Title: Editor's evaluation: Telencephalic outputs from the medial entorhinal cortex are copied directly to the hippocampus
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Complementary actions of the neocortex and the hippocampus enable encoding and long-term storage of experience dependent memories. Standard models for memory storage assume that sensory signals reach the hippocampus from superficial layers of the entorhinal cortex (EC). Deep layers of the EC on the other hand relay hippocampal outputs to the telencephalic structures including many parts of the neocortex. Here, we show that cells in layer 5a of the medial EC send a copy of their telencephalic outputs back to the CA1 region of the hippocampus. Combining cell-type-specific anatomical tracing with high-throughput RNA-sequencing based projection mapping and optogenetics aided circuit mapping, we show that in the mouse brain these projections have a unique topography and target hippocampal pyramidal cells and interneurons. Our results suggest that projections of deep medial EC neurons are anatomically configured to influence the hippocampus and neocortex simultaneously and therefore lead to novel hypotheses on the functional role of the deep EC. Editor's evaluation In this work, the authors combine a variety of novel circuit mapping techniques to characterize a novel projection pathway from layer 5a neurons in the medial entorhinal cortex to region CA1 of the hippocampus. By utilizing cell specific viral labelling techniques, RNA sequencing based projection mapping, and optogenetic-aided in vitro physiology, the authors show evidence that the same neurons in layer 5a of medial entorhinal cortex project to both cortical areas of the telencephalon and the hippocampus. This work raises the possibility that deep layers of the entorhinal cortex coordinate hippocampal-cortical interactions. The manuscript will be of interest to readers in the field of anatomy and hippocampal physiology. https://doi.org/10.7554/eLife.73162.sa0 Decision letter eLife's review process Introduction Interplay between the hippocampus and the neocortex is a central tenet of theories for systems memory consolidation (Marr, 1971; McClelland et al., 1995). The entorhinal cortex (EC) mediates the two-way communication between the neocortex and the hippocampus. Its superficial and deep layers, respectively, channel inputs to and outputs from the hippocampus. During a new experience the superficial layers relay convergent inputs from the cortex to the hippocampus where initial conjunctive representations of everyday experiences are generated. Subsequently during rest or sleep, hippocampal output is redistributed to the brain (Squire et al., 2015; Frankland and Bontempi, 2005). Leading theoretical models consider the deep layers of EC as a relay of hippocampal output that reinstates neocortical activation patterns representing memories, enabling neocortex-wide synaptic changes required for long-term storage (Koster et al., 2018; Kumaran et al., 2016; McClelland et al., 1995; Schapiro et al., 2017). Recent discoveries revealed complex and distinct input–output interactions of molecularly defined subtypes of neurons in the deep layers of medial EC (MEC), suggesting their functions extend beyond passing on hippocampal outputs. Layers 5a (L5a) and 5b (L5b) of the EC are genetically distinct with differential input–output organizations. L5a but not L5b is the sole origin of the long-range telencephalic outputs of the EC (Sürmeli et al., 2015), whereas L5b, but not L5a, is directly influenced by superficial layers of EC (Beed et al., 2020; Sürmeli et al., 2015). Inputs from the dorsal hippocampus preferentially target L5b over L5a (Rozov et al., 2020; Sürmeli et al., 2015; Wozny et al., 2018) whereas ventral hippocampus preferentially targets L5a (Rozov et al., 2020). Moreover, the capacity of deep layers to integrate neocortical inputs with hippocampal output (Czajkowski et al., 2013; Beed et al., 2020; Sürmeli et al., 2015), suggest more complex roles. An intriguing possibility, noted in experiments with classic retrograde tracers, is that deep layers of MEC might also project to the hippocampal CA1 region, which provides the deep EC with hippocampal output (Köhler, 1985; Witter and Amaral, 1991). Such recurrent connectivity between CA1 and the deep MEC could be important in coordinating hippocampal–entorhinal activity during memory consolidation (Ólafsdóttir et al., 2016), provide a shortcut for hippocampal outputs to re-enter the hippocampus (Kumaran and McClelland, 2012) or have a more global coordination function encompassing the hippocampus and the neocortex (Khodagholy et al., 2017). Despite its potential functional significance, the key properties of this projection pathway including whether it shares the same source with the telencephalon projections and which neurons it targets in the hippocampus remain unknown. Here we reveal that, neurons in L5a, but not L5b, of the MEC project to hippocampal CA1. We establish that the telencephalon-projecting neurons copy their outputs directly to pyramidal and subclasses of interneuron populations in CA1. Our data define a novel anatomical framework for layer 5a of the MEC to coordinate neocortical and hippocampal networks. Results To find out whether neurons in either L5a or L5b project to the hippocampus we injected retrograde adeno-associated viral vectors (AAVs) expressing green fluorescent protein (GFP) or mCherry into intermediate hippocampus (Figure 1A, B; Figure 1—figure supplement 1A,B). As well as finding retrogradely labelled cell bodies in layer 3 of MEC, which contains neurons that are well established as a critical input to stratum lacunosum-moleculare of CA1 (Naber et al., 2001; Kitamura et al., 2014), we also found labelled cell bodies in L5a, which we identified by immunostaining against the ETS variant transcription factor-1 (Etv1) protein. Retrogradely labelled neurons were not found in L5b. Figure 1 with 2 supplements see all Download asset Open asset L5a but not layer 5b of the medial entorhinal cortex (MEC) projects to the hippocampus. (A) Schematic showing the injection site of a retrograde adeno-associated viral vector (AAV) in the intermediate hippocampus (light grey) of wild-type mice. The black outline in the horizontal plane roughly corresponds to the dorso-ventral position of the horizontal section shown in B. Corresponding injection site image is shown in Figure 1—figure supplement 1A. (B) Immunostained brain section in the horizontal plane showing retrogradely labelled neurons in L3 and L5a of the MEC. ETS variant transcription factor-1 (Etv1) expression marks L5a (scale bar: 250 μm). Inset: high-magnification image of boxed region in left panel showing co-expression of green fluorescent protein (GFP) and Etv1 (scale bar: 50 μm). PaS, parasubiculum; P, posterior; A, anterior; M, medial; L, lateral. (C) Schematic showing the injection site of an AAV in the deep MEC (dark grey) of Rbp4-Cre mice for Cre-dependent expression of GFP. The black outline in the horizontal plane roughly corresponds to the dorso-ventral position of the slice shown in D. (D) Entorhinal area on an immunostained brain section in the horizontal plane showing the overlapping expression of GFP with Etv1 (scale bar: 250 μm). Inset: high magnification of boxed region in middle panel showing GFP and Etv1 expression confined mainly to L5a in MEC (scale bar: 100 μm). To further investigate this projection, we identified and validated a transgenic mouse line that enables specific genetic access to L5a. In this mouse line, Cre expression is driven by the promoter of the retinol binding protein four gene (Rbp4-Cre; Gerfen et al., 2013). Following injection of a Cre-dependent AAV expressing GFP or mCherry into the deep MEC of Rbp4-Cre mice, fluorescent signal was detected mainly in L5a (Figure 1C, D and Figure 1—figure supplement 1C). At the centre of the injection site, where labelling was densest, the majority of neurons in L5a (71.3% ± 2.2 %, n = 3 mice, 180 neurons) were found to be fluorescently labelled (Figure 1D). Labelled neurons were non-GABAergic (99.5% ± 0.3%, n = 3 mice, 1137 cells; Figure 1—figure supplement 1D), most of which expressed Etv1 (88.6% ± 3.1 %, n = 3 mice, 1866 cells; Figure 1D). This is similar to the proportion of Etv1 expression in all L5a neurons (Sürmeli et al., 2015) and also in retrogradely labelled subpopulations of L5a neurons from the hippocampus and from cortical and subcortical target areas (Figure 1—figure supplement 1E–H). Moreover, axons of L5a neurons labelled in the Rbp4:Cre line were found in the telencephalon (Figure 1—figure supplement 2). Thus, we established that the Rbp4-Cre line provides unbiased genetic access to excitatory projection neurons in L5a. To what extent does the projection from L5a to the hippocampus differ from previously identified projections arising from superficial layers of MEC? In contrast to projections from superficial layers 2 and 3, which, respectively, target stratum lacunosum (SL) and stratum moleculare (SM) of CA1 (Kitamura et al., 2014), axons from L5a neurons were primarily found in deep stratum pyramidale (SP) and SL, but were sparse in SM (Figure 2A–D and Figure 2—figure supplement 1A). Axons from L5a also differed in their proximo-distal organization within CA1. Whereas projections from superficial layers of MEC favour proximal CA1 (Tamamaki and Nojyo, 1995), axons from L5a had highest density in distal CA1 (paired one-tailed Wilcoxon signed rank test, p = 0.0312, n = 5 mice; Figure 2E–H and Figure 2—figure supplement 1B–F) and the subiculum (Figure 2I and Figure 2—figure supplement 1G). We observed the same topography when axons and axon terminals of L5a neurons were labelled with membrane-bound GFP and fluorescently tagged presynaptic proteins (synaptophysin), confirming that the topography is not a result of labelling of passing axons (Figure 2J–O). This distinct topography suggests a unique functional role for the projections from L5a of MEC to CA1. Figure 2 with 1 supplement see all Download asset Open asset Projections to CA1 of L5a neurons of the medial entorhinal cortex (MEC) have distinct topography. (A) Schematic showing the injection strategy of a Cre-dependent virus in the deep MEC of Rbp4-Cre mice. (B) Immunostained coronal brain section showing the distribution of axons of MEC L5a cells in CA1 (scale bar: 500 μm). Inset: high-magnification image of boxed region in left panel (scale bar: 100 μm). Bottom-left schematic indicates the antero-posterior position of the slice. Corresponding injection site image is shown in Figure 2—figure supplement 1A. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SL, stratum lacunosum; SM, stratum moleculare; D, dorsal; V, ventral; M, medial; L, lateral. (C) Quantification of fluorescence intensity of green fluorescent protein (GFP) across the layers of CA1. The area between 0 and 20 in (B) was divided into 20 equally sized bins, and the mean fluorescence intensity was calculated for each bin. Values shown are normalized mean fluorescence intensity values (n = 6 mice, 2 sections per brain). (D) Normalized mean fluorescence density values in deep versus superficial SP (n = 6 mice). Within SP, axon density in deep SP was significantly higher than density in superficial SP (paired one-tailed Wilcoxon signed rank test, p = 0.0156, n = 6). (E) Schematic showing the injection strategy of a Cre-dependent virus in the deep MEC of Rbp4-Cre mice. The black outline in the horizontal plane roughly corresponds to the dorso-ventral position of the horizontal slice shown in (F).( F) Immunostaining of a horizontal brain section showing the distribution of axons of MEC L5a cells in the subiculum (Sub), distal CA1 (dCA1), and proximal CA1 (pCA1; scale bar: 200 μm). Corresponding injection site image is shown in Figure 2—figure supplement 1B. (G) High-magnification images of the pyramidal cell layer in distal (top) and proximal (bottom) CA1 showing axonal labelling (scale bar: 20 μm). (H) Normalized mean fluorescence density values in distal versus proximal CA1 (n = 5 mice). (I) High-magnification image of the subiculum showing axonal labelling (scale bar: 20 μm). (J) Schematic showing the injection strategy of an adeno-associated viral vector (AAV) for Cre-dependent expression of a membrane-bound form of GFP and a synaptophysin–mRuby conjugate in L5a of the MEC in the Rbp4-Cre mice. The black outline in the horizontal plane roughly corresponds to the dorso-ventral position of the slice shown in (K). (K) Immunostained brain section in the horizontal plane showing the distribution of axons (green) and synaptic terminals (red) in the hippocampus as well as the parasubiculum (PaS) and perirhinal cortex (PRh). Injection site in the MEC is distinguishable by the strong fluorescent labelling of L5a cell bodies (scale bar: 400 μm). (L) High-magnification image of the injection site in the MEC showing cell membrane and synaptic labelling specifically in L5a (scale bar: 20 μm). (M) High-magnification image of the subiculum showing axonal and synaptic labelling (scale bar: 20 μm). High-magnification images of the pyramidal cell layer in distal (N) and proximal (O) CA1 showing axonal and synaptic labelling (scale bar: 20 μm). Our findings suggest two intriguing possibilities for the organization of output neurons in L5a of the MEC. The hippocampus-projecting neurons in L5a may be distinct from the telencephalon-projecting neurons, implying separate processing within the deep MEC layers of signals to the hippocampus and telencephalon. Alternatively, the same neurons in L5a may project to both the telencephalon and the hippocampus, implying that telencephalic outputs from MEC are copied back to the hippocampus. To distinguish between these possibilities, we used a combinatorial viral labelling strategy. We restricted reporter gene expression to subpopulations of L5a cells that project to specific targets by injecting a retrograde AAV expressing Cre recombinase in either the retrosplenial cortex (RSC) or nucleus accumbens (NucAcb), and a Cre-dependent fluorescent reporter virus into the MEC (Figure 3A, B and Figure 3—figure supplement 1). With this approach we detected in CA1 fluorescently labelled axons originating from both RSC- and NucAcb-projecting L5a neurons (Figure 3C, D). The axon distribution across layers was similar to when L5a neurons were labelled in bulk using the Rbp4-Cre mouse line (Figure 3E, F). These results establish the principle that axon projections from L5a of MEC to the hippocampus are collaterals of projections to telencephalic targets. However, it is unclear from these experiments whether this principle applies to projections from L5a to all of its many telencephalic targets. Figure 3 with 1 supplement see all Download asset Open asset Retrosplenial cortex (RSC) and nucleus accumbens (NucAcb) outputs of the medial entorhinal cortex (MEC) are copied to CA1. Experimental strategy. In wild-type mice, a retrogradely transported virus expressing Cre was injected in either the RSC (A) or NucAcb (B), while a Cre-dependent reporter virus was injected in the deep MEC. Hippocampal axon collaterals of RSC- (C) or NucAcb-projecting MEC neurons (D; scale bar: 250 μm). Insets: high-magnification images from CA1 showing distribution of axons across CA1 layers (scale bar: 100 μm). Bottom-left schematic indicates the antero-posterior position of the slice. Corresponding injection site images are shown in Figure 3—figure supplement 1A, B. (E, F) Quantification of mean fluorescence intensity across the layers of CA1 when only RSC- (E) or NucAcb-projecting (F) MEC neurons were labelled (n = 3 mice for each group). Axon topography is similar to when L5a neurons are marked globally in the Rbp4-Cre mice (Figure 2C). EC outputs have diverse targets, including the entire cortical mantle as well as parts of basal ganglia and amygdala (Swanson and Köhler, 1986; Sürmeli et al., 2015). To test whether axon collateralization to the hippocampus is a general feature of all telencephalon-targeting neurons, we used Multiplex Analysis of Projections by Sequencing (MAPseq; Kebschull et al., 2016). MAPseq is a high-throughput alternative to single neuron anterograde tracing. In MAPseq, instead of multiple copies of a virus carrying the same genetic material, a virus library is utilized where each virus facilitates expression of a unique 30-nucleotide RNA particle. The vast diversity of unique RNA sequences and relatively small number of neurons infected allows each neuron to be marked with a unique RNA barcode (Kebschull et al., 2016). Because the barcodes are actively transported to axon terminals, their detection can be used to establish connectivity to putative target structures; if a neuron projects to multiple target structures then its barcode should be detected in each one. We injected the MAPseq barcode RNA virus library into the full dorso-ventral extent of the deep MEC (Figure 4—figure supplement 1A) and quantified barcode RNA expression in tissue collected from dorsal and ventral MEC (dMEC, vMEC), dorsal and ventral hippocampus (dHip, vHip), isocortex, cerebral nuclei (CNU), olfactory areas and cortical subplate (Olf/Ctxsp) as well as brainstem and spinal cord as negative controls (see Materials and methods for detailed description and controls, Figure 4A). The majority of barcodes that were detected in any one of the three target divisions (isocortex, CNU, or Olf/Ctxsp) were also detected in the hippocampus, suggesting that collateral axons to the hippocampus are a common feature of telencephalon-projecting neurons (Figure 4B). The results were similar regardless of whether neurons were located in dorsal or ventral MEC (Figure 4—figure supplement 1B), suggesting that collateralization does not depend on the neuron's position in the dorso-ventral axis of the MEC. Using barcode counts as a measure of projection strength (Kebschull et al., 2016) we further found that dorsal MEC neurons preferentially project to dorsal aspects of the hippocampus and ventral MEC neurons to the ventral aspects, indicating that the back-projections to the hippocampus are topographically organized (Figure 4C). Together these results reveal general organizing principles by which all projections from MEC to the telencephalon are copied back to the hippocampus. Figure 4 with 1 supplement see all Download asset Open asset Telencephalic outputs are widely copied onto the hippocampus. (A) Experimental strategy. A MAPseq virus library was injected into deep medial entorhinal cortex (MEC; n = 3 mice). Forty-four hours later, mice were sacrificed and brains were serially sectioned in the sagittal plane at 400 μm thickness. Tissue from five major divisions (isocortex, CNU, Olf/Ctxsp, dHip, and vHip), as well as dorsal and ventral MEC were then dissected and collected in tubes. Brainstem and spinal cord tissue was collected as a negative control. Tissue was further processed for RNA extraction and next generation sequencing. (B) Nearly all barcodes detected in CNU, Olf/Ctxsp, and isocortex were also detected in the hippocampus (isocortex: 98.6% ± 0.06%; CNU: 99.2% ± 0.08%; Olf/Ctxsp: 99.0% ± 0.4%; n = 3 mice, 1603, 3493, and 1677 barcodes from brains 1, 2, and 3, respectively). (C) Dorso-ventral topography of projections revealed by relative barcode counts. For each barcode, counts were normalized to show the relative projection strength of each neuron to dorsal and ventral hippocampus (n = 3 mice). Which cell types within the hippocampus receive signals from L5a of MEC? While the compartmentalized arrangement of axons from L5a of MEC suggests selective targeting of CA1 layers, axonal topography does not necessarily reflect functional connectivity. Therefore, we targeted viral vectors expressing a channelrhodopsin2–mCherry conjugate to L5a neurons in MEC and tested for connectivity using whole-cell ex vivo patch-clamp recordings from CA1 neurons. We focused initially on responses of pyramidal cells. Brief light pulses evoked depolarizing subthreshold postsynaptic potentials (PSPs) in 64% of SP pyramidal neurons recorded at their resting membrane potential (n = 64 cells, 27 mice, Figure 5A–C and Figure 5—figure supplement 1E). The response probability of neurons in distal CA1 was not significantly different than neurons in proximal CA1 (Figure 5D). Although the vast majority of axons occupy the deep portion of the SP we found no significant difference in the response probability of neurons in deep versus superficial SP (Figure 5D). Thus, rather than selecting subtypes of pyramidal neurons axons might target distinct compartments of neurons according to their depth in SP. Figure 5 with 1 supplement see all Download asset Open asset L5a neurons of the medial entorhinal cortex (MEC) provide direct excitatory and indirect inhibitory inputs to pyramidal cells in CA1. (A) Experimental design showing viral expression, placement of patch-clamp electrode, and light delivery over CA1. (B) Examples of biocytin filled pyramidal neurons recorded in CA1. (C) An example electrical recording of a pyramidal neuron at rest (−67 mV) showing depolarizing responses upon 3-ms light stimulation (top) (blue line, scale bar: 1 mV, 10 ms). Train of action potentials upon 200 pA step-current injection (bottom; scale bar: 20 mV, 100 ms). (D) Proportion of responsive pyramidal neurons located in the distal and proximal halves of CA1 (top). The difference in proportions was not significant (X-squared = 1.9071, df = 1, p value = 0.167291, chi-squared test, n = 12 cells in proximal and n = 39 cells in distal CA1). Proportion of responsive pyramidal neurons located in the deep versus superficial locations of CA1 pyramidal cell layer (bottom) (X-squared = 0.2969, df = 1, p value = 0.585804, chi-squared test, n = 8 cells in deep SP and n = 13 cells in superficial SP). (E) Effects of bath application of Gabazine (orange, n = 10 cells, 9 mice) and NBQX (green, n = 5 cells) on postsynaptic potentials (PSPs) recorded from a pyramidal neuron (scale bar: 0.5 mV, 10 ms) and a summary plot of PSP amplitude measurements for all tested pyramidal neurons. Note that some neurons were only treated with Gabazine which did not cause a significant change in amplitudes (p = 0.97, two-tailed Student's t-test, n = 10 cells, 9 mice). (F) An example of ten consecutive PSP responses recorded from a single pyramidal neuron illustrates the short and invariant latency of PSPs (scale bar: 0.5 mV, 10 ms) and a cumulative probability plot of standard deviation of latencies for neurons with PSP responses that were >1 mV in amplitude (n = 20 cells). (G) Effects of bath application of tetrodotoxin (TTX; orange) and 4-aminopyridine (4-AP; green) on PSPs recorded from a pyramidal neuron (scale bar: 0.5 mV, 10 ms) and a summary plot of changes in PSP amplitudes for all tested pyramidal neurons. TTX application abolished responses (n = 5 cells, 5 mice, p = 0.01, two-tailed Student's t-test). (H, I) An example inhibitory PSP response recorded from a pyramidal neuron upon 10 Hz light stimulation (blue bars). Response polarity reversed when the neuron's membrane potential was adjusted to –50 mV and was abolished after application of Gabazine (scale bars: 0.2 mV, 100 ms). Inset shows the long latency (>10 ms) of PSP onset indicating polysynaptic connectivity. The majority of PSPs (20 out of 26) maintained their polarity when the membrane potential was adjusted from rest (Vm = −66.7 ± 0.6 mV) to −50 mV, indicating that they were glutamatergic. In these neurons, EPSPs were maintained when GABAA receptors were blocked with Gabazine, but were abolished by the AMPA receptor antagonist NBQX, confirming that they are glutamatergic (Figure 5E). The responses had short latencies (3.04 ± 0.26 ms, n = 20 cells, 15 mice) that were relatively invariant from trial to trial (Figure 5F) and were independent of the response's position within a train of stimuli, indicating that they are monosynaptic (Figure 5—figure supplement 1D). Consistent with this, responses were sensitive to bath application of tetrodotoxin (TTX) but recoverable after application of 4-aminopyridine (4-AP; Figure 5G; Petreanu et al., 2007). Together, these data demonstrate that projections from L5a of the MEC provide glutamatergic excitatory inputs directly to pyramidal neurons in proximal and distal CA1. A smaller population of pyramidal neurons showed PSPs with characteristics of indirect inhibitory connections (6 out of 26). These responses either reversed polarity when the cell's membrane potential was held above the chloride reversal potential (Figure 5H) or had an early depolarizing and a late hyperpolarizing component (Figure 5—figure supplement 1B). Application of Gabazine either completely blocked these PSPs (Figure 5I) or revealed a larger excitatory component that was sensitive to application of NBQX (Figure 5—figure supplement 1B). Thus, inputs from L5a of the MEC also recruit local interneurons that provide inhibitory input to SP pyramidal neurons. To identify which interneurons in CA1 were targets of L5a projections, we tested responses of interneurons in all layers. In SP, we distinguished interneurons from principal cells either by classifying them based on biophysical properties (fast-spiking: SPFS and non-fast spiking: SPNFS; Figure 5—figure supplement 1A and see Materials and methods), or by recording from fluorescently labelled GABAergic or parvalbumin-expressing (PV+) inhibitory neurons in double transgenic mice (Figure 6A, B and Figure 5—figure supplement 1C). Stimulation of L5a axons elicited both subthreshold and suprathreshold responses primarily in SP, SR, and SL interneurons (Figure 6C, D and Figure 5—figure supplement 1E). Depolarizing PSPs were observed in 64% of the SPFS including PV+ interneurons, 46% of the SPNFS, 64% of SR, and lower proportions of recorded neurons in SL (31%), SM (23%), and SO (16%). Except for interneurons in SO, PSPs in all layers showed characteristics of monosynaptic connectivity (Figure 6G, H, I, J and Figure 5—figure supplement 1G, H) and AMPA receptor-mediated glutamatergic synaptic transmission (Figure 6E, F and Figure 5—figure supplement 1F). Therefore, projections from L5a of MEC recruit CA1 interneurons through monosynaptic inputs driven by excitatory glutamatergic synapses. Figure 6 Download asset Open asset L5a neurons of the medial entorhinal cortex (MEC) provide direct excitatory inputs to interneurons in CA1. (A) Experimental design showing viral expression, patch pipette, and light stimulation. Interneurons are marked by the expression of venus fluorescent protein driven by the vesicular GABA transporter (Vgat) gene's promoter. (B) Biocytin filled neurons in the Rbp4-Cre X Vgat-Venus double transgenic mouse line. Fluorescent labelling facilitated patch-clamp recordings made from GABAergic interneurons and distinguishing SR, SL, and SM layers. (C) Representative examples of depolarizing responses recorded at resting membrane potential following 3ms blue light stimulation (blue line) of L5a axons in CA1 (left) (scale bar: 1 mV, 10 ms). Neurons in SO and SM were typically not responsive (<30%). Responses from SR neurons were on average larger than the responses from neurons in other layers (scale bar: 1 mV, 10 ms). Neurons' spiking response to injecting 200 pA current (right) (scale bar: 10 mV, 100 ms). (D) Proportion of responsive interneurons in all layers of CA1 recorded at resting membrane potential. Green highlighted segment corresponds to the proportion of cells with depolarizing membrane potentials; grey highlighted segment corresponds to neurons with no change in their membrane potential. SO: n = 18 cells, 10 mice; SPFSint: n = 33 cells, 8 Rbp4-Cre X Pvalb-Flp mice, 5 Rbp4-Cre X Vgat-Venus mice, 8 Rbp4-Cre mice; SPNSFint: n = 13 cells, 11 Rbp4-Cre mice, 1 Rbp4-Cre X Vgat-Venus mouse; SR: n = 14 cells, 6 mice; SL: n = 60 cells, 15 mice; SM: n = 21 cells, 10 mice. (E, F) Effects of bath application of Gabazine (orange) and NBQX (green) on postsynaptic potentials (PSPs) recorded from a fast-spiking pyramidal layer interneuron (left) (scale bar: 1 mV, 5 ms). Summary quantification of PSP amplitudes for multiple cells (right). The PSP amplitudes were largely unaffected by application of Gabazine (SPint: n = 6 cells, p = 0.99 two-tailed Student's t-test) but were largely blocked by NBQX (SPint: n = 5 cells, p = 0.03, two-tailed Student's t-test) indicating AMPA receptor-mediated glutamatergic synaptic transmission. (G, H) Effects of bath application of tetrodotoxin (TTX; orange) and 4-aminopyridine (4-AP; green) on the response amplitude PSPs recorded from pyramidal layer interneurons. Example traces from a fast-spiking pyramidal layer interneuron (scale bar: 1 mV, 5 ms). Summary plots of response amplitude measurements from multiple recordings (SPint: n = 4 cells). (I, J) An example of ten consecutive PSP responses recorded from a single fast-spiking interneuron in SP illustrates the short and invariant latency of PSPs. Cumulative probability plots of standard deviation of latencies for neurons with PSP responses that were >1 mV in amplitude (mean latency SP: 2.08 ± 0.08 ms, n = 21 cells). Discussion Together our results suggest a substantial revision to the idea that the entorhinal deep layers unidirectionally convey hippocampal output messages to the neocortex. Using two lines of evidence, combinatorial viral labelling and MAPseq, we show that L5a axons bifurcate to target both the telencephalon and the hippocampus. Our results imply that nearly all telencephalic output is copied onto the hippocampus regard