Our results paint a far more active and dynamic picture of FS-PV IN dendrites than the currently prevailing view. Although the presence of Ca-permeable AMPA and NMDA receptors, as well as voltage-gated Na, Ca, and Kchannels in FS-PV IN dendrites had been demonstrated in earlier studies, it has been proposed that the fast electrical and Casignals provided by these channels are spatially and temporally attenuated and well-compartmentalized (). Such functional dampening and spatial segregation are thought to endow these interneurons with prompt and effective signal integration in the sublinear range. Our results, based on a caged-glutamate compound and 3D imaging methods, challenge this classical view of the functional role of interneurons by demonstrating a network-activity-dependent dynamic switch in dendritic integration mode. We have shown that passive, well-dampened FS-PV IN dendrites can be transiently activated by a high number of spatially and temporally coincident excitatory inputs during SPW-Rs. In this active state, several physiological properties of FS-PV INs are changed ( Table S2 ): (1) AP-associated Caresponses are not compartmentalized to the proximal dendritic regions but also invade distal dendritic segments, (2) dendritic spikes occur, in contrast to the low-activity baseline state (), (3) supralinear dendritic integration with a dual-integration threshold replaces linear or sublinear summation, (4) compartmentalized synaptic Casignals are replaced by broadly propagating Cawaves that are generated at dendritic hot spots, (5) dendritic voltage-gated Nachannels, which are functionally inactive in low activity conditions (), start to generate interneuronal ripple oscillations, which are associated with the dendritic Caspikes, and (6) the integration mode of FS-PV INs changes, AP outputs are tightly coupled to the phase of interneuronal ripple oscillations, and the total time-window of AP outputs becomes broader compared to the submillisecond precision in EPSP-AP coupling that characterizes the low activity state.

These methods yielded several insights into the fine spatiotemporal structure of dendritic hot spot activity. Similar to our previous study in stratum radiatum dendrite-innervating interneurons, here, we have shown that in double-perfused acute slices, the network activity is better preserved and provides spatially and temporally clustered synaptic input patterns to FS-PV INs that activate dendritic hot spots (). In contrast to previous studies in cell cultures where no hot spot activity was found in FS-PV INs (), we showed that SPW-R-associated dendritic hot spots can be intense in FS-PV INs. Moreover, we demonstrated that at a sufficiently high active input number (the second threshold) dendritic Caspikes can emerge from local hot spot activity and can activate long dendritic segments in multiple branches of the apical dendritic tree. Initially, calcium-permeable (∼40%) and nonpermeable (∼35%) AMPA receptors, and NMDA receptors (∼25%) trigger the initial signal in hot spot regions. The second step is for voltage-gated Caand Nachannels to be activated, and propagating dendritic spikes are initiated laterally to the hot spot regions with a significant delay, and spikes then propagate both centripetally and centrifugally dominantly by engaging the available VGCCs (dominantly L-type channels), thereby connecting neighboring hot spots. The VGCCs and Nachannels, which were activated in the second step, also contributed more then 35% to the somatic EPSP amplitude and further increased Caresponses in the central hot spot regions. The propagation speed of dendritic Caspikes was relatively low, much slower than the typical AP backpropagation speed, creating dendritic “delay lines” for signal integration. This could provide a relatively broad temporal window for dendritic integration both between and within hot spots, which may play a crucial role in coincidence detection and synaptic plasticity.

The main difficulty in imaging hot spot activity in complex dendritic arbors is the inadequate temporal and spatial resolution of currently available imaging technologies, which operate at video-rate time resolution and are constrained to 2D image acquisition in order to maintain an appropriate signal-to-noise ratio. Our fast 3D scanning methods overcome these limitations by providing a high temporal resolution up to tens of microseconds (temporal super-resolution microscopy) (), which allows simultaneous measurement of even the fastest regenerative events in multiple dendritic segments of the thin distal dendritic arborization, with a high spatial discretization on the size scale of dendritic hot spots during SPW-Rs (∼4 μm). In order to determine the dendritic active input patterns and underlying ion channel mechanism that are capable of generating the hot spots and the associated dendritic spikes during SPW-Rs, we took advantage of a caged glutamate compound, DNI-Glu⋅TFA. Its ∼7-fold higher uncaging efficiency allowed the more complex spatiotemporal patterns required for this work to be generated: up to 60 active inputs within a short time period (less than one cycle of the ripple oscillation). As phototoxicity increases rapidly and nonlinearly () as a function of the required laser intensity, the increased uncaging efficiency of DNI-Glu⋅TFA provides a significant advantage in neurophysiological experiments compared to the widely used MNI-Glu.

Interneuronal Ripple Oscillation as an Ingredient in the Generation of Population Ripple Oscillations

+ channels) and resonance-amplifying (e.g., persistent Na+ channels or NMDA receptors) conductances, have been observed mainly in the relatively low-frequency theta band, with a rapid decrease in amplitude at higher frequencies ( Hu et al., 2009 Hu H.

Vervaeke K.

Graham L.J.

Storm J.F. Complementary theta resonance filtering by two spatially segregated mechanisms in CA1 hippocampal pyramidal neurons. Johnston and Narayanan, 2008 Johnston D.

Narayanan R. Active dendrites: colorful wings of the mysterious butterflies. Narayanan and Johnston, 2007 Narayanan R.

Johnston D. Long-term potentiation in rat hippocampal neurons is accompanied by spatially widespread changes in intrinsic oscillatory dynamics and excitability. Zemankovics et al., 2010 Zemankovics R.

Káli S.

Paulsen O.

Freund T.F.

Hájos N. Differences in subthreshold resonance of hippocampal pyramidal cells and interneurons: the role of h-current and passive membrane characteristics. Zemankovics et al., 2010 Zemankovics R.

Káli S.

Paulsen O.

Freund T.F.

Hájos N. Differences in subthreshold resonance of hippocampal pyramidal cells and interneurons: the role of h-current and passive membrane characteristics. 2+ responses and electrophysiological data recorded during SPW-Rs have suggested that one unique property of FS-PV INs is that dendritic Ca2+ spikes are associated with intrinsically generated membrane potential oscillations in a much higher, ripple-frequency band. Supporting this, clustered input patterns were able to induce interneuronal ripple oscillations in short distal dendritic segments, even when axosomatic Na+ channels were blocked. In contrast to VGCCs, Na+ channels had a relatively high impact on somatic EPSPs and contributed less to the generation of dendritic Ca2+ signals. The dendritic origin of interneuronal ripple oscillations was further supported by the presence of oscillations in dendritic, but not in axosomatic, juxtacellular recordings, and by the weak dependence of the relative oscillation threshold on somatic membrane potential. It is important to note that the detection of interneuronal ripple oscillations was facilitated by the baseline subtraction method which, in contrast to traditional frequency-based approaches (such as band-pass filtering), better preserved the amplitude and the phase of individual oscillation cycles. Intrinsic membrane resonances, which are mediated by resonating (e.g., HCN-channels or M-type Kchannels) and resonance-amplifying (e.g., persistent Nachannels or NMDA receptors) conductances, have been observed mainly in the relatively low-frequency theta band, with a rapid decrease in amplitude at higher frequencies (). Earlier studies revealed either no resonance, or a resonance at beta-gamma frequencies in fast-spiking hippocampal interneurons (). The correlated Caresponses and electrophysiological data recorded during SPW-Rs have suggested that one unique property of FS-PV INs is that dendritic Caspikes are associated with intrinsically generated membrane potential oscillations in a much higher, ripple-frequency band. Supporting this, clustered input patterns were able to induce interneuronal ripple oscillations in short distal dendritic segments, even when axosomatic Nachannels were blocked. In contrast to VGCCs, Nachannels had a relatively high impact on somatic EPSPs and contributed less to the generation of dendritic Casignals. The dendritic origin of interneuronal ripple oscillations was further supported by the presence of oscillations in dendritic, but not in axosomatic, juxtacellular recordings, and by the weak dependence of the relative oscillation threshold on somatic membrane potential. It is important to note that the detection of interneuronal ripple oscillations was facilitated by the baseline subtraction method which, in contrast to traditional frequency-based approaches (such as band-pass filtering), better preserved the amplitude and the phase of individual oscillation cycles.

+ channels and may reflect the generation of high-frequency trains of action potentials within the distal apical dendrites. In the presence of interneuronal ripple oscillations, synaptic inputs are related to spike output through the following sequence of events. In the first step, ongoing active inputs are integrated and hot spots are generated. The membrane ripple oscillation starts to form a few millisecond-long time windows for signal integration; finally, after some oscillation periods, the AP output is generated. The AP output is synchronized to these interneuronal ripple oscillations, i.e., EPSPs which are in phase synchrony with the oscillations will be amplified and contribute to the APs. Moreover, an ongoing input assembly (or its failure), may shift the AP phase in a positive (or negative) direction relative to the interneuronal ripple oscillations, forming the firing pattern of individual FS-PV INs in the SPW-R associated cell assembly. Our working hypothesis is that there is a bidirectional relationship between dendritic mechanisms and cell assembly firing: the cell assemblies activate dendritic hot spots, while hot spots generate ripples, which finally determine the neuronal outputs of the individual neurons within cell assemblies. According to this bidirectional relationship, the memory information that is thought to be captured in the hippocampus during SPW-Rs as a temporal pattern of cell assembly discharges ( Buzsáki and Silva, 2012 Buzsáki G.

Silva F.L. High frequency oscillations in the intact brain. The ripple oscillations detected in the membrane voltage of FS-PV INs are actively mediated by Nachannels and may reflect the generation of high-frequency trains of action potentials within the distal apical dendrites. In the presence of interneuronal ripple oscillations, synaptic inputs are related to spike output through the following sequence of events. In the first step, ongoing active inputs are integrated and hot spots are generated. The membrane ripple oscillation starts to form a few millisecond-long time windows for signal integration; finally, after some oscillation periods, the AP output is generated. The AP output is synchronized to these interneuronal ripple oscillations, i.e., EPSPs which are in phase synchrony with the oscillations will be amplified and contribute to the APs. Moreover, an ongoing input assembly (or its failure), may shift the AP phase in a positive (or negative) direction relative to the interneuronal ripple oscillations, forming the firing pattern of individual FS-PV INs in the SPW-R associated cell assembly. Our working hypothesis is that there is a bidirectional relationship between dendritic mechanisms and cell assembly firing: the cell assemblies activate dendritic hot spots, while hot spots generate ripples, which finally determine the neuronal outputs of the individual neurons within cell assemblies. According to this bidirectional relationship, the memory information that is thought to be captured in the hippocampus during SPW-Rs as a temporal pattern of cell assembly discharges () should also be present as dendritic active input patterns with a certain phase relative to the interneuronal ripple oscillations.

Buzsáki and Silva, 2012 Buzsáki G.

Silva F.L. High frequency oscillations in the intact brain. Buzsáki and Silva, 2012 Buzsáki G.

Silva F.L. High frequency oscillations in the intact brain. Ellender et al., 2010 Ellender T.J.

Nissen W.

Colgin L.L.

Mann E.O.

Paulsen O. Priming of hippocampal population bursts by individual perisomatic-targeting interneurons. Maier et al., 2003 Maier N.

Nimmrich V.

Draguhn A. Cellular and network mechanisms underlying spontaneous sharp wave-ripple complexes in mouse hippocampal slices. Maier et al., 2011 Maier N.

Tejero-Cantero A.

Dorrn A.L.

Winterer J.

Beed P.S.

Morris G.

Kempter R.

Poulet J.F.

Leibold C.

Schmitz D. Coherent phasic excitation during hippocampal ripples. Nimmrich et al., 2005 Nimmrich V.

Maier N.

Schmitz D.

Draguhn A. Induced sharp wave-ripple complexes in the absence of synaptic inhibition in mouse hippocampal slices. Nimmrich et al., 2005 Nimmrich V.

Maier N.

Schmitz D.

Draguhn A. Induced sharp wave-ripple complexes in the absence of synaptic inhibition in mouse hippocampal slices. In this study, we propose a dendritic hot spot-related mechanism to be integrated into the currently accepted network model of SPW-R activity (). According to our results, membrane oscillations at ripple frequencies can be generated following strong depolarizing events in the dendrites of FS-PV INs. In intact hippocampal circuits, this depolarization can be provided predominantly by the synchronized firing of CA3 cell assemblies, which are directly responsible for the envelope of the SPW events (). Smaller cell assemblies can also provide the required depolarization, as individual CA3 subfields (), and CA1 (or CA3) minislices have also been shown to be capable of generating SPW-Rs (). Moreover, local application of KCl to the dendritic layer, with a complete blockade of GABAergic and glutamatergic synaptic transmission, reproduced SPW events and was also capable of generating the associated network ripple oscillations in CA1 minisclices (). These data also suggest that a single depolarization event in dendrites without any internal pattern is capable of activating intrinsic membrane mechanisms that then generate the ripple-oscillations. In line with this prediction, we showed that a single activation of clustered glutamatergic inputs in the distal dendrites of FS-PV INs, which generate a depolarizing hump and reproduce the hot spots associated with spontaneous SPW-R events, is also capable of generating secondary membrane oscillations in the ripple frequency range. In summary, we can say that the phase-locked firing during SPW-Rs is not a simple reflection of the discharge pattern of presynaptic cell assemblies, but oscillations can be formed actively and intrinsically by the dendritic membrane.