An Approach for Reliably Investigating Hippocampal Sharp Wave-Ripples In Vitro

Among the various hippocampal network patterns, sharp wave-ripples (SPW-R) are currently the mechanistically least understood. Although accurate information on synaptic interactions between the participating neurons is essential for comprehensive understanding of the network function during complex activities like SPW-R, such knowledge is currently notably scarce. counterpart. We show that slice storage in the interface chamber close to physiological temperature is the required condition to preserve network integrity that is necessary for the generation of SPW-R. Moreover, we demonstrate the utility of our method for studying synaptic and network properties of SPW-R, using electrophysiological and imaging methods that can only be applied in the submerged system.The approach presented here demonstrates a reliable and experimentally simple strategy for studying hippocampal sharp wave-ripples. Given its utility and easy application we expect our model to foster the generation of new insight into the network physiology underlying SPW-R

Animal Learning in a Multidimensional Discrimination Task as Explained by Dimension-Specific Allocation of Attention

Reinforcement learning describes the process by which during a series of trial-and-error attempts, actions that culminate in reward are reinforced, becoming more likely to be chosen in similar circumstances. When decisions are based on sensory stimuli, an association is formed between the stimulus, the action and the reward. Computational, behavioral and neurobiological accounts of this process successfully explain simple learning of stimuli that differ in one aspect, or along a single stimulus dimension. However, when stimuli may vary across several dimensions, identifying which features are relevant for the reward is not trivial, and the underlying cognitive process is poorly understood. To study this we adapted an intra-dimensional/ extra-dimensional set-shifting paradigm to train rats on a multi-sensory discrimination task. In our setup, stimuli of different modalities (spatial, olfactory and visual) are combined into complex cues and manipulated independently. In each set, only a single stimulus dimension is relevant for reward. To distinguish between learning and decision-making we suggest a weighted attention model (WAM). Our model learns by assigning a separate learning rule for the values of features of each dimension (e.g., for each color), reinforced after every experience. Decisions are made by comparing weighted averages of the learnt values, factored by dimension specific weights. Based on the observed behavior of the rats we estimated the parameters of the WAM and demonstrated that it outperforms an alternative model, in which a learnt value is assigned to each combination of features. Estimated decision weights of the WAM reveal an experience-based bias in learning. In the first experimental set the weights associated with all dimensions were similar. The extra-dimensional shift rendered this dimension irrelevant. However, its decision weight remained high for the early learning stage in this last set, providing an explanation for the poor performance of the animals. Thus, estimated weights can be viewed as a possible way to quantify the experience-based bias

Basal properties of sharp wave-ripples in submerged recording conditions.

<p>A, example experiment of SPW-R sampled in CA1 stratum pyramidale (upper trace). Below: 150–300 Hz band pass-filtered derivatives of the above signal isolating the SPW-associated ripple oscillation. Right: temporally magnified trace segment as indicated by arrows. B1, as a representative sample, in 15 slices the incidence of sharp wave-ripples was determined. Cumulative probability plots show the individual distributions of inter-SPW-intervals as a measure of SPW incidence and illustrating within- and inter-slice variability. <i>Inset:</i> magnification of cumulative functions as indicated. B2, cumulative probability plot of mean values derived from the same 15 slices. <i>Inset</i> indicates mean incidence (0.81±0.08 Hz). C1, from the same pool of data SPW amplitudes were determined. The distributions of amplitudes from the individual experiments are presented, illustrating within- and inter-slice variability of this parameter. C2, mean SPW amplitudes cumulated from the same dataset. <i>Inset</i> represents the mean SPW amplitude (106.2±11.2 µV). D1, from the same pool of data, 50 sharp wave-ripple events were randomly chosen from each experiment. Power spectrum density (PSD) functions were computed on each of these 750 SPWs and averaged. Plot shows the averaged PSD function ± SEM. <i>Inset</i> shows peak-triggered SPW-average and its derived 150–300 Hz filtered ripple oscillation. Calibration: 100 and 25 µV; 10 ms. D2, on these PSD functions, frequency in the ripple frequency band was determined. Cumulative frequency distribution and mean value are shown (208.9±0.7 Hz, <i>inset</i>).</p

Population imaging of somatic Ca²⁺ transients during sharp wave-ripples.

<p>A, high-resolution overview image of an OregonGreen-BAPTA1 bulk-loaded group of cells in the CA1 pyramidal layer of the hippocampus. The white box refers to the subregion imaged for the time-lapse recordings. Compared to the overview picture, Ca²⁺ measurements were performed on a smaller number of cells limited by the size of the camera chip used for Ca²⁺ imaging. Single cells can be well distinguished. The asterisk indicates a putative astrocyte. B, LFP recording from <i>stratum radiatum</i> (str. rad.) and corresponding time courses of the somatic Ca²⁺ signals indicative of suprathreshold activation and subsequent AP firing from cells labelled 1–4 in A. C, raster plot of significant responses imaged in A. Grey dashed lines correspond to peak negativities of sharp waves. Note that significant somatic Ca²⁺ signals indicative of cellular spiking can be exclusively found temporally coupled to sharp waves.</p

Perfusion rate and recording temperature modulate sharp wave incidence in submerged recordings <i>in vitro</i>.

<p>A, example experiment demonstrating the effect of successive reduction of recording temperature and perfusion rate on sharp wave occurrence. Magnified signals correspond to the events marked by asterisk. B, reduction of recording temperature and perfusion speed impairs but does not preclude the generation of sharp waves. Quantification was done 10–20 min after start of the respective experimental interference.</p

Experimental requirements for the reliable expression of sharp wave-ripples in submerged condition <i>in vitro</i>.

<p>A, slices stored in beakers and recorded from in conventional submerged chambers are viable but only anecdotally express sharp wave-ripples. Stimulus-induced field-EPSPs from these slices, however, revealed normal synaptic responses. 100 µs stimulation pulses of different intensities were applied at CA1 Schaffer collateral inputs. Stimulation artifacts are truncated. B1, submerged type recording chamber used throughout this study. The ovaloid shape of the chamber was chosen to enable flow conditions close to an ‘ideal’ laminar flow profile. B2, <i>‘in situ’</i> image of the used recording chamber. Str. pyr. and str. rad., stratum pyramidale and stratum radiatum, respectively (<i>n</i> = 48 slices for this study).</p

Spatial characteristics of sharp wave-ripples in submerged recordings.

<p>A, serial multisite LFP recordings were performed to reveal the spatial profile of sharp wave-ripples in CA1. A1, infrared-differential contrast (IR-DIC) video image displaying the reference electrode positioned in CA1 stratum radiatum close to CA2 while SPW-R were recorded with a second electrode in CA1 at locations closer to the subiculum (from right, positions as indicated, 10 to 100 µm steps). O, stratum oriens; p, stratum pyramidale; r, stratum radiatum; lm, stratum lacunosum-moleculare; Subic, subiculum. A2, voltage profile of SPW-R of the experiment shown in A1. LFP averages, each representing 20 events, were triggered by the signal sampled with the reference electrode. A3, amplitude depth profile of the example experiment shown in A1-A2. At each recording position with increasing distance to the alveus, the first significant maximum from baseline was determined as the peak of the SPW (see insets for examples regarded as positive and negative events). B, averaged amplitude depth profile (<i>n</i> = 5, solid line) and single depth profile functions (dashed). C, paired LFP recordings in CA3 and CA1 (C1) or subiculum (C2) of the same slice (red and green, respectively). Right: overlay of the indicated events and corresponding ripple signals (150–300 Hz) below. Note the increments of delays of CA1 and subiculum events <i>versus</i> SPWs recorded in CA3. D, normalized crosscorrelation functions to quantify latencies of SPW-R sampled from CA1 and subiculum with respect to the simultaneously recorded reference signal from CA3 (single experiments, dashed; average, bold; red, CA1 (<i>n</i> = 6); green, subiculum (<i>n</i> = 5)). Note increase in latency of subicular <i>versus</i> CA1 sharp waves, representing the spread of SPW-R towards the output structures of the hippocampus.</p