The FOXP2/Foxp2 gene is involved in fine motor control in many vertebrate species; in humans, it is one of the candidate genes thought to play a prominent role in language production. Several studies suggest that in the neocortex, Foxp2 is exclusively expressed in a subset of corticothalamic (CT) pyramidal cells (PCs) in layer 6 (L6). However, the morphological and intrinsic electrophysiological, synaptic and neuromodulatory properties of Foxp2-expressing L6 PCs remain largely unknown. Here we systematically characterise these properties for FOXP2-positive (FOXP2+) PCs across L6 in the rat somatosensory cortex. We find that L6 FOXP2+ PCs are distinct in all of these properties from those of L6 FOXP2-negative (FOXP2-) neuronal cell types. We show that L6 FOXP2+ PCs project exclusively to thalamus. In upper L6 (L6a), FOXP2+ PCs innervate either the first-order thalamus or both first and higher-order thalamic nuclei. FOXP2+ PCs in deep L6 (L6b) project almost exclusively to higher-order thalamus. Synaptic connections established by L6a and L6b FOXP2+ PCs exhibit low synaptic release probability, whereas L6 corticocortical PCs have a high release probability. Both L6a and L6b FOXP2+ PCs respond strongly to acetylcholine (ACh), which in the absence of TTX results in firing of action potential (AP) trains. Notably, L6b but not L6a FOXP2- PCs are highly sensitive to ACh. In addition, L6b FOXP2+ PCs close to the white matter border show strong responses to dopamine that develop into prolonged AP firing. Our data suggest that FOXP2 is a marker for CT PCs in L6 that are strongly controlled by neurotransmitters such as ACh and dopamine. These findings are in line with a pivotal role for both L6a and L6b CT PCs as modulators of thalamic activity.

Background: Genetic variation in the alpha5 nicotinic acetylcholine receptor (nAChR) subunit of mice results in behavioral deficits linked to the prefrontal cortex (PFC). A Single Nucleotide Polymorphisms (SNP) in CHRNA5 imparts a partial loss of function to the alpha5 subunit-containing (alpha5*) nAChRs and have been demonstrated to be associated with psychiatric disorders in humans, including schizophrenia, nicotine dependence, cocaine and alcohol addiction. Methods: We performed single cell-electrophysiology combined with morphological reconstructions on layer 6 (L6) excitatory neurons in the medial PFC (mPFC) of wild type (WT) rats (n = 25), rats carrying the human coding polymorphism rs16969968 in Chrna5 (n = 11) and alpha5 knockout (KO) rats (n = 28). Neuronal and synaptic properties were compared among three rat genotypes. Galantamine was applied to identified L6 neuron populations to specifically boost the nicotinic responses mediated by alpha5*nAChRs in the rat mPFC and human neocortex (n = 6 patients). Results: Compared with neurons in WT rats, L6 regular spiking (RS) neurons in the alpha5KO group exhibited altered electrophysiological properties, while those in alpha5SNP rats remained unchanged. L6 RS neurons in mPFC of alpha5SNP and alpha5KO rats differed from WT rats in dendritic morphology, spine density and spontaneous synaptic activity. Galantamine acted as a positive allosteric modulator of alpha5*nAChRs in RS but not burst spiking (BS) neurons in both rat and human cortical L6. Conclusion: Our findings suggest that dysfunction in the alpha5 subunit gene leads to aberrant neuronal and synaptic properties, shedding light on the underlying mechanisms of cognitive deficits observed in human populations carrying alpha5SNPs. They highlight a potential pharmacological target for restoring the relevant behavioral output.

Adenosinergic modulation in the PFC is recognized for its involvement in various behavioral aspects including sleep homoeostasis, decision-making, spatial working memory and anxiety. While the principal cells of layer 6 (L6) exhibit a significant morphological diversity, the detailed cell-specific regulatory mechanisms of adenosine in L6 remain unexplored. Here, we quantitatively analyzed the morphological and electrophysiological parameters of L6 neurons in the rat medial prefrontal cortex (mPFC) using whole-cell recordings combined with morphological reconstructions. We were able to identify two different morphological categories of excitatory neurons in the mPFC of both juvenile and young adult rats with both sexes. These categories were characterized by a leading dendrite that was oriented either upright (toward the pial surface) or inverted (toward the white matter). These two excitatory neuron subtypes exhibited different electrophysiological and synaptic properties. Adenosine at a concentration of 30 µM indiscriminately suppressed connections with either an upright or an inverted presynaptic excitatory neuron. However, using lower concentrations of adenosine (10 µM) revealed that synapses originating from L6 upright neurons have a higher sensitivity to adenosine-induced inhibition of synaptic release. Adenosine receptor activation causes a reduction in the probability of presynaptic neurotransmitter release that could be abolished by specifically blocking A 1 adenosine receptors (A 1 ARs) using 8-cyclopentyltheophylline (CPT). Our results demonstrate a differential expression level of A 1 ARs at presynaptic sites of two functionally and morphologically distinct subpopulations of L6 principal neurons, suggesting the intricate functional role of adenosine in neuronal signaling in the brain.

Adenosinergic modulation in the PFC is recognised for its involvement in various behavioural aspects including sleep homoeostasis, decision-making, spatial working memory and anxiety. While the principal cells of layer 6 (L6) exhibit significant morphological diversity, the detailed cellspecific regulatory mechanisms of adenosine in L6 remain unexplored. Here, we quantitatively analysed the morphological and electrophysiological parameters of L6 neurons in the rat medial prefrontal cortex (mPFC) using whole-cell recordings combined with morphological reconstructions. We were able to identify two different morphological categories of excitatory neurons, those with a leading dendrite that was oriented either upright (towards the dial surface) or inverted (towards the white matter). These two excitatory neuron subtypes exhibited different electrophysiological and synaptic properties. Adenosine at a concentration of 30 uM indiscriminately suppressed connections with either an upright or an inverted presynaptic excitatory neuron. However, using lower concentrations of adenosine (10 uM) revealed that synapses originating from L6 upright neurons have a higher sensitivity to adenosine-induced inhibition of synaptic release. Adenosine receptor activation causes a reduction in the probability of presynaptic neurotransmitter release that could be abolished by specifically blocking A1 adenosine receptors (A1ARs) using 8-cyclopentyltheophylline (CPT). Our results demonstrate a differential expression level of A1ARs at presynaptic sites of two functionally and morphologically distinct subpopulations of L6 principal neurons, suggesting that they may play distinct roles in the maintenance of sleep homoeostasis by adenosine.

Cortical layer 6b is considered by many to be a remnant of the subplate that forms during early stages of neocortical development, but its role in the adult is not well understood. Its neuronal complement has only recently become the subject of systematic studies, and its axonal projections and synaptic input structures have remained largely unexplored despite decades of research into neocortical function. In recent years, however, layer 6b (L6b) has attracted increasing attention and its functional role is beginning to be elucidated. In this review, I will attempt to provide an overview of what is currently known about the excitatory and inhibitory neurons in this layer, their pre- and postsynaptic connectivity, and their functional implications. Similarities and differences between different cortical areas will be highlighted. Finally, layer 6b neurons are highly responsive to several neuropeptides such as orexin/hypocretin, neurotensin and cholecystokinin, in some cases exclusively. They are also strongly controlled by neurotransmitters such as acetylcholine and norepinephrine. The interaction of these neuromodulators with L6b microcircuitry and its functional consequences will also be discussed.