State-of-the-art two photon guided in vivo whole cell recordings are carried out to characterise the whisker-evoked responses in L2/3 pyramidal neurons under urethane anaesthesia, which maintained a ‘controlled brain state’. Urethane anaesthesia mimics a slow wave sleep like brain state that is characterised by a cortical slow oscillation consisting of synchronous membrane potential fluctuations between a depolarised (‘up’) and hyperpolarised (‘down’) state. ACh strongly influences cortical network activity, and a low cholinergic tone is found during slow wave sleep.
The whisker-evoked responses are elicited by application of an air puff to the whiskers, which resulted in their deflection (i.e. protraction and retraction of whiskers that approximate their natural whisking behaviour). The frequency and number of presented stimuli are varied, e.g. a single stimulus presented at varying frequency or a train of stimuli. Discharge of action potentials (APs) in L2/3 pyramidal neurons is sparse, and <10% of neurons fired APs in response to whisker puff(s) being delivered. The evoked responses in L2/3 pyramidal neurons that did not fire APs to whisker puff(s) are heterogeneous and cortical state dependent. If a cortical down state precedes the stimulus a subthreshold postsynaptic potential in L2/3 pyramidal neurons is evoked. Such subthreshold events are known as dendritic spikes, which are regenerative events produced by non-linear integration of synaptic inputs onto the dendritic arbour of a neuron and at times by backpropagating APs from the soma into dendrites. In contrast if a whisker puff is presented during a cortical upstate the membrane potential of L2/3 pyramidal neuron undergoes hyperpolarisation. The evoked responses to a train of whisker puffs are complex: from eliciting sharply delineated postsynaptic potentials corresponding to each individual whisker deflection to summating of responses with each subsequent whisker deflection.
To localise the specific basal forebrain nucleus that sends cholinergic projections to S1, a combination of anterograde viral tracers and retrograde neuroanatomical tracers is carried out in a transgenic mouse line where site specific expression of cholinergic neurons can be driven. A relatively small proportion of cholinergic neurons from this basal forebrain nucleus sends a large network of diffused axonal projections to S1. The viral injection of channelrhodopsin2, an opsin that is light sensitive, into this nucleus results in the specific expression of channelrhodopsin2 in these cholinergic axons; thereby permitting manipulation of cholinergic tone in S1 using light (i.e. optogenetics) during whisker deflections to examine cholinergic modulation of whisker-evoked responses (characterised above).
Initial findings with pharmacological manipulation of cholinergic tone during in vivo whole cell recordings show that activation of ACh receptors leads to depolarisation and increased rate of firing of L2/3 pyramidal neurons in response to somatic current injections. The application of an ACh receptor agonist also induces a highly desynchronised brain state altering the synchronous cortical up/down state transitions. State-of-the-art in vivo whole cell recording and calcium imaging experiments are currently underway to examine the effects of pharmacological and optogenetic manipulation of cholinergic tone on previously characterised whisker-evoked responses.
The python scripted codes developed in collaboration with James Thomas at Jean Golding Institute, University of Bristol for the analyses of in vivo data generated from this project are currently maintained in a private GitHub repository, which will be made public at the initial publication of the project on the preprint server for biology, bioRxiv. The final publication will be made open access with a link to the GitHub repository included.
