Hippocampal CA1 pyramidal cells form functionally distinct sublayers

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1 r t i l e s Hippompl CA pyrmidl ells form funtionlly distint sulyers Kenji Mizuseki, Kmrn Di,2, Ev Pstlkov,2 & György Buzsáki 2 Nture Ameri, In. All rights reserved. Hippompl CA pyrmidl neurons hve frequently een regrded s homogeneous ell popultion in iophysil, phrmologil nd modeling studies. We found roust differenes etween pyrmidl neurons residing in the deep nd superfiil CA sulyers in rts. Compred with their superfiil peers, deep pyrmidl ells fired t higher rtes, urst more frequently, were more likely to hve ple fields nd were more strongly modulted y slow osilltions of sleep. Both deep nd superfiil pyrmidl ells fired preferentilly t the trough of thet osilltions during mze explortion, wheres deep pyrmidl ells shifted their preferred phse of firing to the pek of thet during rpid eye movement () sleep. Furthermore, lthough the mjority of thet phse-shifting ells fired t the sending phse of gmm osilltions during wking, nonshifting ells preferred the trough. Thus, CA pyrmidl ells in djent sulyers n ddress their trgets jointly or differentilly, depending on rin sttes. Although the moleulr, ntomil nd funtionl diversity of ortil interneurons is well doumented 3, prinipl ells re typilly grouped together on the sis of their ortil lyer nd/or suregion ssignments. However, severl reent oservtions hve suggested tht there re distint sugroups of prinipl neurons with different properties, projetions nd lol intertions, even in the sme region nd ortil lyer 4 4. In the neoortex nd entorhinl ortex, speilized sunetworks of exittory neurons hve een desried in 8 nd ross ortil lyers 2,3. These sunetworks my ssist in segregting different strems of informtion 4,,4. The hippompl CA region is model system tht is frequently used to study plstiity, phrmologil effets nd intrellulr fetures. In the rodent, CA pyrmidl neurons form ompt lyer onsisting of 5 8 superimposed rows of pyrmidl neurons. Eh pyrmidl ell is typilly ssumed to hve similr morphology, inputs, outputs nd iophysil properties 5. In ontrst with this notion, ntomil experiments indite tht the medil nd lterl entorhinl ortex projet preferentilly to proximl nd distl prts of the CA region 6, respetively. Correlted with this topogrphy, pyrmidl ells show grdully inresing propensity of emitting spike ursts 7 nd deresing sptil speifiity in the proximo-distl (CA3 to suiulum) diretion 8. Other physiologil oservtions lso point to funtionl segregtion in CA pyrmidl ells. For exmple, the mgnitude of somtodendriti kpropgtion of tion potentil shows imodlity, perhps s result of sutle morphologil differenes of pyrmidl ells 9. The phse preferene of CA pyrmidl ells to gmm osilltions lso hs imodl distriution 2. Another potentil soure of vriility of CA pyrmidl neurons is the position or depth reltive to the ell lyer 2. Supporting this hypothesis, erly studies hve demonstrted segregtion of lindin immunoretivity 22 nd zin ontent 23 in the deep nd superfiil sustrt of CA pyrmidl lyer, nd, more reently, within-lyer differenes hve eome pprent in gene expression studies 24,25. However, funtionl differenes etween deep nd superfiil CA pyrmidl neurons re not known, nd most physiologil experiments nd modeling studies hve ssumed tht pyrmidl neurons in the CA sulyers re homogeneous. Exploiting the pility of silion proes to lolize the reltive vertil position of neuronl somt in the reorded volume of neuronl tissue 26, we exmined the funtionl properties of the superfiil nd deep CA pyrmidl ells in the ehving rt. We found two suiruits tht were differentilly ontrolled y intr- nd extr-hippompl inputs nd ould distintly ffet their trgets in rin stte dependent mnner. RESULTS Lol field potentils (LFPs) nd unit firing were reorded in the hippompl CA pyrmidl lyer (n = rts; Fig. ). In four of the rts, reordings were mde simultneously in CA nd in multiple lyers of the medil entorhinl ortex 27. Reordings were rried out while the rt rn on n open field (8 m y 8 m, or 2 m y 2 m), liner trk (25 m long), n elevted plus mze ( m y m) or zigzg mze ( m y 2 m), or performed T-mze lterntion tsk or rewrded wheel-running tsk 27 (herefter, thet periods during ehviorl tsks re referred to s ). Reordings were lso rried out during sleep, typilly oth efore nd fter tsks, in the niml s home ge. Loliztion of neuronl somt in the CA pyrmidl lyer The vertil spn (4 µm) nd the preise distriution of the eight reording sites on the proe shnks (2-µm vertil steps) llowed us to determine the reltive depths of the ell odies of the simultneously reorded neurons (Fig. ). The site with the lrgest spike Center for Moleulr nd Behviorl Neurosiene, Rutgers, The Stte University of New Jersey, Newrk, New Jersey, USA. 2 Present ddress: Jneli Frm Reserh Cmpus, Howrd Hughes Medil Institute, Ashurn, Virgini, USA (E.P.), Deprtment of Psyhology, University of Wisonsin t Milwukee, Milwukee, Wisonsin, USA (K.D.). Correspondene should e ddressed to G.B. (uzski@xon.rutgers.edu). Reeived Ferury; epted 3 June; pulished online 7 August 2; doi:.38/nn.2894 nture NEUROSCIENCE dvne online pulition

2 r t i l e s 2 Nture Ameri, In. All rights reserved. Figure Sptil loliztion of neuronl somt in the CA pyrmidl lyer. () Bioytinfilled neuron in the deep CA pyrmidl ell 5 lyer. Surrounding unstined neuronl somt rd 5 µm provide referene for the reording sites of 5 4 the superposed silion proe. Reording 4 sites ( 2 to 5) re olor oded. designtes the middle of the lyer, determined y the pyr 2 mximum ripple power (lk in d). rd, strtum rditum; pyr, strtum pyrmidle; or, mv 2.3 strtum oriens. ( d) Dt from shnk from single session. (), filtered (4 23 Hz) single tre t depth. Bottom,.3 verge LFP tres triggered y ripple power or 2 peks (n = 2,635), reorded t different depths. Top, men integrted ripple power t different depths, normlized y the s.d. Time from ripple power pek (ms) Ripple power ( mv ) of ripple power t depth. Color ode refers 2 ms to reording sites in. () Ripple power t d 5 different reording depths (men ± s.e.m.,, 4 top). The reording site with the lrgest ripple 3 power identifies the middle of the pyrmidl 2 lyer (rrow, referene depth ) nd serves for the determintion of the distne of the reorded ell ody of individul neurons from the middle of the lyer. (d) Averge wveforms of five simultneously reorded neurons 2 ( Hz to 5 khz). Arrows indite the puttive position of the neuron s som reltive to the referene depth, inferred from the lrgest spike mplitude 26. Two superfiil neurons (tht is, ove referene depth ) nd three deep neurons (elow referene depth ) re shown. Polr plots of spike phse distriution of the five neurons referened to the thet osilltion in the CA pyrmidl lyer (pek of thet =, ; trough = 8 ). Normlized ontour plots nd preferred spike phse of the neurons (rrows) during mze running (dotted) nd sleep (solid). Mgent polr plots represent -shifting neurons nd light lue plots nonshifting ells. Positive polrity is up in ll figures. mplitude for eh unit ws regrded s the lotion of the ell ody 26 (Fig. d). The site with the lrgest mplitude of ripples, refleting the middle of the pyrmidl lyer 28 (designted s site ; Fig. ), ws lso determined for eh shnk (Fig. ) nd served s the referene depth for lulting the distne of the ell odies from the middle of the pyrmidl lyer. Although the strtum oriens is stereotxilly ove the pyrmidl lyer in the dorsl CA region of the rodent hippompus, we use the trditionl terminology of ortil ntomy, in whih deep nd superfiil refer to the diretion of the sl nd pil dendrites, respetively 29. For relily seprting neurons ording to depth, the neurons with the lrgest tion potentils t site were termed middle, nd those ove (lose to strtum rditum) nd elow (lose to strtum oriens) reording site were termed superfiil nd deep, respetively. The middle lyer likely ontined mixture of deep nd superfiil neurons nd served s seprtor etween the true deep nd superfiil ells. These three tenttive divisions ontined pproximtely equl numers of reorded neurons (deep,,228; middle,,24; superfiil,,9). Somt ove site 3 nd elow site 3 were rre (<8.5%) nd were therefore lumped together with the somt reorded t sites 3 nd 3, respetively. Shifting thet phse preferene etween wking nd Five well-isolted 3 pyrmidl neurons from single shnk were sptilly distriuted long the vertil (oriens-rditum) xis (Fig. d). During mze explortion, ll five of the neurons fired preferentilly ner the trough (8 ) of the LFP thet reorded in the pyrmidl Depth (2-µm steps) s.d..5 mv lyer. During sleep, the two superfiil neurons ontinued to dishrge ner the trough, wheres the three neurons in the deeper prts of the lyer shifted their preferred firing phse y lmost hlf thet yle 3. To quntify this oservtion ross sessions nd rts, we determined the phse preferene of eh signifintly thet phse modulted pyrmidl neuron during oth explortion nd sleep (see Online Methods). The preferred thet phse of neurons during sleep vried with depth (P <., irulr ANOVA), with deep neurons showing preferene for the pek of the thet wves, s shown y the signifintly different men thet phse preferene of neurons t deeper sites (Fig. 2; for individul rts, see Supplementry Fig. ). To simplify further nlysis, we grouped neurons reorded t different depths into superfiil, middle nd deep sugroups. During sleep, the phse preferene distriutions ross sugroups were signifintly different (P <., Wtson U 2 test; Fig. 2; for individul rts, see Supplementry Fig. ) ut were similr during. In the deep sulyer, pproximtely hlf of the pyrmidl ells fired preferentilly ner the pek of thet during sleep. Given tht thet pek preferring ells during my themselves omprise funtionlly seprte group tht does not segregte perfetly in depth (Fig. 2), we lso seprtely ompred the properties of phse shifting nd nonshifting ells, exploiting the imodl distriution of phse preferenes during (Fig. 2,). Pyrmidl ells tht were signifintly phse-loked (P <., Ryleigh test, see Online Methods) to thet osilltions during sleep (68.3%,,937 of 2,838 CA pyrmidl neurons tht fired t lest 5 spikes during ) were rodly divided into -shifting (<2 or >3 Depth (2-µm steps).5 mv dvne online pulition nture NEUROSCIENCE

3 r t i l e s 2 Nture Ameri, In. All rights reserved. Figure 2 Preferred thet phse of spikes during sleep depends on the position of the som in the CA pyrmidl lyer. () Preferred thet phse for eh neuron (dots) reorded t different depths of the lyer during sleep. Solid lines indite men (±95% onfidene intervls) preferred thet phse (green, ). For omprison, the men preferred thet phse during (gry) is lso shown. The few neurons ove nd elow reording sites 3 nd 3 were dded to vlues t 3 nd 3, respetively. () Distriution of the preferred thet phse of pyrmidl ells in the superfiil (reording sites ove ), middle (site ) nd deep (sites elow ) depths during mze running () nd sleep. Note the unimodl distriution of thet phse preferene in the deep lyer group during nd imodl distriution during sleep. Top gry tres indite idelized referene thet yle in CA pyrmidl lyer. () Distriution of preferred thet phses during nd sleep. Only neurons tht showed signifint thet-phse modultion during oth nd sleep re inluded here. Top nd right, thet phse preferene histogrms for sleep nd. Note the imodl distriution of preferred thet phse during sleep. Neurons with <2 or >3 preferred thet phses during (mgent) were designted s -shifting ells, wheres those etween 2 to 3 (lue) were designted s nonshifting ells. (d) Left, depth distriution of -shifting nd nonshifting neurons (±95% Clopper-Person onfidene intervls). Right, distriution of the frtion of -shifting ells s funtion of depth in the pyrmidl lyer. (e) Perent of neurons with >9 phse shift etween nd (±95% Clopper-Person onfidene intervls) in different regions. EC2, EC3 nd EC5 orrespond to neurons in lyers II, III nd V of the entorhinl ortex, respetively. Interneurons did not shift phse preferene ross sttes. Thet phse of LFP reorded from the CA pyrmidl ell lyer ws used for ll of the ell types. preferred thet phses during ) nd nonshifting groups (2 to 3 preferred thet phses; Fig. 2). Overll, 35.% (679 ells) elonged to the -shifting tegory. As expeted, the depth distriutions of -shifting nd nonshifting neurons were signifintly different (P <., two-smple Kolmogorov-Smirnov test, one-til; Fig. 2d). The rtio of -shifting ells douled from the superfiil middle sulyers to the deep sulyer (Fig. 2d). To ompre these oservtions with CA interneurons nd with neurons in the entorhinl ortex, we lulted the frtion of these ells tht shifted their preferred phse from to y more thn 9 (Fig. 2e). In ontrst with CA pyrmidl ells, CA interneurons did not shift their preferred phse etween wke nd sttes. Furthermore, neurons with >9 phse shift etween nd in the vrious lyers of the entorhinl ortex (EC2, EC3 nd EC5) were rre (Fig. 2e nd Supplementry Fig. 2). Thus, the thet phse shift of CA pyrmidl neurons during sleep nnot simply e explined y ltered timing from the entorhinl input (Supplementry Figs. 2 nd 3). Phse-loking of CA pyrmidl ells y slow osilltions To exmine whether the CA sulyer speifi groups differed in other ehviorl sttes, we exmined their firing ptterns during non- sleep. A prominent physiologil feture of non- sleep in neoortil-pleoortil strutures is the presene of slow ortil osilltions, ssoited with UP nd DOWN sttes of neurons 32. Slow osilltions lso ffet hippompl network ptterns 33. To detet slow osilltions, we summed the spiking tivity of ll of the simultneously reorded entorhinl ortex neurons nd identified more thn 5-ms silent periods, followed y more thn 2 ms of tivity, s DOWN-UP trnsitions 33 (Fig. 3). When segregted ording to depth, neurons in superfiil, middle nd deep CA Depth (2-µm steps) Preferred thet phse () Superfiil P <. Proportion (deg) Preferred thet phse () Preferred thet phse (deg) Preferred thet phse () d Depth (2-µm steps) Superfiil P < Preferred thet phse (deg) sugroups showed signifintly different mgnitudes of modultion y the entorhinl slow osilltion (P <., F = 22.9, ANOVA; Fig. 3, results in individul rts re shown in Supplementry Fig. 4). When segregted on the sis of thet-phse shift during sleep, neurons in the -shifting group, reltive to the nonshifting group, showed signifintly stronger phse modultion y the slow osilltion (P <., t test; Fig. 3). Together, these findings indite tht the entorhinl input n differentilly tivte superfiil nd deep sugroups of CA pyrmidl ells during slow-wve sleep. We lso investigted the reltionship etween shrp wve ripple ptterns 28 nd the depth of CA pyrmidl ells. The frtion of spikes during ripples (reltive to ll spikes during slow-wve sleep episodes) ws not signifintly different etween superfiil nd deep neurons (superfiil = 8.7 ± 5.33%, deep = 8.2 ± 4.95%, P >.2, t test, ut the middle group ws signifintly higher thn oth sulyers; middle = 8.9 ± 4.93, men ± s.d., P <.). On the other hnd, -shifting ells were more strongly ssoited with ripple tivity, s evidened y oth the higher frtion of spikes during ripples (8.94 ± 4.93% versus 7.8 ± 4.2%, P <., t test) nd higher perentge of ripples ssoited with spiking, reltive to nonshifting ells (7.2 ± 2.% versus 5.4 ± 2.8%, men ± s.d., P <.5). These findings indite tht -shifting neurons respond more vigorously to CA3 inputs during shrp wve ripples. Firing rte, urst nd tivity-dependene of thet phse Next, we exmined whether CA pyrmidl ells in the different sulyers differ in their spiking properties nd how these differenes might relte to their thet phse preferene. Neurons in the deeper prt of the pyrmidl lyer hd signifintly higher overll firing rtes thn did superfiil neurons (P <., rnk sum test; Fig. 4 nd Supplementry Fig. 5). In ddition to rte, lultion of spike-urst shifting e Perentge (- thet phse shift >9 ) Proportion Rtio of -shifting ells 3 2 Superfiil P <. Prinipl ells Interneurons CA EC2 EC3 EC5 nture NEUROSCIENCE dvne online pulition

4 r t i l e s 2 Nture Ameri, In. All rights reserved. Figure 3 Phse-loking of CA pyrmidl ell spikes y slow osilltions is lotion dependent. Top, peri-event firing rte histogrms of CA pyrmidl neurons in the superfiil, middle nd deep sulyers during slow osilltions in sleep. The firing rtes of single ells were normlized y pek firing rtes. Men ± s.e.m. is shown for eh group..u., ritrry units., rte histogrms of -shifting nd nonshifting neurons. Bottom, entorhinl ortex unit firing histogrm. Time zero is the DOWN-UP trnsition of slow osilltion. index, defined s the frtion of spikes with <6-ms interspike intervls (ISIs) 27,3, reveled tht superfiil neurons were signifintly less ursty thn their deeper peers (P <., rnk sum test; Fig. 4 nd Supplementry Fig. 6). Firing rte nd urst propensities were lso signifintly different etween -shifting nd nonshifting ells (Supplementry Figs. 5 nd 6). -shifting ells deresed their firing rtes signifintly more during sleep ompred with (P <., t test; Fig. 4), nd were lso signifintly more ursty during sleep ompred with (P <., t test; Fig. 4d). Beuse of the known orreltion etween dishrge frequeny nd the thet phse of spikes 27,34,35, we exmined how firing pttern hnges, from single spikes to urst firing, ffeted the thet phse of spiking. Spikes of eh neuron were sorted into eight ISI tegories, nd the signifine of thet modultion, preferred thet phse nd modultion depth were lulted for eh neuron in eh tegory 27 (Fig. 5, Supplementry Fig. 7 nd Online Methods). During, oth -shifting nd nonshifting neurons preferred the phse ner the trough, with mostly wek dependene on the ISI. An exeption ws for <6-ms ursts in -shifting neurons, whih showed reltively wide phse preferene (Fig. 5,). In ontrst, during sleep, nerly ll ursts of -shifting neurons preferred the pek of the thet yle (<5-ms ISI groups, P <., Wtson-Willims test, - omprison; Fig. 5 nd Supplementry Fig. 7), wheres most single spikes (>2-ms ISIs) ontinued to fire fter the trough. In the nonshifting group, oth single spikes nd ursts ontinued to fire fter the trough during sleep (Fig. 5d). Proportion.5..5 (Hz) Proportion.5..5 P <. P <.. Firing rte (Hz) (Hz) Burst index. Normlized firing rte (.u.) Count During, lrge perentge of oth -shifting nd nonshifting neurons were signifintly phse-modulted y thet (P <., Ryleigh test, see Online Methods), reltively independently of the ISIs (Fig. 5e). However, the perentge of signifintly thetmodulted neurons ws lrger for oth ursts (ISI < ms) nd single spikes (ISI > 3 ms) in the -shifting group thn in the nonshifting ells. During sleep, the perentge of signifintly thet-modulted neurons during ursting Superfiil P <. 4 5 P <. P <. CA pyrmidl ells Entorhinl neurons Superfiil -shifting N =,938 ells Time from DOWN-UP trnsition (ms) ws signifintly higher in the -shifting thn in the nonshifting group (P <., χ 2 independent test), wheres the perentge of signifintly thet-modulted neurons for single spikes ws signifintly higher in the nonshifting group thn in the -shifting group (P <.; Fig. 5f). In the signifintly phse-modulted groups, we lso nlyzed the mgnitude of thet phse-modultion (men resultnt d. -shifting... (Hz) shifting (Hz) Proportion Proportion ( )/( + ).2 P < ( )/( + ) Figure 4 Firing rtes nd ursting properties of CA pyrmidl ells. (,) Distriution of overll firing rtes (Hz, log sle, ) nd urst index (frtion of spikes with <6-ms ISIs, either preeding or following spikes, ) for neurons loted in the superfiil, middle nd deep sulyers. () Firing rtes of -shifting (left) nd nonshifting neurons (middle) during mze running () nd sleep. -shifting neurons were signifintly less tive during sleep thn during. Right, distriution of the mgnitude nd diretion of firing rte hnges ross sttes. (d) Burst index of shifting nd nonshifting neurons during mze running () nd sleep. -shifting neurons were signifintly more ursty during sleep thn during. dvne online pulition nture NEUROSCIENCE

5 r t i l e s 2 Nture Ameri, In. All rights reserved. Proportion Proportion.4.2 -shifting Preferred thet phse during (deg) length). Brin stte hnge exerted differentil effet on the mgnitude of thet phse-loking of spikes ross the -shifting nd nonshifting neurons (Fig. 5e,f). During, nonshifting neurons were more strongly phse-loked thn were -shifting ells, during oth ursts (ISI < ms) nd single-spike firing (ISI > 3 ms) ptterns (Fig. 5e). During, this differene remined the sme for single spikes ut ws reversed for ursts. In ft, the thet phsemodultion of urst events (<-ms intervls) in -shifting neurons ws lmost twie s powerful during s during (Fig. 5e,f). Comprison of ISI groups from -shifting neurons reveled tht the sme ells shifted their preferred phse from trough (ISI > 3 ms) to pek (ISI < ms) nd inresed their phse modultion during urst events (Fig. 5g,h). In summry, deep nd superfiil neurons hve different firing rtes nd ursting properties. During sleep, -shifting (tht is, minly deep) neurons urst more, s ompred with, nd ursts of spikes of -shifting ells were more strongly modulted y thet nd showed stronger thet pek preferene thn nonshifting neurons. Thus, the intertions mong instntneous firing rte, ursting, thet modultion nd preferred thet phse depend on the joint effet of rin stte nd ell lotion in the CA pyrmidl lyer. d.4.2 -shifting Preferred thet phse during (deg) Figure 5 ISI-thet phse reltionship of CA pyrmidl ells. ( d) Distriution of the preferred thet phse of -shifting nd nonshifting neurons in eh ISI tegory. In eh ISI tegory, only neurons tht were signifintly modulted in tht tegory re shown. Note the different thet phse preferene during () nd rupt thet phse shift during sleep () in -shifting neurons in reltion to the hnges of ISIs from <5 ms to >2 ms. The thet phse preferene of nonshifting neurons (,d) ws less sensitive to hnges in ISIs. Top gry tres, idelized referene thet yle in CA pyrmidl lyer. (e) Perentge of signifintly thet phse modulted neurons (±95% Clopper-Person onfidene intervls) nd the strength of thet phse-loking (men resultnt length, men ± s.d.) in the <-ms nd >3-ms ISI tegories.4.2 g preferred thet phse >3 ms <6 ms <8 ms < ms <5 ms >2 ms >3 ms >5 ms > ms e Thet-modulted ells (%) f Thet-modulted ells (%) preferred thet phse < ms * * < ms >3 ms * * * *.4 < ms >3 ms 2 4 This ws lso the se when we used sptil oherene greter thn.7 s the riteri for defining ple fields 37,38 (Supplementry Fig. 8). The distriutions of pek firing rtes nd within-field men rtes were lso signifintly different ross the depth groups (P <.; Supplementry Figs. 9 nd ), onsistent with the overll rte differene desried ove. Informtion ontent 39 4 (oth its per spike nd its per seond) ws lso signifintly different ross the CA sulyers nd etween -shifting nd nonshifting ells (P <.; Supplementry Figs. 9 nd ), with informtion ontent per spike eing higher in the superfiil nd nonshifting groups thn in the deep nd -shifting groups. Other exmined fetures of ple ells Men resultnt length -shifting h men resultnt length >3 ms 4 2 Men resultnt length < ms >3 ms * * < ms >3 ms men resultnt length < ms during. -shifting (mgent) nd nonshifting (lue) neurons re shown seprtely. (f) Dt presented s in e for sleep. All omprisons etween -shifting nd nonshifting groups were signifint (*P <., χ 2 test for perent of modulted ells, t test for men resultnt length). (g) Comprison of preferred thet phse etween <-ms nd >3-ms tegories of the sme neurons during sleep. Only -shifting ells signifintly modulted for oth ISI tegories re shown. (h) Dt presented s in g for omprison of men resultnt length. Cells with ple fields >2 Hz (%) Open field (.8 m.8 m) Liner trk (2.5 m) Superfiil -shifting Higher inidene of ple ells in the deep CA sulyer Hippompl neurons in rodents show lotion-seletive firing 36. Neurons with pek firing rtes exeeding 2 Hz on the liner trk or on the open field (see Online Methods) were defined s ple ells 8. The proportion of ple ells ws signifintly higher in the deep sulyer thn in the superfiil lyer (P <., χ 2 independent test; Fig. 6). Figure 6 Lrger frtion of ple ells in deep lyer neurons nd -shifting group. () Proportion of neurons with ple fields, defined y pek firing rte (>2 Hz, Online Methods), in different sulyers nd in the -shifting nd nonshifting groups during open field explortion. Clopper-Person onfidene intervls (P <.5) re shown. () Dt presented s in for neurons reorded on the liner trk (for other sptil fetures, see Supplementry Figs. 8 ). nture NEUROSCIENCE dvne online pulition

6 r t i l e s 2 Nture Ameri, In. All rights reserved. Figure 7 Reltionship etween thet nd gmm phse preferenes of CA pyrmidl ells. () Distriution of preferred phse of gmm osilltion (3 8 Hz) during mze running () nd sleep, shown seprtely for -shifting nd nonshifting neurons. Note tht oth groups fired preferentilly t the trough of lol gmm wves (~8 ) during sleep, wheres the mjority of -shifting neurons were phse-loked to the rising phse (gmmr) of the gmm yle during. The referene site for LFP gmm ws the middle of the pyrmidl lyer (site in Fig. ). Two gmm yles re shown for etter visiility. Top gry tres represent the idelized referene gmm yle in CA pyrmidl lyer. (,) Modultion of pyrmidl ells y oth gmm nd thet. Note tht most gmm-modulted neurons during were nonshifting neurons (, light lue) nd most gmmr neurons during elonged to the -shifting group (, mgent). The side histogrms in nd (right) re the sme s the plots in. Only neurons tht were signifintly modulted y oth thet nd gmm osilltions re inluded in the plots in nd. (ple field size, sptil oherene 37 nd stility 4 ) were not different ross the groups (Supplementry Figs. 9 nd ). In ddition, the slopes of phse preession 42 on the liner trk were similr ross depth groups (Supplementry Fig. ). Consistent with the results of the depth nlysis, the proportion of ple ells ws signifintly higher in the -shifting group thn in the nonshifting neurons in oth the liner trk nd the open field (P <., χ 2 independent test; Fig. 6). On the other hnd, the slopes of phse preession 42 on the liner trk were similr etween the shifting nd nonshifting groups (Supplementry Fig. ). In summry, lthough the proportion of tive ple ells in given environment ws higher in the deep (-shifting) group, the mjor sptil fetures nd thet phse dynmis of ple ells were not different ross groups. Thet phse (deg) Thet phse (deg) efore Fmilir Thet phse (deg) fter Novel Gmm phse Proportion Thet phse (deg) fter.2. P <. -shifting Preferred gmm phse (deg) Preferred gmm phse (deg) Thet phse d Fmilir e Novel f Thet phse (deg) fter Thet phse (deg) fter 2 4 Proportion Proportion Gmm phse Thet phse Thet phse Reltionship etween thet nd gmm phse preferene The hippompus lso displys prominent gmm osilltions 43 45, nd it ws reently found tht spikes of pyrmidl ells in the wking rt show phse preferene to either the trough (gmmt neurons, 3 24 ) or the rising phse of the gmm wve 2 (gmmr neurons, 3 or ). We onfirmed this imodl gmm phse distriution during (Fig. 7) nd found tht, during, virtully ll of the signifintly gmm-modulted pyrmidl ells were phseloked to the trough of gmm wves 2 (Fig. 7,). The preferred gmm phse during showed depth-dependent shift, with preferene of deep ells for the rising phse of the gmm yle (Supplementry Fig. ), lthough this effet ws less roust thn the depth-dependent thet-phse preferene during. On the other hnd, the mthes etween gmmr nd -shifting nd etween Thet phse differene (deg) etween nd.2. Fmilir Novel Fmilir Novel P >.2 P > thet phse differene (deg) etween efore nd fter gmmt nd nonshifting ells were strong (Fig. 7), prtiulrly in the middle nd deep neurons (Supplementry Fig. ). During, signifintly fewer pyrmidl ells were signifintly phse-loked to gmm osilltions ( =.4% versus = 26.7%, P <., χ 2 independent test) or modulted y oth thet nd gmm osilltions Figure 8 The thet phse of spikes during is not ffeted y novel experiene. (,) Reltionship etween preferred thet phse during nd fter sessions in fmilir () nd novel () tsks. () Distriution of thet phse shifts etween nd fter sessions. Fmilir nd novel tsks re shown seprtely. (d,e) Preferred thet phse of CA pyrmidl neurons during sleeps efore nd fter fmilir tsk (d) nd novel tsk (e). (f) Distriution of preferred thet phse differenes etween sleeps efore nd fter tsk. Note the similr phse preferene, independent of tsk novelty (Wtson U 2 test, P >.2). dvne online pulition nture NEUROSCIENCE

7 r t i l e s 2 Nture Ameri, In. All rights reserved. ( = 7.2% versus = 2.8%, P <.; Fig. 7,) thn during. Signifintly fewer -shifting ells were gmm-modulted during sleep thn during (P <., χ 2 independent test), wheres omprle frtion of nonshifting ells were gmmmodulted nd fired t the trough of gmm oth during nd (Fig. 7,). The few gmmr pyrmidl ells, whih were signifintly gmm phse-loked in oth nd sttes, shifted their phse preferene to the trough during (Supplementry Fig. 2). In ontrst, puttive CA interneurons mintined their gmm phse preferene ross ehviorl sttes 2 (Supplementry Fig. 3). Novelty does not ffet thet phse preferene during Finlly, we ompred the thet phse preferene of pyrmidl ells during sleep episodes oth efore nd fter explortion of novel (n = 9 sessions in 4 rts) or fmilir environment (n = 6 sessions). We exmined whether the wke- shift in thet phse preferene of CA pyrmidl ell spikes ws experiene dependent, s previous study 3 suggested tht, fter exposure to novel rm of the mze, the phse preferene of novel ple field ells remins the sme s tht oserved during, wheres sleep relted shift of phse preferene emerges only fter multiple experienes. In our experiments, for the mjority of CA pyrmidl ells (tht is, nonshifting ells), the preferred thet phse during remined similr to tht during, even fter the fmilir tsk (Fig. 8 ). Furthermore, the distriutions of thet phse differene etween nd, tested fter sessions, were similr regrdless of the fmilirity of the tsk (P >.2, Wtson U 2 test; Fig. 8 ), suggesting tht environmentl novelty did not signifintly ffet preferred thet phse of spiking during sleep. In support of this oservtion, the preferred thet phses of the sme neurons during sleep episodes efore nd fter tsks were similr regrdless of whether the tsk ws novel or fmilir (Fig. 8d f). DISCUSSION We found supopultions of pyrmidl ells with distint responses to inputs, spiking properties nd output influene, segregted in the superfiil nd deep prts of CA strtum pyrmidle. Neurons residing in different sulyers differed in multiple properties, inluding thet phse preferene nd phse-modultion strength during sleep, modultion y slow osilltions nd shrp wve ripples during non- sleep, gmm phse preferene during wking stte, firing rte, ursting propensity nd the proportion with ple fields. Although the CA pyrmidl ells hve often een titly ssumed to e homogeneous popultion 5, the within-lyer segregtion of funtionl groups tht we found is onsistent with the reltively distint strt of superfiil nd deep pyrmidl ells 29. The strtifition of funtion in the CA pyrmidl lyer is supported y numer of ntomil nd moleulr oservtions. Severl surfe nd hnnel genes show sulyer-speifi expression in the pyrmidl lyer 24,25. In prtiulr, oth zin- nd lindinontining pyrmidl ells reside predominntly in the superfiil lyer (nerly ll lindin-immunoretive neurons in this sulyer re zin-positive 23 ). Zin mplifies AMPA reeptor medited urrents nd suppresses NMDA reeptor medited responses 46. In ddition, the C 2+ uffer lindin my e involved in synpti plstiity, s long-term potentition of Shffer ollterl synpses onto CA neurons nd sptil lerning re impired in ntisense trnsgeni lindin-defiient mie 47. Furthermore, the neurogenesis in the superfiil lyer ours 2 d lter in development thn it does in the deep pyrmidl ell lyer 22. Indeed, distint supopultions of prinipl neurons in eh sufield (dentte gyrus, CA3 nd CA) of the hippompus hve similr gene expression profiles, distint time windows of neurogenesis nd synptogenesis, nd my form seletive suiruits 4. Finlly, neurons tht projet to the lterl septum re found lrgely in the deep CA sulyer 48, suggesting n output segregtion of the distint sulyers desried here. The roust orreltion etween the position in the CA pyrmidl lyer nd their physiologil fetures my hve multiple explntions. The simplest ount is tht intrinsi properties of pyrmidl ells (morphology, distriution of ion hnnels, reeptors, et.) re responsile for most of the oserved differenes etween sulyers. For exmple, morphologil differenes etween the deep nd superfiil neurons 2,29 my differentilly ffet the ursting, firing rte nd other iophysil properties of neurons, s hs een oserved in the neoortex 6,7 nd the CA3 region 49. In turn, these intrinsi fetures would indue the different firing ptterns nd phse preferenes tht we oserved during vrious LFP ptterns, even if ll of the neurons were uniformly innervted y fferents nd lol interneurons. In support of this possiility, we found relile differenes in firing rtes nd the ursting propensities of neurons in different sulyers. During sleep, ursts of -shifting (deep) ells (<5 ms ISIs) displyed stronger thet phse oupling thn single spikes (>2 ms), nd ursts nd single spikes preferred opposite phses of the thet yle. Another possile explntion is tht deep nd superfiil neurons re trgeted y differentil sets of fferents nd the oserved hnges simply reflet the distint influene of their upstrem prtners. Supporting this hypothesis, deep CA neurons were more effetively entrined y slow osilltion of non- sleep, potentil indition tht these ( shifting) neurons re more strongly driven y the entorhinl input. During thet osilltions, the CA region is under the ompeting influenes of CA3 nd entorhinl inputs. Agin, the oserved shifts of thet phse preferenes of deep CA neurons n e explined y stronger influene of the diret entorhinl input on these ells, given tht during sleep EC3 prinipl neurons fired 25 ms efore the -shifting CA neurons (Supplementry Figs. 3 nd 4). Most likely, iruit nd intrinsi properties intert with eh other during ontogenesis so tht the sptil position of neurons my orrelte with oth their integrtion into the iruitry nd their intrinsi properties 2,4. Neither intrinsi nor iruit properties lone n dequtely explin the two distint ell groups, -shifting nd nonshifting ells, euse physiologil fetures defining the two groups re dependent on rin stte. A fundmentl differene etween the wking stte nd sleep is the mrkedly redued toni relese of severl suortil neurotrnsmitters, inluding serotonin, norepinephrine nd histmine, during sleep 5. Suortil neuromodultors my distintly ffet the -shifting nd nonshifting ells y differentilly hnging oth their intrinsi properties nd their synpti intertions in the network in whih they re emedded, therey produing distint hnges in firing rtes, ursting propensity nd thet phse of spiking. In summry, funtionlly distint sulyers re present in the CA hippompl region. These my serve different funtions, suh s melding strems of informtion or segregting them, depending on rin stte. In the wking niml, the inputs my e integrted y the o-firing of superfiil nd deep neurons t the thet trough nd stremlined to their joint trgets. In ontrst, during sleep, the different inputs my e kept seprte, or perhps the sme inputs my ddress oth deep nd superfiil neurons ut n then e routed to different trgets nd/or t different phses of the thet yle. Although they represent minority, the -shifting deep neurons nture NEUROSCIENCE dvne online pulition

8 r t i l e s 2 Nture Ameri, In. All rights reserved. fire ursts of spikes on the pek of thet during sleep nd my therefore exert s powerful downstrem effet s the nonshifting, trough-preferring mjority. The ehviorl importne of suh rin stte dependent integrtion nd segregtion of neuronl informtion y the CA region remins unknown. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Neurosiene wesite. Aknowledgments We thnk E. Cel, S. Fujisw, P.M. Hihe, S. Ozen, A. Sirot, E. Strk nd Y. Wng for omments on the mnusript, nd D. Sullivn for vlule suggestions. This work ws supported y the US Ntionl Institutes of Helth (NS34994, MH5467), the Ntionl Siene Foundtion, the J.D. MDonnell Foundtion, the Uehr Memoril Foundtion, the Astells Foundtion for Reserh on Metoli Disorders, the Jpn Soiety of Promotion for Sienes, nd the Roert Leet nd Clr Guthrie Ptterson Trust. AUTHOR CONTRIBUTIONS K.M. nd G.B. designed the experiments. K.M., K.D. nd E.P. olleted dt. K.M. nlyzed the dt. K.M. nd G.B. wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprints/index.html.. Freund, T.F. & Buzski, G. Interneurons of the hippompus. Hippompus 6, (996). 2. Mrkrm, H. et l. Interneurons of the neoortil inhiitory system. Nt. Rev. Neurosi. 5, (24). 3. Kluserger, T. & Somogyi, P. Neuronl diversity nd temporl dynmis: the unity of hippompl iruit opertions. Siene 32, (28). 4. Nelson, S.B., Sugino, K. & Hempel, C.M. The prolem of neuronl ell types: physiologil genomis pproh. Trends Neurosi. 29, (26). 5. Connors, B.W., Gutnik, M.J. & Prine, D.A. Eletrophysiologil properties of neoortil neurons in vitro. J. Neurophysiol. 48, (982). 6. Chgn-Amiti, Y., Luhmnn, H.J. & Prine, D.A. 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Dihotomy of tion-potentil kpropgtion in CA pyrmidl neuron dendrites. J. Neurophysiol. 86, (2). 2. Senior, T.J., Huxter, J.R., Allen, K., O Neill, J. & Csisvri, J. Gmm osilltory firing revels distint popultions of pyrmidl ells in the CA region of the hippompus. J. Neurosi. 28, (28). 2. Bnnister, N.J. & Lrkmn, A.U. Dendriti morphology of CA pyrmidl neurones from the rt hippompus. I. Brnhing ptterns. J. Comp. Neurol. 36, 5 6 (995). 22. Bimridge, K.G., Peet, M.J., MLennn, H. & Churh, J. Bursting response to urrent-evoked depolriztion in rt CA pyrmidl neurons is orrelted with luifer yellow dye oupling ut not with the presene of lindin-d28k. Synpse 7, (99). 23. Slomink, L. Neurons of origin of zin-ontining pthwys nd the distriution of zin-ontining outons in the hippompl region of the rt. Neurosiene 48, (992). 24. Thompson, C.L. et l. Genomi ntomy of the hippompus. Neuron 6, 2 (28). 25. Dong, H.W., Swnson, L.W., Chen, L., Fnselow, M.S. & Tog, A.W. Genomintomi evidene for distint funtionl domins in hippompl field CA. Pro. Ntl. Ad. Si. USA 6, (29). 26. Csisvri, J. et l. Mssively prllel reording of unit nd lol field potentils with silion-sed eletrodes. J. Neurophysiol. 9, (23). 27. Mizuseki, K., Sirot, A., Pstlkov, E. & Buzski, G. Thet osilltions provide temporl windows for lol iruit omputtion in the entorhinl-hippompl loop. Neuron 64, (29). 28. Ylinen, A. et l. Shrp wve ssoited high-frequeny osilltion (2 Hz) in the intt hippompus: network nd intrellulr mehnisms. J. Neurosi. 5, 3 46 (995). 29. Lorente de Nó, R. Studies on the struture of the ererl ortex. II. Continution of the study of the mmoni system. J. Psyhol. Neurol. (Lpz) 46, 3 77 (934). 3. Hrris, K.D., Hirse, H., Leinekugel, X., Henze, D.A. & Buzski, G. Temporl intertion etween single spikes nd omplex spike ursts in hippompl pyrmidl ells. Neuron 32, 4 49 (2). 3. Poe, G.R., Nitz, D.A., MNughton, B.L. & Brnes, C.A. Experiene-dependent phse-reversl of hippompl neuron firing during sleep. Brin Res. 855, 76 8 (2). 32. Steride, M., Nunez, A. & Amzi, F. A novel slow (< Hz) osilltion of neoortil neurons in vivo: depolrizing nd hyperpolrizing omponents. J. Neurosi. 3, (993). 33. Isomur, Y. et l. Integrtion nd segregtion of tivity in entorhinl-hippompl suregions y neoortil slow osilltions. Neuron 52, (26). 34. Hrris, K.D. et l. Spike trin dynmis predits thet-relted phse preession in hippompl pyrmidl ells. Nture 47, (22). 35. Meht, M.R., Lee, A.K. & Wilson, M.A. Role of experiene nd osilltions in trnsforming rte ode into temporl ode. Nture 47, (22). 36. O Keefe, J. & Dostrovsky, J. The hippompus s sptil mp. Preliminry evidene from unit tivity in the freely-moving rt. Brin Res. 34, 7 75 (97). 37. Muller, R.U. & Kuie, J.L. The firing of hippompl ple ells predits the future position of freely moving rts. J. Neurosi. 9, 4 4 (989). 38. Hfting, T., Fyhn, M., Bonnevie, T., Moser, M.B. & Moser, E.I. Hippompus-independent phse preession in entorhinl grid ells. Nture 453, (28). 39. Skggs, W.E., MNughton, B.L., Gothrd, K.M. & Mrkus, E.J. An informtiontheoreti pproh to deiphering the hippompl ode. in Advnes in Neurl Informtion Proessing Systems, vol. 5 (Hnson, S.J., Cown, J.D. & Giles C.L., eds.) 3 37 (Morgn Kufmnn, 993). 4. Skggs, W.E., MNughton, B.L., Wilson, M.A. & Brnes, C.A. Thet phse preession in hippompl neuronl popultions nd the ompression of temporl sequenes. Hippompus 6, (996). 4. Mrkus, E.J., Brnes, C.A., MNughton, B.L., Gldden, V.L. & Skggs, W.E. Sptil informtion ontent nd reliility of hippompl CA neurons: effets of visul input. Hippompus 4, 4 42 (994). 42. O Keefe, J. & Ree, M.L. Phse reltionship etween hippompl ple units nd the EEG thet rhythm. Hippompus 3, (993). 43. Brgin, A. et l. Gmm (4 Hz) osilltion in the hippompus of the ehving rt. J. Neurosi. 5, 47 6 (995). 44. Whittington, M.A., Tru, R.D. & Jefferys, J.G. Synhronized osilltions in interneuron networks driven y metotropi glutmte reeptor tivtion. Nture 373, (995). 45. Csisvri, J., Jmieson, B., Wise, K.D. & Buzski, G. Mehnisms of gmm osilltions in the hippompus of the ehving rt. Neuron 37, (23). 46. Peters, S., Koh, J. & Choi, D.W. Zin seletively loks the tion of N-methyl-dsprtte on ortil neurons. Siene 236, (987). 47. Molinri, S. et l. Defiits in memory nd hippompl long-term potentition in mie with redued lindin D28K expression. Pro. Ntl. Ad. Si. USA 93, (996). 48. Sørensen, J.C., Tonder, N. & Slomink, L. Zin-positive fferents to the rt septum originte from distint supopultions of zin-ontining neurons in the hippompl res nd lyers. A omined fluoro-gold tring nd histohemil study. Ant. Emryol. (Berl.) 88, 7 5 (993). 49. Bilkey, D.K. & Shwrtzkroin, P.A. Vrition in eletrophysiology nd morphology of hippompl CA3 pyrmidl ells. Brin Res. 54, (99). 5. Pe-Shott, E.F. & Hoson, J.A. The neuroiology of sleep: genetis, ellulr physiology nd suortil networks. Nt. Rev. Neurosi. 3, (22). dvne online pulition nture NEUROSCIENCE

9 ONLINE METHODS Animls nd surgery. Ten mle Long-Evns rts (25 4 g) were implnted with 4- or 8-shnk silion proe in the right dorsl hippompus under isoflurne nesthesi (.5%) nd reorded from dorsl CA pyrmidl lyers. In four of the rts, nother 4-shnk silion proe ws lso implnted in the right dorsoudl medil entorhinl ortex 27. The silion proes were tthed to miromnipultors nd moved slowly to the trget. Eh shnk hd eight reording sites (6 µm 2 eh site, 3-MΩ impedne) nd inter-shnk distne ws 2 µm. Reordings sites were stggered to provide two-dimensionl rrngement (2-µm vertil seprtion). The entorhinl ortex proe ws positioned so tht the different shnks reorded from different lyers 27. Histologil nd eletrophysiologil loliztion of reording sites in the entorhinl ortex ws desried previously 27. Two stinless steel srews inserted ove the ereellum were used s indifferent nd ground eletrodes during reordings. All protools were pproved y the Institutionl Animl Cre nd Use Committee of Rutgers University. DOWN-UP trnsitions in the entorhinl ortex were deteted y using spiking tivity of entorhinl ortex neuron popultion during slow wve sleep. All the simultneously reorded single entorhinl ortex neurons were omined s multiunit tivity (MUA), nd smoothed with Gussin kernel (s.d. = ms, kernel size = 6 ms). Upstte onsets were deteted if the following riteri were ll fulfilled. First, the smoothed MUA exeeded the upstte threshold, defined s the geometri men of ll nonzero MUA. Seond, the men MUA in 5-ms window efore the onset of the ndidte event ws elow the downstte threshold, whih ws defined s.6 times the upstte threshold. Third, the men MUA in oth -ms nd 2-ms window fter the onset ndidte ws ove the upstte threshold. The frequeny of the deteted DOWN-UP trnsitions ws.45 ±.4 Hz (men ± s.d.). To ompre the mgnitude of DOWN-UP modultion etween ell groups, we omputed the modultion index for eh neuron. NspikesUP NspikesDOWN Modultion Index = NspikesUP + NspikesDOWN 2 Nture Ameri, In. All rights reserved. Behviorl testing. Physiologil signls during wking were reorded during six different tsks: tsk on the liner trk (25 m 7 m), tsk on the open field (8 m 8 m, or 2 m 2 m), wheel running tsk, n lterntion tsk in the T mze ( m 2 m) with wheel running dely (desried previously 27 ), n elevted plus mze ( m m) in whih the rts were motivted to run to the ends of four orridors, where wter ws given every 3 s, nd zig-zg mze ( m 2 m) with orridors, in whih the rts lerned to run k nd forth etween two wter ups pled in the first nd lst orridors. Thet periods from ll mze ehviors were lumped together s. The tsk ws regrded s eing novel when the niml performed it for the first time nd fmilir fter t lest three testing sessions. Dt olletion nd ell-type lssifition. Detiled informtion out the reording system nd spike sorting hs een desried 27. Briefly, signls were mplified (, ), ndpss-filtered ( Hz to 5 khz) nd quired ontinuously t 2 khz. LFP ws down-smpled to,25 Hz for dditionl nlysis. Spike sorting ws performed y KlustKwik ( net/), followed y mnul djustment of the lusters (Klusters softwre pkge, CA pyrmidl ells nd interneurons were seprted s desried 27. Cell-type lssifition of entorhinl ortil neurons ws desried previously 27. A totl of 3,54 (CA), 49 (EC2), 576 (EC3) nd 559 (EC5) prinipl neurons nd 468 (CA), 85 (EC2), 27 (EC3) nd 94 (EC5) interneurons were identified nd used for nlyses. Medin isoltion distnes 3, refleting the qulity of luster isoltion, were similr ross the deep, middle nd superfiil CA pyrmidl ells (28.7, 28.3 nd 28.7, respetively; P >.3, Wiloxon rnk sum test). Detetion of rin sttes. Thet periods during tsk performne (), epohs nd slow-wve sleep (SWS) were deteted using the rtio of the power in thet nd (5 Hz) to delt nd ( 4 Hz) of LFP, followed y mnul djustment with the id of visul inspetion of whitened power spetr nd the rw tres 27. periods were ross-vlidted with experimenter notes tken while oserving thet tivity on-line in sleep session nd verifying tht the rt ws sleeping. To detet gmm epohs, we used reording site with the highest ripple power in given shnk. LFPs were nd-pss filtered (3 8 Hz) nd the power (r.m.s.) of the filtered signl ws lulted in 8-ms time windows. Gmm phse of spiking ws omputed in gmm osilltory periods. Gmm osilltory periods were defined s epohs with gmm power lrger thn the 85 perentile of gmm power. If the gp etween epohs ws shorter thn 3 ms, these flnking epohs were omined, generting single epoh. Epohs less thn 4 ms were disrded. The deteted gmm periods represented 9.8 ± 3.4% of, 3.2 ± 3.3% of sleep, 2.8 ± 2.% of SWS nd 7.9 ± 2.8% of wke nonthet epohs (men ± s.d.). To detet ripple events, LFPs in CA pyrmidl lyer during nonthet periods were nd-pss filtered (4 23 Hz), nd the power (r.m.s.) ws lulted in 7-ms time windows. Ripple epohs were defined s periods during whih ripple power ws ontinuously greter thn men + 3 s.d., nd pek of power in the periods were greter thn men + 7 s.d. Events shorter thn 5 ms were disrded. where N spikesdown is the numer of spikes from 2 to ms, nd N spikesup is the numer of spikes from to 2 ms (time = DOWN-UP trnsition). One-wy ANOVA or t test ws used to test the signifine. Loliztion of neuronl somt in the CA pyrmidl lyer. For eh silion proe shnk, one reording site ws hosen first nd used to detet ripple epohs s desried ove. LFP reorded from eh reording site ws nd-pss filtered (4 23 Hz) nd the power (r.m.s.) ws lulted in 7-ms time windows. The site with the lrgest men power during ripple epohs, refleting the middle of the pyrmidl lyer 28, ws determined for eh shnk for eh session nd served s the referene depth. The site with the lrgest verge spike mplitude for eh unit ws regrded s the pproximte lotion of the ell ody 26. The error of ell ody ssignment ws 2 µm, due to the limittion of the vertil distne etween reording sites. Thet nd gmm phse modultion. Three methods were used to determine the phse of nd-pss filtered thet (5 Hz) or gmm (3 8 Hz) wves y(t) 27. First, instntneous phse ws derived from Hilert trnsform of y(t). Seond, peks ( ) of filtered wves were identified s the positive to negtive zero rossings of the derivtive dy/dt, nd phse ws linerly interpolted etween the peks. Third, troughs (8 ) of filtered wves were identified s the negtive-to-positive zero rossings of the derivtive dy/dt, nd phse ws linerly interpolted etween the troughs. A phse vlue ws ssigned to eh tion potentil using liner interpoltion. Peks re t nd 36 nd troughs t 8 throughout the pper. The results otined y these three methods were onsistent nd the pek method ws used for oth thet nd gmm phses. The men diretion nd men resultnt length of the phses of given neuron s spikes were tken s the preferred phse nd modultion depth of tht neuron, respetively. For the preferred phse nd modultion depth, only neurons tht were signifintly modulted y the osilltions re shown. For the irulr sttistis of thet phse of single ells, only neurons with t lest 5 spikes during thet epohs ( or ) were used, nd P <. (Ryleigh test) ws used to define signifintly thet-modulted neurons. For gmm modultion of single ells, only neurons with t lest 2 spikes during gmm osilltory periods in relevnt rin stte (,, SWS or wke nonthet) were used nd P <.5 (Ryleigh test) ws used to define signifintly gmm-modulted neurons 2. To void gmm phse vriility s funtion of depth 45, the reording site with the lrgest ripple power (tht is, the middle of the CA pyrmidl lyer) for eh proe shnk ws used for deteting gmm phse. ISI nlysis. Burst index ws defined s the rtio of ursting spikes to ll spikes. A ursting spike ws defined s spike ssoited with ISI t lest either efore or fter tht spike smller thn 6 ms. To ompute the irulr sttistis of thet phse t different ISIs, we first sorted the spikes of neuron ording to their ISIs. A spike ssoited with n ISI either efore or fter tht spike less thn 6, 8,, 5 ms ws sorted into <6-ms, <8-ms, <-ms or <5-ms ISI groups, llowing less stringent groups to redundntly ontin spikes in more stringent groups. Therefore, the <5-ms ISI group ontins the <6-ms, <8-ms nd <-ms groups. A spike ssoited with ISIs oth efore nd fter tht spike lrger thn 2, 3, 5 or ms ws sorted into >2-ms, >3-ms, >5-ms or >-ms ISI groups. doi:.38/nn.2894 nture NEUROSCIENCE