Cortical Up states induce the selective weakening of subthreshold synaptic inputs

Abstract

Slow-wave sleep is thought to be important for retuning cortical synapses, but the cellular mechanisms remain unresolved. During slow-wave activity, cortical neurons display synchronized transitions between depolarized Up states and hyperpolarized Down states. Here, using recordings from LIII pyramidal neurons from acute slices of mouse medial entorhinal cortex, we find that subthreshold inputs arriving during the Up state undergo synaptic weakening. This does not reflect a process of global synaptic downscaling, as it is dependent on presynaptic spiking, with network state encoded in the synaptically evoked spine Ca²⁺ responses. Our data indicate that the induction of synaptic weakening is under postsynaptic control, as it can be prevented by correlated postsynaptic spiking activity, and depends on postsynaptic NMDA receptors and GSK3β activity. This provides a mechanism by which slow-wave activity might bias synapses towards weakening, while preserving the synaptic connections within active neuronal assemblies.Slow oscillations between cortical Up and Down states are a defining feature of deep sleep, but their function is not well understood. Here the authors study Up/Down states in acute slices of entorhinal cortex, and find that Up states promote the weakening of subthreshold synaptic inputs, while suprathreshold inputs are preserved or strengthened.

Fig. 1

Pairing subthreshold inputs with Up states causes synaptic weakening. a Schematic of the experimental set-up, with whole-cell patch-clamp recordings obtained from a LIII principal neuron, and extracellular stimulation electrodes placed in LIII and the border of the deep layers, to evoke local Up states and single EPSPs, respectively. b Current-clamp recording from a layer III principal cell shows reliable induction of Up states following electrical stimulation in LIII (white arrowheads), resulting in a typical bimodal distribution of the membrane potential (right histogram; percentage of time on a logarithmic scale). c Simultaneous whole-cell current-clamp (upper trace) and LFP (lower trace) recordings from layer III showing an induced Up state. d Single EPSPs were evoked every 15 s during Down state periods in order to measure synaptic strength during baseline, and following three pairing protocols with 5 Hz stimulus trains: Down state pairing (total of 50 individual EPSP pairings; n = 6; left panel), Up state pairing (total of 50 individual EPSP pairings; n = 9; middle panel), and Up state pre-post spike pairing (total of 50–100 individual EPSP pairings; n = 6; right panel). Top: example recordings during pairing protocols, showing the timings of the synaptic stimuli (black arrowheads), Up state induction (white arrowheads), and the brief somatic current injections used to evoke spiking following each synaptic stimulation in the Up state pre-post pairing protocol (grey steps). Spontaneous spikes did occur during Up states, and the example traces with no background spiking are chosen to emphasize that responses to synaptic stimulation were predominantly subthreshold during pairing, which is further detailed in Supplementary Fig. 1. Middle: representative EPSP traces from the baseline period (1; blue) and the end of recording (2; orange) (mean of five traces). Bottom: pooled data of normalized single EPSP slope across experiments for each pairing protocol (1 min bins). Inset: summary statistics, showing that only the subthreshold Up state pairing induced synaptic weakening. **P < 0.01, One-Way ANOVA followed by Tukey’s multiple comparisons test. Error bars show the SEM

Fig. 2

Pairing subthreshold inputs with Up states does not reverse synaptic potentiation. Pairing synaptic input with spike bursts during Up states induced synaptic potentiation (Pairing I: stimulus trains at 5 Hz, 100 pairings in total; each extracellular stimulation was followed by a depolarizing current step evoking >3 postsynaptic spikes). In one group of experiments, synaptic responses were measured for >40 min after input-burst pairing, and in the second group an additional subthreshold Up state pairing (Pairing II: stimulus trains at 5 Hz, 50 pairings in total; same as applied in Fig. 1d) was applied 20 min after input-burst pairing. Top, left: representative recordings during Pairing I (upper) and Pairing II (lower), showing the timings of Up state induction (white arrowheads), synaptic stimulation (black arrowheads), and the brief somatic current injections used to evoke spike bursts (grey steps). The inset shows one magnified input-burst pairing (scale bar: 40 mV). Top, right: EPSPs from the baseline (1), 20 min after Pairing I (2) and > 40 min after Pairing I (3), for an experiment with Pairing I only (upper) and Pairing I + II (lower; average of five traces each). Bottom: pooled data of normalized single EPSP slope across experiments for each pairing protocol (1 min bins), showing a decrease in synaptic potentiation over time, but no significant effect of pairing paradigm or significant interaction between time and pairing paradigm (results of mixed measures ANOVA reported in text). Error bars show the SEM

Fig. 3

Spontaneous spike patterns generated during Up states (US) induce synaptic weakening in a frequency-dependent manner. a Two-photon reconstruction of two synaptically coupled mEC LIII principal cells labeled with biocytin. A putative synapse formed between a presynaptic axonal bouton (highlighted in red) and a postsynaptic basal dendrite (blue) is indicated in the magnified region below (white arrowhead). Scale bars: 40 μm/10 μm. b Presynaptic spikes were triggered by brief (3–5 ms) depolarizing current steps and the postsynaptic EPSP amplitudes were measured. Following the recording of a 10-min baseline, Up states were also induced once per sweep for a period of ~15 min. When necessary, a constant hyperpolarizing current injection was applied to the presynaptic neuron to keep Up state spiking activity low or absent. Top: representative traces of evoked presynaptic (upper) and postsynaptic (lower) activity, during the baseline period (left, averages of ten traces), Up state induction (middle; timing of Up state induction shown with white arrowhead), and at the recording end (right, averages of ten traces; baseline EPSP trace included in blue for comparison). Bottom: time course of changes in normalized EPSP amplitude (n = 5; 5 min bins). Period of Up state induction is highlighted in green, with numbers indicating the time points used to calculate the average traces. c As in b, except with presynaptic Up state depolarization sufficient to produce self-generated spike patterns of varying frequencies (n = 6). d Summary statistics. *P < 0.05, unpaired t-test. e Plot of the relationship between synaptic weakening and presynaptic Up state spike rate (circles: control group with subthreshold/sparsely spiking presynaptic Up states; triangles: group with presynaptic Up state spiking). For the group with spontaneous presynaptic Up state spiking, the degree of synaptic weakening correlated significantly with presynaptic spike rate (Pearson linear correlation test). f For the group with spontaneous presynaptic Up state spiking, there was a significant correlation between postsynaptic and presynaptic Up state spike rates (dotted line, Pearson linear correlation test). Bold line indicates unity correlation. Inset: distribution of postsynaptic Up state spike timing relative to presynaptic activity from recording shown in c. Error bars show the SEM

Fig. 4

Boost of the mean synaptically evoked spine Ca²⁺ response during Up states. a Experimental set-up for spine Ca²⁺ imaging, local synaptic activation and Up state induction (stimulation electrode for the latter not in the field of view). Scale bar: 40 μm. b In wide-field (WF) imaging experiments, the cell was loaded with Alexa 594 (40 μM) and OGB (200 μM), for structural and Ca²⁺ imaging, respectively. The inset shows the regions of interest for imaging Ca²⁺ responses in the spine head (h) and neighboring dendritic shaft (d). Scale bars: 1.5 μm. Somatic voltage responses (top traces), and corresponding relative changes in OGB fluorescence (ΔF/F) in the spine head (h; middle traces), and adjacent dendritic shaft (d; bottom traces) evoked by subthreshold Up states (left panel), local synaptic stimulation during Down states (middle panel), and local synaptic stimulation during Up states (right panel). c Comparison of the mean spine ΔF/F responses (400 ms window) evoked by local synaptic stimulation in Down states vs. Up states across different neurons in wide-field imaging experiments (n = 8). d In two-photon (2P) imaging experiments, the cell was loaded with Alexa 594 (40 μM) and Fluo-5F (400 μM), for structural and Ca²⁺ imaging, respectively. The inset shows the line-scan path. Example images of line-scans were filtered with a 3 × 3 Gaussian kernel. Scale bar: 2.5 μm. Somatic voltage responses (top traces), and line scans and ΔF/F for Fluo-5F signal in the spine head (h; middle traces), and adjacent dendritic shaft (d; bottom traces) evoked by spontaneous Up state spike burst (left panel), local synaptic stimulation during Down states (middle panel), and local synaptic stimulation during Up states (right panel). The line-scan plots are scaled between the minimum (min) and maximum (max), and color-coded according to the inset key. e Comparison of the mean spine ΔF/F responses (400 ms window) evoked by local synaptic stimulation in Down states vs. Up states across different neurons in two-photon imaging experiments (n = 5). *P < 0.05, paired t-test. Error bars show the SEM

Fig. 5

Synaptically evoked spine Ca²⁺ transients depend on NMDAR. a Wide-field imaging experiments were used to pharmacologically examine the contribution of NMDAR to spine Ca²⁺ transients evoked by local synaptic stimulation, with the cells loaded with Alexa 594 (40 μM) and OGB (200 μM), for structural and Ca²⁺ imaging, respectively. a To mitigate the potential effects of run-down over the time course of drug application, synaptically evoked Ca²⁺ transients were imaged during a baseline period (B), and following treatment (T) with either control aCSF (left) or bath-applied D-AP5 (40 μM; right). Top: regions of interest for imaging Ca²⁺ response in the spine head. Middle: relative changes in OGB fluorescence (ΔF/F) in the spine head following local synaptic stimulation during baseline and treatment conditions. Scale bars: 1 μm. Bottom: corresponding EPSPs evoked by local synaptic stimulation. Scale bars: 50 ms/1 mV (control) and 40 ms/2 mV (D-AP5). b Pooled data showing the mean spine ΔF/F responses (400 ms window) in the treatment condition relative to baseline. **P < 0.01, paired t-test. Error bars show the SEM

Fig. 6

Up state-induced synaptic weakening depends on postsynaptic NMDAR and GSK3β. Single-cell pharmacology was used to explore the mechanisms of synaptic weakening induced by subthreshold Up state pairing, using extracellular stimulation protocols analogous to those in Fig. 1d (stimulus trains at 5 Hz; total of 50 individual EPSP pairings per recording). Both a intracellular blockade of NMDA receptors with MK-801 (200 μM in internal solution, n = 6 vs. vehicle control, n = 5) and b inhibition of GSK3β SB415286 (10 μM in internal solution + 0.01% DMSO, n = 6 vs. vehicle control, n = 6), significantly reduced synaptic weakening. Top, middle: representative recordings during pairing protocols, showing the timings of Up state induction (white arrowheads) and the synaptic stimuli (black arrowheads). Top, right: representative average EPSP traces from the baseline period (1; blue) and the end of recording (2; orange) (average of five traces). Bottom: pooled data of normalized single EPSP slope across experiments for each pairing protocol (1 min bins). **P < 0.01, *P < 0.05, unpaired t-test. Error bars show the SEM