Enhanced Dendritic Action Potential Backpropagation ... - Springer Link

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Nov 3, 2010 - Ferenc Erdélyi • Gábor Szabó • Hannah Monyer • Attila Csákányi •. E. Sylvester Vizi • Balázs Rózsa. Accepted: 30 September 2010 / Published ...
Neurochem Res (2010) 35:2086–2095 DOI 10.1007/s11064-010-0290-4

ORIGINAL PAPER

Enhanced Dendritic Action Potential Backpropagation in Parvalbumin-positive Basket Cells During Sharp Wave Activity Bala´zs Chiovini • Gergely F. Turi • Gergely Katona • Attila Kasza´s • Ferenc Erde´lyi • Ga´bor Szabo´ • Hannah Monyer • Attila Csa´ka´nyi • E. Sylvester Vizi • Bala´zs Ro´zsa

Accepted: 30 September 2010 / Published online: 3 November 2010 Ó Springer Science+Business Media, LLC 2010

Abstract In this study two-photon imaging and single cell electrophysiological measurements were carried out in PV? hippocampal interneurons to compare the dendritic calcium dynamics of somatically evoked backpropagating action potentials (BAPs) and in vitro sharp wave oscillation (SPW) activated BAPs at different distances from the soma. In the case of 300 lm thick, non-oscillating slices, the BAP-evoked Ca2? (BAP-Ca2?) influx propagated along the dendritic tree in a non-uniform manner and its amplitude gradually reduced when measured at more distal regions. In contrast to the evoked BAP-Ca2?s, the spontaneous SPWinduced Ca2? influx had only a small distance-dependent decrement. Our results suggest that similarly to nicotinic acetylcholine receptor activation, synaptic activity during hippocampal SPWs increases AP backpropagation into distant dendritic segments. Bath application of Nimodipine, a specific Ca2? channel blocker and tetrodotoxine decreased the amplitude of the somatically evoked Ca2? influx, which suggests that L-type Ca2? channels play an important role both during somatically evoked and SPW-induced BAPs.

Special Issue: In Honor of Dr. Abel Lajtha. Gergely Katona, E. Sylvester Vizi, Bala´zs Ro´zsa, are owners of Femtonics Ltd. B. Chiovini  G. F. Turi  G. Katona  A. Kasza´s  F. Erde´lyi  G. Szabo´  A. Csa´ka´nyi  E. S. Vizi  B. Ro´zsa (&) Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony str. 43, 1083 Budapest, Hungary e-mail: [email protected] H. Monyer Department of Clinical Neurobiology, University of Heidelberg, Heidelberg, Germany

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Keywords Two-photon  Sharp wave  Backpropagating action potential  Parvalbumin-positive basket cell  Hippocampus

Introduction Backpropagating action potentials (BAPs) play a crucial role in neuronal Hebbian plasticity [1] by changing synaptic gains following different time-dependent local dendritic interactions with ongoing synaptic activity [2]. Backpropagation has been shown to be cell-type dependent; for example, it is strong in mitral cell dendrites in the olfactory bulb [3] and in stratum radiatum interneurons of the hippocampus [4]. However, backpropagation is weak in dendrites of Purkinje cells [5] and parvalbumin-expressing (PV?) neurons [6], where backpropagation has been shown to become passive due to the distance-dependent decrease of sodium channel density relative to potassium channel density [7]. Fast spiking (FS), PV? basket cells (BCs) as the clockworks for neuronal oscillations are important elements of hippocampal neuronal networks [8]. All types of the PV? interneurons in the CA1 area of the hippocampus, such as axo-axonic, bistratified, oriens-lacunosum moleculare and BCs, have extended dendritic arborization spreading even to the lacunosum moleculare [9]. A strong, distance-dependent decrement of action potential (AP) backpropagation has been demonstrated in cortical and hippocampal PV? cells [6, 7], suggesting that a large part of their dendritic arborization is not reached by BAPs, leaving out these regions from receiving somatic feedback. However, ongoing coincident synaptic inputs could slightly modify AP backpropagation as in cortical and hippocampal stratum radiatum interneurons [4, 10, 11]. Synchronous network activity—such as sharp wave oscillations (SPWs)—may be

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a better candidate for BAP modification, because during SPWs synchronous neuronal activity generates extended synaptic activation within a narrow time window [12]. To test this assumption, we have compared AP backpropagation in thin slices in which the spontaneous synaptic activity is relatively low and in conditions when maintained network activity is able to generate SPWs. Using a novel method, we found that AP backpropagation is enhanced during SPWs suggesting that active network states may change global dendritic coincidence detection rules measured during basal states.

Experimental Procedures Slice Preparation and Electrophysiology All experiments were carried out in accordance with the Hungarian Act of Animal Care and Experimentation (1998; XXVIII, section 243/1998.). Acute hippocampal slices were prepared from 16 to 22-day-old transgenic mice expressing enhanced green fluorescent protein controlled by the PV promoter [13]. The animals were deeply anesthetized using isoflurane and decapitated. After the decapitation, the brain was quickly removed and dipped into ice-cold cutting solution containing (in mM): 2.8 KCl, 1 MgCl2, 2 MgSO4, 1.25 NaH2PO4, 1 CaCl2, 10 D-glucose, 26 NaHCO3 and 206 sucrose. Horizontal brain slices (300, 450 and 800 lm) were cut with a vibratome (Vibratome 3000). Slices were then stored at room temperature in artificial cerebrospinal fluid (ACSF) that consisted of (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose [4]. For electrophysiological recordings, optical oxygen measurements and for two-photon imaging two types of submerged chambers were used. Firstly, a regular chamber was used, equipped with single perfusion tubing (Luigs and Neumann, Ratingen, Germany). Secondly, a chamber equipped with a double perfusion system improved oxygenation of the slices by simultaneously perfusing both the top and the bottom surfaces of the slices at a high perfusion rate (11.2 ml/min) [14]. This dual perfusion recording chamber was optimized for optical recordings by exchanging the metal mesh with a thin nylon slice supporting grid (Warner Instruments; thickness: 100 lm; mesh size: 1.0 mm 9 1.0 mm;) (Fig. 1a, left). Furthermore a pair of bubble traps was also inserted between the perfusion pump and the chamber to prevent gas bubbles from entering into the recording chamber (Fig. 1a, right). The bubble trap also served as a pulse dampener for the increased perfusion rate that was necessary for the high oxygen saturation of the slices. Finally, all recordings were performed at 32–34°C. Whole-cell current-clamp recordings and spontaneous field potentials were acquired using a MultiClamp 700B

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amplifier and digitized with the Digidata 1440 Data Acquisition System (Molecular Devices, Sunnyvale, CA, USA). The detection of local field potentials (LFPs) from CA3 was performed with glass electrodes (6–9 MX) filled with ACSF containing 1 M NaCl. The recorded LFP was band-pass filtered offline (1–3 kHz and 100–300 Hz) using the built-in filtering function of the MES software package (Femtonics Ltd., Budapest, Hungary). PV? interneurons in CA1 stratum pyramidale were visualized using 880 nm infrared oblique illumination and two-photon imaging (830 nm). For whole-cell recordings, glass electrodes (6–9 MX) were filled with a potassium-based intracellular solution containing (in mM): 125 K-gluconate, 20 KCl, 10 HEPES, 10 Di-Tris-salt phosphocreatine, 0.3 Na-GTP, 4 Mg-ATP, 10 NaCl, 0.06 Oregon Green BAPTA-1 (OGB1), 0.008 biocytin. For the physiological and optical recordings, cells with a resting membrane potential more negative than -50 mV were accepted. The recorded interneurons represented the typical electrophysiological properties of fast spiking interneurons [15] (maximum firing frequency = 206.66 ± 43.33 Hz; firing adaptation = 7.8 ± 1.7%; AP amplitude = 52.6 ± 12.4 mV; resting membrane potential = -63.9 ± 7.6 mV). BAPs were induced by somatic current injections (700 pA for 5 ms; five BAPs were evoked at 140 Hz). Step depolarization was also induced by somatic current injections (1,500–1,700 pA for 100 ms). Data acquisition was performed using the pClamp10 (Molecular Devices, Sunnyvale, CA, USA) and MES (Femtonics Ltd., Budapest, Hungary) software package. Drug Application All of the drugs were applied in the bath except for acetylcholine (ACh 1 mM), which was injected by a continuous-flow, motion artifact-free, rapid perfusion system [16]. Tetrodotoxin (TTX) (1 lM), atropine (1 lM) and bicuculline (20 lM) was purchased from Sigma–Aldrich, the voltage-gated calcium channel (VGCC) blockers Nimodipine (20 lM) and Mibefradil (10 lM) and the glutamatergic synaptic blockers (CNQX, 10 lM; D,L-AP5, 40 lM) were purchased from Tocris Bioscience. Two-photon Imaging Two-photon imaging started 15–20 min after obtaining the whole-cell configuration on a two-photon laser-scanning system (Femto2D, Femtonics Ltd., Budapest) equipped with a femtosecond laser tuned to 830 nm (Mai Tai HP, SpectraPhysics, Mountain View, CA, USA). The block scheme of the light path can be seen in Fig. 1b. Multiple regions of interest (ROIs) were scanned with constant speed, while intermediate sections were jumped over within 60 ls, using

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Fig. 1 a Top, Illustration of the modified recording chamber equipped with the dual perfusion system and bubble trap (right). Bottom, Schematic diagram of the fluid flow in the dual perfusion chamber designed for the wide field of view of two-photon imaging. The fluorescent signal was detected through a high NA condenser (cond.) and objective (obj.) lenses. Whole-cell patch-clamp and field recordings were performed simultaneously. Slices were placed on a thin polypropylene mesh. b Schematic drawing of the light path of the two-photon laser scanning microscope system used for imaging (PMT photomultiplier, dic dichroic mirror, IR light source, TIR transmission infra red detector). c Oxygen concentration and saturation are shown

as a function of depth from the surface of the slice in the dual perfusion chamber with a fast flow rate (11.2 ml/min; black trace and symbols) and in a regular chamber with a normal flow rate (3.5 ml/ min; gray trace and symbols). Inset, CCD image of a hippocampal slice with the optode used for oxygen concentration measurements. d Left, Representative local field potentials recorded in a regular chamber (top) and in the dual perfusion chamber (bottom). Right, Sharp wave oscillations (SPWs) could be observed only in the dual perfusion chamber. Normal ACSF was used in all experiments. Scale bars: c 100 lm; d 500 ms, 0.05 mV; inset: 20 ms, 0.05 mV

a spline-interpolated path (Multiple Line Scanning) [17]. This scanning mode increases the signal-to-noise (S/N) ratio, accelerates data collection, and therefore abolishes the effect of the time-dependent amplitude decrease of the Ca2? transients, which results in a smaller variability during measurements of distance dependence. Measurement control, real-time data acquisition and analyses were performed with the Matlab- and C??-based MES program package (Femtonics Ltd., Budapest). Fluorescence traces are expressed as relative fluorescence changes [DF/F = (F - F0)/F0], where F0 is the background-corrected pre-stimulus fluorescence. To obtain the amplitude and decay of the transients, three sweeps were averaged. Unless otherwise indicated, data are presented as the mean ± SEM.

hippocampal CA1 region (Fig. 1c). The sensor was calibrated using 2–3 mM of sodium sulfite (Na2SO3) to eliminate the dissolved oxygen from the non-bubbled ACSF (defined as 0% oxygen), while *95% oxygen was set in ACSF, bubbled for 1 h with 95% O2/5% CO2. The recording was started 200 lm above the slice. Thereafter, the sensor tip was lowered diagonally into the tissue with 50 lm steps using a micromanipulator. The oxygen level was recorded at each step for 30 s at 1 Hz. The raw data of this period were averaged and expressed as oxygen saturation % and lmol/l dimensions. The thin (300 lm) slices were measured in a regular, submerged-type chamber with single perfusion and a low flow rate (3.5 ml/min), while the 450–800 lm thick slices were recorded in dual perfusion chamber with a high perfusion rate (11.2 ml/min).

Measurement of Oxygen Concentration in Slices Histology The oxygen saturation both for the 300 lm and the 800 lm brain slices was measured with an optode (tip diameter *50 lm; Microx TX3, PreSens GmbH, Germany) in the stratum radiatum—stratum pyramidale layers of the

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After recording, slices were fixed in a mixture of 4% paraformaldehyde and 15% saturated picric acid dissolved in phosphate buffered saline (PBS; pH 7.4). For biocytin

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visualization, the slices were washed three times in PBS and subsequently incubated for 30 min in 0.3% TritonX100 (SigmaAldrich) in PBS and overnight in 1/200 dilution of Fluorescein (DTAF) streptavidin complex (Jackson ImmunoResearch Europe Ltd.). The slices were then washed in PBS, mounted on glass slides in Vectashield Mounting Medium (Vector Laboratories, Inc. Burlingame, CA, USA) and stored at 4°C. Sections were examined and archived with a confocal laser-scanning microscope (FV1000, Olympus). Statistics Data are presented as the mean ± SEM. Statistical comparisons were performed using the Student’s paired t-test.

Results Dual Perfusion Chamber and Fast Perfusion Assist to Generate Sharp Wave Oscillations In Vitro Although neuronal oscillations can be observed in interface type chambers [18], it is difficult to reproduce these phenomena in submerged chambers ideal for imaging and pharmaceutical experiments. One of the main reasons for this difference could be the poor oxygenation of the slices in the submerged chambers with single perfusion [14, 19]; nevertheless, network activity has been shown to be maintained in a chamber with dual perfusion [14, 19]. To record simultaneous fluorescent signals with high sensitivity, we made the following modifications to the dual perfusion chamber. First, the z-level of the slice-supporting mesh was minimized in order to match the working distance of a high numeric aperture (NA) condenser while keeping half of the perfusion fluid flowing below the slice (Fig. 1a). Moreover, gas bubbles that normally entered into the recording chamber were kept out of the chamber by a custom-made, dual-channel bubble trap. Finally, the optical aperture of the chamber was increased to be optimized for multi-channel recordings and for the application of high NA objectives with small working distances (Fig. 1a, c). To confirm the effect of the higher flow rate on the brain tissue oxygenation, we measured oxygen saturation and concentration with an optode in the slices and compared recordings in a regular chamber with normal flow rate (3.5 ml/min) to those in a dual perfusion chamber with an increased flow rate (11.2 ml/min). Oxygen concentration decreased rapidly with the depth of the slice in the regular chamber because of the slow (3.5 ml/min), single-flow perfusion (Fig. 1c). Conversely, in the dual perfusion chamber the oxygen saturation was high enough for the generation of spontaneous SPWs (Fig. 1d). Therefore,

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according to the previous observations [14], the dual perfusion chamber with an increased flow rate provides better oxygenation, which can maintain physiological network oscillations such as SPWs. Action Potential Backpropagation Decreases as a Function of Distance From the Soma in Slices Without SPW Activity To eliminate the time-dependent decrease of Ca2? signals in long-term comparative measurements, we used the Multiple Line Scanning method (Fig. 2a) [17]. This means that instead of acquiring frames, the two-photon laser spot moves along user-defined line segments (Fig. 2a). With this method, depending on the length and geometry of the scanning line, one can achieve a faster data acquisition speed (in the kHz range) with a high S/N ratio and less photodamage to the non-scanned areas. To study the kinetics of the Ca2? transients in the dendrites of fast spiking PV? BCs, we evoked dendritic Ca2? influx by one or five somatically induced BAPs (BAP-Ca2? and BAP5-Ca2?, respectively). In the case of 300-lm-thick, non-oscillating slices, the Ca2? influx propagated in a non-uniform manner along the dendritic tree, and its amplitude reduced gradually when measured in more distal regions (Fig. 2). The average amplitude of the BAP-Ca2? was 0.14 ± 0.03 (DF/F) in the proximal dendritic regions (\50 lm from the soma), while the amplitude showed significant reduction in the proximal dendrites 50–100 lm from the soma (0.07 ± 0.03, DF/F, n = 4). No Ca2? influx could be detected in the regions more distal to the soma ([100 lm). In contrast, when the measurements were repeated with five BAPs (delivered at 140 Hz), the average amplitude increased in the proximal dendritic regions (\50 lm, 0.30 ± 0.05, DF/F), in the range of 50–100 lm (0.15 ± 0.04, DF/F n = 5) and at more distal regions ([100 lm, 0.06 ± 0.03, n = 5) where the single BAPs could not evoke Ca2? responses. However, no local Ca2? transients could be measured in the dendritic tree beyond a distance of 150 lm.

Action Potential Backpropagation is Enhanced During Hippocampal SPW Activity in CA1 PV? Interneurons To test AP backpropagation in the presence of enhanced network activity, we combined two-photon imaging, whole-cell recordings and CA3 LFP measurement (Fig. 1a). PV? and EGFP? BCs located in stratum pyramidale of the CA1 region were filled with OGB-1 via the somatic recording pipette. Multiple ROIs or long dendritic segments were selected for two-photon imaging of BAPs

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Fig. 2 Action potential backpropagation decreases as a function of distance from the soma in slices without SPW activity. a Maximum intensity two-photon image stack projection of a parvalbumin (PV)positive interneuron located in CA1 stratum pyramidale filled with Oregon Green BAPTA-1 (60 lM). White line segments were scanned in a numbered order at a constant speed (4 lm/ms), while black segments were jumped over rapidly (within 60 ls) in order to increase the measurement speed and S/N ratio. b Five backpropagating action potential (BAP5)-evoked normalized multiple line scan Ca2? image measured in regions indicated in (a). Black triangle indicates the

timing of the BAP5 (BAP5—140 Hz, Ca2? response—average of five traces). c Evoked dendritic Ca2? transients from numbered regions of (b). d Representative somatic voltage trace of an action potential induced by somatic current injection. e Amplitude of single BAPinduced Ca2? transients as a function of distance from the soma. The different symbols denote DF/F values recorded from the same cells. f Average of Ca2? responses in four cells measured at different dendritic distances. g–i Same as d–f, but five BAPs delivered at 140 Hz. Scale bars: a 20 lm; b 200 ms; c 200 ms, 0.1 DF/F; d 20 mV, 25 ms; g 10 mV, 10 ms

(Fig. 3b). We found that spontaneous SPWs detected in the CA3 field recording were able to induce APs in CA1 PV? basket cells which then propagated reliably to the imaged dendritic segments located at different distances from the soma (Fig. 3c–g). However, somatic depolarization (induced by a 5 ms, 200–400 pA current injection) elicited a BAP-Ca2? with a smaller amplitude in the same region (Fig. 3c, d). In contrast to somatic depolarizationinduced BAP-Ca2?s, spontaneous SPW-induced BAPCa2? had only a small distance-dependent decrement (compare Figs. 3g to 2f, i). The amplitude difference between somatic depolarization and SPW-induced BAPCa2? was largest at somatic distances above 100 lm (compare Figs. 3g to 2f, i). The average amplitude (DF/F) of the calcium accumulation at \50 lm was 0.19 ± 0.05 (n = 3), at 50–100 lm was 0.2 ± 0.02 (n = 5) and was 0.14 ± 0.02 (n = 2) at distances [100 lm. In the remaining dendritic segments, we found that the spatial distribution of individual Ca2? transients induced by spontaneous SPWs or somatic depolarization was similarly homogeneous (Fig. 3d, bottom), suggesting that the local summation of synaptic inputs with BAPs may not contribute to the globally enhanced AP backpropagation.

Our results suggest that synaptic activity during hippocampal SPWs increases AP backpropagation into distant dendritic segments.

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Voltage-gated Calcium Channels (VGCCs) Mediate Dendritic Ca2? Signals in PV? Interneurons Action potentials initiated in the BC axon have been shown to propagate passively into the dendrites of PV? BCs [7]. These properties might be explained by a distancedependent rapid decrease in Na? conductance and Na? to K? conductance ratio [7]. Due to the low distal Na? conductance density, we hypothesized that VGCCs activated by passive AP backpropagation may play an important role in the generation of the BAP-Ca2? in distant dendritic segments. To test this hypothesis we injected somatic depolarizing current steps (100 ms, 0–1,700 pA) into the somas of PV? BCs (Fig. 4a, b) in the presence of TTX (1 lM), which induced large, well propagating Ca2? transients (Fig. 4c). Mibefradil (10 lM), which selectively blocks T-type Ca2? channels at this low concentration [20] did not decrease the step depolarization-induced dendritic Ca2? accumulation in PV? BCs (104.40 ± 1.63%, n = 4

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Fig. 3 Action potential backpropagation in CA1 interneurons is enhanced during hippocampal SPW activity. a Schematic diagram of the experimental arrangement. b Maximum intensity image stack projection of a dendritic segment of a PV? interneuron loaded with 60 lM OGB-1. The black line indicates the scanned dendritic region. c Top, Amplitude of the somatic current step injected into the soma of an interneuron. Middle, Local field potential recorded in the CA3 region shows SPW activity. Bottom, Simultaneously recorded somatic voltage trace and d the corresponding Ca2? transient (Top) with the normalized image of the raw Ca2? trace (Bottom) measured from the

region in (b). d The black triangles indicates a BAP-Ca2? transient that was smaller in amplitude than the SPW-induced BAP-Ca2? transients. e Enlarged view of a SPW ripple (Top) and simultaneously recorded voltage (Middle) and Ca2? transients (f). g The amplitude of the SPW evoked BAP-Ca2? transients was only modestly reduced as a function of distance (n = 5). Scale bars: b 5 lm; c timescale 1,000 ms; horizontal scales: (top) 500 pA, (middle) 0.05 mV, (bottom) 20 mV; (d) 0.05 DF/F, 1,000 ms, colorbar: 0–0.8 DF/F; (e) (top) 0.05 mV, 20 ms; (bottom) 20 mV, 20 ms; (f) 0.05 DF/F, 200 ms

P = 0.07, Fig. 4c, d). In contrast, the L-type calcium channel blocker Nimodipine (20 lM) induced significant reduction (41.66 ± 7.75%, n = 4, P = 0.0024) in Ca2? transients under the same conditions (Fig. 4c, d).

(see the composition of the cocktail in the legend of Fig. 5) elicited suprathreshold responses with APs (Fig. 5a) or subthreshold responses at more hyperpolarized membrane potentials (Fig. 5b) in randomly selected CA1 interneurons from stratum radiatum to oriens. The somatic depolarization amplitudes were in the range observed during spontaneous SPW events. Therefore, we asked whether depolarization induced by the activation of the non-synaptic nicotinic receptors induced similar changes in BAPs compared to synaptic activation during SPW activity. The co-occurrence of a7-nAChR activation and BAPs was studied by performing somatic current injections in order to induce BAPs during rapid ACh injection. nAChR activation followed by high amplitude responses summed sublinearly with BAPs. In contrast, the small, more physiological nAChR responses summed supralinearly with BAPs (Fig. 5c, d).

Non-synaptic Nicotinic Receptor Activation and SPWs Induce Similar Enhancement of Action Potential Backpropagation Hippocampal nicotinic acetylcholine receptors (nAChRs) serve as potential targets of endogenous acetylcholine (ACh) released from mainly nonsynaptic nerve terminals [21] and nicotine inhaled with tobacco smoking. Neurochemical [22–25] and electrophysiological [16, 26, 27] evidence has shown that GABAergic interneurons are equipped with nicotinic receptors. As we have shown in our earlier study [16], these receptors are of a7 subtype. Therefore, we studied the effect of nAChR stimulation on BAPs. The stimulation of a7-nAChRs by rapid ACh application (1 mM for 200 ms) in the presence of muscarinic receptor antagonist atropine and glutamatergic synaptic blockers

Discussion The hippocampus is an area important in learning and memory [28]. The medial septum/diagonal band area

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Fig. 4 VGCCs mediate dendritic Ca2? signals in PV? basket cells. a Confocal image stack of a representative PV? basket cell developed with DTAF-conjugated avidine after the physiological recording. Axonal processes of the recorded neuron in the stratum pyramidale (SP) show the typical axonal arborization of the basket cells. Left inset shows the location of the recorded PV? basket cell in the hippocampus (CA1). Right inset, enlarged view of the measured dendritic segment. b Somatic voltage trace from a cell in response to a 500 ms, 800 pA somatic current injection (bottom) (AP frequency: 200 Hz, adaptation: 6.45%). c Two-photon measurement of dendritic Ca2? transients evoked by somatically injected current steps (1,700

pA) in the presence of TTX and VGCC blockers (TTX, red trace). T-type VGCCs were blocked by 10 lM Mibefradil (TTX ? M, orange trace), while L-type VGCCs were blocked by 20 lM Nimodipine (TTX ? M ? N, blue trace). Average of three traces. d (Left) Changes in the average peak amplitude of Ca2? responses of the interneuron in (a) TTX (red bar), TTX ? M (orange bar) and TTX ? M ? N (blue bar). (Right) Pooled Ca2? responses in the percentage of evoked Ca2? responses in the presence of TTX (n = 4 cells; P \ 0.01). Scale bars: a 30 lm, left inset: 200 lm, right inset 10 lm; b timescale: 100 ms vertical scale, top: 20 mV, bottom: 1,000 pA c top: 200 ms, 5 lm, middle 0.2 DF/F, 200 ms; bottom: 500 pA

provides both cholinergic and GABAergic projections to the hippocampus. They are involved in oscillatory activity required for cognitive function and working memory [29]. In contrast to the fact that PV? interneurons have long dendritic segments [30], APs have only been shown to propagate passively with an attenuated amplitude into proximal dendritic segments [7]. The lack of backpropagation would dissociate distal synaptic input sites from somatic feedback by decreasing coincident detection mediated by BAPs. High frequency AP bursts have been shown to break through this BAP distance barrier at physiological temperatures in pyramidal cells [31]; however, in PV? basket cells, bursts of APs were unable to amplify distance-dependent decrease of BAPs (Fig. 2) [6]. Mechanisms underlying enhanced distance-dependent decrease of backpropagation may be the decreasing Na? channel density and the Na? to K? channel ratio [7]. Similar to pyramidal cells, the decreasing Na? channel density might be compensated by distal VGCCs in interneurons [32] which could be opened by local depolarization. Indeed, a short step depolarization, which

approximately mimics the depolarization window during SPWs, induced propagating Ca2? transients that were sensitive for L-type VGCC blockers. These data suggest that high-threshold L-type VGCCs located at distant dendritic segments could not be activated by single BAP- or five BAP-induced depolarization, but could be opened by larger coincident dendritic depolarization induced by synchronous network activity such as SPWs. Here we have found that AP backpropagation during SPWs breaks down the distance threshold and induces large homogeneous Ca2? signals in distant dendritic segments. Therefore, feedback information from AP firing may reach distant dendritic segments. In our earlier study we have shown [16] that functional extrasynaptic a7 subtype-containing nAChRs are abundant on GABAergic interneurons and play an important role in the regulation of the spike timing-dependent plasticity and modulation of BAPs. Here we show that nAChR activation—similarly to SPWs—induced similar depolarization and enhancement in AP backpropagation in randomly selected hippocampal interneurons. Taking into account the

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Fig. 5 Action potential backpropagation is enhanced during nAChR activation. a Top, Dendritic Ca2? transients elicited by rapid ACh injections (1 mM for 200 ms) in the presence of atropine (1 lM), bicuculline (20 lM), AP5 (40 lM) and CNQX (10 lM). Bottom, Corresponding somatic membrane potential with a brief train of APs. b Subthreshold voltage and Ca2? responses at more hyperpolarized membrane potential. c Top, Backpropagating action potential induced Ca2? transients in control conditions (BAP5, thick black traces) and in the presence of rapid nAChR activation (BAP5 ? ACh, thick gray traces). In case of small nAChR activation the difference of BAP5 ? nACh and BAP5 (Diff. thin black traces) was larger as compared to the nAChR-induced transient, suggesting supralinear summation of the response; however, summation was sublinear (Bottom) when nAChR activation was large. d The difference of BAP5 ? nACh and BAP5 transients as a function of nAChR activation. Each point represents different cells

diversity of GABAergic interneurons [33], it remains to be revealed whether PV? interneurons in the hippocampus express nAChRs. However, it has been shown in the rostral ventral medulla [34] that PV? GABAergic interneurons in this area exhibit immunoreactivity for the a4 and a7 nAChR subunits [34]. Taken together, these findings suggest the direct effect of stimulation of nAChR stimulation on dendrites of GABAergic interneurons. The difference between small and large nicotinic effects (Fig. 5d) can be explained by the desensitization process of nAChRs [35]. The importance of these findings is that the phenomenon of nonsynaptic nAChRs acting through ligand gated ionotropic channels located on GABAergic interneurons [36] is a potential alternative for metabotropic muscarinic modulation of hippocampal plasticity [16], which exhibits a rather slow action [37]. Regarding methodological aspects, we modified the double perfusion chamber designed by Hajos et al. [14] by increasing the optical aperture, trapping bubbles from

entering into the recording chamber and decreasing the slice holding mesh z-level to make it compatible with high NA lenses. These modifications allowed two-photon fluorescence imaging with dual (upper and lower) whole field detection, providing highly sensitive dendritic measurements under more physiological conditions. The combination of the new chamber with whole-cell patch clamping, field recordings and Multiple Line Scan Imaging provides a new and effective method for the investigation of dendritic computation in different neuronal network states such as network oscillations. One of the main advantages of this combined method is that, in contrast to in vivo measurements, network activity-driven dendritic computation can be studied without motion artefacts. Acknowledgments This work was supported by the grants OM-00131/2007, OM-00132/2007, GOP-1.1.1-08/1-2008-0085, NK 72959, Grant of Hungarian Academy of Sciences.

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2094 Comments Dr. Lajtha was one of the founders of modern neurochemistry and a founding member of several journals and societies. Over the past 60 years, he has earned his reputation as a first-class scientist; editor of several books and journals, and his Handbook of Neurochemistry. But, when we asked him what pleases him most about his scientific accomplishments, he answered, and that is characteristic of him, ‘‘most rewarding was the feeling that I was helping young scientists from all over the world experience the pleasure of performing creative research.’’ In his life there has been no change whatsoever in his commitment to quality science. That is what he stood for, stands for, and that is what he shall remain. Neurochemical research at the bench level and collaboration with other colleagues has always been important to him. It was an additional pleasure for him to have informal contacts with foreign scientists of different cultural backgrounds. Such contacts and collaborations carried out in several laboratories have fostered many long-lasting friendships.

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