Investigation of Molecular Mechanism of Chronic Pain in the Anterior Cingulate Cortex Using Genetically Engineered Mice

Recent advances into the understanding of molecular mechanism of chronic pain have been largely developed through the use of genetic manipulations. This is in part due to the scarcity of selective pharmacological tools, which can be readily solved by creating knockout or transgenic mice. By identifying new genes that are of import, our efforts can then be aimed at studying relevant signaling pathways, and combination of pharmacological manipulations with genetic models can be used to further examine the specific mechanisms involved in chronic pain. In this review, we will examine the genetic models that are currently in use to study chronic pain in the anterior cingulate cortex: knockout mice; transgenic mice; and the strength of combining pharmacology with these genetic models

Translational investigation and treatment of neuropathic pain

Neuropathic pain develops from a lesion or disease affecting the somatosensory system. Translational investigations of neuropathic pain by using different animal models reveal that peripheral sensitization, spinal and cortical plasticity may play critical roles in neuropathic pain. Furthermore, descending facilitatory or excitatory modulation may also act to enhance chronic pain. Current clinical therapy for neuropathic pain includes the use of pharmacological and nonpharmacological (psychological, physical, and surgical treatment) methods. However, there is substantial need to better medicine for treating neuropathic pain. Future translational researchers and clinicians will greatly facilitate the development of novel drugs for treating chronic pain including neuropathic pain

In vivo whole-cell patch-clamp recording of sensory synaptic responses of cingulate pyramidal neurons to noxious mechanical stimuli in adult mice

The anterior cingulate cortex (ACC) plays important roles in emotion, learning, memory and persistent pain. Our previous in vitro studies have demonstrated that pyramidal neurons in layer II/III of the adult mouse ACC can be characterized into three types: regular spiking (RS), intermediate (IM) and intrinsic bursting (IB) cells, according to their action potential (AP) firing patterns. However, no in vivo information is available for the intrinsic properties and sensory responses of ACC neurons of adult mice. Here, we performed in vivo whole-cell patch-clamp recordings from pyramidal neurons in adult mice ACC under urethane anesthetized conditions. First, we classified the intrinsic properties and analyzed their slow oscillations. The population ratios of RS, IM and IB cells were 10, 62 and 28%, respectively. The mean spontaneous APs frequency of IB cells was significantly greater than those of RS and IM cells, while the slow oscillations were similar among ACC neurons. Peripheral noxious pinch stimuli induced evoked spike responses in all three types of ACC neurons. Interestingly, IB cells showed significantly greater firing frequencies than RS and IM cells. In contrast, non-noxious brush did not induce any significant response. Our studies provide the first in vivo characterization of ACC neurons in adult mice, and demonstrate that ACC neurons are indeed nociceptive. These findings support the critical roles of ACC in nociception, from mice to humans

Calcium/calmodulin-dependent kinase IV contributes to translation-dependent early synaptic potentiation in the anterior cingulate cortex of adult mice

Calcium/calmodulin-dependent kinase IV (CaMKIV) phosphorylates the major transcription factor, cyclic AMP-responsive element binding protein (CREB), which plays key roles in synaptic plasticity and memory consolidation. Our previous study showed that long-term potentiation (LTP) in the anterior cingulate cortex (ACC) was significantly enhanced in transgenic mice overexpressing CaMKIV. Considering that the CaMKIV-CREB pathway plays a central role in the protein synthesis-dependent LTP, it is possible that upregulation of CaMKIV contributes to enhancement of LTP by promoting protein synthesis. To test this possibility, we examined the effects of transcription and translation inhibitors on synaptic potentiation induced by pairing of synaptic activity with postsynaptic depolarization (paired training) in ACC pyramidal neurons of wild-type and CaMKIV transgenic mice. We found that synaptic potentiation induced by paired training was partially inhibited by transcription or translation inhibitors both in wild-type and CaMKIV transgenic mice; the extent of inhibition was markedly larger in the CaMKIV transgenic mice than in the wild-type mice. Biochemical and immunohistochemical studies revealed that CaMKIV was distributed in the membrane, cytosol and nucleus of ACC neurons. Our results reveal in the first time a transcription- and translation-dependent component of early synaptic LTP in adult ACC synapses, and demonstrate that CaMKIV enhances early synaptic potentiation by activating new protein synthesis

Glutamate acts as a neurotransmitter for gastrin releasing peptide-sensitive and insensitive itch-related synaptic transmission in mammalian spinal cord

Itch sensation is one of the major sensory experiences of human and animals. Recent studies have proposed that gastrin releasing peptide (GRP) is a key neurotransmitter for itch in spinal cord. However, no direct evidence is available to indicate that GRP actually mediate responses between primary afferent fibers and dorsal horn neurons. Here we performed integrative neurobiological experiments to test this question. We found that a small population of rat dorsal horn neurons responded to GRP application with increases in calcium signaling. Whole-cell patch-clamp recordings revealed that a part of superficial dorsal horn neurons responded to GRP application with the increase of action potential firing in adult rats and mice, and these dorsal horn neurons received exclusively primary afferent C-fiber inputs. On the other hands, few Aδ inputs receiving cells were found to be GRP positive. Finally, we found that evoked sensory responses between primary afferent C fibers and GRP positive superficial dorsal horn neurons are mediated by glutamate but not GRP. CNQX, a blocker of AMPA and kainate (KA) receptors, completely inhibited evoked EPSCs, including in those Fos-GFP positive dorsal horn cells activated by itching. Our findings provide the direct evidence that glutamate is the principal excitatory transmitter between C fibers and GRP positive dorsal horn neurons. Our results will help to understand the neuronal mechanism of itch and aid future treatment for patients with pruritic disease

Interplay of Amygdala and Cingulate Plasticity in Emotional Fear

The amygdala is known to be a critical brain region for emotional fear. It is believed that synaptic plasticity within the amygdala is the cellular basis of fear memory. Recent studies demonstrate that cortical areas such as the prefrontal cortex (PFC) and anterior cingulate cortex (ACC) may also contribute to the formation of fear memory, including trace fear memory and remote fear memory. At synaptic level, fear conditioning also triggers plastic changes within the cortical areas immediately after the condition. These results raise the possibility that certain forms of synaptic plasticity may occur within the cortex while synaptic potentiation takes place within synapses in the hippocampus and amygdala. This hypothesis is supported by electrophysiological evidence obtained from freely moving animals that neurons in the hippocampus/amygdala fire synchronous activities with cortical neurons during the learning. To study fear-related synaptic plasticity in the cortex and its functional connectivity with neurons in the amygdala and hippocampus will help us understand brain mechanisms of fear and improve clinical treatment of emotional disorders in patients

Neurabin in the anterior cingulate cortex regulates anxiety-like behavior in adult mice

Affective disorders, which include anxiety and depression, are highly prevalent and have overwhelming emotional and physical symptoms. Despite human brain imaging studies, which have implicated the prefrontal cortex including the anterior cingulate cortex (ACC), little is known about the ACC in anxiety disorders. Here we show that the ACC does modulate anxiety-like behavior in adult mice, and have identified a protein that is critical for this modulation. Absence of neurabin, a cytoskeletal protein, resulted in reduced anxiety-like behavior and increased depression-like behavior. Selective inhibition of neurabin in the ACC reproduced the anxiety but not the depression phenotype. Furthermore, loss of neurabin increased the presynaptic release of glutamate and cingulate neuronal excitability. These findings reveal novel roles of the ACC in anxiety disorders, and provide a new therapeutic target for the treatment of anxiety disorders

Genetic enhancement of behavioral itch responses in mice lacking phosphoinositide 3-kinase-γ (PI3Kγ)

Phosphoinositide 3-kinases (PI3Ks) are important for synaptic plasticity and various brain functions. The only class IB isoform of PI3K, PI3Kγ, has received the most attention due to its unique roles in synaptic plasticity and cognition. However, the potential role of PI3Kγ in sensory transmission, such as pain and itch has not been examined. In this study, we present the evidence for the first time, that genetic deletion of PI3Kγ enhanced scratching behaviours in histamine-dependent and protease-activated receptor 2 (PAR-2)-dependent itch. In contrast, PI3Kγ-deficient mice did not exhibit enhanced scratching in chloroquine-induced itch, suggesting that PI3Kγ selectively contributes to certain types of behavioal itch response. Furthermore, PI3Kγ-deficient mice exhibited normal acute nociceptive responses to thermal and mechanical noxious stimuli. Behavioral licking responses to intraplantar injections of formalin and mechanical allodynia in a chronic inflammatory pain model (CFA) were also not affected by PI3Kγ gene deletion. Our findings indicate that PI3Kγ selectively contributes to behavioral itching induced by histamine and PAR-2 agonist, but not chloroquine agonist

Roles of KChIP1 in the regulation of GABA-mediated transmission and behavioral anxiety

K+ channel interacting protein 1 (KChIP1) is a neuronal calcium sensor (NCS) protein that interacts with multiple intracellular molecules. Its physiological function, however, remains largely unknown. We report that KChIP1 is predominantly expressed at GABAergic synapses of a subset of parvalbumin-positive neurons in the brain. Forced expression of KChIP1 in cultured hippocampal neurons increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), reduced paired pulse facilitation of autaptic IPSCs, and decreases potassium current density. Furthermore, genetic ablation of KChIP1 potentiated potassium current density in neurons and caused a robust enhancement of anxiety-like behavior in mice. Our study suggests that KChIP1 is a synaptic protein that regulates behavioral anxiety by modulating inhibitory synaptic transmission, and drugs that act on KChIP1 may help to treat patients with mood disorders including anxiety