Locally sequential synaptic reactivation during hippocampal ripples

Abstract

The sequential reactivation of memory-relevant neuronal ensembles during hippocampal sharp-wave (SW) ripple oscillations reflects cognitive processing. However, how a downstream neuron decodes this spatiotemporally organized activity remains unexplored. Using subcellular calcium imaging from CA1 pyramidal neurons in ex vivo hippocampal networks, we discovered that neighboring spines are activated serially along dendrites toward or away from cell bodies. Sequential spine activity was engaged repeatedly in different SWs in a complex manner. In a single SW event, multiple sequences appeared discretely in dendritic trees, but overall, sequences occurred preferentially in some dendritic branches. Thus, sequential replays of multineuronal spikes are distributed across several compartmentalized dendritic foci of a postsynaptic neuron, with their spatiotemporal features preserved.

Fig. 1. Cultured hippocampal networks spontaneously emit SWs.

(A) An example trace of LFPs recorded from the CA1 stratum pyramidale during a SW event (top) was analyzed using the wavelet transform (bottom). (B) Three representative 125 to 250 Hz filtered SW traces and the average of all 1048 SWs recorded in a slice. (C) Left: A SW event was captured in LFPs of the CA1 stratum oriens (SO), the stratum pyramidale (SP), the stratum radiatum (SR), and the stratum lacunosum moleculare (SLM). Right: The current source density (CSD) was calculated for the average traces of 20 consecutive SWs. (D) Representative traces of SWs recorded from the CA1 stratum pyramidale before and 10 min after bath application of 10 μM carbachol. (E) Carbachol reduced the event frequency of SWs (n = 5 slices; *P = 0.015, t₄ = 4.1, paired t test).

Fig. 2. SW-participating CA1 pyramidal cells receive clustered synaptic inputs during SWs.

(A) Representative traces for simultaneous recordings of LFPs from the stratum pyramidale and spikes from a cell-attached recording of a CA1 pyramidal cell in a cultured hippocampal slice. Red tick marks in the top row indicate the SW times detected in the LFP trace. (B) The participation rates, i.e., the probabilities that SWs were accompanied by spikes of a given neuron, are plotted versus their chance levels estimated from 10,000 surrogates, in which the Poisson point process generated the same number of spikes in each cell. Each circle indicates the mean value of a single cell, and its error bar represents the 95% confidence interval of the surrogates. (C) Z scores of the spike participation rates in SWs of individual cells. Green dots indicate SW participants, and black dots indicate nonparticipants. (D and E) Peri-SW time histograms of the mean spike rates of 12 SW participants (D) and 9 nonparticipants (E). (F) Stack image of basal dendrites of a CA1 pyramidal cell filled with Fluo-4. (G) Representative raster plot of calcium transients emitted by a total of 385 imaged spines. Each dot indicates a single event of a single spine. Red dots indicate spine activity that occurred within 300 ms (red shades) before the SW onset (top ticks). A SW event is time-expanded in the right inset. (H and I) Peri-SW time histograms of the mean rates of calcium activity per spine in SW participants (n = 14 movies from 12 cells) (H) and nonparticipants (n = 9 movies from 9 cells) (I). (J) Peri-SW time histograms of the difference in the frequency of calcium activity from the baseline. Green bars indicate SW participants, and black bars indicate nonparticipants. The black line represents the time frames in which the difference of the calcium activity frequency between participants and nonparticipants is larger than the 95% confidential interval estimated by 10,000 bootstrap surrogates, in which the calcium activity was resampled randomly from the data pooled from both participants and nonparticipants. (K and L) Peri-SW time histogram of the mean geometric energies (spatial clustering) of synchronously activated spines in 12 SW participants (K) and in 9 nonparticipants (L). The gray histogram indicates the 95% confidence intervals (CI) of 10,000 surrogates, in which the spine locations were randomly exchanged within the videos.

Fig. 3. SW participant cells receive a rich repertoire of repeated sequences of synaptic inputs during SWs.

(A) All sequences detected in a representative raster plot are shown in different colors. Five sequences are temporally expanded (lower). (B) SW participants exhibited sequences more frequently than nonparticipants (n = 14 videos from 12 SW participants and n = 9 videos from 9 nonparticipants; *P = 0.0015, U = 14, Mann-Whitney U test). (C) Peri-SW time histograms of the mean event frequency of sequences in 12 SW participants. (D) Representative combinatorial patterns of individual sequences participating in individual SWs (left). Sequences and SWs were individually sorted along their dendrogram based on Ward’s method (n = 143 SW events and n = 85 sequences). Representative spatial distributions of spines involved in sequences #15′ (blue), #16′ (purple), #17′ (green), and #29′ (red), which were observed in SW events #71′, #74′, and #80′ (right).

Fig. 4. Synaptic sequences appear nonuniformly in dendritic trees.

(A) Locations of spines involved in a total of 76 sequences detected from a representative neuron are superimposed on the spine map. Each color indicates a single sequence. (B) The cumulative density function of the geometric energy (spatial clustering) of spines involved in individual sequences was compared to the 95% confidence interval (gray area) of its chance distribution estimated from 10,000 surrogates, in which the spine locations were randomly exchanged within the videos. All 313 sequences are pooled. (C) A topographic tree plot indicates the locations of spines that participated in sequences in a representative video. The heights of individual green bars on the tree represent the total number of events in sequences in which the corresponding spines participated. The ordinate indicates the path distance from the cell body. On the basis of this tree plot, we calculated entropy as the spatial bias of sequence-relevant spine activity across dendritic branches (right). The 95% confidence intervals (gray) were estimated from 10,000 surrogates, in which the numbers of sequence participations were randomly exchanged among the recorded spines.

Fig. 5. Sequences vectorially activate adjacent spines in the IN or OUT directions along the dendritic axis.

(A) Representative local sequences that had different directions, IN (inbound, red) and OUT (outbound, blue), relative to the cell body. Relative locations of the spines in these two local sequences are plotted as a function of relative times of their activity (t). In these coordinate planes, we calculated the coefficients of determination (R²) and the slopes (b₁) of the regression lines (black). (B and C) The cumulative density functions of R² (B) and b₁ (C) of all 350 local sequences were compared to the 95% confidence intervals (gray areas) of 10,000 bootstrap surrogates. (D) Peri-SW time histograms of the event frequencies of IN (red) and OUT (blue) sequences (top) and their differences (bottom).

Fig. 6. Distributed clusters of sequential synaptic activation during SWs.

During activity propagation in SWs, sequential spikes are sorted into discrete sequential synaptic inputs to locally clustered spines of a downstream neuron. The sequential synaptic inputs are vectorially toward (IN) or away from (OUT) the cell body. Thus, sequential replays of neuronal ensembles may converge on specific target dendritic zones (hotspots) in a spatiotemporally preserved fashion, thereby augmenting nonlinear dendritic computation.