Characterization of synaptic connections and cholinergic modulation of layer 6A microcircuitry in rat barrel cortex

Of all neocortical layers, layer 6 is the least studied layer that shows a relatively high neuronalheterogeneity and low intralaminar connectivity. Layer 6A provides direct projections from corticothalamic(CT) pyramidal cells to both VPM and POM nucleus of the thalamus and are thus integral part of a thalamo-cortical-thalamic feedback loop that controls sensory signalling. In this study, we investigated anatomical and functional properties of L6A intra-laminar excitatory and inhibitory connections in L6A of rat barrel cortex by performing dual whole-cell recordings with simultaneousbiocytin fillings. The neuronal morphology was subsequently reconstructed and putative CT andcortico-cortical (CC) pyramidal cells were distinguished based on their distinct axonal projection patterns. An unsupervised cluster analysis were performed to classify FS and nFS interneurons based on their electrophysiological properties such as membrane properties and firing properties. Thus, different types of connections were identified based on pre- and postsynaptic neuronalsubtypes. The intralaminar connectivity of L6A neurons is low with a connectivity ratio of 6.5% (n=79).There is a much higher probability of a CC-like rather than a CT-like pyramidal cell being presynaptic as well as postsynaptic. In response to presynaptic APs elicited in CT-like pyramidal cells, EPSPs showed remarkably smaller amplitude, larger PPR, CV and failure rate recorded than those elicited by presynaptic APs in CC L6A pyramidal cells. For excitatory-inhibitory connections between CC cells and interneurons, FS and non-FS interneuron exhibited short-term depression and facilitation, respectively; while for connections between CT cells and interneurons, facilitated EPSPs were observed regardless of interneuron type. Moreover, we found that FS interneurons trigger a ‘fast’ postsynaptic response with short rise time and latency in excitatory neurons, whereas nFS interneurons display ‘slow’ kinetics by generating IPSPs with significantly longer rise time and latency. Notably, reciprocal connections were found only between two CC-like pyramidal cells orCC-interneurons, but not for connections involving a CT pyramidal cell. Acetylcholine (ACh) is released from the basal forebrain during different behavioural states, e.g. wakefulness and attention and differentially modulates neocortical neurons via both nicotinic and muscarinic ACh receptors. Here we investigated the cholinergic modulation of CT and CCpyramidal cells in layer 6A of the barrel cortex. We found that ACh differentially modulates theL6A microcircuitry by persistently depolarizing CT but hyperpolarizing CC L6A pyramidal cells, effects that are concentration-dependent and are mediated via M1 and M4 mAChRs, respectively. ACh application increases frequency of miniature EPSCs via presynaptic nAChRs in L6A CT but not CC pyramidal cells. To better understand the effects of ACh on intralaminar synaptictrans mission, recordings were performed from synaptically coupled L6A pyramidal cell pairs. We found that ACh suppresses presynaptic release in neuronal microcircuits with a presynaptic CCpyramidal cell via activation of M4Rs. In marked contrast, ACh increased the release probability inL6A connections with a presynaptic CT neuron through activating nAChRs. Our results reveal that two functionally and morphologically distinct subpopulations of L6A pyramidal cells are affected differentially by ACh indicating that intra- and subcortical signaling is subject to behavioural modulation