R E S E A R C H A R T I C L E
Chrna2-OLM interneurons display different membrane
properties and h-current magnitude depending on
dorsoventral location
Markus M. Hilscher
1,2,3| Ingrid Nogueira
1| Sanja Mikulovic
3| Klas Kullander
3|
Richardson N. Leão
1,3| Katarina E. Leão
11
Brain Institute, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
2
Institute for Analysis and Scientific Computing, Vienna University of Technology, Vienna, Austria
3
Unit of Developmental Genetics, Department of Neuroscience, Uppsala University, Uppsala, Sweden
Correspondence
Markus M. Hilscher and Katarina E. Leão, The Brain institute, Federal University of Rio Grande do Norte, Av. Nascimento de Castro 2155, CEP: 59056-045, Natal, RN, Brazil. Email: markus.hilscher@neuro.ufrn.br (M. M. H.) and
Email: katarina.leao@neuro.ufrn.br (K. E. L.)
Funding information
Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES); Brazilian National Council of Technological and Scientific Development (CNPq); Swedish Foundation for International Cooperation in Research and Higher Education (STINT)
Abstract
The hippocampus is an extended structure displaying heterogeneous anatomical cell
layers along its dorsoventral axis. It is known that dorsal and ventral regions show
dif-ferent integrity when it comes to functionality, innervation, gene expression, and
pyramidal cell properties. Still, whether hippocampal interneurons exhibit different
properties along the dorsoventral axis is not known. Here, we report
electrophysio-logical properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from
coronal sections of the Chrna2-cre mouse line. We found dorsal OLM cells to exhibit
a significantly more depolarized resting membrane potential compared to ventral
OLM cells, while action potential properties were similar between the two groups.
We found ventral OLM cells to show a higher initial firing frequency in response to
depolarizing current injections but also to exhibit a higher spike-frequency adaptation
than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags
in response to negative current injections correlating with our results showing that
dorsal OLM cells have more hyperpolarization-activated current (I
h) compared to
ventral OLM cells. Immunohistochemical examination indicates the h-current to
cor-respond to hyperpolarization-activated cyclic nucleotide-gated subunit 2 (HCN2)
channels. Computational studies suggest that I
hin OLM cells is essential for theta
oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a
higher membrane resonance frequency than ventral OLM cells. Thus, our results
highlight regional differences in membrane properties between dorsal and ventral
OLM cells allowing this interneuron to differently participate in the generation of
hip-pocampal theta rhythms depending on spatial location along the dorsoventral axis of
the hippocampus.
K E Y W O R D S
Chrna2-cre, dorsoventral, HCN, H-resonance, hyperpolarization-activated current, OLM cell, septotemporal
DOI: 10.1002/hipo.23134
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| I N T R O D U C T I O N
There is emerging evidence that the hippocampus demonstrates spe-cializations in signal processing along its dorsoventral (or septotemporal) axis (Dougherty et al., 2013; Dougherty, Islam, & Johnston, 2012; Fanselow & Dong, 2010; Marcelin, Lugo joaquin, et al., 2012b; Strange, Witter, Lein, & Moser, 2014). For example, the dorsal hippocampus (DH) appears involved in cognitive functions and spatial orientation (Buzsáki & Moser, 2013; Jung, Wiener, & McNaughton, 1994; Morris, Garrud, Rawlins, & O'Keefe, 1982) while the ventral hippocampus (VH) relates to emotions and stress (Contreras, Pelc, Llofriu, Weitzenfeld, & Fellous, 2018; Felix-Ortiz et al., 2013; Mikulovic et al., 2018; Tendler & Wagner, 2015; Winne et al., 2018). Such specializations reflect differences in connectivity, but interestingly, also single cell gene expression of hippocampal neu-rons show regional differences (Bienkowski et al., 2018; Cembrowski et al., 2016). Thereby, dorsal CA1 cell types exhibit a more homoge-neous gene profile when compared to ventral CA1 cells (Shah, Lubeck, Zhou, & Cai, 2016). Additionally, synaptic and membrane properties of CA1 pyramidal cells (PCs) have been shown to follow a dorsoventral organization (Dougherty et al., 2012; Dubovyk & Manahan-Vaughan, 2017; Malik, Dougherty, Parikh, Byrne, & John-ston, 2016; Marcelin, Lugo joaquin, et al., 2012b; Milior et al., 2016; Papaleonidopoulos, Trompoukis, Koutsoumpa, & Papatheodoropoulos, 2017). Specifically, the hyperpolarization-activated current (Ih) is dif-ferentially expressed in PCs along the CA1 dorsoventral axis (Dougherty et al., 2012; Marcelin, Lugo joaquin, et al., 2012b). Ihis known to contribute to differences in subthreshold resonance of hip-pocampal PCs and interneurons (Dougherty et al., 2013; Zemankovics, Káli, Paulsen, Freund, & Hájos, 2010) and shapes membrane reso-nance frequency, causing cells to preferably fire in response to inputs around theta frequency (Pike et al., 2000; Zemankovics et al., 2010).
Interestingly, type 1 theta oscillations (theta1, 7–12 Hz) that origi-nate in the dorsal hippocampus during locomotion and other types of voluntary movements, are shown to “travel” as waves along the septotemporal axis of the hippocampus and display progressively lower amplitude and speed-relationship toward VH (Lubenov & Siapas, 2009; Patel, Fujisawa, Berényi, Royer, & Buzsáki, 2012). Spike time recordings of several distinct hippocampal interneurons showed traveling theta waves to rely on temporal shifts between different interneurons within the dorsal and intermediate hippocampus (Forro, Valenti, Lasztoczi, & Klausberger, 2015). Furthermore, one study has shown that VH generates slightly slower theta oscillations, resembling cholinergic-dependent type 2 theta (theta2, 4–9 Hz), in anxiety-related behavior (Adhikari, Topiwala, & Gordon, 2010). Our group has recently demonstrated that these oscillations can coexist with the type 1 theta activity and are driven by a specific subpopulation of VH interneurons (Mikulovic et al., 2018). However, whether hippocampal theta oscillations rely on dorsoventral gradients in interneuron inte-grative properties is still not known.
Modeling studies have suggested that oriens lacunosum moleculare (OLM) interneurons are essential to hippocampal theta rhythmogenesis through feedback inhibition (Hummos, Franklin, & Nair, 2014; Neymotin et al., 2013; Tort, Rotstein, Dugladze, Gloveli, & Kopell, 2007). OLM cells, together with bistratified cells that target different dendritic compartments of PCs than OLM cells (Müller & Remy, 2014), contribute to behaviors where gating of information is important for the functional output of the hippocampus (Katona et al., 2014; Leão et al., 2012; Lovett-Barron et al., 2014; Mikulovic et al., 2018; Siwani et al., 2018). The OLM neuron is characterized based on its morphological features and slow accommodating firing (Leão et al., 2012), and recently several genetic markers have been evaluated for targeting OLM cells (Chittajallu et al., 2013; Leão et al., 2012; Mikulovic, Restrepo, Hilscher, Kullander, & Leão, 2015; Taniguchi et al., 2011). Chittajallu and colleagues, show that OLM cells can be separated based on expression of either 5-HT3A receptors (5HT3AR) or the transcription factor Nkx2-1 (Chittajallu et al., 2013). They dem-onstrated that OLM cells expressing either 5-HT3AR or Nkx2-1 were differentially involved in network oscillations, with 5-HT3AR expressing OLM cells being regulated by serotonin and not displaying phase locked firing to the kainite-induced gamma activity in vitro. In contrast, Nkx2-1-expressing OLM cells fired phase locked to kainate-induced gamma oscillations (Chittajallu et al., 2013). Recently, Leão and others used the mouse Cre-line for nicotinic acetylcholine recep-tor subunit alpha 2 (Tg(Chrna2-cre)1Kldr mice) that labels OLM cells of the CA1 (OLMα2 cells) (Leão et al., 2012). They found ventral Chrna2-cre OLMα2cells to drive slow, type 2 theta oscillations that relate to increased risk-taking in response to predator odor (Mikulovic et al., 2018). The presence of Ihin interneurons of the stratum oriens has been shown (Griguoli et al., 2010; Lupica, Bell, Hoffman, & Watson, 2001; Maccaferri & McBain, 1996) and investigations of knockout mice for the hyperpolarization-activated cyclic nucleotide-gated channel subunit 2 (HCN2) indicate that HCN2 modulates OLM cell firing (Matt et al., 2011). Whether OLMα2cells show different membrane properties, such as Ih, in dorsal or ventral regions of the CA1 is not known. Computationally, Ihhas been shown to allow the tuning of both theta and gamma oscillations in a CA3 circuit model (Neymotin et al., 2013). On the other hand, a recent study found CA1 OLM cells to follow theta-frequency inputs independently of Ih (Kispersky, Fernandez, Economo, & White, 2012). However, the aforementioned studies do not report the specific location of investi-gated OLM cells along the dorsoventral hippocampal axis, while a recent study (Siwani et al., 2018) demonstrated that OLMα2cells from the intermediate hippocampus show larger depolarization in response to nicotine than DH OLMα2interneurons. These results indicate dif-ferential biophysical properties of OLM interneurons along the septotemporal hippocampal axis. Hence, the electrophysiological properties, and specifically the role of OLM cell Ihin generating theta oscillations is not well understood.
Here, we compare active and passive membrane properties of dorsal and ventral CA1 OLMα2cells of the Chrna2-cre mice hippo-campi. We show that dorsal OLMα2cells display membrane resonance resembling faster type 1 theta frequency, while ventral OLMα2cells
resonate in a lower frequency, resembling type 2 theta frequency. We demonstrate that dorsal CA1 OLMα2cells have larger Ihthan ventrally located OLMα2cells, which contributes to the more depolarized rest-ing membrane potential in dorsal OLMα2cells. Finally, we discuss how these findings relate to different rhythmogenesis observed in the DH and VH.
2
| M A T E R I A L S A N D M E T H O D S
2.1 | Mice
Adult transgenic Tg(Chrna2-cre)1Kldr mice were crossed with the fluorescence reporter line Gt(ROSA)26Sortm14(CAG-tdTomato)Hze (R26tom; Allen Brain Institute) to generate Chrna2-Cre/R26tommice (Leão et al., 2012; Mikulovic et al., 2015). We refer to these mice as Chrna2-cre/tdTomato positive (+), where the promoter of the cholin-ergic nicotine receptor alpha 2 (chrna2) gene controls the expression of the red fluorescent protein tandem dimer tomato (tdTomato). The Chrna2-cre animals were created at Uppsala University by the group of K. Kullander and stock animals were kindly donated to the Brain institute, Natal, Brazil. Mice were housed in the animal facilities of Uppsala University and the Federal University of Rio Grande do Norte under 12:12 light/dark cycles. Experiments were approved by the Swedish Animal Welfare authorities and followed Uppsala University guidelines for the care and usage of laboratory animals (ethics permits C132/13 and C135/14), and the Animal Ethics Committee (CEUA) of the Federal University of Rio Grande do Norte (Protocol number 052/2015). Effort was made to minimize suffering and discomfort of animals and to reduce the number of animals used.
2.2 | Electrophysiology
Coronal slices from 13 Chrna2-Cre/R26tommice (P19-23) from both sexes were used, and dorsal and ventral cells were mostly recorded from the same slices. In summary, brains were rapidly removed and placed in ice-cold sucrose/artificial cerebrospinal fluid (ACSF) con-sisting of the following (in mM): KCl, 2.49; NaH2PO4, 1.43; NaHCO3, 26; glucose, 10; sucrose, 252; CaCl2, 1; MgCl2, 4 (from Sigma Aldrich, MO). Slices were cut using a vibratome (VT1200, Leica, Microsystems) and were subsequently moved to a submerged holding chamber con-taining normal ACSF (in mM): NaCl, 124; KCl, 3.5; NaH2PO4, 1.25; MgCl2, 1.5; CaCl2, 1.5; NaHCO3, 30; glucose, 10, constantly bubbled with 95% O2and 5% CO2and kept at 35C for 1 hr then maintained at room temperature. The slices were transferred to a submerged chamber under an upright microscope equipped with DIC optics (Olympus, Japan) and perfused with 30C oxygenated ASCF (1–1.25 mL/min). Patch pipettes from borosilicate glass capillaries (GC150F-10, Harvard Apparatus, MA) were pulled on a vertical puller (PC-10, Narishige, Japan). Pipette resistances varied from 4 to 7 MΩ (series resistance was >13 MΩ and compensated by 80%). There was no significant difference in pipette/series resistance in recordings from dorsal and ventral cells. Pipettes were filled with internal solu-tion containing (in mM): K-gluconate, 130; NaCl, 7; MgCl2, 2; ATP, 2;
GTP, 0.5; HEPES, 10; EGTA, 0.1 (from Sigma Aldrich, MO). The pH was adjusted to 7.2 using KOH. Whole cell current and voltage clamp recordings were acquired using a Multiclamp 700B amplifier (Axon Instruments, CA), or Axopatch 200B amplifier (Axon instruments, CA) and digitized with a Digidata 1440A data acquisition card (Axon Instruments, CA), or a BNC-2111 panel block (National instruments,
TX). WinWCP and WinEDR softwares implemented by
Dr. J. Dempster (University of Strathclyde, Glasgow, UK) were used to record electrophysiological signals. Some experiments required bath application of the Ihblocker ZD7288 (Tocris Cookson Inc., Bristol, UK, Cat #: 1000; 20μM) and tetrodotoxin (TTX, Tocris Cookson Inc., Bris-tol, UK, Cat #: 1078; 1μM). When ZD7288 was applied to the bath only one cell per slice was patched, as ZD7288 binds intracellularly and is therefore difficult to wash out from the cytosol. Alexa Fluor (Invitrogen, MA, Cat #: A10436; 488 nm), or biocytin (Sigma Aldrich, MO, Cat #: B4261; 1.5 mg/mL) counterstained with Streptavidin-Alexa-Fluor (Invitrogen, MA, Cat #: 49937; 488 nm), was routinely added to the intracellular solution to visualize OLMα2 cells and to group cells as dorsal or ventral based on the presence in the upper or lower region similar to the Allen Institute mouse reference atlas (Lein et al., 2007). Images were collected on a confocal microscope (Zeiss LSM 510 Meta or AxoExaminer A1, Jena, Germany) and stacked.
2.3 | Analysis
Electrophysiological data was analyzed in MATLAB (versions 2016b and 2018b, MathWorks, MA). Membrane input resistance was calcu-lated from small positive current injections (10 pA) and measuring the steady-state membrane potential. Action potentials (APs) were trig-gered by 500 ms depolarizing current injections (10–100 pA, 10 pA increments). The first AP generated upon positive current injections was analyzed for AP amplitude (peak to afterhyperpolarization [AHP] potential), threshold (where the change in membrane potential reaches 20 mV/ms), half-width (halfway between threshold voltage and peak), and first spike latency (time between stimulus onset and the AP threshold of the first spike). Phase-plane plots were also car-ried out on the first APs following positive current injections. The inflection rate was estimated by fitting a linear regression to the three data points closest to the AP threshold (Felix, Vonderschen, Berrebi, & Magnusson, 2013). AHPs were analyzed for depth (minimum of the voltage peak between two consecutive APs). Firing frequency was recorded as response to 1 s depolarizing current injections (0–200 pA, 10 pA increments). Spike-frequency adaptation was calculated as the inverse of the mean of the last three interspike intervals (steady-state frequency) divided by the inverse of the first interspike interval (initial firing frequency) in response to 150 pA current injections and sub-tracted from 100% (no adaptation = 0%). Peak and steady-state volt-age were examined in response to hyperpolarizing current injections (0 to−100 pA, 10 pA decrements, 500 ms). The first rebound AP was described by amplitude and half-width. Sag response at different peak voltages was quantified for cells (held at−60 mV and subjected to negative current steps [0 to−100 pA, 10 pA decrements, 500 ms]) by measuring the difference between peak and steady-state voltage
(ΔVm). For voltage clamp recordings, cells were held at−60 mV and submitted to negative voltage steps (−60 to −130 mV, 10 mV decre-ments, 1 s) and steady-state h-current was measured as the average current at 500–1,000 ms. Tail currents were analyzed to examine voltage dependency of Ih.Tail currents were measured at−60 mV fol-lowing negative voltage steps (−65 to −120 mV, 5 mV decrements, 1 s). To decrease variability in tail values we averaged 5–15 ms after the voltage step. Normalized tail currents (I/Imin) were fitted to a Boltzmann function (I/Imin= [1 + exp (V1/2− V)/k]−1, where V1/2is the half-activation voltage and k is the slope factor).
For electrical resonance of dorsal and ventral OLMα2 cells, the impedance (Z) amplitude profile (ZAP) was obtained (Hu, Vervaeke, & Storm, 2002). The membrane potential was first manually clamped to −60 mV by direct current injection through the recording electrode. A sinusoidal current with constant amplitude (three conditions of peak to peak value: ±25 pA, ±50 pA, and ±100 pA with a minimum delay of 10 s between the different amplitudes) and linearly increasing fre-quency (0–20 Hz over 20 s) was injected to dorsal and ventral OLMα2 cells and their voltage response was measured. TTX (1μM) was rou-tinely applied to the perfusate to prevent cells from spiking. For ZD7288 experiments, the membrane potential was manually clamped to−73 mV and after ZD7288 application again adjusted to compen-sate for the ZD7288-induced hyperpolarization. To measure the mag-nitude of the cell impedance as a function of frequency, the magnitude of the fast Fourier transform (FFT) of the voltage response was divided by the magnitude of the FFT of the input current (Hu et al., 2002).
2.4 | Immunohistochemistry
Mice were perfused with saline and cold 4% paraformaldehyde and 35μm thick coronal or horizontal slices were cut on a cryostat (Leica or Thermo Scientific Microm HM550) (1–2 months old). Fresh antibody/blocking solution (10 mL phosphate buffer [0.1M], 500μL normal donkey serum or goat serum, 30μL Triton-X-100) was used to block sections for 1 hr and also to dilute primary antibodies (Rabbit [Rb] anti HCN1 [1:100, Alamone Labs], Rb anti HCN2 [1:100, Alamone Labs], Rb anti HCN4 [1:100, Alamone Labs], mouse anti parvalbumin [1:1,000, Chemicon]) and sections were incubated at 4C for 3 days. After washing 3 times in PBS sections were incubated with secondary antibodies (donkey anti mouse 1:400 [647 nm], donkey anti rabbit 1:400 [488 nm] or goat anti rabbit 1:1,000 [488 nm], from Thermo Fisher Scientific, MA) diluted in antibody solution. Incubations with only secondary antibodies were carried out as controls. Images were collected on a confocal microscope (Zeiss LSM 510 Meta or Zeiss AxoExaminer A1, Jena, Germany) with a 20x/0.8 Plan Apochromate objective and adjusted for contrast and brightness in ImageJ 1.52i.
2.5 | Statistics
Data is reported as mean ± SEM. Statistical comparisons between dor-sal and ventral OLMα2cells were done in MATLAB (version 2016b
and 2018b, MathWorks, MA). The statistical significance of patch clamp experiments was evaluated with the two-tailed Student's t test when the datasets were normally distributed. Normal distribution was assessed by the Lilliefors test. Comparisons of dependent variables, such as before and after ZD7288 application, were determined by a paired t test. When data were not normally distributed, or the vari-ances were unequal, the nonparametric Wilcoxon rank-sum (RS) test was used.
3
| R E S U L T S
We recorded electrophysiological properties of OLMα2 cells (Leão et al., 2012; Mikulovic et al., 2015) in coronal brain slices of Chrna2-Cre/tdTomato mice (Figure 1). Visually identified OLMα2cells, expressing red fluorescent protein (tdTomato) located in the CA1 stra-tum oriens were grouped as dorsal or ventral in post hoc analysis of images of Alexa488- or biocytin-filled OLMα2 cells (Figure 1a), by dividing elongated, ventrocaudal, and intermediate CA1 coronal section similar to the Allen Institute mouse reference atlas (Lein et al., 2007) in a dorsal and a ventral part (Figure 1b). We did not assess cor-onal sections of the most dorsorostral CA1, as this region contains few OLMα2cells (Mikulovic et al., 2015, 2018). All filled OLMα2cells had axonal projections extending to the lacunosum moleculare layer, where they branched profusely and created a dense red fluorescent signal (Figure 1b,c). There was no apparent difference in morphology or axonal length between dorsal and ventral OLMα2 cells in ventrocaudal and intermediate coronal sections (Figure 1c and Mikulovic et al., 2018). Filled dorsal and ventral OLMα2 cells from thick sections showed similar axonal bifurcation and dendritic distribu-tion (Figure 1d). OLMα2cells are a distinct, concise OLM cell popula-tion that have been described previously (Leão et al., 2012; Mikulovic et al., 2015; Nichol, Amilhon, Manseau, Badrinarayanan, & Williams, 2018). Here, we provide additional information that OLMα2cells sel-dom show overlap with antibodies against parvalbumin (Pvalb) (0.43%, 3/691 Chrna2-cre/tomato+ cells were Pvalb immuno positive, n = 3 mice, horizontal sections, Figure S1).
Whole-cell patch clamp recordings showed all OLMα2cells to dis-play consistent regular spiking (32/32, Figure 2a) as reported previ-ously (Mikulovic et al., 2015). Comparing dorsal and ventral OLMα2 cells showed a difference in mean resting membrane potential with dorsal OLMα2cells being more depolarized than ventral OLMα2cells (dorsal OLMα2cells: −60.65 ± 0.73 mV, n = 16 cells; ventral OLMα2 cells:−68.53 ± 1.89 mV, n = 16 cells; p = .038, Student's t test). The somatic mean input resistance at rest was not significantly different (dorsal OLMα2cells: 395.24 ± 42.93 MΩ, n = 16 cells; ventral OLMα2 cells: 318.27 ± 58.16 MΩ, n = 16 cells; p = .302, Student's t test). Also the average steady-state depolarization in response to depolarizing current injections (with 10 pA increments) was not significantly differ-ent for dorsal OLMα2cells (2.91 ± 0.28 mV, n = 16 cells) compared to ventral OLMα2cells (3.50 ± 0.29 mV, n = 16 cells; p = .119, Student's t test). Examining firing frequency properties of dorsal and ventral OLMα2cells showed that ventral cells had a shorter first interspike
interval (23.31 ± 1.96 ms, n = 16 cells) compared to dorsal cells (29.60 ± 2.37 ms, n = 16 cells, p = .012, Wilcoxon RS test) in response to a 150 pA, 1 s pulse (Figure 2a). Although initial firing frequency was higher for ventral OLMα2cells (43.22 ± 2.51 Hz, n = 16 cells) com-pared to dorsal OLMα2cells (34.30 ± 1.71 Hz, n = 16 cells; p = .048, Wilcoxon RS test), there was no difference in average steady-state fir-ing frequency at the end of the stimulation (Figure 2a,b, Table 1). Thus, the spike-frequency adaptation rate, that is, the decline of firing frequency, was higher for ventral OLMα2cells (47%, n = 16 cells) than for dorsal OLMα2cells (34%, n = 16 cells; p = .040, Wilcoxon RS test). Current clamp recordings further revealed no differences in action potential features between dorsal and ventral OLMα2cells (10–100 pA, 10 pA increments, 500 ms) but AHP depth was more prominent in ventral OLMα2cells (dorsal OLMα2cell: −66.51 ± 1.52 mV, n = 16 cells; ventral OLMα2cells:−74.78 ± 2.08 mV, n = 16 cells; p = .027, Student's t test, Figure 2c, Table 1). AHP depth voltages of repeated spikes of both dorsal and ventral OLMα2 cells exposed a small but gradual depolarization of AHP depth in consecutive spikes. Addition-ally, also the phase portrait of action potentials for both dorsal and ventral OLMα2cells, were examined. The phase-plane plots, which show the derivative of membrane potential (dV/dt) as a function of instantaneous membrane potential, exhibited a biphasic shape for both cells (Figure 2d). The inflection rate at AP threshold showed simi-lar values for dorsal and ventral OLMα2cells (Table 1). Significant elec-trical properties from Table 1 were also examined for potential
relationships with cell position. While resting membrane potential and afterhyperpolarization depth of dorsal and ventral OLMα2 cells only showed weak correlation with the location of the cells, initial firing frequency, and spike-frequency adaptation correlated well with cell position (Figure S2).
In response to negative current injections (0 to−100 pA, 10 pA decrements, 500 ms) dorsal OLMα2cells produced more prominent membrane sags than ventral OLMα2 cells, with ventral cells only showing membrane depolarizing sags in response to the most nega-tive current steps (Figure 3a). At the termination of the hyp-erpolarizing step rebound spikes were often elicited in dorsal OLMα2 cells (11 out of 16 cells) while ventral OLMα2cells were less likely to elicited rebound spikes (3 out of 16 cells) (Figure 3a, Table 1). Sags are an indicative of hyperpolarization-activated currents (Ih) that can shape membrane resonance frequency and allow cells to preferably fire in response to inputs around theta frequency (Zemankovics et al., 2010). Comparing peak and steady-state voltage for dorsal OLMα2 cells showed a significant difference at −60 to −100 pA steps (Figure 3b, left, black lines; n = 9 cells) but no difference for ventral OLMα2cells (Figure 3b, right, black lines; n = 9 cells). After applying the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (20μM) to the artificial cerebrospinal fluid perfusate the sag-response of dorsal OLMα2 cells diminished (Figure 3b, left, gray lines) whereas the response in ventral OLMα2cells was mainly unaltered (Figure 3b, right, gray lines). While the average resting F I G U R E 1 Dorsal and ventral OLMα2cells of the CA1 hippocampal region show similar morphology. (a) Representative images of
Chrna2-Cre/tdTomato positive OLM cells (red, top: dorsal, bottom: ventral) with examples of biocytin-filled OLMα2cells (green) for identification of spatial location in a 300μm thick slice are shown. Scale bar 50 μm. (b) Confocal images showing the distribution of Chrna2-cre/tdTomato+ OLMα2cells along CA1 in a coronal section. The lacunosum moleculare region is strongly labeled from the dense axonal ramifications. The outer layer of labeled neurons represents cortical Martinotti cells (Hilscher, Leão, Edwards, Leão, & Kullander, 2017). Inset: Schematic image showing the approximate location of patched dorsal and ventral OLMα2cells (dashed lines mark the separation between dorsal and ventral regions). (c) Representative confocal stacks (collapsed z-stack, 35μm section) from dorsal (top) and ventral (bottom) regions of the CA1 show similar axonal extensions and similar axonal length. Dorsal OLMα2cells are more clustered while ventral OLMα2cells appear more banded. (d) Representative images of biocytin filled OLMα2cells (green) from a dorsal region (top) and ventral region (bottom) show similar axonal bifurcations and dendritic extensions in the vertical direction. Scale bar 50μm. OLM, oriens lacunosum moleculare [Color figure can be viewed at wileyonlinelibrary.com]
membrane potential of dorsal and ventral OLMα2 cells was signifi-cantly different before ZD7288 (p = .030), this difference was abolished following ZD7288-application (dorsal OLMα2cells:−64.42 ± 2.84 mV, n = 5 cells; ventral OLMα2cells:−66.11 ± 3.11 mV, n = 5 cells; p = .760, Student's t test) (Table 2). As dorsal and ventral OLMα2 cells showed differences in resting membrane potential, some cells were held at−60 mV by injecting a bias current, before responses to negative current steps were examined. Here, we plot the peak voltage over the difference between peak and steady-state voltage (ΔVm) to quantify the differences in sag between dorsal and ventral OLMα2 cells, for responses that reached similar peak voltages (Figure 3c). Horizontal error bars indicate variations in peak voltages. At a mean peak voltage of−107.31 ± 1.92 mV dorsal OLMα2 cells exposed a ΔVmof 9.82 ± 1.71 mV while ventral OLMα2cells had aΔVmof 4.72 ± 1.62 mV at−104.20 ± 1.61 mV peak voltage (n = 8 cells; p = .003, Student's t test).
Next, we quantified OLMα2cell Ihaccording to dorsal or ventral cell location following the response to negative voltage steps (−60 mV to −130 mV, 10 mV decrements, 1 s). Dorsal OLMα2cells generated a greater inward current compared to ventral OLMα2cells
and this difference was abolished by ZD7288 (20μM) application (Figure 4a). On average, dorsal OLMα2cells generated a steady-state current of−317.99 ± 14.91 pA in response to a −130 mV voltage step which significantly diminished to−217.43 ± 16.15 pA following ZD7288 application, resulting in an average Ih-dependent current of −100 pA (subtracted current; n = 5 cells; Figure 4a, left). Dorsal OLMα2 cell h-current was significantly different before and after ZD7288 application at voltage steps more negative than−100 mV (n = 5 cells; p < .05, paired Student's t test, Figure 4b, left and Table 2). Ventral OLMα2cells did not show a significantly smaller cur-rent upon blocking h-curcur-rent, neither was resting membrane potential altered by ZD7288 (n = 5 cells, paired Student's t test, Figure 4a,b, right and Table 2). The voltage dependency of the Ihwas examined by tail currents generated by hyperpolarizing current steps (−65 to −120 mV, 5 mV decrements). After a −90 mV test step, the average dorsal OLMα2 cell tail current (measured at −60 mV) was −52.31 ± 3.40 pA (n = 9 cells). For comparison we also measured hyperpolarization-generated tail currents from ventral OLMα2 cells, despite them being small (−22.92 ± 10.37 pA, n = 9 cells) with magni-tude of ventral tail currents different from dorsal tail currents
F I G U R E 2 Spike-frequency adaptation is different between dorsal and ventral OLMα2cells. (a) Current clamp traces of a dorsal (left) and a ventral (right) OLMα2cell in response to a 150 pA, 1 s long stimulus. Gray boxes highlight maximum frequency (inverse of the first interspike interval) and steady-state frequency (inverse of the mean of the last three interspike intervals.) resp. dashed lines denote the baseline voltages. (b) The maximum frequency (left) and steady-state firing frequency (right) for dorsal (●) and ventral (○) OLMα2cells. Data presented as mean ± SEM.*p < .05. (c) Representative example of the first action potential in response to somatic current injection for a dorsal (solid line) and a ventral (dashed line) OLMα2cell highlighting difference in AHP depth. (d) Graph showing the phase-plot of a dorsal (solid line) and a ventral (dashed line) OLMα2cell. OLM, oriens lacunosum moleculare
(p = .016, Student's t test). Fitting a Boltzmann equation to quantify the voltage-dependency of the tail currents, indicating Ih, showed no difference in half-activation voltage or slope factor between dorsal (n = 9 cells) and ventral (n = 9 cells) OLMα2cells (Figure 4c,d, Table 2). Immunohistological examination did not show HCN1 staining col-ocalized with Chrna2-cre/tdTomato+ cell bodies, instead immune-positive puncta was more apparent in the region of the stratum pyr-amidale (n = 3 mice, 9 slices, Figure S3). We found antibodies against the HCN2 subunit to show overlap with Chrna2-cre/tdTomato+ cells in dorsal regions (Figure 5a,b) and weaker somatic colabeling in ven-tral areas (n = 3 mice, 9 slices, Figure 5c). Immunostaining with HCN4 antibodies did not generate any detectable signal (Figure S4, n = 2 mice, 6 slices). This suggests the h-current of dorsal OLMα2cells to be generated mainly through HCN2 channels.
Furthermore, we examined the resonance frequency of dorsal and ventral OLMα2cells in the presence of TTX (1μM). OLMα2cells were stimulated with a sinusoidal current (a“ZAP” current giving the imped-ance (Z) Amplitude Profile) in the form of a chirp stimulus of three dif-ferent amplitudes (±25 pA, ±50 pA, and ±100 pA). Chirps consisted of a linearly increasing (1 Hz/s) sine wave with constant amplitude dur-ing 20 s. Voltage responses for dorsal and ventral OLMα2cells were recorded (Figure 6a) and next the magnitude of the impedances as a function of frequency was compared. This was done by performing the fast Fourier transform (FFT) of the voltage responses (Figure 6b, top) as well as the FFT of the ZAP input current (Figure 6b, middle). The impedance magnitude (Z) was calculated by dividing FFT(V) by FFT(I) and a peak in the impedance profile highlights the underlying
resonance frequency (fR). We compared the fRvalues obtained at the peak impedance for dorsal and ventral OLMα2 cells and found that they were significantly different (dorsal OLMα2cells: 2.75 ± 0.12 Hz, n = 5 cells; ventral OLMα2cells: 1.85 ± 0.10 Hz, n = 5 cells; p = .037; Student's t test; Figure 6b, bottom) in response to a 100 pA chirp sig-nal, highlighting the distinct membrane resonance properties of dorsal and ventral OLMα2 cells in the hippocampus. Similar to (Hu et al., 2002), resonance frequency was also examinated on hyperpolarized OLMα2cells and the application of ZD7288 showed a decreased volt-age peak-to-peak-amplitude in response to the different ZAP current inputs (±25 pA, ± 50pA, and ±100 pA, Figure S5).
Taken together, we found the dorsal and ventral distribution of OLM cell properties to be opposite, yet complementary, to what has been reported for CA1 PCs (Dougherty et al., 2012, 2013) when com-paring basic membrane properties, Ih and resonance frequency (Figure S6).
4
| D I S C U S S I O N
The hippocampus is a large and complex structure, comprised of numerous cell types organized in distinct layers, involved in memory formation, locomotion mapping, and emotional responses. Recently, several dorsoventral/septotemporal gradients of hippocampal charac-teristics have been reported, with most studies focusing on PC prop-erties (Cembrowski et al., 2016; Dougherty et al., 2012, 2013; Malik et al., 2016; Marcelin, Lugo joaquin, et al., 2012b; Milior et al., 2016) and others on regional gene expression (Bienkowski et al., 2018; T A B L E 1 Summary of membrane and AP properties of dorsal and ventral Chrna2-cre+ OLM cells
Dorsal Ventral p value
Vrest(mV) −60.65 ± 0.73 −68.53 ± 1.89 .038* (n = 32) Rinpat Vrest(MΩ) 395.24 ± 42.93 318.27 ± 58.16 .302 SSdep(mV) 2.91 ± 0.28 3.50 ± 0.29 .119 finitat 150 pA (Hz) 34.30 ± 1.71 43.22 ± 2.51 .048* fssat 150 pA (Hz) 22.53 ± 1.08 22.92 ± 1.16 .407 SFA at 150 pA (%) 34.30 ± 2.71 46.50 ± 3.40 .040* APampl(mV) 86.11 ± 2.52 91.76 ± 3.26 .184 APthres(mV) −40.07 ± 1.05 −44.34 ± 1.84 .208 APhalf-width(ms) 1.27 ± 0.19 1.24 ± 0.14 .723 AHPdepth(mV) −66.51 ± 1.52 −74.78 ± 2.08 .027*
Infl. rate at APthres(ms−1) 13.06 ± 0.45 12.00 ± 0.63 .182
Re_AP (% cells) 69 (11/16) 19 (3/16)
-Re_APampl(mV) 81.71 ± 4.33 85.26 ± 1.39
-Re_APhalf-width(ms) 1.23 ± 0.24 1.20 ± 0.10
-P (days) 20.6 ± 0.27 20.4 ± 0.26 .742
Note: Data is reported as mean ± SEM. Statistical significance was determined with either the Student's t test or the Wilcoxon rank-sum test with*p < .05 (see Section 2).
Abbreviations: AHPdepth, afterhyperpolarization depth; APampl, action potential amplitude; APhalf-width, action potential half-width; APthres, action potential threshold; finit, initial firing frequency; fss, steady state firing frequency; Infl. rate, inflection rate; OLM, oriens lacunosum moleculare; P, postnatal age; Rinp, input resistance; Re_AP, occurrence of rebound action potentials; Re_APampl, rebound action potential amplitude; Re_APhalf-width, rebound action potential half-width; SFA, spike-frequency adaptation; SSdep, steady-state depolarization; Vrest, resting membrane potential.
Dubovyk & Manahan-Vaughan, 2017; Fanselow & Dong, 2010; Shah et al., 2016). Here, we divided Chrna2-positive OLM cells into groups of dorsal and ventral OLMα2cells and carried out a first study of com-paring membrane properties of a distinct population of interneurons depending on spatial location within coronal section of the CA1 region. In general, the dorsal hippocampus is often accessed for in vivo recordings, while ventral areas are usually sampled in slices for in vitro studies (Dougherty et al., 2012). Here, we patched cells in dor-sal or ventral regions of coronal sections, and we did not include an intermediate region, to aim our findings on gross differences between dorsal or ventral OLMα2cells. This, in turn, can reflect discrepancies between in vivo and in vitro studies. We found dorsal OLMα2cells to have a more depolarized membrane potential compared to ventral OLMα2cells, but no significant difference in input resistance (Table 1), although there was a trend of dorsal OLMα2cells to have a higher input resistance than ventral OLMα2cells. Mean input resistance of OLM cells is diverse, which might reflect differences in sampling from more dorsal or ventral horizontal/transverse sections (Chittajallu et al., 2013; Leão et al., 2012; Nichol et al., 2018). A pre-vious work have shown that OLM cells can be embryonically
derived from two different eminences and this reflects on the dif-ferential expression of 5-HT3AR (Chittajallu et al., 2013). Ventral OLMα2 cell properties appear similar to the resting membrane potential and input resistance of both 5-HT3AR and Nkx2-1 expressing OLM cell populations recorded from transverse slices (Chittajallu et al., 2013). However, we have not tested if dorsal or ventral OLM cells are differentially affected by 5HT3AR agonists and future studies should address whether dorsal and ventral OLM cells can be differentiated.
Recent single-cell RNA sequencing has found the hippocampus to show a large genetic variability among gene profiles of hippocampal interneurons (Harris et al., 2018; Zeisel et al., 2015). Thereby, three classes of OLM cells have been identified by expressing somatostatin (Sst), glutamate metabotropic receptor 1 (Grm1), prepronociceptin (Pnoc), and reelin (Reln) (Harris et al., 2018). While two of these clas-ses contain parvalbumin (Pvalb)-expressing OLM cells (Katona et al., 2014; Klausberger et al., 2003), the third class contains the non-Pvalb expressing OLM cells (Harris et al., 2018). This cluster also includes Chrna2+ OLM cells and is distinct from the hippocamposeptal cells as well as the bistratified neurons (Harris et al., 2018).
F I G U R E 3 Dorsal OLMα2cells show more prominent depolarization sag in response to negative current injections compared to ventral OLMα2cells. (a) Representative current clamp traces of a dorsal (left) and a ventral (right) OLMα2 cell in response to depolarizing (left: 60 pA, 500 ms; right: 100 pA, 500 ms) and hyperpolarizing current injections (0 pA to−100 pA, 20 pA decrements, 500 ms) are shown. (b) Current–voltage relationships between peak voltage (peak, o) and steady-state membrane voltage (SS,■) before (black) and after (gray) ZD7288 (20μM) application for dorsal (left) ventral (right) OLMα2cells. (c) Representative traces from a dorsal and a ventral OLMα2cell, held at−60 mV using a bias current, responding to negative current injections (0 pA to−100 pA, 10 pA decrements, 500 ms). The plot to the right shows peak voltage over the difference in peak and steady-state voltage (ΔVm) for dorsal (●) and ventral (○) OLMα2cells. Data presented as mean ± SEM.*p < .05. OLM, oriens lacunosum moleculare
4.1 | Dorsal OLM
α2cells express more
hyperpolarization-activated current than ventral
OLM
α2cells
Despite OLMα2 cells showing similar AP properties across the CA1 dorsoventral axis, differences in passive membrane properties can potentially allow them to be differently recruited into hippocampal network output. Here, we found that the more depolarized resting membrane potential of dorsal OLMα2 cells compared to ventral OLMα2cells was due to Ih, as application of the HCN-blocker ZD7288 abolished this difference (Table 2). The small hyperpolarization-induced current of ventral OLMα2cells did not significantly contribute to setting the resting membrane potential. Analyzing the tail-currents showed dorsal and ventral OLMα2 cells to share similar voltage-dependency of Ih, despite the difference in magnitude of the current. This suggests that similar types of HCN channels are contributing to generating the h-current, although dorsal OLMα2cells may have stron-ger membrane expression than ventral OLMα2cells. Studies of HCN2 knockout mice suggest OLM cell Ihto mostly consist of HCN2 sub-units and that this current regulates resting membrane potential (Matt et al., 2011). We found somatic HCN2 expression in OLMα2cells, and the immunoreactivity appeared stronger in dorsal areas compared to ventral areas. Due to the strong genetic expression of tdTomato we had to narrow the bandwidth of detection spectra on the confocal microscope to avoid bleed-through of tdTomato signal in the green channel, and therefore we did not quantify the fluorescent signal of HCN2 antibodies in this study. However, it is interesting that the rest-ing membrane potential of OLM cells in Matt et al. was similar to dor-sal OLMα2cells in our study, and HCN2 knockout mice showed a Vrest similar to after ZD7288 application for dorsal OLMα2cells reported here (Matt et al., 2011). Also, Bender et al. show overlap between somatostatin (Sst) and HCN2 mRNA of stratum oriens interneurons, while a lack of overlap between HCN1 and Sst-expression in stratum oriens interneurons from rats (Bender et al., 2001). This is in agree-ment with OLM cells expressing HCN2 channels, as Sst is expressed by OLM cells (Baude et al., 1993; Klausberger et al., 2003; Maccaferri,
Roberts, Szucs, Cottingham, & Somogyi, 2000; Naus, Morrison, & Bloom, 1988) and OLMα2cells (Mikulovic et al., 2018; Nichol et al., 2018). Also, OLM cell recordings from HCN1 knockout mice showed no difference in hyperpolarization-activated currents compared to recordings from control mice (Matt et al., 2011). HCN4 subunits have been shown to label cell somas of interneurons of the CA1 stratum oriens (Bender et al., 2001; Hughes et al., 2013); however, Hughes et al. report HCN4 positive cells of the stratum oriens as Pvalb-positive (Hughes et al., 2013). We did not detect any HCN4 immunoreactivity in OLMα2cells, and as we hardly observed any Pvalb+ OLMα2cells (0.43%) it is unlikely that OLMα2cells express the HCN4 subunit to any greater extent. In a study by Notomi and Shigemoto, examining HCN1-4 channel distribution in the rat brain, only HCN1 and HCN2 immunoreactivity was reported for the CA1stratum oriens (Notomi & Shigemoto, 2004). The differential expression of HCN channels on OLM cell dendrites has been suggested by a multicompartment com-putational model (Sekulic, Chen, Lawrence, & Skinner, 2015; Sekulic, Lawrence, & Skinner, 2014) but remains to be examined for OLM cells in vitro. For PCs, Ihin dendritic recordings shows a gradient along the dorsoventral axis (Marcelin, Lugo joaquin, et al., 2012b), in agreement with immunohistochemical studies of HCN subunit expression on PC dendrites (Dougherty et al., 2012). In addition, Marcelin et al. showed VH CA1 PCs to be more sensitive to cyclic AMP (cAMP) (Marcelin, Liu, et al., 2012a) that shifts the voltage-dependency of HCN4 chan-nels, but less so of HCN2 and HCN1 chanchan-nels, to more positive potentials (Robinson & Siegelbaum, 2003; Santoro & Baram, 2003). Dendritic expression of HCN channels on OLM cells, and whether OLM cell Ihis modulated by cAMP, remains to be studied.
4.2 | Dorsal and ventral OLM
α2cells have different
resonance frequencies
Membrane resonance, an important feature often linked to oscillatory properties of cells, is shaped by interactions between passive mem-brane properties and additional currents with slow time constants, T A B L E 2 Contribution of the hyperpolarization-activated current to Chrna2-cre+ OLM cell membrane properties
Dorsal Ventral p value
Vrest(mV) −60.65 ± 1.09 −68.53 ± 3.20 .038* (n = 32) + ZD7288 (20μM) −64.42 ± 2.84 −66.11 ± 3.11 .760 (n = 10) Rinpat Vrest(MΩ) 392.89 ± 37.00 321.11 ± 44.58 .328 (n = 32) + ZD7288 (20μM) 452.87 ± 62.01 349.14 ± 43.22 .402 (n = 10) Iss(pA) −317.99 ± 14.91 −238.11 ± 20.05 .085 (n = 10) + ZD7288 (20μM) −217.43 ± 16.15 −215.75 ± 19.11 .953 (n = 10) Itail(pA) −52.31 ± 3.40 −22.92 ± 10.37 .016* (n = 18) V1/2(mV) −92.72 ± 1.12 −94.85 ± 1.21 .216 (n = 18) k 7.52 ± 0.35 8.10 ± 0.66 .451 (n = 18)
Note: Data is reported as mean ± SEM. Statistical significance was determined with either the Student's t test or the Wilcoxon rank-sum test with*p < .05 (see Section 2).
Abbreviations: Iss, steady state current; Itail, tail current; k, slope factor; OLM, oriens lacunosum moleculare; Rinp, input resistance; V1/2, half-activation voltage; Vrest, resting membrane potential.
such as Ih(Hu et al., 2002). Here, we show that both dorsal and ven-tral OLMα2cells possess membrane resonance behavior and dorsal OLMα2cells to express distinct somatic h-current compared to ventral OLMα2cells. In our preparation, we found dorsal OLMα2cells to reso-nate close to 3 Hz while ventral OLMα2cells resonated close to 2 Hz.
The existence of two different types of hippocampal theta oscillations in vivo was postulated decades ago (Kramis, Vanderwolf, & Bland, 1975). The higher frequency, movement-related, cholinergic-independent type 1 theta originates in the dorsal hippocampus (Lubenov & Siapas, 2009; Patel et al., 2012) and it is associated with cognition and spatial information processing (Bender et al., 2015; Colgin, 2013; O'Keefe, 1993). Differently, lower frequency, cholinergic-dependent type 2 theta is involved in emotional processing and suggested to originate in the ventral hippocampus (Ciocchi, Passecker, Malagon-Vina, Mikus, & Klausberger, 2015; Mikulovic et al., 2018; Winne et al., 2018). Recently two studies have found slow type 2 theta to correlate to anxiety behavior (Mikulovic et al., 2018; Winne et al., 2018). Thereby, ventral OLMα2cells were shown to control type 2 theta oscillations and activation of ventral OLMα2cells deceased anxiety response to predator odor (Mikulovic et al., 2018). Our in vitro study adds to the results that OLMα2cells contribute to different theta oscillations depending on location within the hippocampus, and show that dorsal OLMα2cells have higher reso-nance frequency, possibly due to the larger Ihin dorsal OLMα2cells. Two studies from horizontal slices of the rat CA1 have examined whether OLM cells show resonance behavior. Zemankovics et al. reported 10 out of 15 OLM cells to resonate in a frequency range of 1–3 Hz (Zemankovics et al., 2010) while Kispersky et al. found no reso-nance peak in impedance measurements of OLM cells (Kispersky et al., 2012). This difference of OLM cell resonance properties may result from diverse preparations and locations of the investigated cells.
Furthermore, it has been shown that type 1 theta is driven by the glutamatergic input from the medial septum onto putative OLM cells in DH (Fuhrmann et al., 2015) as well as Pvalb+ cells of the medial septum (Boyce, Glasgow, Williams, & Adamantidis, 2016), while Mikulovic et al. found that cholinergic input onto VH OLMα2cells drive type 2 theta activity (Mikulovic et al., 2018). This implies that DH and VH OLM cells also receive different afferent inputs, further supporting their differential involvement in behavior. Interestingly, a recent study shows how ventral, but not dorsal, inactivation of the hippocampus impairs reward memory (Riaz, Schumacher, Sivagurunathan, Van Der Meer, & Ito, 2017). In addition, a computa-tional study demonstrated that OLM cells can be recruited at high or low theta frequencies depending on the presence or absence of h-currents on their dendrites and coregulation with the slow delayed rectifier channel (Sekulic & Skinner, 2017). The authors suggest that this system of channels could represent the“switch” that allows two different OLM cell subpopulations to be theta1 or theta2 spiking reso-nators. Given the larger Ihin VH PCs when compared to DH PCs, and the opposite relationship for VH and DH OLMα2 interneurons, it is plausible to speculate that Ihin DH OLMα2cells has a greater impact for type 1 theta, while Ihin VH PCs for type 2 theta rhythmogenesis (see Figure S6). Thus, our data support the emerging evidence that OLM cells play distinct roles in type 1 and type 2 theta oscillations and that this distinct contribution may result from the anatomical location along the dorsoventral axis of the hippocampus, the differ-ence in passive membrane properties and the magnitude of Ih. F I G U R E 4 Dorsal OLMα2cells display more
hyperpolarization-activated current (Ih) than ventral OLMα2cells. (a) Example of a dorsal (left) and a ventral (right) OLMα2cell response to negative voltage steps (−60 mV to −130 mV, 10 mV decrements, 1 s) before and after ZD7288 application. (b) Average current responses to negative voltage steps showing the current before (control,●) and the current remaining after ZD7288 application (■) for dorsal (left) and ventral (right) OLMα2cells. Digital subtraction of these currents shows the ZD7288-sensitive current (Δ). (c) Left; tail currents response to negative voltage steps for dorsal (●) and ventral (○) OLMα2cells. Right; Boltzmann curves illustrating the normalized current in response to voltage (−65 mV to −120 mV, 5 mV decrements) for dorsal (●) and ventral (○) OLMα2cells. Data presented as mean ± SEM.*p < .05, **p < .01. (d) Boxplots of half-activation voltage (left) and slope factor (k) (right) for dorsal and ventral OLMα2cell tail currents. OLM, oriens lacunosum moleculare
F I G U R E 6 Dorsal OLMα2cells have a higher resonance frequency than ventral OLMα2cells. (a) Example of voltage responses of a dorsal (left) and ventral (right) OLMα2cell in response to a chirp stimulus (20 s, linear frequency increase from 0 to 20 Hz, top: ±25 pA, middle: ±50 pA, bottom: ±100 pA). Dashed lines denote the baseline voltages. (b) Top: Fast Fourier transform (FFT) of membrane potential responses as shown in (a) for dorsal (left) and ventral (right) OLMα2cells. Middle: Fast Fourier transform of the impedance amplitude function (ZAP) input for ±25 pA (blue), ±50 pA (red), ±100 pA (black). Bottom: Impedance amplitude profiles for dorsal and ventral OLMα2cells in response to ±25 pA (blue), ±50 pA (red), ±100 pA (black). The vertical line shows the mean resonance frequency (fR) in response to each chirp frequency. The resonance frequency value in response to a 100 pA chirp is specified in the figure (black line) and the gray box shows a zoom-in of the resonance profile. OLM, oriens lacunosum moleculare [Color figure can be viewed at wileyonlinelibrary.com]
F I G U R E 5 OLMα2cells colocalize with somatic HCN2 immunoreactivity in the stratum oriens CA1 region. (a, b) Coronal sections (35μm) from Chrna2-cre/tdTomato+ mice show tdTomato+ somas colocalizing with HCN2 immunoreactivity (green) in the dorsal region of the CA1 (see inset). (c) An example from the ventral region of the CA1 (see inset) is shown. Arrows indicate tdTomato+ cell bodies that also show HCN2 somatic expression. All images show split channels (red and green) and the merged image to the right. Scale bar 50μm. OLM, oriens lacunosum moleculare [Color figure can be viewed at
A C K N O W L E D G M E N T S
This work was supported by the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) www.stint.se, the Brazilian National Council of Technological and Scientific Devel-opment (CNPq) www.cnpq.br, and the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) www.capes. gov.br.
C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.
A U T H O R C O N T R I B U T I O N S
M.M.H., I.N., and R.N.L. conceived and conducted the experiments. M.M.H., R.N.L., and K.E.L. analyzed the results and wrote the paper with inputs from S.M. and K.K.
O R C I D
Markus M. Hilscher https://orcid.org/0000-0001-7782-0830 Richardson N. Leão https://orcid.org/0000-0001-8496-1965 Katarina E. Leão https://orcid.org/0000-0001-7295-1233
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S U P P O R T I N G I N F O R M A T I O N
Additional supporting information may be found online in the Supporting Information section at the end of this article.
How to cite this article: Hilscher MM, Nogueira I, Mikulovic S, Kullander K, Leão RN, Leão KE. Chrna2-OLM interneurons display different membrane properties and h-current magnitude depending on dorsoventral location. Hippocampus. 2019;1–14.https://doi.org/10.1002/hipo. 23134
