diagonal band complex in the mouse

Brain Research 1006 (2004) 74 – 86 www.elsevier.com/locate/brainres

Research report

Characterisation of hyperpolarization-activated currents (Ih) in the medial septum/diagonal band complex in the mouse Neil P. Morris a,*, Robert E.W. Fyffe b, Brian Robertson c b

a School of Biomedical Sciences, University of Leeds, Worsley Building, Leeds LS2 9NQ, UK Department of Anatomy, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA c Department of Pharmacology and Physiology, University of Strathclyde, Glasgow G4 ONR, UK

Accepted 30 January 2004

Abstract Hyperpolarization-activated cyclic nucleotide gated (HCN) channel subunits are distributed widely, but selectively, in the central nervous system, and underlie hyperpolarization-activated currents (Ih) that contribute to rhythmicity in a variety of neurons. This study investigates, using current and voltage-clamp techniques in brain slices from young mice, the properties of Ih currents in medial septum/diagonal band (MS/ DB) neurons. Subsets of neurons in this complex, including GABAergic and cholinergic neurons, innervate the hippocampal formation, and play a role in modulating hippocampal theta rhythm. In support of a potential role for Ih in regulating MS/DB firing properties and consequently hippocampal neuron rhythmicity, Ih currents were present in around 60% of midline MS/DB complex neurons. The Ih currents were sensitive to the selective blocker ZD7288 (10 AM). The Ih current had a time constant of activation of around 220 ms (at 130 mV), and tail current analysis revealed a half-activation voltage of 98 mV. Notably, the amplitude and kinetics of Ih currents in MS/DB neurons were insensitive to the cAMP membrane permeable analogue 8-bromo-cAMP (1 mM), and application of muscarine (100 AM). Immunofluoresence using antibodies against HCN1, 2 and 4 channel subunits revealed that all three HCN subunits are expressed in neurons in the MS/DB, including neurons that express the calcium binding protein parvalbumin (marker of fast spiking GABAergic septo-hippocampal projection neurons). The results demonstrate, for the first time, that specific HCN channel subunits are likely to be coexpressed in subsets of MS/DB neurons, and that the resultant Ih currents show both similarities, and differences, to previously described Ih currents in other CNS neurons. D 2004 Elsevier B.V. All rights reserved. Theme: Excitable membrane and synaptic transmission Topic: Other ion channels Keywords: GABAergic; Voltage clamp; Immunocytochemistry; HCN channel; Parvalbumin; Current clamp

1. Introduction The rhythmic activity of many central neurons is regulated by various ionic conductances, including the hyperpolarization-activated cation current, Ih [36,41,53]. The Ih current activates upon membrane hyperpolarization from resting potentials, producing an inward, depolarizing current [23,27,56]. Blocking Ih currents with externally applied caesium or the bradycardic agent (4-N-ethyl-N-phenyamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloAbbreviations: HCN, hyperpolarization-activated cyclic nucleotide gated; MS/DB, medial septum/diagonal band * Corresponding author. Tel.: +44-113-343-7014; fax: +44-113-3434228. E-mail address: [email protected] (N.P. Morris). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.01.062

ride (ZD7288) results in alterations in rhythmicity in various networks [12,16,37,66]. For example, it has been demonstrated that Ih currents can control rhythmic activity in thalamocortical relay cells during sleep [41 –43,61]. Rhythmic pacemaker activity may also contribute to electroencephalographic rhythms such as the hippocampal theta rhythm [6], which may be crucial in short-term memory formation and learning processes. Theta rhythm is thought to be initiated and/or modulated by several brain regions, including the medial septum/diagonal band (MS/ DB) complex [63,69,70]. Cholinergic and GABAergic neurons in the MS/DB complex form powerful synaptic connections with both pyramidal cells and GABAergic interneurons in the hippocampal formation, through the septo-hippocampal pathway [3,15,28,30,71]. Septo-hippocampal neurons display rhythmic bursting activity in vivo

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that is tightly coupled to the frequency of the theta rhythm [21,54,65]. Lesion studies reveal that cholinergic MS/DB neurons modulate the amplitude of hippocampal theta rhythm, while GABAergic neurons influence the frequency [31]. Previous current-clamp recordings from putative GABAergic MS/DB neurons in vitro suggest the presence of a hyperpolarization-activated Ih current [1,20,22,25,50]. Recently, a family of four genes has been identified and cloned which encode Ih currents [26,33,34,48,57,58,60,68]. Hyperpolarization-activated cyclic nucleotide gated (HCN) channels (HCN1 –4) have been expressed heterologously, revealing currents with features characteristic of native Ih currents. Also, HCN channels are differentially modulated by direct binding of intracellular cAMP [26,33,34,58]. HCN channels are distributed differentially in the CNS, and sometimes overlap in specific neuronal populations. In particular, in situ hybridisation studies suggest that HCN1, 2 and 4 channel subunits are expressed, at different levels, in the MS/DB complex, although it is not known to what extent expression overlaps in individual neurons [45,59]. The aims of this study were to investigate the electrophysiological properties of hyperpolarization-activated currents in fast spiking neurons in the MS/DB complex, and to examine the expression of HCN1, HCN2 and HCN4 channel subunits in specific neuronal populations in that region. Although sensitive to pharmacological blockade by ZD7288, the Ih currents in the MS/DB were insensitive to cAMP and muscarine. Immunocytochemistry revealed that parvalbumin expressing (presumed GABAergic) neurons in the MS/DB express all three tested isoforms, with HCN1 and HCN2 being most prevalent.

2. Materials and methods 2.1. Tissue preparation and solutions Three- to five-week-old male TO mice (Charles River) were killed humanely by cervical dislocation and decapitated in accordance with UK Home Office regulations [UK Animals (scientific procedures) Act, 1986]. The brain was rapidly removed and transferred to a chilled, oxygenated, sucrose-based artificial cerebro-spinal fluid (ACSF) solution. Parasaggital slices (250 Am thick) of the MS/DB complex were prepared in sucrose-based ACSF solution using a Vibroslicek (Campden Instruments). The standard ACSF contained (in mM): NaCl 124, KCl 3, NaHCO3 26, NaH2PO4 2.5, MgSO4 2, CaCl2 2, D-glucose 10, bubbled with 95% O2/5% CO2. The sucrose-based ACSF used for dissection and slicing was identical apart from replacement of NaCl with iso-osmotic sucrose (74.5 g/l). 2.2. Electrophysiological recording During recording, slices were maintained in a recording chamber perfused at 2 –4 ml per min with standard oxy-

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genated ACSF. Individual neurons were visualized with infrared differential interference contrast (DIC) optics using an Axioskop FS microscope (Zeiss, Germany). Recordings were made at room temperature (20 –24 jC) using electrodes fabricated from borosilicate glass capillaries (GC150F10, Harvard Apparatus, Kent, UK). Electrodes had a resistance of between 3 and 7 MV when filled with an intracellular solution containing (in mM): KCl 140; MgCl2 1; CaCl2 1; EGTA 10; HEPES 10; ATP-Mg 3 and GTP 0.3 (pH 7.3). Whole cell patch clamp recordings were made using an EPC-9 amplifier (HEKA), controlled by Pulse software (HEKA) with a Macintosh (7500/100) computer. Series resistance was typically 10– 15 MV, and was compensated by 60 –95%. Whole cell current-clamp recordings were obtained using an Axopatch 1D amplifier, and recorded using Pulse software on a Macintosh (7500/100) computer. Data were filtered at one third of the appropriate sampling frequency (typically 7 kHz). All drugs (Sigma, Tocris, Alomone) were bath applied. Tetrodotoxin (1 AM) was applied in some voltage-clamp experiments to aid measurement of the kinetics of the Ih current, but had no effect on the amplitude or kinetics of the current. Electrophysiological recordings were restricted to visually identified, medium-sized neurons in midline regions of the MS/DB complex. This is the region in which parvalbumin-immunoreactive GABAergic septo-hippocampal neurons are predominantly localized [28], although our population of recorded neurons likely included a variety of other functional neuronal types. 2.3. Immunohistochemistry The distribution of HCN channel protein in the MS/DB complex was analysed using specific antibodies raised against different channel isoforms (HCN1, 2, and 4; Alomone, rabbit polyclonal antibodies diluted 1:25 – 1:50). Young adult mice (C57 black strain) were euthanized by overdose of barbiturate anaesthetic using animal protocols in accordance with NIH guidelines and approved by the local Laboratory Animal Use Committee. Animals were perfused transcardially with cold vascular rinse (0.1 M phosphate-buffered saline, PBS) followed by 4% paraformaldehyde in PBS. The forebrain was removed and postfixed in the same fixative for 1– 2 h at room temperature. Forty-micrometer-thick coronal sections were cut on a freezing sliding microtome, blocked in normal goat serum, and incubated, on slides, for 24 h in primary antibodies against one of the HCN channels, followed by visualization using FITC- or Cy3-conjugated secondary antibodies (Jackson Immuno Research, diluted 1:50). Additional experiments involved dual labelling of HCN channels and a variety of cellular and synaptic markers including anti-parvalbumin (Chemicon, mouse IgG diluted 1:2000) and anti-GAD 65 (Boehringer Mannheim, mouse IgG diluted 1:1000), and a fluorescent Nissl stain (NeuroTrace Green, Molecular Probes). Immunofluorescence was

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viewed using standard epifluorescence and laser scanning confocal microscopy (Olympus Fluoview) on an Olympus BX50 microscope. Images were usually obtained using 60  (dry, N.A. 0.9 or oil immersion N.A. 1.4) objectives, and were printed on a Tektronix Phaser 450 printer using CorelDraw 3.0 or ImagePro Plus version 3.0. Specificity of the HCN channel antibodies was checked by confirmation that immunostaining was blocked when each antibody (at 1:50 dilution) was preabsorbed by incubation with 10 Ag of antigenic peptide (Alomone). Immunostaining in these control experiments was visualized using standard avidin– biotin complex protocols (Vector Laboratories) using diaminobenzidine as substrate for the peroxidase histochemistry. In each case, antibody staining was abolished by the specific peptide against which the particular anti-HCN antibody was directed, but not by the inappropriate peptides. Staining was also abolished by omission of the primary antibody. 2.4. Data analysis Data analysis was carried out using Pulsefit (HEKA), Axograph (Axon Instruments), Igor (Wavemetrics) and Kaleidagraph software. Data are presented as mean F S.E.M., where n = number of cells. Statistical significance was determined using Student’s t-test unless specified, and a value of P < 0.05 was deemed significant. Action potential parameters were measured as described earlier [50], and depolarizing sag was measured as the difference between the peak deflection and the basal response during a hyperpolarizing current pulse. The steady state activation curve was determined from the amplitude of instantaneous tail currents after hyperpolarizing steps to 130 mV following voltage steps from 50 to 130 mV. Tail current amplitudes were measured after the decay of capacative current transients, and were plotted as a function of the step voltage. Data were fitted with a Boltzmann equation: IMAX = 1/ {1 + exp[(V1/2 V)/k]}, where IMAX is the maximum current amplitude, V the step potential, V1/2 the half-activation potential, and k is the slope factor.

3. Results Whole cell current- or voltage-clamp recordings were obtained from visually identified, medium-sized neurons (approximately 20-Am diameter) in the midline portion of the MS/DB complex. Twenty neurons were recorded under current-clamp conditions, and 104 neurons were recorded under voltage-clamp conditions. For current-clamp analysis of neuronal firing properties, neurons were held at a holding potential of approximately 60 mV for the duration of the experiment using injection of D.C. current, and depolarizing and hyperpolarizing current pulses (600-ms duration unless stated) were applied to the neurons. For voltage-clamp analysis, neurons were held at 70 mV and had stable

series evoke jected from

resistance for the duration of the experiment. To inward currents, voltage-clamped neurons were subto 500-ms hyperpolarizing steps (unless specified) 70 to 130 mV (in 10-mV increments).

3.1. Firing properties of MS/DB neurons Fifteen (75%) of the neurons recorded under currentclamp conditions exhibited depolarizing sag in their voltage response to hyperpolarizing current pulses (Fig. 1A). The depolarizing sag had an average amplitude of 14.0 F 1.6 mV following a step of 0.3 nA. The properties of neurons not exhibiting depolarizing sag were not examined further in this study. The input resistance of neurons displaying depolarizing sag was 258 F 59 MV (n = 5), determined using a small hyperpolarizing current pulse (50 pA). Depolarizing sag was blocked by ZD7288 (10 AM), a selective blocker of the hyperpolarization-activated current (Ih), with around 2 – 3 min required to reach maximal block (Fig. 1A). ZD7288 also increased the input resistance to 352 F 85 MV ( P < 0.05, n = 5), and increased the latency of rebound action potentials following a hyperpolarizing current pulse ( 0.3 nA), from 64.1 F 20.7 to 122.2 F 10.2 ms ( P < 0.05, n = 5) (Fig. 1A). Application of 8-bromo-cAMP had little effect on the amplitude of the depolarizing sag (a slight reduction was observed, from 15.3 F 2.6 to 14.0 F 2.2 mV; P >0.05, n = 3). Depolarizing current pulses were used to elicit repetitive firing in MS/DB neurons that displayed depolarizing sag. The majority (73.3%) of neurons showed non-adapting (fast-spiking) action potential firing patterns (n = 11/15; Fig. 1B) as described earlier in parvalbumin-immunoreactive neurons [25,50], and we applied ZD7288 to four of these fast spiking neurons. In the control situation, the maximum frequency of these neurons was 120.1 F 14.2 Hz, and the steady firing frequency (measured between the last two spikes) was 76.6 F 9.4 Hz (n = 4). The firing properties of non-adapting neurons were not significantly altered by the blockade of the hyperpolarization-activated current (Fig. 1B); in the presence of ZD7288 maximum frequency was 109.1 F 10 Hz ( P >0.05; n = 4) and steady firing frequency was 78.8 F 13.3 Hz ( P >0.05; n = 4), and action potential parameters were unaffected (data not shown). In an earlier study, ZD7288 has been found to reduce spontaneous firing rates in retrogradely labelled GABAergic septo-hippocampal neurons [74]. 3.2. Characterisation of the hyperpolarization-activated currents Under voltage-clamp conditions, the presence of a hyperpolarization-activated inward current was observed as a slow onset, non-inactivating, current (Fig. 2A). The Ih current was observed in 67 of the 104 neurons recorded (64%). The current – voltage relationship for 13 MS/DB neurons is illustrated in Fig. 2B, which also depicts the Ih

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Fig. 1. Depolarizing sag in neurons of the MS/DB complex. (A) Hyperpolarizing current pulses (600 ms) in a representative MS/DB neuron, recorded using whole-cell current-clamp configuration, induce depolarizing sag from a holding potential of 60 mV. Notice the rebound action potentials following cessation of the current pulse. In the presence of the Ih blocker ZD7288 (10 AM), depolarizing sag is blocked (lower trace), and the rebound latency is increased. (B) Depolarizing current pulses induce high frequency train of action potentials in a subgroup of MS/DB neurons. Notice the lack of adaptation in the firing frequency during the train. ZD7288 (10 AM, lower trace) has little effect on the firing frequency, or discharge pattern. (C) The cAMP analogue 8-bromo-cAMP (1 mM) had no effect on the amplitude or kinetics of the depolarizing sag in MS/DB neurons.

Fig. 2. Electrophysiological properties of the Ih current in the MS/DB complex. (A) Hyperpolarizing steps of 500-ms duration from a holding potential of 70 mV in an MS/DB cell reveal inward currents following steps to 130 mV in 10 mV increments (Inset, not to scale). II, point where instantaneous current was measured; Iss, point where steady state current was measured. (B) Current – voltage relationship following hyperpolarizing steps indicating II, Iss and the difference current (diff) which is the Ih current (n = 13; mean F S.E.M.). (C) Hyperpolarizing steps in another MS/DB cell before (grey trace) and following addition of the selective Ih blocker ZD7288 (10 AM; black trace). (D) ZD7288 sensitive Ih current revealed by digital subtraction of control traces from traces in the presence of ZD7288. Notice the small amplitude and the inward tail following the step. (E) Time course of action of ZD7288 (10 AM) indicating the % block of Ih current over time (half-time for block f 130 s).

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current as the difference between the instantaneous current and the steady state current. 3.2.1. Properties of the Ih currents in the MS/DB complex The selective Ih current blocker ZD7288 (10 AM) completely blocked the Ih current (Fig. 2C,D), but took several minutes to achieve a maximal effect (half-time for full block f 130 s; Fig. 2E), as noted previously [16,24,35]. Application of external caesium (2 mM) also blocked the Ih current (n = 3; data not shown; [74]). The subtracted Ih current revealed following block with ZD7288 was used to measure the amplitude and kinetics of this current (Fig. 2D). These measurements were obtained from test pulses up to 130 mV, since pulses beyond this often resulted in instability in MS/DB neurons, and were therefore avoided. The threshold for activation of the Ih current was 70 to 80 mV, being slightly negative to the holding potential of 70 mV. Direct measurement of the reversal potential of the Ih current was not attempted since voltage-gated potassium currents activated at potentials positive to 60 mV (see also Ref. [62]). The amplitude of the subtracted Ih current in MS/DB cells ranged from 18.6 to 68 pA at 130 mV, with a mean amplitude of 43.5 F 5.2 pA at 130 mV (n = 10). The ZD7288-sensitive Ih current time course (over a 500-ms period) could be adequately fitted with a single exponential function (Fig. 3A; step to 130

mV), providing an initial lag period of around 10 ms was excluded from the analysis (see Refs. [2,9]). It is possible that exclusion of the early phase from analysis may preclude identification of a very rapidly activating component, although we think this unlikely. The mean time constant of activation (sa) of the Ih current was 221.3 F 30.2 ms at 130 mV (n = 9). Longer duration pulses of 1500 ms were applied to nine MS/DB cells to determine if there was an additional slower component; for these longer steps, the mean sa at 130 mV was 232.1 F 29.5 ms (n = 9) when fitted with a single exponential function. Double exponential functions applied to these traces produced an equivalent fit to that obtained using a single exponential (n = 5/6). The activation time constant was faster at hyperpolarized potentials, ranging from 402.4 F 64.5 ms at 110 mV to 171.3 F 18.4 ms at 140 mV, and had a linear dependence on voltage over the range of 110 to 140 mV, changing e-fold for a 35-mV change in membrane potential (n = 6; Fig. 3B). The voltage dependence of the Ih current in MS/ DB cells was examined using analysis of instantaneous tail currents (see Refs. [13,38,47]). Cells were pre-pulsed to potentials between 50 and 130 mV for 1500 ms, followed by a step to 130 mV for 500 ms (Vhold 50 mV; Fig. 3C). Peak tail currents at 130 mV were measured, normalised, and plotted against the pre-pulse potential ( 50 to 130 mV). The activation curve was

Fig. 3. Kinetics of the MS/DB Ih current. (A) Single exponential fit (dotted line) imposed on ZD7288 sensitive current trace at 130 mV gives a time constant of activation of 180 ms in this example. (B) Voltage dependence of activation time constant (semi-logarthmic scale; n = 6), with an exponential fit to the data showing an e-fold change for 35 mV. (C) Long duration hyperpolarizing steps (1500 ms) to potentials from 50 to 130 mV, followed by a step to 130 mV (Vh 50 mV for these experiments) to measure the magnitude of tail currents (arrow). (D) Tail current amplitude normalised and plotted versus prepulse potential ( 50 to 130 mV) and fitted with a Boltzmann function (see Materials and methods) (n = 8). Half-activation voltage (V1/.2) 98.7 F 2.4 mV and slope factor (k value) 12.0 F 1.3 mV.

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Fig. 4. Effect of raised external potassium on Ih current in MS/DB neurons. (A) Hyperpolarizing steps from 70 to 130 mV (1500 ms) in a representative cell in control conditions, where the dotted line indicates zero current. (B) Identical protocol in the same cell in the presence of ACSF containing 20 mM K+. Notice the shift in holding current and the increase in spontaneous IPSCs. (C) Current – voltage relationship for the subtracted Ih current in six cells (mean F S.E.M.).

Fig. 5. Effect of muscarine on the Ih current in MS/DB neurons. (A) Current – voltage relationship showing the effect of muscarine (100 AM) on the subtracted 70 mV or 130 mV) followed by a step to 130 mV to measure Ih current in five cells. (B) Representative traces of 1500-ms hyperpolarizing steps ( tail current amplitude in the control situation and the presence of muscarine. (C) Normalised tail current amplitude plotted against prepulse potential fitted with a Boltzmann function, control (n = 5, filled circles, mean F S.E.M.), muscarine (open circles). Note the very slight depolarizing shift in the presence of muscarine. Control: V1/2 = 96.7 mV, k = 11.2. Muscarine: V1/2 = 95.1 mV, k = 9.9.

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fitted to a single Boltzmann function, which revealed a mean half-activation voltage (V1/2) of 98.7 F 2.4 mV and a slope factor (k value) of 12.0 F 1.3 mV (n = 8; Fig. 3D). Ih current deactivation was best described by a single exponential function, following a delay, with a time constant of 30.4 F 9.0 ms at 70 mV (n = 4; Fig. 2D). A delay was required because of a ‘hump’ before full deactivation, which has been observed in both cloned and native Ih channels (e.g. Refs. [39,58,62]). To establish the relative contribution of potassium ions to the size of the Ih current, the ACSF solution was adjusted by raising the concentration of external potassium from the control level of 3 mM to a concentration of 20 mM. There was a large increase in the number and size of spontaneous synaptic events during the application of ACSF containing 20 mM K+, and thus 1 AM tetrodotoxin (Alomone, Israel) was included for some of these experiments. In the presence of 20 mM external K+, there was a shift in the holding current, and a reversible increase in hyperpolarization-activated inward currents (Fig. 4A, B). The effect on the Ih current was assessed by subtracting the steady state current from the instantaneous current (digitally subtracted Ih current). The subtracted Ih current increased significantly in amplitude from 58.8 F 11.7 to 101.6 F 17.2 pA (n = 6)

at 130 mV in the presence of raised external potassium ( P < 0.05; n = 6; Fig. 4C). The time constant of activation at 130 mV was also increased from 320 F 43.2 to 578 F 73.2 ms ( P < 0.01, n = 6), in the presence of ACSF containing 20 mM K+. 3.2.2. Modulation of Ih by muscarine Cholinergic transmission plays an important role in the MS/DB complex, and connections between cholinergic neurons and GABAergic neurons have been observed anatomically [8,32]. Furthermore, cholinergic agonists have been shown to increase excitability in GABAergic septohippocampal neurons [73]. Therefore, we sought to assess any effects of muscarine on the magnitude and kinetics of the Ih current. Application of muscarine (100 AM) induced a large increase in inward currents, although there was no significant effect on the Ih current. The digitally subtracted Ih current was not significantly changed in the presence of muscarine ( 55.3 F 27.1 to 63.0 F 35.8 pA at 130 mV; P>0.05, n = 5; Fig. 5A). There was also an insignificant shift in the activation curve measured from instantaneous tail currents (1.5 mV depolarizing shift in V1/2; Fig. 5B, C). Muscarine produced a reversible increase in the holding

Fig. 6. Effect of 8-Br-cAMP on the kinetics of the Ih current. (A) Current – voltage relationship indicating the effect of 8-Br-cAMP (1 mM) on the subtracted Ih current in five MS/DB neurons (mean F S.E.M.). Inset, 500-ms hyperpolarizing step to 130 mV in a representative neuron. (B) Effect of 8-Br-cAMP on normalised tail current amplitude (1500-ms hyperpolarizing steps from 50 to 130 mV followed by step to 130 mV). Boltzmann function fitted to control (filled circles) and 8-Br-cAMP (open circles) traces gave V1/2 93.3 F 4.2 mV (control), 91.3 F 5.1 mV (8-Br-cAMP).

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current of 47.6 F 7.5% (n = 5). Application of muscarine to MS/DB complex neurons also produced an increase in spontaneous IPSC amplitude and frequency (see Fig. 5B). 3.3. Modulation of Ih by cyclic nucleotides Homomeric HCN1– 4 channel subunits expressed in heterologous systems are differentially sensitive to modulation by cyclic nucleotides such as cAMP and cGMP [9,33,34,58]. In particular, HCN2 is extremely sensitive to cAMP, resulting in a large shift in activation voltage, whereas HCN1 is relatively insensitive. In order to investigate the sensitivity of the Ih current in MS/DB neurons to cyclic nucleotides, various approaches were taken. Firstly, external 8-bromo-

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cAMP was applied to neurons exhibiting an Ih current at a concentration of 1 mM (sufficient to evoke a maximal shift in voltage sensitivity and amplitude in other systems; [9,11, 17]). Application of 8-bromo-cAMP (1 mM) produced a small decrease in digitally subtracted Ih current amplitude at 130 mV from 39.2 F 6.0 to 28.6 F 5.6 pA ( P>0.05; paired T-test; n = 5; Fig. 6A). Tail current analysis revealed an insignificant shift in the half-activation voltage when data was fitted with a Boltzmann function (V1/2: 93.3 F 4.2 to 91.3 F 5.1 mV; n = 5; Fig. 6B). Additionally, there was no alteration in the amplitude or frequency of spontaneous IPSCs in the presence of 8-bromo-cAMP. Further experiments were conducted with 8-bromo-cAMP (1 mM) added to the internal pipette solution, which again resulted in no

Fig. 7. Localization of HCN channel subunits in the MS/DB complex. (A – B) Neurons in the MS/DB complex labelled using Nissl stain (green) and HCN immunoreactivity (red) visualized using scanning confocal microscopy. (A) Notice HCN1 labelling localized to somatic and proximal dendritic surface membranes. (B) Widespread HCN2 labelling in the medial septum. (C – E) Low power photomicrographs illustrating the distribution of HCN immunoreactive neurons (green) and parvalbumin-immunoreactive neurons (red) in the midline region of the MS/DB complex (colocalization is indicated by yellow colour). (C) HCN1 labelling is extensively colocalized with parvalbumin immunoreactivity in midline regions of the medial septum. (D) HCN2 immunolabelling is present in every parvalbumin-immunoreactive neuron in the medial septum, and in a large proportion of parvalbumin negative neurons. (E) HCN4 is extensively distributed in the medial septum and diagonal band, and shows a high degree of colocalization with parvalbumin immunoreactivity. Scale bar in A is 10 Am, B is 30 Am and C – E is 50 Am.

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alteration in the amplitude or kinetics of the Ih current (n = 3; data not shown). In a similar study Xu et al. [74] also reported that the MS/DB Ih current is insensitive to cAMP. 3.3.1. Molecular identity of the Ih current in the MS/DB To investigate the potential molecular correlates of the Ih currents observed in medial septal cells, we used commercially obtained polyclonal antibodies to HCN1, 2 and 4. Earlier studies using in situ hybridisation suggested the presence of HCN1, HCN2 and HCN4 mRNA in the medial septum/diagonal band complex [45,59], prompting us to investigate the distribution of these channel proteins using immunolabelling. All three HCN channels were visualized by immunohistochemistry in the medial septum and diagonal band nuclei, in overlapping and complementary neuronal populations. Neurons displayed variable levels of immunoreactivity largely confined to somatic and proximal dendritic regions of the labelled cells. Punctate, relatively uniformly distributed immunolabelling, was discernible in association with the surface of many cells (see below), as well as a variable amount of immunoreactivity distributed throughout the cytoplasm. HCN1 immunoreactivity was localized to the somatic and proximal dendritic surface membrane of neurons (Fig. 7A), with fainter, granular, staining within the cytoplasm of the cell body region, especially close to the surface membrane. Most HCN1-immunoreactive neurons were located in the midline region of the medial septal nuclei, with sparser labelling of cells located in the diagonal band (Fig. 7C). HCN1 immunolabeled neurons were typically surrounded by a dense plexus of GAD 65 immunoreactive (i.e. GABAergic) terminal structures, although it was not possible to determine if HCN1 or other HCN subunits were present in the presynaptic terminals (see above) as well as in the postsynaptic cell. Labelling using the HCN2 antibody [55] presented a similar regional distribution (i.e. primarily throughout the medial septum, with fewer labelled cells in the diagonal band), and although the cytoplasmic staining was prominent, there was less distinct cell surface labelling than for HCN1 (Fig. 7B). HCN4 was expressed in a more widespread fashion, including many neurons in the diagonal band region as well as in the medial septal nuclei (Fig. 7E). In both areas, neurons were quite intensively labelled, with much granular labelling distributed towards the periphery of the soma, close to or associated with the surface membrane. In contrast to HCN1, there was little HCN4 immunoreactivity in dendritic profiles. In order to study cellular distribution in more detail, dual immunostaining was performed with antibodies against the calcium binding protein parvalbumin, which labels a set of GABAergic septo-hippocampal neurons close to the midline of the medial septal region [14,28]. As described previously, parvalbumin-IR was also evident in terminal-like structures and many fibres coursing through the MS/DB complex [50]. A significant majority of parvalbumin-positive cells expressed HCN1 (ranging from about 50% to 90% in

different sections), but it is not known if the variable incidence of double labelling was due to sampling of different levels of the MS/DB complex or other reasons. Nevertheless, even in single sections, it was clear that the level of HCN1 expression in parvalbumin-positive neurons could vary even between adjacent cells; the intensity of HCN1 staining did not appear to be related to the intensity of parvalbumin immunostaining (Fig. 7C). HCN2 immunolabelling was present in every parvalbumin containing cell in the medial septum (Fig. 7D), and, in addition, was expressed in parvalbumin-negative neurons intermingled with the parvalbumin-positive population (Fig. 7D). HCN4 was expressed in virtually all parvalbumin containing cells (Fig. 7E) in the medial septum. Colocalization of HCN isoforms and parvalbumin was also examined in the neocortex and basal ganglia. Although many parvalbumin containing cells are present in these areas, none of them expressed appreciable amounts of HCN1, 2 or 4.

4. Discussion We have examined the prevalence and properties of the hyperpolarization-activated currents (Ih), using voltage- and current-clamp techniques in a slice preparation of the mouse MS/DB complex. The main findings of this study are: (1) that a large proportion of neurons (f 60%) in the MS/DB complex exhibit a hyperpolarization-activated current, and (2) that antibody labelling for HCN1, 2 and 4 reveals relatively widespread distribution of these channel proteins in the MS/DB. Each HCN channel protein is found in parvalbumin containing (presumed GABAergic) neurons in the MS/DB, suggesting that the GABAergic neurons may coexpress different isoforms of the HCN channel subunits. These observations, when combined with earlier findings, suggest that parvalbumin-containing septo-hippocampal GABAergic neurons express an Ih current, which may have functional implications for the modulation of the hippocampal theta rhythm. 4.1. The Ih current displays biophysical features reminiscent of HCN1 and HCN2 subunits The Ih currents in the MS/DB complex share some characteristics with those in other areas of the CNS, namely: block by the bradycardic agent ZD7288 [7,24], block by caesium ions, exponential activation/deactivation time courses and external K+ dependence. However, the Ih current was not significantly affected by the second messenger cAMP using our recording conditions, which may have important implications when suggesting the potential molecular correlates underlying this conductance (see below). The Ih current activates in a voltage-dependent manner, with a time constant of around 220 ms at 130 mV at room temperature. Current activation was best fitted with a single exponential function, although double exponential fits are

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commonly required to describe native Ih currents at hyperpolarized potentials (i.e. 130 mV) in thalamic and hippocampal neurons [59]. The time constant of activation decreased exponentially with hyperpolarization, and had a modest voltage sensitivity (e-fold change for 35 mV), as noted by others [58,62]. The time constant of activation is markedly different between the four heterologously expressed homomeric HCN subunits [47]. Our activation data most closely approximates to HCN2 channel subunits, which show activation kinetics of around 200 – 500 ms at potentials between 100 and 140 mV (at room temperature). Ludwig et al. [34] report a time constant of activation of 179 ms at 140 mV, while Moosmang et al. [47] quote 184 ms, which is close to our figure of 171 ms. Santoro et al. [59] used a voltage range more depolarized than ours, but extrapolation of their data reveals a time constant of 225 ms at 130 mV, which also compares well with our experimental data [59]. The activation kinetics for heterologously expressed HCN1 subunits are more rapid, in the range of 30 –300 ms (at 100 to 140 mV) [47,58,59]. 4.2. Modulation of the Ih current in the MS/DB complex The Ih current in MS/DB neurons was unaffected by an externally applied membrane-permeable analogue of cAMP, suggesting that the HCN subunits underlying this conductance are insensitive to modulation by cyclic nucleotides (see also Ref. [74]). A recent study of Ih currents in the Calyx of Held, utilising both internal cAMP and external perfusion of 8-Bromo-cAMP, described a significant shift in the activation curve suggesting sensitivity of their Ih current to cAMP [11]. However, heterologously expressed HCN subunits are differentially sensitive to cyclic AMP, and its analogues. The voltage dependence of activation of HCN2 subunits is strongly modulated by cAMP (positive shift of 10 – 20 mV), while HCN1 subunits display only weak sensitivity (1 – 2 mV) [33,34,58,72]. Our evidence suggests that cAMP induces a small (1 –2 mV) shift in half-activation in MS/DB complex neurons, which is consistent with the possibility that HCN1 subunits are involved in the functional Ih channel complex in this region. However, the possibility that basal levels of cAMP are sufficient to produce a near maximal shift in half-activation cannot be excluded [9] and will require additional evaluation. Recent studies suggest that functional heteromultimers of HCN1 and HCN2 subunits in heterologous systems show cAMP sensitivity which is intermediate between the two subunits [9,67]. The time constant of activation in expression system cells coexpressing HCN1 and HCN2 subunits is intermediate between the homomeric channels at around 100 ms (at 105 mV, [9]; at 125 mV, [67]). A recent study suggested that native Ih currents may be comprised of HCN channel subunits formed of heteromultimers ([11]; see also [13]). Furthermore, evidence from single cell RT-PCR and in situ hybridisation studies suggests that multiple HCN

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isoforms may be coexpressed in the same cell [13,59], a finding supported by our immunolocalization studies (see below). The correlation of specific isoform expression (and subcellular distribution) with specific Ih current properties remains an outstanding issue. Recent studies have suggested that muscarinic activation increases excitability in GABAergic neurons in the MS/DB complex [73], produces continuous hippocampal theta rhythm and facilitation of learning and memory processes [18,19,40]. We therefore tested whether this effect could be mediated through a shift in Ih current activation. However, our data suggest that muscarine has no effect on the activation of the Ih current in the MS/DB complex. In other systems, the Ih current is modulated by neurotransmitters, including facilitation by norepinephrine, serotonin and muscarinic agonists [10,38,44] and inhibition by A-opioid receptor activation [64]. It remains to be determined whether other neurotransmitters in the MS/ DB complex may influence the activation of the Ih current, and thus alter neuronal excitability via that mechanism, although preliminary evidence from Xu et al. [74] suggest that the MS/DB Ih current is insensitive to application of 5-HT and norepinephrine. 4.3. Localization of HCN channels in the MS/DB complex Three members of the HCN channel subunit family that could contribute to Ih currents are present in neurons in the MS/DB complex. In keeping with our direct immunohistochemical evidence for the presence of HCN1, 2 and 4 channel proteins in MS/DB neurons, previous studies utilising in situ hybridisation have demonstrated the presence of HCN1, HCN2 and HCN4 mRNA in similar patterns in the MS/DB complex [45,46,59]. In the present study, HCN1 protein is prominently localized in the soma and proximal dendrites, whereas HCN2 and 4 appear to be more restricted to the somatic region. Our observation of prominent membrane localization of HCN1 is also consistent with recent studies using single cell RT-PCR methods and electrophysiological characterisation of Ih, which suggest that only HCN1 is discriminatively expressed in neurons with rapidly activating Ih [13]. Functional aspects of Ih current properties measured in the present study, including relative cAMP insensitivity, are also consistent with HCN1 subunits contributing to currents in the MS/DB complex. Nevertheless, all HCN channel subunits are present in parvalbumin-positive neurons in the medial septal area, and virtually all parvalbumin-positive cells express at least one of the HCN channel proteins. Furthermore, the respective incidence of each HCN channel within the parvalbuminpositive neuronal population suggests that HCN2 and HCN4 must be coexpressed in the majority of these neurons. Interestingly, HCN2 mRNA is also closely associated with HCN4 message in neurons where prominent rhythmic activity is seen, such as thalamocortical relay neurons [59]. Very few other (parvalbumin negative) cells in the medial

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septal nuclei express HCN channels, with the exception of a few cells that express HCN2. Although recorded cells were not subsequently immunostained, the predominance of HCN channels in GABAergic parvalbumin-containing septo-hippocampal neurons suggests that these neurons comprised the majority of recorded cells displaying Ih or depolarizing sag, due to the high correlation between fast spiking (parvalbumin-containing) phenotype and incidence of depolarizing sag observed in this study and in previous studies [29,49,50]. Parvalbumin negative cells which express HCN2 channels are likely to consist of regular spiking (presumed GABAergic) neurons which also display depolarizing sag [50], although these neurons were not investigated in this study. Cholinergic neurons in the MS/DB complex do not display Ih currents, but are thought to possess inward rectifier-like currents [20,22,29]. 4.4. Functional significance of the Ih current in MS/DB neurons Earlier studies have shown that septo-hippocampal neurons display rhythmic bursting activity in vivo (see introduction), which may be controlled in part by activation of the Ih current, which is known to confer pacemaker properties on many central neurons [36]. Indeed, preliminary evidence suggests that blockade of the Ih current in behaving rats reduces hippocampal theta rhythm, highlighting the importance of this pacemaker current [74]. The selective Ih blocker ZD72888 utilised in this study resulted in an increase in the rebound action potential latency in MS/DB neurons, as has been described in substantia nigra neurons [51]. The Ih current may have an important role in controlling action potential firing, and thus further studies of network activity in this region are warranted. A recent study indicates that Ih currents contribute to spike frequency preference in hippocampal CA1 neurons [52]. Also, other studies have illustrated the role of the I h current in modulating synaptic neurotransmission; in particular in the cerebellar cortex [62], the hippocampal formation [35,39] and crayfish neuromuscular junction [5]. However, a recent study in calyx of Held neurons suggested that the effect of presynaptic Ih current on neuronal excitability was small [11]. In conclusion, our data suggests that Ih currents in the MS/DB complex may be composed of a variety of HCN channel subunits. Electrophysiological findings provide support for this notion, based on the similarities of the Ih current kinetics to values obtained in heterologous systems, and our immunocytochemical data provides evidence for the presence of HCN1, 2 and 4 subunits in parvalbumin-containing neurons. In general, the Ih currents are relatively small, at least under our recording conditions, and their regulation appears to be complex. A key goal now is to assess the functional role of this current in the MS/DB complex, both in terms of potential contributions to the resting potential of the GABAergic neurons and to their firing properties.

Acknowledgements We gratefully acknowledge the financial support of The Wellcome Trust, UK, and the National Institute of Health (NS25547 to REWF).

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