Quantitative Fluorescence Analysis Reveals Dendrite-Specific Thalamocortical Plasticity in L5 Pyramidal Neurons during Learning

Abstract

High-throughput anatomic data can stimulate and constrain new hypotheses about how neural circuits change in response to experience. Here, we use fluorescence-based reagents for presynaptic and postsynaptic labeling to monitor changes in thalamocortical synapses onto different compartments of layer 5 (L5) pyramidal (Pyr) neurons in somatosensory (barrel) cortex from mixed-sex mice during whisker-dependent learning (Audette et al., 2019). Using axonal fills and molecular-genetic tags for synapse identification in fixed tissue from Rbp4-Cre transgenic mice, we found that thalamocortical synapses from the higher-order posterior medial thalamic nucleus showed rapid morphologic changes in both presynaptic and postsynaptic structures at the earliest stages of sensory association training. Detected increases in thalamocortical synaptic size were compartment specific, occurring selectively in the proximal dendrites onto L5 Pyr and not at inputs onto their apical tufts in L1. Both axonal and dendritic changes were transient, normalizing back to baseline as animals became expert in the task. Anatomical measurements were corroborated by electrophysiological recordings at different stages of training. Thus, fluorescence-based analysis of input- and target-specific synapses can reveal compartment-specific changes in synapse properties during learning.SIGNIFICANCE STATEMENTSynaptic changes underlie the cellular basis of learning, experience, and neurologic diseases. Neuroanatomical methods to assess synaptic plasticity can provide critical spatial information necessary for building models of neuronal computations during learning and experience but are technically and fiscally intensive. Here, we describe a confocal fluorescence microscopy–based analytical method to assess input, cell type, and dendritic location-specific synaptic plasticity in a sensory learning assay. Our method not only confirms prior electrophysiological measurements but allows us to predict functional strength of synapses in a pathway-specific manner. Our findings also indicate that changes in primary sensory cortices are transient, occurring during early learning. Fluorescence-based synapse identification can be an efficient and easily adopted approach to study synaptic changes in a variety of experimental paradigms.

Nanoscale analysis of functionally diverse glutamatergic synapses in the neocortex reveals input and layer-specific organization

“…The large postsynaptic nanoscale features of L5 TC synapses are consistent with work showing relatively strong TC synaptic currents on basal dendrites of L5 neurons [55]. Selective enlargement of L5A inputs and associated PSD-95 has been recently linked to experience-dependent plasticity [71]. It will be essential to determine whether TC synapses on basal dendrites lack mGluR nanodomains, a characteristic feature of glutamatergic drivers [70].…”

Stimulus-reward contingencies drive long-lasting alterations in neocortical somatostatin inhibition during learning

“…Over the course of several days, animals learn to associate a whisker stimulus with a water reward, measured by an increase in lick frequency to stimulus compared to “blank” trials (Figure 1F). Prior studies have shown that this sensory association training (SAT) is whisker-dependent (Bernhard et al, 2020) and also drives rapid changes in thalamocortical synaptic strength (Audette et al, 2019; Ray et al, 2023), indicating that this training is sufficient to initiate both synaptic and behavioral plasticity. Across the population of trained SST-Cre animals, mice showed a significantly greater lick frequency in stimulus versus “blank” trials after just 1 day of SAT (Figure 1G; 6.3±0.5Hz versus 5.6±0.5Hz).…”

“…It has been proposed that increasing inhibition is critical during excitatory synaptic plasticity in order to maintain some balance of excitation and inhibition (Barron, 2021; Okun and Lampl, 2008; Vogels et al, 2011), but these hypotheses have not articulated a source or a target for this inhibition, nor the timescale at which this balancing plasticity should occur. Prior studies have shown that SAT drives the potentiation of both thalamocortical and intracortical excitatory synapses (Audette et al, 2019; Ray et al, 2023). These new data indicate that at least at the early stages of learning, in this task and for L2 Pyr neurons, the potentiation of excitatory synapses is not accompanied by an increase in SST-mediated inhibition.…”

Control of neuronal excitation–inhibition balance by BMP–SMAD1 signalling

“…To test whether pyramidal-cell-derived BMP2 modifies the innervation of PV interneurons, we generated Bmp2 conditional knockout mice in whi ch Bmp2 is selectively ablated in upper-layer glutamatergic neurons ( Cux2 creERT2 ::Bmp2 fl/fl ; referred to as Bmp2 ΔCux2 mice). We then adopted genetically encoded intrabodies (fibronectin intrabodies generated by mRNA display; FingRs) to quantitatively map the synaptic inputs to PV interneurons 39 , 40 (Extended Data Fig. 6a–c and Supplementary Video 1 ).…”