The present study is the first to systematically characterize the action potential properties of ACC pyramidal cells and their alterations after nerve injury in adult mice. Considering the cumulative reports of studies using transgenic and gene knockout mice [27, 30], the current study provides important information of intrinsic properties for mouse ACC neurons. Three main electrophysiological classes of ACC cells were distinguished according to their firing pattern: (i) RS cells; (ii) IM cells; and (iii) IB cells. From single labeled cell morphological analyses, we found that these cells send its branches or terminals to layer I as well as layer V/VI, suggesting that they form broad neuronal connections with neurons in superficial and deeper layers within the ACC. Finally, after neuropathic pain, we found that the firing rates in IM neurons were increased compared with control IM cells. Our work provides the basic map for future investigations of molecular mechanism for long-term plastic changes in neuronal properties after nerve injury.
ACC, synaptic transmission, plasticity and spike
Previous spike studies were recorded from PFC neurons in young rats [31, 32] and pyramidal neurons located in layer V, major cortical output neurons to other cortical and subcortical areas. For example, Yang et al.  reported that there are four major pyramidal cells found in the Layer V of the rat PFC, regular spiking, intrinsic bursting, repetitive oscillatory bursting and intermediate cells. To our knowledge, the present report is the first systemic studies of pyramidal neurons in the layer II/III of ACC areas. Although the PFC in previous reports often contains some of rostral part of ACC neurons, most of current recordings are performed from neurons located caudal ACC to the typical PFC area. Although many anatomic and functions of the PFC-ACC are similar between rats and mice, we feel that it is necessary to study the adult mouse ACC in a systemic manner. Taking advantage of transgenic and gene knockout mice, recent studies reveal novel molecular and synaptic mechanisms for synaptic transmission and plasticity in the ACC [13, 15, 27, 33, 34] For example, using genetic deletion of GluR5, Glur6, or GluR5&6, Wu et al (2005a) demonstrated that glutamate kainate receptor GluR5 and 6 contribute to excitatory synaptic transmission in the synapses of layer II/III ACC. By using AC1 and AC8 gene knockout mice, Zhao et al (2006) and Xu et al (2008) showed that calcium-stimulated AC1 contribute to long-lasting synaptic changes within the ACC after peripheral inflammation or nerve injury. These studies would be impossible using traditional pharmacological methods, since there is no selective inhibitor for these target proteins. Unlike synaptic transmission and plasticity in the ACC, little work has been done on spike analyses in the ACC. In the present studies we focused on layer II/III, because (i) our previous work has mostly focused on synaptic transmission and plasticity of ACC layer II and III; (ii) most of cells located in layer II/III are pyramidal cells; (iii) neurons in layer II/III receive sensory inputs [27, 34]; and finally, (iv) neurons in the layer II/III are activated by peripheral sensory stimuli and injury [14, 35]. Our current spike work is the first study in adult mouse ACC, and provides basic information about the intrinsic properties of cingulate pyramidal cells. We are currently studying inhibitory neurons in the layer II/III as well as neurons located in other layers of the ACC in adult mice.
Classification of three types of ACC pyramidal cells
This study has identified three main types of pyramidal cells in adult mouse ACC, based on their specific discharge patterns in response to depolarizing current pulses. To our knowledge, this is the first report in the ACC region. The classification of the different electrophysiological types of ACC pyramidal cells proposed in the present study has taken into account the previously reported classifications of cortical pyramidal cells. The three types of cells are mainly classified by whether they can generate burst and whether there is ADP following their first action potential. In young rat PFC, previous studies have revealed four classes of pyramidal neurons, mainly on the basis of their response to application of prolonged intracellular current pulses and the shape of their action potentials . As compared with rat PFC, we have found mostly similar results in adult mouse ACC. There are three major classes of pyramidal cells found in rats and mice, RS, IB and IM. In rat PFC region, Yang et al (1996) identified repetitive oscillatory bursting cells (ROB) at about 13% of the total cells recorded. However, we did not detect any ROB cell in mouse ACC so far. One possible explanation is the region and animal difference, since previous reports were from layer VI of the rat PFC. It will be important to investigate if ROB cell may be found in the layer VI of the mouse ACC in future studies.
Synaptic and nonsynaptic plasticity
Recent studies have indicated that sensitization at different levels along somatosesnory pathways are likely contributing to chronic pain including neuropathic pain; these include sensitization at peripheral, spinal cord, amygdala and cortex such as ACC [27, 34, 36, 37]. At sensory synapses between afferent fibers and dorsal horn neurons, it has been believed that LTP as well as heterosynaptic facilitation are triggered by peripheral injury [38, 39]. Similar LTP were proposed in the amygdala and ACC. In the amygdala, it has been reported that peripheral inflammation or nerve injury triggered long-lasting changes in excitatory synaptic transmission in the central nucleus of the amygdala . In the ACC, Zhao et al (2006) and Xu et al (2008) reported that peripheral nerve injury or inflammation caused long-lasting, LTP-like synaptic changes in the ACC layer II/III synapses [16, 17]; and both presynaptic increases of glutamate releases and enhancement of postsynaptic AMPA receptor mediated responses are thought to contribute to the injury-triggered potentiation.
In addition to synaptic plasticity, it has been noted that nonsynaptic plasticity may also play important roles in learning, memory and other brain functions [40–43]. Typically, such nonsynaptic plasticity contains the changes in spike threshold, spike accommodation, amplitude of burst-evoked after hyperpolarization. It has been reported that learning produced long-lasting changes in the intrinsic excitability of central neurons that are known to contribute to the behavioral learning tasks such as cortical, hippocampal and cerebellum neurons . A recent study nicely showed that different neurotransmitter receptors and intracellular signaling pathways are contributing to synaptic potentiation and nonsynaptic plasticity in the subicular pyramidal neurons, respectively . In sensory or nociceptive system, it has been known for many years that peripheral tissue or nerve injury triggered enduring changes in neuronal spike responses to sensory stimulation [44–46]. However, most of previous studied did not distinguish the contribution of synaptic plasticity (e.g., LTP) and/or intrinsic plasticity  to such increases, in part, due to the limit of recording technique (i.e., extracellualr recordings in vivo).
However, some important findings have been reported in the DRG cells, first sensory neurons in the CNS. It has been reported that rat nerve injury tended to reduce rheobase and increase the number of APs in response to the same depolarizing currents injection, the AP amplitude and spike width [47–49], many of which mimic those nonsynaptic plasticity found in learning models . In amygdala, the neurons from both visceral pain model and arthritis pain model showed an increased action potential firing rate compared with control neurons [50, 51]. In the present study, we have found that nerve injury induced an increased firing rate in IM cells, suggesting that not all neurons are involved in processing the injury information by changes in intrinsic properties. The alterations of the expressions and/or properties of ion channels underlying the action potentials may account for the changes of firing rates. In summary, the results of this study suggest that the plastic change of intrinsic plasticity of ACC neurons is involved in the central processing of neuropathic pain.