The project's fundamental working hypothesis was that the 'target specific projections of L6b are the key to eliciting specific behaviours'. Therefore, all project work packages aimed to answer specific questions: What are the electrophysiological properties and afferent connections of L6b neurons of the mPFC? Where do L6b neurons project in general, do L6b neurons project specifically to fear-relevant structures? Etc. Throughout the project, the plan was to exploit as many state-of-the-art technologies as possible to understand the structural and functional properties of L6b neurons in the infralimbic area of the medial prefrontal cortex (IL-mPFC). Therefore, our experiments used a Cre-dependant Tg (Drd1a-Cre) FK164Gsat/Mmucd (Drd1a-Cre) mouse strain that selectively expresses Cre on L6b neurons throughout the cortical mantle. To reach our goals, we have followed multiple strategies; for instance, to investigate L6b neurons for electrophysiological properties, we crossed the Drd1a-Cre mouse line with a reporter mouse strains like Ai-14, so that all L6b neurons expressed robust tdTomato fluorescence following the cre-mediated recombination. To trace the connections of L6b of the IL-mPFC to the fear-relevant structures, such as the basolateral complex (BLA) and hippocampus, we used adeno-associated viruses (AAV). This AAV can specifically infect L6 b neurons and express channel rhodopsin and the anterograde tracing properties that can be detected by EYFP fluorescence.
This project's most crucial part was characterising the L6B neurons for their electrophysiology and correlating their electrophysiological activities with the recorded neuron morphology. Our L6 b electrophysiological characterisation using a patch-clamp helped obtain multiple intrinsic neuron features. Our recording pipette contained an intracellular recording solution supplemented with 0.5% biocytin; therefore, we could collect the morphologies of the recorded neurons. In addition to the basic intrinsic properties of the L6b neurons, we have also applied orexin agonists to study the pharmacological responses of L6b neurons. Our electrophysiological recording indicated that most L6b neurons had a membrane potential of -65-70mV during the baseline recordings. When we applied pharmacological agents such as orexin agonists YNT-185, there was an apparent effect of orexin-agonist on L6b pyramidal neurons. The orexin application increased the input resistance of the neuron significantly. The input resistance of a neuron reflects the extent to which membrane channels are open; a low resistance (high conductance) indicates many channels are open, while a high input resistance implies many channels are closed. The orexin agonist applications also caused neurons to depolarise more (-50mV), and when we applied agonists longer duration (10 minutes), this caused a complete absence of neuron spiking. Subsequent washing off the orexin agonists by the recording media reversed this effect significantly.
Furthermore, drug applications caused the shift in the neuronal input/output (I/O)I/O curve to the left side. The neuronal input/output (I/O) is a function that determines the likelihood that a neuron elicits an action potential in response to synaptic input of a given strength. The curve indicated that the orexin agonist applications made neurons more sensitive to the current injections; they could elicit spikes even at lower current injections. We extended these experiments one step further using a high-density microelectrode array (HD-MEA) system containing nearly 4100 recording channels, and we observed similar results. We made another interesting observation, not all neurons from IL-mPFC were not responding to the agonist application; approximately 30-35% of neurons were not responding to the agonist application. As a next step, we have decided to look for the morphological differences between these two categories. Unfortunately, the slicing truncation and incomplete labelling due to the thick slicing hampered the morphological characterisation of L6b neurons.
To trace the long-range projections of the L6b neurons, we used AAV injected into the infralimbic area of the medial prefrontal cortex. The infected neurons expressed channelrhodopsin-2 and EYFP fluorescence for anterograde axon tracing. Interestingly, although we found several red fluorescent axons (indicating L6 b projections) in both BLA and hippocampus, we did not find any projections originating from L6b of the IL-mPFC to the BLA and hippocampus. To elucidate the source of L6b inputs onto the BLA, we will use retrograde tracing techniques in the next step; subsequently, we may find the origins of those L6 b cortical inputs. Following the anatomical finding, we did not find the effect of optogenetic stimulations in both BLA and CA1 areas of the hippocampus. In another set of experiments, we raised cortical activity with intra-cerebroventricular (icv) injections of orexin-b and recorded L6b responses from the IL-mPFC. The direct icv application of orexin-b indicated that orexin-b alters the brain states with a prevalent shift from slower rhythms towards higher frequencies in the infralimbic prefrontal cortex in vivo. Our results indicate that the orexinergic system can modulate infralimbic mPFC L6b neurons. These results have a high potential impact on manipulating hyperarousal states such as fear and schizophrenia.
