Target selectivity of septal cholinergic in the medial and lateral

Srinidhi Desikana,b,1, David E. Kosera,c,1, Angela Neitza,c,2, and Hannah Monyera,c,3

aDepartment of Clinical Neurobiology, German Cancer Research Center, 69120 Heidelberg, Germany; bFaculty of Biosciences, Heidelberg University, 69120 Heidelberg, Germany; and cDepartment of Clinical Neurobiology, Medical Faculty, Heidelberg University, 69120 Heidelberg, Germany

Edited by Nathaniel Heintz, The Rockefeller University, New York, NY, and approved January 31, 2018 (received for review September 20, 2017) The entorhinal cortex (EC) plays a pivotal role in processing and leading to deficits (23–25). The septo-entorhinal conveying spatial information to the . It has long been pathway comprises glutamatergic, GABAergic, and cholinergic known that EC neurons are modulated by cholinergic input from the projections (26). The target selectivity of septal glutamatergic medial septum. However, little is known as to how synaptic release and GABAergic projections in LII of MEC has been previously of affects the different cell types in EC. Here we studied. While the former targets pyramidal cells (PCs) and fast- combined optogenetics and patch-clamp recordings to study the spiking (FS) interneurons (27), the latter selectively inhibits in- effect of cholinergic axon stimulation on distinct neurons in EC. We terneurons in LII of MEC (13, 28). Selective lesions of septal found dense cholinergic innervations that terminate in layer I and II cholinergic neurons or their optogenetic activation have in- (LI and LII). Light-activated stimulation of septal cholinergic projec- dicated that acetylcholine (ACh) plays an important role in tions revealed differential responses in excitatory and inhibitory regulating theta rhythmic activity in the hippocampus, thereby neurons in LI and LII of both medial and lateral EC. We observed augmenting the dynamics of memory encoding (29–32). Opto- depolarizing responses mediated by nicotinic and muscarinic recep- genetic activation of septal cholinergic fibers in the CA1 region tors primarily in putative serotonin receptor (p5HT3R)-expressing in- of the hippocampus evoked responses in both PCs and in a va- terneurons. Hyperpolarizing muscarinic receptor-mediated responses riety of interneurons (33–35), but the identity of cholinergic MS/ were found predominantly in excitatory cells. Additionally, some DBB target cells in EC has remained elusive. In vitro investi- excitatory as well as a higher fraction of inhibitory neurons received gations using bath application of cholinergic receptor agonists mono- and/or polysynaptic GABAergic inputs, revealing that medial (36, 37) gave first indications as to differential effects of ACh in septum cholinergic neurons have the capacity to corelease GABA alongside acetylcholine. Notably, the synaptic effects of acetylcho- EC neurons. However, technical limitations precluded specific line were similar in neurons of both medial and lateral EC. Taken axonal activation that is nowadays offered by virus-supported together, our findings demonstrate that EC activity may be differen- optogenetics, enabling both clear-cut identification of source tially modulated via the activation or the suppression of distinct cells and mimicking of endogenous release. subsets of LI and LII neurons by the septal cholinergic system. Here we analyzed the septal cholinergic projections to MEC and LEC, focusing on the identification of target cells and char- medial septum | optogenetics | nicotinic receptor | muscarinic receptor | acterization of optogenetically induced responses. We character- corelease ized the projections based on antero- and retrograde tracing. Following virus-mediated ChR2-mCherry expression in cholinergic he superficial layers of entorhinal cortex (EC) play a quin- MS/DBB neurons, we electrophysiologically studied cholinergic Ttessential role in processing and conveying sensory in- formation from the neocortex to the hippocampus via the Significance perforant and temporoammonic pathways (1, 2). Commonly, EC is subdivided into medial EC (MEC) and lateral EC (LEC), Acetylcholine is a key modulator of hippocampal and entorhinal which display both functional and organizational differences (3– cortex (EC) function. The majority of cholinergic projections 5). MEC conveys mostly spatial information (6, 7), while LEC targeting these structures originate in the basal forebrain com- mediates nonspatial inputs to the hippocampus (8–10). Based on plex, specifically the medial septum. Many studies focused on anatomical and electrophysiological studies, distinct principal cell the behavioral effects involving these projections, but there still types have been described in layer II (LII) of MEC and LEC (11– is a paucity regarding their connectivity in the target area. Here 14). These principal cell types, together with local interneurons, we provide this missing link. By combining optogenetics with constitute the complex local microcircuit in LII of EC (13, 15), whole-cell recordings in superficial EC layers, we identified the which received increasing attention ever since the discovery of grid synaptic target cells of septal cholinergic neurons. This level of cells and other spatially tuned cells in the superficial layers of analysis is an important step toward a better understanding of MEC (6, 7). In vivo recordings from this area in freely moving the modulatory action of acetylcholine in EC in vivo. animals reveals that the most striking rhythmic activity is theta – Author contributions: H.M. conceived the research; S.D. and D.E.K. designed and per- oscillations (4 12 Hz) (16), which is a prerequisite for the tem- formed the experiments; A.N. performed pilot experiments; D.E.K. wrote the MATLAB poral organization of neurons within local networks. Of note, scripts; S.D. and D.E.K. analyzed the data; and S.D., D.E.K., and H.M. wrote the manuscript. MEC exhibits stronger theta rhythmicity than LEC, and so far, it The authors declare no conflict of interest. appears that in contrast to MEC, neurons in LEC are only weakly This article is a PNAS Direct Submission. modulated by theta (17). Although local networks can support the This open access article is distributed under Creative Commons Attribution-NonCommercial- generation and maintenance of theta oscillations (18, 19), the NoDerivatives License 4.0 (CC BY-NC-ND). main source for theta oscillations in EC is the medial septum/ 1S.D. and D.E.K. contributed equally to this work. – vertical limb of the (MS/DBB) (19 21). 2Present address: Quality Department Spiriva, Böhringer Ingelheim, 55216 Ingelheim am It has been known for decades that MS/DBB, which is a part of Rhein, Germany. the basal forebrain complex, is the main external pacemaker of 3To whom correspondence should be addressed. Email: [email protected]. hippocampal-EC theta rhythm, thereby coordinating several This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. distant brain areas (19, 22). Pharmacological inactivation of MS 1073/pnas.1716531115/-/DCSupplemental. strongly reduces theta oscillations and grid cell firing in MEC, Published online February 27, 2018.

E2644–E2652 | PNAS | vol. 115 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1716531115 Downloaded by guest on October 3, 2021 responses in identified neurons in acute slices and demonstrate from 4 mice). We found that mCherry was virtually exclusive in PNAS PLUS that they activate or suppress distinct subsets of LI/LII neurons. MS/DBB cholinergic neurons, as 98.92 ± 0.22% of the cells + Measurements in MEC and LEC were directed toward answering positive for mCherry expression were also ChAT (Fig. 1C, + + the question of whether common connectivity rules hold true in 573 double cells from a total of 579 mCherry cells in 12 slices the two brain areas. This indeed appears to be the case. However, from 4 mice). Furthermore, within MEC and LEC, we observed + based on our anatomical and electrophysiological data, we infer mCherry cholinergic axons predominantly in LI/LII, with fewer that cell type-specific septal cholinergic modulation is more pro- axons in LEC (Fig. 1G and Fig. S1). To corroborate the results of nounced in MEC than in LEC neurons. our anterograde tracing experiments and to estimate the per- centage of EC projecting septal cholinergic neurons, we injected Results the retrograde tracer fluorogold (FG; 0.5%) unilaterally into Septal Cholinergic Neurons Project to the MEC and LEC. To in- MEC and LEC (Fig. 1D). We found more MS/DBB cholinergic vestigate the extent of MS/DBB cholinergic projections to EC, neurons projecting to MEC (5.70 ± 0.75%) than LEC (3.37 ± + we injected AAV-DIO-ChR2-mCherry virus (in four instances 0.51%) (Fig. 1 E and F, 93 double cells from a total of 1,786 + + AAV-DIO-eGFP virus was used; for details see SI Materials and ChAT cells in 14 slices from 5 mice for MEC and 83 double + Methods) into MS/DBB of 8-wk-old male ChATCre mice (Fig. cells from a total of 2,270 ChAT cells in 15 slices from 1A). Viral expression, as indicated by mCherry labeling of cell 6 mice for LEC; P < 0.05). As indicated by vesicular acetylcholine + bodies, was observed across MS/DBB (Fig. 1B), with an infection transporter (vAChT) stainings, the mCherry axons within the EC rate of 50.76 ± 2.32% within MS/DBB cholinergic cells (Fig. 1C, can pack and release ACh (Fig. 1H). To test whether there is a + + 573 double cells from a total of 1,148 ChAT cells in 12 slices difference in synapse density, we quantified the number of vAChT

A AAV-DIO- D1 D2 G ChR2-mCherry FG in rLEC LII MEC

LI

FG in rMEC B E1 E2 LII

rMEC rLEC LEC LI H MS MS MS MEC

DBB DBB DBB LEC

Merge ChAT mCherry Merge ChAT FG Merge ChAT FG

Merge vAChT mCherry C F FG in rEC I 100 Double+/mCherry+ 10 * 0.50 * Double+/ChAT+

50 5 0.25 µm of axon No. of vAChT/ Percentage [%] Percentage [%] 0 0 0 MEC LEC MEC LEC

Fig. 1. MS/DBB cholinergic neurons project to EC. (A) Schematic representation indicating the injection site (red) in MS/DBB of a ChATCre mouse for an- terograde tracing experiments with AAV-DIO-ChR2-mCherry.(B) Maximum intensity confocal image showing selective expression of ChR2-mCherry in ChAT- + + expressing neurons upon stereotaxic virus injection into MS/DBB. Higher magnification of a representative mCherry /ChAT cell from the boxed area is shown + below. (C) Percentage of double cells relative to mCherry (gray) and ChAT (green) expression (n = 12 slices from 4 mice). (D) Schematic representation of a

horizontal section indicating the unilateral injection site into MEC (D1, blue) and LEC (D2, orange) for retrograde FG (0.5%) tracing experiments. (E) Maximum intensity confocal image of MS/DBB showing retrograde FG labeling in ChAT-expressing neurons injected unilaterally in MEC (E1)andLEC(E2). Higher + + + magnification of a representative FG and ChAT cell is shown as below. (F) Percentage of double cells relative to ChAT expressions after unilateral injection + of the retrograde tracer FG into MEC (red; n = 14 slices from 5 mice) and LEC (blue; n = 15 slices from 6 mice). (G) Confocal images showing mCherry axonal projections in superficial layers of MEC (Top) and LEC (Bottom). Red dashed lines indicate the border between LI and LII as ascertained based on DAPI staining. + (H) vAChT punctae are localized within mCherry cholinergic axons in MEC (Top) and LEC (Bottom). Images below show higher magnification of the boxed

areas. (I) Number of vAChT punctae per micrometer of axon length is shown for MEC (red; n = 32 axon segments from 5 mice) and LEC (blue; n = 20 axon NEUROSCIENCE segments from 4 mice). Error bars indicate mean ± SEM. *P < 0.05. [Scale bars: (B and E) 150 μm, (G)50μm, and (H)10μm.]

Desikan et al. PNAS | vol. 115 | no. 11 | E2645 Downloaded by guest on October 3, 2021 punctae per axon length. Indeed, the synapse density with 2.1 ± and depolarizing (Fig. 3A2) responses—were elicited in superficial 0.1 vAChT punctae/10 μm of axon segment was significantly higher EC neurons. The resulting EPSCs or IPSCs were verified by bath- in MEC compared with LEC, with 1.5 ± 0.2 vAChT punctae/10 μm application of atropine (nonselective mAChR blocker, n = 8/8 for of axon segment (Fig. 1I, n = 32 axon segments from 5 mice for hyperpolarizing and n = 7/7 for depolarizing responses). A third MEC and n = 20 axon segments from 4 mice for LEC, respectively; type of mAChR-mediated response (biphasic) was rarely observed P < 0.05). (Fig. 3A3, 2/488 cells patched) and was not further analyzed. In MEC and LEC, hyperpolarizing responses were predominantly Identification and Characterization of Postsynaptic Targets in LI/LII of observed in LII excitatory cells [Fig. 3C, excitatory cells (58/ MEC and LEC. We employed acute slices and performed optogenetic 151 and 44/98; MEC and LEC) vs. interneurons (5/107 and 2/73): − − stimulation of MS/DBB-derived cholinergic axons and patch- P < 10 10 and P < 10 10], whereas depolarizing responses were clamped adjacently located neurons. We recorded from LII EC preferentially elicited in interneurons [Fig. 3D, interneurons excitatory and LI/LII EC inhibitory neurons (223 and 289 cells, (20/107 and 5/73; MEC and LEC) vs. excitatory cells (0/151 and − respectively). In MEC, the four principal cell types—namely, PCs, 0/98): P < 10 8 and P < 0.05]. Different from other interneurons, + intermediate pyramidal cells (IMPCs), stellate cells (SCs), and pPV interneurons in MEC exhibited a hyperpolarizing and not a intermediate stellate cells (IMSCs)—were classified based on their depolarizing response (P < 0.05). Latency to peak, peak ampli- intrinsic electrophysiological properties as described before (ref. tude, and charge for all hyperpolarizing responses were 1.7 [1.2, 13; Fig. S2 A–F and Table S1; estimated classification error of 1.9] s, 2.8 [2.1, 4.7] pA, and 6.9 [4.1, 13.0] pC (Fig. 3B1) and for ∼5%; see Materials and Methods). In LEC, in line with previous depolarizing responses were 2.8 [1.4, 4.0] s, 2.0 [1.1, 2.9] pA, 4.5 −3 −3 reports, the three major principal cell types—that is, PCs, fan cells [3.2, 9.7] pC (Fig. 3B2), respectively (P < 10 , P < 10 ,andP < (FCs), and multiform cells (MFCs)—cannot be distinguished 0.05, respectively). based on electrophysiological properties (Fig. S2 G–J and Table In a subset of experiments, we stimulated with 10 stimuli not S2; estimated classification error of ∼50%) but purely by mor- only at 5 Hz but also at 2, 10, and 20 Hz to test for a frequency phology (11, 14). Therefore, only biocytin-filled and visually preference (Fig. S4A). While peak amplitude plateaued in the identified LEC principal cells were used in this study. LI inter- theta range, the overall transferred charge did not appear to be neurons can be morphologically segregated into neurogliaform dependent on the stimulation frequency (Fig. S4B). Thus, we used cells (LI NGCs) and single bouquet-like cells (LI SBCs), which a simple model (linear rise and decay of each individual EPSC and constitute the two major cell types (38–40). We could clearly dis- no short-term plasticity) to predict the observed behavior. As the tinguish them based on their intrinsic electrophysiological prop- prediction fit our data nearly perfectly (Fig. S4B), it is most likely erties (Fig. S3 A–C and Table S3; estimated classification error of that there is no strong frequency preference at this synapse. ∼5%). As in many other brain areas, EC LII interneurons can be Notably, previous studies performed in the MEC (36, 37) and subdivided into three by-and-large nonoverlapping subgroups (13, other brain regions (43–45) reported opposite results to ours. 41, 42). Their electrical signature corresponds to the expression of Exogenous bath application of carbachol (CCh; nonselective distinct neurochemical markers, which are hence considered in this AChR agonist) induced slow mAChR-mediated depolariza- + study as putative parvalbumin (pPV ), putative somatostatin tions, whereas we find that optogenetic stimulation evoked + (pSOM ), and putative serotonin receptors (p5HT3R)-expressing hyperpolarizing responses in excitatory cells. To follow up on interneurons (Fig. S3 D–G and Table S4, estimated classification this apparent contradiction, we conducted a set of experiments error of ∼2%; see Materials and Methods). in which we tested optogenetically and CCh-induced responses on the same cell. Nine out of 22 LII MEC excitatory cells showed Nicotinic Receptor-Mediated Responses Are Elicited in LI and LII a mAChR-mediated hyperpolarizing response upon optogenetic + p5HT3R Interneurons. Nicotinic acetylcholine receptor (nAChR)- stimulation, as opposed to 21 of 22 cells that exhibited mAChR- mediated inputs were tested by stimulating septal cholinergic mediated depolarizations after puff application of 10 mM CCh axons with 5-ms LED pulses, while clamping the cells at their ap- (Fig. S5). We also tested responses evoked by longer stimulations— proximate resting membrane potential of −70 mV. The nicotinic that is, 20 Hz for 2 s. All eight tested LII MEC excitatory cells nature of the resulting excitatory postsynaptic currents (EPSCs) showed a hyperpolarizing response. was verified by mecamylamine application (Fig. 2A, nonselective nAChR blocker, n = 17/17). For some cells, before mecamylamine, MS/DBB Cholinergic Neurons Corelease GABA. Of note, in a sub- we applied CNQX/D-AP5 (glutamatergic receptor antagonists), stantial fraction of cells (61/488 cells recorded), we observed time- which did not block the response (n = 12/12; amplitude comparison locked fast IPSCs that corresponded most likely to GABAA P > 0.1). The latency, peak amplitude, rise, and decay time constants receptor (GABAAR) activation. Hence we wondered whether the for all responding cells in MEC and LEC were 4.1 ± 0.2 ms, 3.2 IPSCs reflected indirect recruitment of local interneurons, in [1.8, 7.2] pA, 1.5 [1.1, 2.0] ms, and 5.9 [3.5, 8.7] ms, respectively (Fig. analogy to what was shown for the hippocampus (34), or if MS/ 2B,reportedasmean± SEM for normally distributed data and DBB cholinergic neurons corelease GABA in EC, as previously median [25th, 75th percentile] for other cases; no difference was suggested in the neocortex (46). We first performed fluorescence in observed between MEC and LEC). Cell type-specific response situ hybridization for Gad1/Gad2 and ChAT mRNA and found that probabilities were statistically estimated using a Bayesian ap- 88.84 ± 3.73% of MS/DBB cholinergic neurons express Gad2 (Fig. + + proach and are depicted as violin plots (for details, see SI Materials 4 A and B, 215 double cells from a total of 242 ChAT cells in four + and Methods). Responses were elicited in LI and LII p5HT R slices from two mice) but not Gad1 mRNA (Fig. 4 A and B, seven 3 + + interneurons of MEC (Fig. 2C). In LEC, we observed only a few double cells from a total of 266 ChAT cellsinfourslicesfromtwo responses in LI/LII interneurons (Fig. 2C). Hence septal cholin- mice, P < 0.0001). Moreover, MS/DBB cholinergic axons expressed + ergic neurons excite LI and LII p5HT3R interneurons via acti- vesicular GABA transporter (vGAT) along with vAChT in EC, vation of nAChRs. indicating that GABA may be coreleased (Fig. 4C). We pursued this idea and tested electrophysiologically whether Septal Cholinergic Neurons Evoke Different Muscarinic Receptor- optogenetic stimulation of cholinergic axons in EC directly evokes Mediated Responses. Muscarinic acetylcholine receptor (mAChR)- GABAAR-mediated responses. Cells were voltage-clamped at mediated inputs were tested by stimulating cholinergic axons with −50 mV, and a single 5-ms LED stimulus was applied. Resulting + ten 5-ms LED stimuli at 5-Hz frequency, while clamping the cells at IPSCs were preferentially elicited in LI and LII pSOM and + a subthreshold membrane potential of −50 mV. Two main types of p5HT3R interneurons (Fig. 4F) and were not blocked by CNQX/ mAChR-mediated responses—namely, hyperpolarizing (Fig. 3A1) D-AP5 (n = 12/12; amplitude comparison P > 0.05), mecamylamine

E2646 | www.pnas.org/cgi/doi/10.1073/pnas.1716531115 Desikan et al. Downloaded by guest on October 3, 2021 A A A B PNAS PLUS 1 2 3 10 100 MEC LEC

+ CNQX/D-AP5 + mecamylamine 5 5 pA 10 Amplitude [pA] 20 ms Latency [ms] 0 1 C CEM CEL 1/36 0/73 20/71 0/22 10/23 0/26 1/45 1/15 0/8 1/67 0/12 6/13 2/22 0/28 1/22 1/16 1/13

+ + + + + + PC SC R PC FC R 3 MFC 3 IMPC IMSC pPV pPV LI SBC LI SBC LI NGC pSOM LI NGC pSOM p5HT p5HT

Fig. 2. MS/DBB cholinergic neurons elicit nicotinic receptor-mediated responses in interneurons of EC. (A1) Representative average current trace of a cell held at −70 mV showing nAChR-mediated EPSC upon single 5-ms LED stimulation. (A2) Response was not blocked by glutamatergic receptor antagonists (CNQX/D- AP5; n = 12/12) (A3) but was completely blocked by a nonselective nAChR antagonist (mecamylamine; n = 12/12). Baselines and LED stimulations are indicated by red dashed lines and blue lines, respectively. (B) Box and jittered-dot plots showing the latency and peak amplitude (on a logarithmic scale) of EPSCs elicited in all responding cells in MEC (red) and LEC (blue). (C) Cell type-specific responses in MEC (Left)andLEC(Right) are depicted as violin plots that reflect the number of sampled cells of a given cell type and the number of responding cells within that group of cells (for details, see SI Materials and Methods). Red lines indicate the median of the distribution. Vertical dashed line in each plot segregates excitatory (Left) from inhibitory (Right) cell types. Numbers above the violin plots denote responding and patched cells for every cell type.

(n = 21/21; amplitude comparison P < 0.05, increased amplitude), following 4-AP revival is still detectable. Furthermore, the bi- or atropine (n = 17/17; amplitude comparison P > 0.05), but only by modal latency distribution (Fig. 4E) might also reflect presynaptic gabazine (Fig. 4 D1–D7,GABAAR antagonist; n = 21/21). In six differences in evoked GABA release from MS/DBB axonal ter- cells, we tested whether the evoked IPSCs resulted from mono- minals. Using latency as a criterion resulted in an approximation synaptic input. We employed consecutive pharmacological appli- of 41–73%. Additionally, we found a clear tendency of more cation of tetrodotoxin (TTX; voltage-gated sodium channel frequent putative monosynaptic responses in interneurons (22/45) blocker) and 4-aminopyridine (4-AP; voltage-gated potassium compared with excitatory cells (6/24; P > 0.05), which was not the channel blocker), thereby allowing selective optogenetic depolar- case for putative polysynaptic responses (Fig. 4G, P > 0.25). ization only in ChR2-expressing cells (Fig. 4 D5 and D6,6/6cells). Finally, GABAAR-mediated responses were verified by gabazine MS/DBB Cholinergic Cells Elicit Differential Responses in Specific EC application (Fig. 4D7, 5/5 cells; one cell was lost during the wash in Populations. Next, we questioned whether nAChR-, hyper-/ process of gabazine). depolarizing mAChR-, and mono-/polysynaptic GABAAR-mediated Next, we tested if all recorded responses (n = 69) could be responses occurred within the same neuron. Fig. 5 A1–A3 shows subdivided into mono-/polysynaptic responses based on the latency sample current traces from a cell in which both nAChR- and of pharmacologically tested monosynaptic responses (n = 6). The GABAAR-mediated EPSC and IPSC, respectively, were observed. mean latency of confirmed monosynaptic inputs was found to be Similarly, Fig. 5 B–F shows sample current traces depicting different 3.62 ± 0.24 ms and was significantly faster than the overall re- response combinations. Note the individual time-locked EPSCs and sponse latency 4.89 ± 0.18 ms (P < 0.05). Moreover, the overall IPSCs corresponding to nAChR- and GABAAR-mediated re- latency distribution showed a partially overlapping bimodal distri- sponses, respectively, observed at 5-Hz LED stimulation (Fig. 5 A3, bution (Fig. 4E, Akaike Information Criterion smallest for a bi- C3, E3,andF3). Fig. 5G shows sample current traces of a cell modal mixed Gaussian model; uni- to decamodal tested). Thus, we responding to nAChR and GABAAR activation. At −50 mV, a used a k-mean clustering algorithm (run 1,000 times) to divide the clear and distinct biphasic response mediated via both nAChR and dataset into three groups: putative monosynaptic (41%; 28/69 GABAAR was observed (Fig. 5G2). The nAChR-mediated response cells; latency: ≤4.1 ms), putative polysynaptic (28%; 19/69 cells; was abolished not by atropine but only by mecamylamine, and the latency: ≥5.8 ms), and either mono-/polysynaptic (32%; 22/69 cells; GABAAR-mediated response was abolished only after the appli- latency: between 4.1 and 5.8 ms). The mean latency, rise, and cation of gabazine (Fig. 5G), thus reconfirming the specificity of the decay time constants, as well as the median peak amplitude, and responses. The relative frequency of the different responses elicited charge were 3.40 ± 0.10 ms, 2.11 ± 0.26 ms, 9.35 ± 1.01 ms, 6.3 in the indicated cell types of MEC and LEC is shown in Fig. 5H. [4.2, 12.3] pA, 0.082 [0.033, 0.137] pC, and 6.53 ± 0.14 ms, 3.70 ± The estimated overall response rate of LI (LII) cells was higher than 0.44 ms, 16.93 ± 2.72 ms, 2.7 [2.1, 4.9] pA, 0.037 [0.016, 0.115] pC 55% (40%) in MEC and more than 25% (35%) in LEC. Please for putative mono- and polysynaptic responses, respectively. Not note the more restricted repertoire of response patterns of excit- only was the latency shorter for putative monosynaptic responses atory cells in contrast to the large variety of response combinations −23 + (P < 10 ), but the rise and decay time constant and the ampli- in interneurons, especially in LI and LII p5HT3R interneurons. tude were also larger (P < 0.01 for all) compared with putative polysynaptic responses. This difference in amplitude might account Discussion for the divergent estimates for monosynaptic responses based on Using whole-cell patch-clamp recordings combined with opto- the pharmacological approach or latency. Thus, in all pharmaco- genetics, we demonstrate here that the synaptic release of ACh logically tested cells, responses were monosynaptic. However, a from MS/DBB neurons selectively recruits specific subclasses of

bias was introduced, as all six cells had been selected for high superficial EC neurons via the activation of different AChRs. NEUROSCIENCE amplitude to ensure that the frequently observed small amplitude Viral tracing experiments revealed dense MS/DBB cholinergic

Desikan et al. PNAS | vol. 115 | no. 11 | E2647 Downloaded by guest on October 3, 2021 A1 Hyperpolarization A2 Depolarization

+ atropine + atropine

A3 Biphasic B1 Hyperpolarization B2 Depolarization 6 6 MEC LEC 10 pA 4 10 4 10 2 s 2 2 Amplitude [pA] 1 Amplitude [pA] 1 Latency to peak [s] Latency to peak [s] 0 0 C MEC - Hyperpolarization LEC - Hyperpolarization 14/34 22/57 0/54 4/15 1/14 7/26 24/44 1/14 1/8 19/48 3/12 0/13 0/11 13/28 0/22 0361/ 1/0

D MEC - Depolarization LEC - Depolarization 0/34 0/57 8/54 0/15 3/14 0/26 0/44 2/14 0/8 0/48 0/12 6/13 3/11 0/28 2/22 1361/ 1/0

+ + + + + + PC SC R PC FC R 3 MFC 3 IMPC IMSC pPV pPV LI SBC LI SBC LI NGC pSOM LI NGC pSOM p5HT p5HT

Fig. 3. MS/DBB cholinergic neurons evoke hyperpolarizing or depolarizing muscarinic receptor-mediated responses in a cell type-specific fashion.

(A) Representative average current traces (gray) of cells held at −50 mV showing mAChR-mediated hyperpolarizing (A1, Left), depolarizing (A2, Left), and biphasic (A3) response upon ten 5-ms LED stimulations at 5 Hz. In black, Savitzky–Golay-filtered data are shown. Baselines and LED stimulations are indicated by red dashed lines and blue lines, respectively. (A1, Right; A2, Right) Responses were completely blocked by a nonselective mAChR antagonist (atropine; n = 8/8 and 7/7, respectively). (B) Box and jittered-dot plots depicting the latency to peak and peak amplitude (on a logarithmic scale) of currents elicited by all MEC

(red) and LEC (blue) cells that show either (B1) a hyperpolarizing response or (B2) a depolarizing response. (C and D) Cell type-specific responses are depicted as violin plots that reflect the number of sampled cells of a given cell type and the number of responding cells within that group of cells (for details, see SI Materials and Methods). The violin plots indicate the response probability for all target cells in superficial layers of MEC (Left) and LEC (Right) that show (C)a hyperpolarizing response and (D) a depolarizing response. Red lines indicate the median of the distribution. Vertical dashed line in each plot segregates excitatory (Left) from inhibitory (Right) cell types. Numbers above the violin plots denote responding and patched cells for every cell type.

projections in the superficial layers of MEC and to a lesser extent Given the response kinetics, one can infer that the majority of in LEC. Upon optogenetic stimulation of these axons locally in evoked nicotinic responses were very likely mediated by fast α7- EC, we observed both fast nAChR- and slow mAChR-mediated subunit–expressing nAChRs (48). + + responses in LI/LII neurons. While pSOM and p5HT3R in- Second, it is striking that the slow mAChR-mediated depo- terneurons are depolarized via the activation of mAChRs and/or larizations affect only GABAergic neurons in EC. Cholinergic + nAChRs, principal cells and pPV are hyperpolarized via the excitation through mAChRs has been previously reported for hip- activation of mAChRs. In a fraction of cholinergic axons, light pocampal interneurons (53, 54). Although the function at the net- stimulation led to corelease of GABA, which activates GABAARs work level remains to be established, the implication of mAChRs in and evokes inhibitory currents predominantly in LI/LII non-FS theta oscillations was often highlighted in the past (19, 55, 56). In + + interneurons (pSOM and p5HT3R ). CA1 hippocampal slices, mAChR-induced depolarization in inter- How can the plethora of MS/DBB cholinergic actions affect neurons is essential for generating and sustaining theta oscillations, EC neuronal activity? First, we observed depolarizing nAChR- presumably by changing their active conductances, making them + mediated responses specifically in p5HT3R interneurons and responsive to theta frequency input (57). This in turn would allow + + not in pPV and pSOM interneurons. This scenario is akin to interneurons to provide rhythmic inhibition onto PCs (58). what was reported for the cortex (45, 47, 48). Notably, nAChR- Finally, slow optogenetically induced mAChR-mediated hyper- + activated LI and LII p5HT3R interneurons in the cortex were polarizing responses were predominantly observed in LII excitatory repeatedly shown to inhibit preferentially, if not exclusively, neurons of EC. Of note, the same cells responded to bath/puff GABAergic interneurons (39, 40, 49). Some studies identified application of ACh/CCh with a membrane depolarization, as + + these GABAergic interneurons as pSOM (50, 51) or pPV in- was often reported by others before (36, 37, 43–45). For instance, terneurons (48, 52). Based on our results, we envisage a scenario Widmer et al. (59) reported that hippocampal interneurons that + in which LI and LII p5HT3R interneuron activation via nAChRs are unresponsive to synaptically released ACh could be recruited causes disynaptic disinhibition in neighboring excitatory cells. following bath application of CCh. We can only speculate that this

E2648 | www.pnas.org/cgi/doi/10.1073/pnas.1716531115 Desikan et al. Downloaded by guest on October 3, 2021 A PNAS PLUS B 100 *** D1 D2 D3 + CNQX/ + mecamylamine 50 D-AP5

0 + + 5 pA Percentage [%] Gad1 / Gad2 / D D D MS MS ChAT+ ChAT+ 4 5 6 40 ms C + atropine + TTX + 4-AP MEC D7 E DBB DBB + gabazine LEC density Probability

AT AT ry Merge Gad1Merge Gad2 Merge Latency [ms] Ch Ch vAChTmCher vGAT F MEC LEC 5/34 7/59 20/55 2/15 6/14 0/26 1/43 2/15 0/8 8/51 1/12 9/13 5/11 0/28 3/22 0261/ 1/1

G MEC LEC

+ + + + + + PC SC R PC FC R 3 MFC 3 IMPC IMSC pPV pPV LI SBC LI SBC LI NGC pSOM LI NGC pSOM p5HT p5HT

Putative monosynaptic Mono-/polysynaptic Putative polysynaptic response response response

Fig. 4. MS/DBB cholinergic neurons elicit GABAAR-mediated responses in EC neurons. (A) Maximum intensity confocal images of fluorescence in situ hybrid- + ization showing expression of Gad1/Gad2 (Left/Right)andChAT mRNA in MS/DBB neurons. Higher magnification images of the boxed areas below show ChAT + + + cells that do not express Gad1 but express Gad2.(B) Percentage of ChAT cells that are Gad1 /Gad2 in MS/DBB (n = 4 slices from 2 mice). (C)vGATpunctae + colocalizing with vAChT punctae inside mCherry axonal termini in MEC (Top)andLEC(Bottom). Images below show higher magnification of the boxed areas.

(D1) Representative average current traces of a cell held at −50 mV showing GABAAR-mediated IPSCs upon single 5-ms LED stimulation. (D2–D4) Response was not blocked by CNQX/D-AP5, mecamylamine, and/or atropine but was blocked by application of the GABAAR antagonist (gabazine, n = 21/21). In six cells, mono- synaptic inputs were tested before gabazine application: (D5) First, the response was abolished by the voltage-gated sodium channel blocker (TTX) and (D6) subsequently revived by the voltage-gated potassium channel blocker (4-AP). (D7) The recovered response was again fully blocked by gabazine (n = 5/5). Baselines and LED stimulations are indicated by red dashed lines and blue lines, respectively. (E) Histogram of the response latency with a bimodal behavior is shown. Red line is a fit of two weighted Gaussian distributions, which come close to the data. (F) Cell type-specific responses in MEC (Left)andLEC(Right) are depicted as violin plots that reflect the number of sampled cells of a given cell type and the number of responding cells within that group of cells (for details, see SI Materials and Methods). Red lines indicate the median of the distribution. Vertical dashed line in each plot segregates excitatory (Left) from inhibitory (Right)celltypes. Numbers above the violin plots denote responding and patched cells for every cell type. (G) The relative frequencies of putative mono- (red), mono-/poly- (blue),

and putative polysynaptic (yellow) GABAAR-mediated responses, estimated from the latencies of the responses obtained from D1 experimental conditions, are shown by stacked bar graphs for all cell types in MEC (Left)andLEC(Right). Error bars indicate mean ± SEM. ***P ≤ 0.0001. [Scale bars: (A)150μmand(C)10μm.]

difference might result from different concentrations of agonist ac- serve a robust and direct synaptic mechanism selective for theta, tivating predominantly synaptic rather than extrasynaptic mAChRs the different kinetics, and therefore timing, of hyperpolarizing or due to different ACh sources (e.g., MS/DBB versus local cholin- excitatory and depolarizing inhibitory cells could potentially ergic interneurons). enhance and/or restrict the overall network oscillation to a cer- The muscarinic depolarizing and hyperpolarizing responses tain frequency (e.g., in the theta range). were likely mediated by M1- and M2-type receptors, respectively There is ample evidence that septal lesions affect not only theta (60, 61), that, as indicated by our functional results, appear to be in both EC and hippocampus (24, 25) but also spatial learning differentially expressed in EC interneurons and excitatory cells. (23). Less clear though appears the contribution of the different The distinct excitatory cell types in the superficial layers of MEC septal projections in sustaining theta and in particular the mech- and LEC did not differ with respect to the strength and fre- anisms underlying different forms of theta. MS inactivation and quency of hyperpolarizing mAChR-mediated responses. Hence cholinergic blockade led to the differentiation of two forms of our results cannot explain the in vivo report of stronger theta theta—namely, urethane- and movement-related theta (21, 63).

rhythmicity in PCs compared with that in SCs (62), and other Theta activity under urethane anesthesia is slower than that seen in NEUROSCIENCE factors must be considered. Furthermore, while we did not ob- freely moving animals, but most importantly, the two forms differ

Desikan et al. PNAS | vol. 115 | no. 11 | E2649 Downloaded by guest on October 3, 2021 nAChR, nAChR, nAChR, GABAAR, GABAAR, nAChR, GABAAR, GABAAR mAChR-hyp mAChR-dep mAChR-hyp mAChR-dep mAChR-dep A1 B1 C1 D1 E1 F1 Nicotinic response A2 B2 C2 D2 E2 F2 15 pA 30 ms response GABAergic A3 B3 C3 D3 E3 F3

10 pA

response 5 s Muscarinic

H 31 44 53 12 133 1015135 nAChR, GABAAR - Drug application G1

15 pA

Nicotinic 30 ms response

+ atropine/ + + + + gabazine PC SC R G mecamylamine 3 2 IMPC IMSC pPV LI SBC LI NGC pSOM p5HT 26 28 43 22 124 16 8 1 response GABAergic G3

10 pA response Muscarinic 5 s + + + PC FC R MFC 3 pPV LI SBC LI NGC pSOM p5HT No response GABAAR nAChR nAChR, GABAAR GABAAR, mAChR-hyp mAChR-hyp nAChR, mAChR-hyp GABAAR, mAChR-dep mAChR-dep nAChR, mAChR-dep nAChR, GABAAR, mAChR-dep

Fig. 5. EC neurons exhibit different receptor-mediated response combinations. (A–F) Combinations of nAChR-, GABAAR-, and hyperpolarizing and depo- + + larizing mAChR-mediated responses. Every column depicts measurements from a single cell (A and F—p5HT3R interneuron; B—MFC; C—pPV interneuron; D—SC; E—LI SBC). Representative average current traces of (A1–F1) nicotinic (cells held at −70 mV), (A2–F2) GABAergic (cells held at −50 mV), and (A3–F3) hyperpolarizing or depolarizing muscarinic responses (cells held at −50 mV). In A3–F3, Savitzky–Golay filtered data are shown in black. In A3, C3, E3, and F3, note the individual time-locked EPSCs/IPSCs (seen in overlaid gray traces) corresponding to nAChR/GABAAR-mediated responses. Baselines and LED stimu- lations are indicated by red dashed lines and blue lines, respectively. (G) Representative average current traces of a cell, in which nAChR responses (recorded + at −70 mV) and GABAAR-mediated responses (recorded at −50 mV) are elicited. This cell was identified as a p5HT3R interneuron. (G1) The nAChR-mediated response is not blocked by atropine but is completely blocked by mecamylamine. (G2) The GABAAR-mediated response is blocked neither by atropine nor by mecamylamine but is blocked by gabazine. (H) Stacked bar graphs depicting the relative response frequency of individual cell types, in which either a single response or combinations of responses in superficial neurons of MEC (Top) and LEC (Bottom) was detected. The number of patched cells for every cell type is

mentioned above. GABAAR-mediated responses include both mono- and polysynaptic responses. Responses are specified based on the receptors mediating the corresponding currents. mAChR-dep, depolarizing mAChR-mediated response; mAChR-hyp, hyperpolarizing mAChR-mediated response.

with respect to their response following cholinergic receptor The finding that some septal neurons corelease ACh and GABA blockade. Atropine, for instance, abolishes urethane-related theta further increases the number of neuronal projections exhibiting a and theta-correlated activity but has only a minor or no effect on dual neurotransmitter phenotype. The notion of a neuron releasing movement-related theta (64, 65). Notably, cholinergic and non- one fast-acting neurotransmitter has been questioned lately by cholinergic components of theta have been observed both in hip- several studies (46, 68–71). For example, supramamillary nucleus to pocampus (31, 64, 65) and in MEC (16, 21).The latter may be and basal ganglia to lateral projections have supported by septal GABAergic neurons that project to both brain the capacity to corelease glutamate and GABA (72, 73). More areas. Indeed, pharmacological interventions support the notion relevant to this study, some basal forebrain cholinergic neurons that septal GABAergic neurons may contribute to both forms of projecting to the neocortex (46) and hippocampus (74) were theta (29, 66, 67). These septal GABAergic neurons can be sub- demonstrated to corelease GABA and ACh. Here we provide both + divided into pPV neurons that inhibit preferentially FS interneurons anatomical and electrophysiological evidence that MS/DBB cho- + and calbindin-positive neurons that inhibit pSOM interneurons linergic neurons corelease GABA in superficial EC layers. It is very (13). We show here that MS/DBB cholinergic neurons can also likely that the two are packed into separate corelease GABA, thereby adding a third GABAergic input that vesicles within the same axonal terminals, as was already shown for could potentially be involved in theta rhythmicity. The exact the supramamillary nucleus to dentate gyrus projections (72) and contribution of any of these projections to EC theta remains to the MS to hippocampus projection (74). Even more interesting and be addressed by cell type-specific genetic manipulations. functionally relevant is the question of if and how signaling via the

E2650 | www.pnas.org/cgi/doi/10.1073/pnas.1716531115 Desikan et al. Downloaded by guest on October 3, 2021 two coreleased neurotransmitters is regulated. It is of note in this (13). For retrograde tracer injections, 100 nL FG (0.5%) was injected in MEC or PNAS PLUS context that, at least for the basal ganglia to lateral habenula pro- LEC of one hemisphere in 8-wk-old wild-type male mice. For this retrograde jection, it was shown that in a mouse model of depression, the labeling, surgical procedures were followed as described previously (13, 14). balance of coreleased GABA and glutamate is altered and can be Please see SI Materials and Methods for further information. restored by antidepressant treatment (73). Immunohistochemistry, Fluorescence in Situ Hybridization, and Reconstruction Upon activation of cholinergic axons, we observed a similar re- of Biotin-Labeled Cells. These methods involved standard procedures de- sponse profile in LEC, but the response probability was much lower scribed in the SI Materials and Methods. than that in MEC. This is in line with the anatomical difference in our study, as we found fewer cholinergic projections in LEC. Al- Imaging and Image Analysis. Images for immunohistochemistry or in situ ternatively, AChR expression may be lower in LEC than MEC. In hybridization experiments were taken according to a standard protocol using addition, the number of vAChT punctae per micrometer of axon a Zeiss LSM 700 confocal microscope (Carl Zeiss) and analyzed using ImageJ was significantly lower in cholinergic fibers projecting to LEC than software (ImageJ; US National Institutes of Health). Please see SI Materials that on fibers targeting MEC, indicating fewer ACh release sites. and Methods for further information. Indeed, anatomical evidence suggests that the extent of cholinergic innervation can vary substantially between different cortical struc- In Vitro Electrophysiology. Acute coronal, horizontal, and sagittal sections μ tures (20, 75). Nevertheless, tracing studies revealed an organiza- containing MS/DBB, LEC, and MEC, respectively, were 300 m thick following tion of basal forebrain neurons that supports concerted cholinergic slicing on a vibratome (Slicer HR2; Sigmann Elektronik). The different planes of sectioning were used to increase the area of the region of interest. Also the regulation of cortical areas that are spatially apart but functionally axonal projection patterns did not indicate that the plane of sectioning linked (75). Given the physical closeness of MEC and LEC, but in influenced the results (Fig. S1). Sections were incubated at ∼30–32 °C in car-

particular the distinct yet overlapping function of the two brain bogenated extracellular solution containing (in mM) 125 NaCl, 25 NaHCO3, structures in spatial memory, it is not surprising that upon antero- 1.25 NaH2PO4,2.5KCl,2CaCl2, 1 MgCl2, and 25 glucose. For patch-clamp ex- grade labeling of septal cholinergic neurons, we could visualize periments, cells in EC were viewed with DIC optics, and recordings were per- axons in both areas. At this point, one can only speculate that the formed using HEKA PatchMaster EPC 10 amplifier (HEKA). The injection site quantitative difference in cholinergic innervation might account for and the extent of axonal fibers were visualized based on neuronal mCherry expression. Recording pipettes with a tip resistance of ∼4–6MΩ were filled the more pronounced theta oscillations and the stronger theta − modulation of neurons in MEC compared with LEC (17). with a low Cl potassium-based solution containing (in mM) 130 K-gluconate, Yet another interesting and related question pertains to the 10 Na-gluconate, 10 Hepes, 10 phosphocreatine, 4 NaCl, 4 Mg-ATP, and 0.3 GTP, and sometimes 3 mg/mL biocytin with pH adjusted to 7.2 with KOH. issue as to what extent several downstream areas are simulta- The LED optic fiber (200 μm in diameter, cat. no. M470F1; Thorlabs) was neously coordinated by the same septal projections. Thus, in the placed above LI to stimulate long-range cholinergic projections with blue context of this study, one wonders whether an individual cholin- light (wavelength, 470 nm). After obtaining a whole-cell configuration, a ergic projection targets both MEC and LEC. At least for septal “test pulse” (TP) and “firing pattern” (FP) were recorded. TP and FP were GABAergic projections targeting the hippocampus and MEC, we analyzed offline by a custom-written MATLAB script (The Mathworks) to showed that this was the case. Thus, injecting cholera toxin sub- obtain passive and active properties of the cell. unit B and FG in the hippocampus and MEC, respectively, we To test for nicotinic receptor (nAChR)-mediated responses, cells were found septal GABAergic neurons that were positive for the two voltage-clamped at −70 mV and a single 5-ms, 470-nm LED stimulus was ap- retrograde tracers (13). plied to depolarize ChR2-containing axonal fibers. To test for muscarinic re- In sum, based on viral tracing and optogenetically aided patch- ceptor (mAChR)-mediated responses, cells were voltage-clamped at −50 mV clamp recordings, we here reported and characterized cell type- and ten 5-ms LED stimuli at 5 Hz (in some cases also at 2, 10, and 20 Hz) were applied. To test for indirect and direct GABAergic responses upon cholinergic specific septal cholinergic synaptic input to MEC and LEC. − — — fiber stimulation, cells were voltage-clamped at 50 mV and a single 5-ms, Functional data at this level of analysis that is, in small circuits 470-nm LED stimulus was given. Please see SI Materials and Methods for are a prerequisite if we are to understand the action of ACh in further information regarding this section. these brain areas in vivo. Thus, our data bridge the gap that links neuronal and behavioral levels and provide the necessary in- Identification of Cell Types. LI/LII EC excitatory and inhibitory neurons were formation to further probe the contribution and function of dif- classified based on their morphological characteristics and/or electrophysi- ferent ACh-mediated effects for the generation of rhythmic theta ological properties similar to previous descriptions (12–14, 38–40, 78). Please and gamma activity in EC. see SI Materials and Methods for further information.

Materials and Methods Violin Plots for the Visualization of Proportions. To get an estimation of the Experiments were carried out on 8–11-wk-old male wild-type C57/BL6 and response probability, we used a Bayesian approach. Please see SI Materials ChATCre mice [cre recombinase expressed in all choline acetyltransferase and Methods for further information. (ChAT) positive cells; Chattm2(cre)Lowl/J, purchased from The Jackson Labora- tory]. Animals were housed in a 12/12 h light/dark cycle with food and water Quantification and Statistical Analysis. Standard statistical tests were used to ad libitum. All experiments were performed according to the German pro- analyze the data. Please see SI Materials and Methods for further information. tection of animals act and after obtaining approval from the Regierung- spräsidium Karlsruhe, Germany. All chemicals were obtained from Sigma- ACKNOWLEDGMENTS. We thank E. Fuchs, A. Caputi, K. Yaqubi, and T. Kuner Aldrich unless mentioned otherwise. for helpful discussions; U. Amtmann and R. Hinz-Herkommer for technical as- sistance; and K. Deisseroth, H. Zeng, and the Allen Brain Institute for making the AAVs available through Penn Vector Core. This work was supported by the Surgical Procedures. Animals were anesthetized with isoflurane, mounted on a Helmholtz Association (PhD scholarship to S.D.), German Cancer Research Cen- stereotaxic apparatus, and kept under isoflurane anesthesia during surgery. ter (DKFZ Postdoctoral Fellowship to D.E.K.), Medical Faculty of Heidelberg Briefly, for AAV injections, AAV-DIO-ChR2-mCherry and AAV-DIO-eGFP viral University (Physician Scientist Fellowship to D.E.K.), and the German Research vectors were obtained from Penn Vector Core (76, 77). Eight-week-old ChATCre Foundation [DFG; funding as part of the DFG Collaborative Research Centers mice were subjected to 300 nL viral injections in the MS/DBB as reported before (CRC) 1134 (to H.M.)].

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