evolu on of parameters evoked by op cal s mula on (current or poten al amplitudes, spike probability) was analyzed with Friedman's test followed by mul ple comparison Dunn's test. Šidák's mul ple comparison test was also used to compare linear and observed responses to ATN and RSC s mula ons. Significance levels are given as p values. E, F, Injec on sites in ATN (E) and RSC (F). AD: anterodorsal thalamic nucleus, AV: anteroventral thalamic nucleus, CA1: field of the hippocampus, cc: corpus callosum, cg: cingulum, CPu: caudate putamen, DG: dentate gyrus, MD: thalamic medial dorsal nucleus, MEC: medial entorhinal cortex, PaS: parasubiculum, PVA: paraventricular thalamic nucleus, anterior, Rt: thalamic re cular nucleus, sm: stria medullaris, Sub: subiculum, VL: thalamic ventrolateral nucleus. G, H, Five sequen al 100-µm horizontal slices of the parahippocampal region show ATN (G) and RSC (H) axons expressing Chronos-GFP (green). Dashed lines show limits of the parahippocampal region to the le and of the dentate gyrus to the right. Numbers show dorsoventral level with respect to bregma. Scale bars 100 µm. I, Normalized profiles of fluorescent intensity for ATN and RSC projec ons to the presubiculum, from white ma er (WM) to pia (PIA). Mean (green) ± SEM (grey), n = 8. J, Ventral (V) to dorsal (D) normalized distribu on of ATN and RSC projec ons to the presubiculum, n = 3 mice. K, ATN axon labeling (green) is segregated from calbindin labeling (white) in the PrS. Scale bar 100 µm. See also Figure 1-figure supplement 1. -Thalamic nuclei: anterodorsal (AD) +++, anteroventral (AV) +++, laterodorsal (LD) +++, lateroposterior ++, dorsal lateral geniculate ++, reuniens ++, anteromedial -, ventrolateral --Retrosplenial cortex: dysgranular (dRSC) +++, granular +++, dysgranular contralateral ++ -Visual cortex: primary (V1) ++, secondary (V2L) +++ -Entorhinal cortex: lateral (LEnt) +++, medial (MEnt) +++ -Hippocampus/parahippocampus: parasubiculum (PaS) +++, subiculum (Sub) +++, controlateral PrS ++, CA1 ++, CA3 -, dentate gyrus --Other: claustrum ++, perirhinal cortex ++, temporal associa on cortex ++ ADN: anterodorsal thalamic nucleus; AM: anteromedial thalamic nucleus; APT(V): anterior pretectal nucleus (ventral part); AVDM: anteroventral thalamic nucleus, dorsomedial part; AVVL: anteroventral thalamic nucleus, ventrolateral part; CA: cornu ammonis; cc: corpus callosum; Cl: Claustrum; Cg1,2: cingular cortex area 1,2; coll. sup: colliculus superior; CPu: caudate putamen (striatum); dhc: dorsal hippocampal commissure; DG: dentate gyrus; DLG: dorsal lateral geniculate nucleus; LEnt: lateral entorhinal cortex; LDDM: laterodorsal thalamic nucleus, dorsomedial part; LDVL: laterodorsal thalamic nucleus, ventrolateral part; LMN: lateral mammillary nucleus; LS: lateral septal nucleus; M1,2: primary, secondary motor cortex; MEnt: medial entorhinal cortex; MMN: medial mammillary nucleus; PAG: periaqueductal gray; PaS: Parasubiculum; PF: parafascicular thalamic nucleus; PH: posterior hypothalamic area; PMCo: posteromedial cor cal amygdaloid nucleus; Po: posterior thalamic nuclear group; PRh: perirhinal cortex; PrS: presubiculum; PVA: paraventricular thalamic nucleus, anterior part; S1BF, S1FL, S1ULp, S1Tr: primary somatosensory cortex, barrel field, forelimb region, upper lip region, trunk region; S2: secondary somatosensory cortex; sm: stria medullaris of the thalamus; Sub: subiculum; Subst. Nigra: substan a nigra; Re/Reuniens: reuniens thalamic nucleus; RSA = dRSC, dysgranular retrosplenial cortex; RSC: retrosplenial cortex; RSG = gRSC: granular retrosplenial cortex; Rt: re cular thalamic nucleus; V1: primary visual cortex; V2M: secondary visual cortex, medial part; V2L: secondary visual cortex, lateral part; VA: ventral anterior thalamic nucleus; VDB: nucleus of the ver cal limb of the diagonal band; VL: ventrolateral thalamic nucleus; VPL: ventral posterolateral thalamic nucleus; VPM: ventral posteromedial thalamic nucleus A, Variance of latency (ji er) in oEPSP onset following Chronos-expressing ATN or RSC fiber s muli. RSC input s mula on gives similar varia on in latency as ATN inputs. B, Ji er as a func on of latency for ATN and RSC s muli shows a similar coefficient of varia on. C, Decay me constants of EPSCs evoked in layer 3 neurons by s mula ng ATN or RSC inputs. D, E, First oEPSC amplitude as a func on of oEPSC half-width (D) and oEPSC rise me (E) for both inputs. We found no clear segrega on of oEPSC shapes according to the origin of afferent fibers. F, oEPSPs in a presubicular layer 3 cell, induced by blue or red light s mula on of Chronosexpressing ATN or Chrimson-expressing RSC afferents, respec vely. G-J, Comparison of oEPSPs evoked by s mula ng ATN or RSC axons, recorded from a same postsynap c layer 3 presubicular neuron. G, Mean amplitude. H, Maximum rising slope of oEPSPs. I, oEPSPs rise me. p < 0.05, Wilcoxon test. J, oEPSPs decay me constants. oEPSPs following ATN and RSC fiber s mula on recorded from the same cell are connected by lines. Dashed lines indicate reversed experimental design (expressing Chronos in RSC and Chrimson in ATN). K, Responses to 10 light s muli at 20 Hz for ATN and RSC inputs to the same cell. L, Amplitudes of oEPSPs evoked by trains of ATN (le ) or RSC (right) fiber s muli for 11 cells. Dashed lines indicate the reversed experimental design (expressing Chronos in RSC and Chrimson in ATN). M, Summary data from L. On average, the amplitudes of oEPSPs evoked by ATN fiber s mula on were smaller for the fi h than for the first s mulus. oEPSPs evoked by RSC fiber s mula on were similar across five s muli. Full line, median; Short line, mean; Min to max and quar les. * p < 0.05, Friedman's test and post-hoc Dunn's test comparing each s mulus with the first. A, Blue light s mula on of ATN Chronos-expressing fibers evoked oEPSPs in presubicular layer 3 neurons at an intensity of 0.25mW, and 0.5 ms dura on of illumina on. B, Blue light s mula on at the same intensity and dura on did not evoke oEPSPs from RSC Chrimson-expressing fibers. C, D, Response probabili es of presubicular layer 3 cells to s mula on of fibers expressing Chronos or Chrimson with red (C) or blue (D, le ) light. E, Experimental design. Injec on of AAV5-Chronos-GFP (green) in the ATN, followed by photos mula on combined with patch recording from a presubicular layer 3 cell. F, Amplitudes of oEPSCs evoked in layer 3 cells by blue (0.5 ms, 0.25 mW) or red light (2 ms, 0.6 mW) s muli. G, Representa ve responses to light s mula on of ATN inputs. H, Injec on of AAV5-Chrimson-tdTomato (red) in the RSC, followed by photos mula on combined with patch recording from a presubicular layer 3 cell. I, Amplitudes of oEPSCs evoked in layer 3 cells by blue (0.5 ms, 0.25 mW) or red light (2 ms, 0.6 mW) s muli. J, Representa ve responses to light s mula on of RSC inputs K, Values and sta s cal analysis for different s mula on configura ons (Panels F and I). F, As in E, for dual ATN and RSC oEPSP integrals. Two-way ANOVA, p = 0.0128. G, Excitatory postsynap c responses to photos mula on of ATN (blue light, cerulean traces) or RSC axons (red light, purple traces) or both (blue and red light, black traces) at res ng membrane poten al (-65 mV) or at a depolarized holding poten al (-55 mV). Synap c excita on led to ac on poten als when dual ATN and RSC s muli reached firing threshold. H, Ac on poten al (AP) probability for either or both s muli at -65 and -55 mV. Data are presented as mean ± SEM. I, Ac on poten als were induced in presubicular layer 3 neurons by near coincident ac va on of ATN axons (red light) and RSC axons (blue light). Time delays varied from -50 to +50 ms. J, Firing probability was maximal for short delays between -2 to +5 ms (RSC preceding ATN). Figure 5. EPSP amplifica ons in layer 3 pyramidal neurons. A, Op cal EPSPs evoked in layer 3 pyramidal neurons following the s mula on of ATN axons (blue light, cerulean traces), RSC axons (red light, purple traces) or both (blue and red light, black traces). Recording pipe e contained a cesium gluconate based internal solu on and the Na + channel blocker QX-314. A large all-or-none EPSP amplifica on occurred for dual s muli at 20 Hz, on some trials. B, In the presence of the GABA A receptor antagonist gabazine (10 µM), dual EPSP amplifica on occurred earlier in the train. C, The addi onal presence of the NMDA receptor antagonist APV (100 µM) abolished dual EPSP amplifica on (black trace). EPSP amplifica on was par ally restored by increasing red light intensity x2 (pale pink traces). Figure 6 with 2 supplements. Presubicular LMN-projec ng layer 4 neurons avoid thalamo-recipient layer 3 and receive li le direct input from ATN and RSC. A, Expression of Chronos-GFP in ATN and retrograde labeling of neurons that target LMN. B, Thalamic axons (green) in superficial layers 1 and 3 of presubiculum. Retrobeads label cell bodies of presubicular layer 4 cells (red). C, Retrograde rAAV2-tdTomato label cell bodies and dendrites of layer 4 LMN projec ng neurons (red). Apical dendrites of layer 4 pyramidal neurons avoid layer 3 where thalamic axons ramify. D, Presubicular slice containing two layer 3 and two layer 4 neurons filled with biocy n (white) and GFP expressing thalamic axons (green). Scale bar 100 µm. Inset, retrobeads (red) in the soma of a biocy n filled LMN-projec ng layer 4 neuron. Scale bar 10 µm. E, Layer 3 neurons are regular spiking and layer 4 neurons are burst firing, ini ally and at rebound, in reponse to current injec on. Black trace, rheobase. F, T-type Ca 2+ channel blocker TTA-P2 (1 μM) suppressed burst firing in presubicular layer 4 neurons, while single ac on poten als were preserved. G. Representa ve oEPSCs in layer 3 (le ) and layer 4 (right) pyramidal cells, in response to s mula on of ATN (cerulean)

Figure titles and legends

Cross-laminar ac va on of LMN-projec ng layer 4 burs ng neurons.

A, Simultaneous records of a layer 4 and a layer 3 neuron during photos mula on of ATN afferents. EPSP onset was delayed in the layer 4 neuron. Right panel, expanded view of the boxed area. B, Simultaneous records of a layer 4 and a layer 3 neuron during photos mula on of RSC afferents. As in A, layer 4 neurons responded with a delay. Right panel, expanded view of the boxed area. C, Latencies of synap c ac va on indicated by the do ed lines in A, B (ATN s mula on, n = 4 cells; RSC s mula on, n = 1). EPSP onset and AP peak from layer 3 neurons (n = 5). Do ed lines link to the EPSP onset in simultaneously recorded layer 4 neurons (n = 5). p < 0.05, Kruskal-Wallis mul ple comparison test. D, Biocy n labeled layer 3 and layer 4 pyramidal neurons, in a presubicular slice containing thalamic (green) and retrosplenial (red) axons. The apical dendrite of one layer 4 neuron makes a U-turn (arrowhead), away from layer 3, where the thalamic axons ramify. The neighboring layer 4 neuron's apical dendrite crosses the thalamo-recipient layer 3 for a short distance before arborizing outside of ATN targeted area, towards the subiculum, on the right. Another biocy n filled layer 4 neuron's dendrites extend toward the deep layers. Scale bar, 100µm. E, Simultaneous records from a layer 3 and a layer 4 cell to ATN input s mula on (top, 0.25 mW, blue light), ATN and RSC input s mula on (middle, 0.25 mW blue and 0.25 mW red light; light intensi es compa ble with independent photos mula on) or non-specific ATN and RSC input s mula on (bo om, 3.25 mW blue and 0.25 mW red light). ATN fibers expressed Chronos-GFP (green) and RSC fibers expressed Chrimson-tdTomato (red). F, Top, oEPSPs and bursts of ac on poten als in a layer 4 neuron, evoked by dual wavelength s mula on of ATN and RSC afferents at 20 Hz. Amplifica ons of dual oEPSPs led to firing. Bo om, the NMDA receptor antagonist APV (100 µM) reduced EPSP amplifica on and prevented ac on poten al firing. G. Layer 4 burs ng neurons are sensi ve to the acetylcholine receptor agonist carbachol (10 µM). Ac on poten al firing in response to step current injec ons in control (black), in the presence of carbachol (grey), and a er wash-out (black). Bo om graph, membrane poten al depolariza on during a 2-minute carbachol applica on. The number of ac on poten als increased during the depolarizing steps (i) and on the baseline (ii). See also Figure 7-figure supplement 1.

Figure 1

 1 Figure 1 with 1 supplement. ATN and RSC send strong axonal projec ons to the superficial layers of dorsal Presubiculum A, Retrograde labeling of cor cal and subcor cal regions projec ng to the presubiculum (PrS) with retrobeads. B, Ipsilateral anterodorsal and anteroventral thalamic nuclei are labeled with beads. C, Ipsilateral granular (gRSC) and dysgranular (dRSC) retrosplenial cortex labeling. Scale bars B, C, 200 µm. D, Anterograde labeling of thalamic and retrosplenial projec ons to PrS with AAV-Chronos-GFP.E, F, Injec on sites in ATN (E) and RSC (F). AD: anterodorsal thalamic nucleus, AV: anteroventral thalamic nucleus, CA1: field of the hippocampus, cc: corpus callosum, cg: cingulum, CPu: caudate putamen, DG: dentate gyrus, MD: thalamic medial dorsal nucleus,

Figure 1 -

 1 figure supplement 1. Brain regions providing input to the Presubiculum. Semi-quan ta ve es mate of retrobeads labelling in serial coronal slices following injec on in the presubiculum. Color intensity represents beads density (mouse #69).

Figure 2

 2 with 2 supplements. Responses of layer 3 presubicular cells to photoac va on of ATN and RSC fibers. A, Expression of AAV5-Chronos in ATN or RSC. B, Biocy n labeled layer 3 cells (white) and GFP posi ve axons (green) from the ATN or RSC. Scale bar 10 µm. C, Firing pa ern of two layer 3 cells receiving inputs from the ATN (cerulean) and the RSC (purple). Insets show current commands. D, Cluster analysis of physiological parameters for cells tested by s mula ng ATN or RSC fibers. E, Representa ve EPSCs evoked in layer 3 cells by light s mula on (blue bar) of ATN or RSC inputs. Right, propor on of cells receiving ATN or RSC inputs. F, Average amplitudes of ATN and RSC induced synap c currents. G, Latency of EPSCs evoked by light-s mula on of ATN (n = 24 cells) or RSC fibers (n = 27 cells). H, EPSPs induced in layer 3 cells (single traces) by s mula ng ATN or RSC inputs in absence and presence of 1 µM TTX and 100 µM 4-AP. I, EPSCs induced by s mula ng ATN or RSC fibers in the absence and presence of 100 µM APV and APV + 10 µM NBQX. Holding poten al +40 mV. J, Voltage-clamp responses of layer 3 cells to 20 Hz train s mula ons of ATN (le ) and RSC (right) inputs. Insets show EPSCs in response to the first two s muli. K, oEPSC amplitudes for 10 ATN or RSC fiber s muli at 20 Hz. n = 15 neurons. Short middle line, mean; Min to max and quar les. L, Paired-pulse ra o (PPR) and 10/1 ra o (ra o between 10 th and 1 st event amplitudes) for ATN or RSC inputs. Wilcoxon matched-pairs signed rank test: ATN PPR vs 10/1 **** p < 0.0001, RSC PPR vs 10/1 *** p = 0.0001. M, Current clamp traces showing ac on poten als and EPSPs evoked by 10 s muli at 20 Hz. N, Spiking probability during trains of 10 s muli. ATN, n = 6, RSC, n = 12. Full line, median; short line, mean; Min to max and quar les. In K and N, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 from Friedman's test followed by Dunn's post-hoc test. O, oEPSP amplitudes for trains of 5 s muli at 20 Hz. See also Figure 2-figure supplements 1 and 2.

Figure 2 -

 2 figure supplement 1. Table showing the intrinsic electrophysiological physiological proper es of layer 3 cells tested for responses to s mula ng ATN or RSC fibers. Figure 2-figure supplement 2. Comparison of evoked synap c events in presubicular layer 3 neurons following photos mula ons of ATN or RSC axons.

Figure 3

 3 with 1 supplement. ATN and RSC axons converge in dorsal presubiculum and contact single layer 3 pyramidal neurons. A, Expression of the blue light sensi ve opsin Chronos in ATN and the red light sensi ve opsin Chrimson in RSC. B, Axons from ATN (GFP labeled, green) and RSC (tdTomato labeled, red) overlap in layer 1 and 3 of dorsal presubiculum. Layer 2 possesses patches of axon-dense and axon-poor zones. RSC fibers avoid axon-dense microstructures formed by ATN fibers in upper layer 3. Scale bar, 200 µm. C, Independent dual wavelength optogene c s mula on of light sensi ve afferent fibers in presubicular slices. D, Two biocy n labeled PrS layer 3 pyramidal cells surrounded by ATN (green) and RSC (red) axons. E, Patch clamp recording from a layer 3 neuron shows op cal EPSCs following photos mula on of ATN axons (blue light) and RSC axons (red light). F, 76% of layer 3 pyramidal neurons tested (n = 17) received both ATN and RSC input. G, Distribu ons of puta ve synap c contacts from ATN (green) and RSC (red) on the dendrites of three layer 3 neurons. Scale bar 50 µm. The boxed area on Cell 2 is shown in panel I. H, Normalized number of green and red spots for 6 neurons as a func on of the distance from soma. Paired values are indicated by do ed lines for the 3 cells in G. I, Examples of ATN-labeled (le ), RSC-labeled (middle) and both (right) synapses closely apposed to dendrites of a biocy n-filled layer 3 pyramidal cell. Scale bar 20 μm. Insets show representa ve high-magnifica on images. Scale bar 2 µm. See also Figure 3-figure supplement 1.

Figure 3 -

 3 figure supplement 1. Calibra on of blue and red light s mula on of Chronos and Chrimson.

Figure 4 .

 4 Figure 4. Supralinear summa on of EPSPs and ac on poten al firing following photos mula on of ATN and RSC axons. A, Op cal EPSPs in layer 3 neurons in response to blue light ac va on of ATN axons (cerulean traces), red light ac va on of RSC axons (purple traces). Supralinear summa on of EPSPs following coincident ac va on of both ATN and RSC axons (black traces). The pink broken line indicates the calculated linear sum of oEPSPs evoked by s mula on of either ATN or RSC axons. Records from cell 1, Fig 3D. B, Amplitudes of dual ATN and RSC oEPSPs plo ed as a func on of the sum of separate ATN and RSC s mula ons (le ). Each circle is a cell (n=11), Pink line (± 10%) indicates linearity. Pie charts give the number of tested layer 3 neurons with supralinear (grey), linear (pink) or sublinear (white) summa on. oEPSPs normalized to linear sum (bo om right). Solid circle, Chronos in ATN/Chrimson in RSC, empty circle, Chronos in RSC/Chrimson in ATN. * p < 0.05, ** p < 0.01, from Wilcoxon test. C, As in B, for dual ATN and RSC oEPSP integrals. D, oEPSPs induced by 20 Hz s mula on of ATN (cerulean), RSC (purple) or both (black). The pink broken line indicates the calculated linear sum of oEPSPs evoked by separate ATN and RSC photos mula on. E, Amplitudes of dual oEPSPs compared to those of the sum of ATN and RSC oEPSPs show that supralinearity increases across 5 s mula ons. Two-way ANOVA, p = 0.0117. * p < 0.05, **** p < 0.0001, Šidák's mul ple comparison test. # p < 0.05, ## p < 0.01, Friedman's and post-hoc Dunn's test.

  or RSC (purple) inputs. H, oEPSC latencies in layer 3 and layer 4 cells, for ATN inputs (le , cerulean), or RSC inputs (right, purple). Each dot is a cell. Same layer 4 cells are indicated by connec ng lines, to show the difference in latency for direct and indirect synap c responses. I, oEPSPs in layer 4 neurons in response to s mula on of ATN (cerulean) or RSC (purple) inputs in control and in the presence of TTX (1 µM) and 4-AP (100 µM). Dashed lines indicate the ming of the large disynap c component of the responses (i, indirect), and the small monosynap c response (d, direct), isolated in TTX-4AP. See also Figure 6-figure supplements 1 and 2.

Figure 6 -

 6 figure supplement 1. Apical dendrites of presubicular layer 4 neurons avoid the thalamorecipient layer 3. A, Retrograde AAV2retro-tdTom was injected in LMN. B, PrS L4 neuron cell bodies and dendrites were labeled. Scale bar, 50 µm. Full arrowheads, L4 neurons apical dendrites crossing L3; Empty arrowheads, L4 neurons apical dendrites skir ng L3. C, Anterograde AAV5-Chronos-GFP was injected in ATN to label thalamic axons, delimi ng PrS layer 3. D, Examples of biocy n filled layer 4 presubicular neurons. (1) The apical dendrite of this neuron avoided the densely packed ATN afferents and it didn't show responses to thalamic s mula ons. (2) This neuron's apical dendrite crossed trough a short stretch of layer 3 containing ATN afferents, and it responded with low amplitude indirect events. (3) The apical dendrite of this neuron crossed layer 3 and the zone containing ATN afferents, and was directly recruited. Scale bar 10µm. E, AAV-Chronos-GFP injected in ATN, and AAV-Chrimson-tdTom injected in RSC. F, Image from Figure 7 panel D. Scale bar 100µm. G, (Le ) Enlarged view of do ed area in A, with two labelled L4 neurons. The apical dendrite of top neuron makes a U-turn to ramify in deep layers and seems to cross path with the axon of the bo om neuron, poten ally forming a synap c contact. (Right) The dendrite of the top neuron and axon of bo om neuron are overlayed with pink and green lines respec vely. Scale bar 20µm.

Figure 6 -

 6 figure supplement 2. Electrophysiological passive and ac ve intrinsic proper es of layer 3 vs. layer 4 neurons. Data for layer 3 are summarized in Figure 2-figure supplement 1 and are shown here for comparison. Sta s cal differences are indicated as levels of p values (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) obtained from Mann-Whitney tests between layer 4 (n = 19) and layer 3 (n = 33) for each parameter. Box plots indicate maximum and minimum values, quar les, full line is the median, short line is the mean.

Figure 7

 7 with 1 supplement.

Figure 7 -

 7 figure supplement 1. Regular firing layer 3 vs. intrinsically burs ng layer 4 neurons responded to high-intensity light s mula ons of ATN or RSC afferents. Layer 3 neurons respond with single spikes following each light s mula on while layer 4 neurons respond with bursts of ac on poten als at the beginning of the s mula on train.

Acknowledgements

This work is supported by the Centre Na onal de la Recherche Scien fique and the Université Paris Cité. DF received funding from the Agence Na onale de la Recherche (ANR-DFG Program, ANR-18-CE92-0051 BURST), from the ERA-NET NEURON Program (ANR-20-NEUR-0005 VELOSO), and the FLAG-ERA HBP Program (ANR-21-HBPR-0002 VIPa ract). This work has benefited from support by the BioMedTech Facili es at Université Paris Cité (Ins tut Na onal de la Santé et de la Recherche Médicale Unité S36/Unité Mixte de Service 2009). We thank Dr. Li-Wen Huang for help with analysis rou nes. We thank Dr. Kate Jeffery and the Royal Society COST-share program, technical assistance from Fabrice Licata from the UPC Microscopy plateform, Claire Lovo from ICM Quant, Dr. Boris Lamo e d'Incamps, and Dr. Richard Miles for helpful comments on the manuscript.

Author contributions

Declaration of Interests

The authors declare no compe ng financial interests.