Using whole-cell patch-clamp approach, we characterized neurons in LI of the ACC from adult mice. Based on the electrophysiological data and unsupervised clustering analysis, the neurons in LI can be classified into Spontaneous firing neuron; Delay-sAHP neurons, Delay-fAHP neurons, neurons with ADP were separated into two clusters, named ADP1 and ADP2, respectively. To our knowledge, this is the first report to characterize the intrinsic properties of neurons in LI of the ACC of adult mice. To further characterize the synaptic inputs to the LI neurons, we performed pharmacological experiments. The results showed that AMPA, kainate and NMDA receptors mediate excitatory synaptic transmission, while GABAA receptors mediate the inhibitory synaptic inputs to LI neurons. Furthermore, this is the first report that adult LI neurons express kainate receptors.
Different types of neurons in cortical LI
Here, we studied the firing properties of LI neurons from the ACC of adult mice (8–10 weeks). All the recorded neurons showed continuous firing properties, among them, 8 neurons fired APs spontaneously. Based on the firing patterns and petilla classification scheme , the others were classified as delay adapting and classical continuous adapting neurons, unlike Wozny C and SR Williams results from rats, we did not found the bursting neurons in LI of the ACC. Spontaneous firing neurons were characterized as displaying the highest input resistance, smallest membrane capacitance, and highest resting membrane potentials. To our knowledge, this is the first time to describe SF neurons in the cortical LI of adult mice. We further performed unsupervised clustering to classify the neurons in LI of the ACC by using different electrophysiological parameters. As shown in Figure3A, four of the SF neurons with sAHP were clearly classified as one group by Ward’s method, the silhouette values suggest that quality of the classification are high. The majority of the neurons with ADP were separated into two clusters by Ward’s method, the neurons of one cluster (ADP1) showed highest APs amplitude and narrowest half width and lowest duration increase, these properties were similar as that the FS interneurons recorded from LI (Wozny C and SR Williams), and LII-V of frontal cortex of rats [47, 48]. However, the neurons in ADP1 in our sample also showed highest amplitude of early frequency adaptation, suggesting the frequency adaptation of these neurons was high, which was different from the classical FS neurons. Previous study showed that Late spiking neurons were major cell types in LI , here we found that about 28% of adult LI neurons exhibited late spiking characteristics in LI of the ACC, which were named as delay adapting neurons. Similar as previous report , the delay adapting neurons had widest spikes and most positive spike threshold compared to other types of neurons.
Postsynaptic targets that mediate synaptic inputs to LI neurons
Previous studies have shown that GABAA, AMPA and NMDA receptors are the major targets of synaptic inputs to LI non-CR neurons in young rats . Here we studied the receptors which mediated synaptic inputs to LI neurons through pharmacological interventions. In our experiment, about 81% of excitatory currents recorded at −60 mV were sensitive to SYM 2206, the rest were blocked by CNQX, which suggests that AMPA receptors are the major receptors which mediate excitatory synaptic input. Following the change of membrane potentials to +10 mV, we recorded another evoked PSCs which were blocked by PTX, indicating the involvement of GABAA receptors to the synaptic inputs to LI. Our data showed that LI neurons received both excitatory and inhibitory inputs from other layers. By recording sEPSCs, we found that nearly all of the neurons in LI receive synaptic input (data not shown), which is inconsistent with previous work showing that neurogliaform cells, did not receive monosynaptic excitatory input from LII/III pyramidal neurons . These inconsistencies may be a result of recording from different species, as well as the use of different recording methods. Since LI neurons also receive projections from thalamus [14, 49], it is possible that some of the neurons in LI may just receive thalamic innervations, and therefore no responses can be recorded from LI neurons with the stimulation of neurons in LII/III.
About 19% of CNQX sensitive EPSCs were resistant to SYM 2206, which suggest that this current was mediated by kainate receptors. The percentage of kainate receptor mediated currents recorded on LI neurons was similar as that from the pyramidal neurons in LII/III of the ACC (SYM 2206 resistant, 19%) . However, both the rise and decay time of the kainate receptors mediated currents were slower than that recorded from pyramidal neurons. This may come from the subunit component differences of the kainate receptors. It has been shown that the activities of GluK1 in LII/III of the ACC modulate GABA release , whether kainate receptors expressed on LI neurons display similar functions needs to be further studied. To our knowledge, this is the first report that cortical LI neurons of the adult moue express kainate receptors.
NMDA receptors mediate the excitatory synaptic inputs to LI neurons
NMDA receptors play an important role in brain function and neuronal plasticity. Typically, functional NMDA receptor channels are heterotetrameric assemblies of two GluN1 subunits plus two GluN2 or GluN3 subunits; GluN2A-2D subunits provide the glutamate-binding sites and modifie channel properties such as current kinetics and channel conductance . GluN2B subunit containing NMDA receptors have been detected in LII/III of the ACC . Previously, Soda et al. reported that non-CR cells in LI expressed NMDA receptors . Later on, Radnikow et al. found that GluN1/GluN2B subunit containing NMDA receptors were expressed by CR cells of LI on day 5-11 rats . Here we showed that in LI of the adult ACC, neurons with GluN1/GluN2B subunit containing NMDA receptors mediated about 32% of the NMDA currents. Due to the lack of selective blockers for other subunits of GluN2, we were unable to investigate which subunit mediated the Ro 25-6981 resistant currents. The involvement of NMDA receptors to the plasticity of synaptic inputs in the CNS has been well demonstrated . Previous studies have shown that GluN2B subunit of the NMDA receptor in the prefrontal cortex is critically involved in both contextual memory  and chronic pain [37, 38]. Whether the GluN2B subunit of NMDA receptors is involved in the modulation of synaptic strengthen to LI neurons should be further studied.
Functional role of LI neuron in ACC
Most of pyramidal neurons in the neocortex project apical tufts to LI, where they receive excitatory inputs from distant cortical areas or thalamus inputs [49, 54]. Under synaptic stimulation, the apical dendrites generate Ca2+ spikes or NMDA spikes , which are important for the computation of pyramidal neurons [54, 56] and synaptic plasticity [57, 58]. LI neurons inhibit the apical dendritic Ca2+ spikes of layer V neurons, which are mediated by GABAB1b receptors ; therefore LI neurons are involved in the modulation of synaptic plasticity received by pyramidal neurons. Different types of LI neurons may provide special inhibition to the tuft branches of pyramidal neurons . For example, Spontaneous Firing L1 neurons may provide a long lasting inhibition to the targeted pyramidal neurons, and FS neurons may be involved into neuronal synchrony . Whether periphery nerve injury or inflammation cause changes in the intrinsic properties of LI neurons needs to be further investigated.
The contributions of cortical over-excitability to chronic inflammatory and neuropathic pain have been well demonstrated . LTP of synaptic transmission has been proposed as a cellular model for the chronic pain. In the ACC, it has been found that the synaptic transmissions were enhanced by periphery nerve injury or inflammation [36, 40, 62]. Through a combination of pharmacological approaches, we first identified that AMPA, Kainate and NMDA receptors mainly mediate the excitatory synaptic inputs to L1 neurons in the adult mice. Our previous studies have shown that the membrane expression of AMPA receptors was enhanced by nerve injury [36, 41]. In addition, NMDA receptor NR2B subunit mediated responses were also enhanced in animal models of chronic inflammatory pain . It is important to investigate possible changes of synaptic inputs to LI neurons of the ACC in chronic pain conditions in future studies. The present findings provide basic information for our understanding of cortical network and functions in physiological and/or pathological conditions such as learning & memory as well as chronic pain.