The Anterior Cingulate Cortex is Critical for Acute Stress-induced Hypersensitivity in Mice.

Stress can be categorized according to physical, psychological and social factors. Exposure to stress produces stress-induced hypersensitivity and forms negative emotions such as anxiety and depression. For example, acute physical stress induced by the elevated open platform (EOP) causes prolonged mechanical hypersensitivity. The anterior cingulate cortex (ACC) is a cortical region involved in pain and negative emotions. Recently, we showed that mice exposed to the EOP changed spontaneous excitatory, but not inhibitory transmission in layer II/III pyramidal neurons of the ACC. However, it is still unclear whether the ACC is involved in the EOP induced mechanical hypersensitivity, and how the EOP alters evoked synaptic transmission on excitatory and inhibitory synaptic transmission in the ACC. In this study, we injected ibotenic acid into the ACC to examine if it was involved in stress-induced mechanical hypersensitivity induced by EOP exposure. Next, by using whole-cell patch-clamp recording from brain slice preparation, we analyzed action potentials and evoked synaptic transmission from layer II/III pyramidal neurons within the ACC. Lesion of the ACC completely blocked the stress-induced mechanical hypersensitivity induced by EOP exposure. Mechanistically, EOP exposure mainly altered evoked excitatory postsynaptic currents such as input-output and paired pulse ratio. Intriguingly, the mice exposed in the EOP also produced low-frequency stimulation induced short-term depression on excitatory synapses in the ACC. These results suggest that the ACC plays a critical role in the modulation of stress-induced mechanical hypersensitivity, possibly through synaptic plasticity on excitatory transmission.

TRPA1 as a O2 sensor detects microenvironmental hypoxia in the mice anterior cingulate cortex.

Transient receptor potential ankyrin 1 (TRPA1) is a member of the TRP channel family and is expressed in peripheral and central nervous systems. In the periphery, TRPA1 senses cold and pain. However, the functions of TRPA1 in the CNS are unclear. Here, we examined the roles of TRPA1 on neural activity and synaptic transmission in layer II/III pyramidal neurons from mice anterior cingulate cortex (ACC) by whole-cell patch-clamp recordings. The activation of Cinnamaldehyde (CA), which is TRPA1 agonist produced inward currents and these were blocked by the TRPA1 antagonists. Furthermore, activating TRPA1 changed the properties of action potentials such as the firing rate, rise time and decay time. In contrast, stimulating TRPA1 did not alter the spontaneous synaptic transmission. Finally, we examined the functional role of TRPA1 on neurons in a hypoxic environment. We induced an acute hypoxia by substituting nitrogen (N2) gas for oxygen (O2) in the external solution. N2 produced biphasic effects that consisting of inward currents in the early phase and outward currents in the late phase. Importantly, blocking TRPA1 reduced inward currents, but not outward currents. In contrast, a KATP channel blocker completely inhibited outward currents. These results suggest that TRPA1 acts on postsynaptic neurons in the ACC as an acute O2 sensor.

Acute elevated platform triggers stress induced hyperalgesia and alters glutamatergic transmission in the adult mice anterior cingulate cortex.

Pain is composed of both physiological and affective/emotional components which potentiate one another. In addition, exposure to stress modulates pain and affective behaviors including, anxiety-like behavior and/or depression-like behaviors. Indeed, chronic exposure to stress has been known to enhance stress-induced hyperalgesia (SIH). The anterior cingulate cortex (ACC) is critically involved in pain sensation and emotions. Animal models of chronic pain, but not acute nociception have been found to induce synaptic plasticity on glutamatergic and GABAergic transmission in the rodent ACC. However, it is unclear whether acute stress exposure could produce SIH and cause synaptic plasticity in the ACC. Accordingly, we studied how acute exposure of stress by the elevated open platform (EOP) could affect mechanical threshold, thermal and cold latency in the adult mice. Thirty minutes of the EOP produced mechanical hypersensitivity lasting for 60 min and thermal hypersensitivity immediately after the exposure. Next, we tested whether the stress could alter the excitatory and inhibitory synaptic transmission in the ACC. We performed whole-cell patch-clamp recordings from layer II/III pyramidal neurons in the ACC and analyzed both glutamatergic and GABAergic transmission in mice following the EOP. Thirty minutes of the EOP altered the rise and decay time of spontaneous glutamatergic AMPA/GluK receptors mediated currents, but did not change the frequency or amplitude of excitatory transmission. By contrast, the kinetics of inhibitory synaptic currents were not altered by the EOP. These results suggest that acute stress by the elevated platform produces SIH and causes synaptic plasticity on excitatory transmission, but not inhibitory transmission in the ACC.

Nicotine Exposure during Adolescence Leads to Changes of Synaptic Plasticity and Intrinsic Excitability of Mice Insular Pyramidal Cells at Later Life.

To find satisfactory treatment for nicotine addiction, synaptic and cellular mechanisms should be investigated comprehensively. Synaptic transmission, plasticity and intrinsic excitability in various brain regions are known to be altered by acute nicotine exposure. However, it has not been addressed whether and how nicotine exposure during adolescence alters these synaptic events and intrinsic excitability in the insular cortex in adulthood. To address this question, we performed whole-cell patch-clamp recordings to examine the effects of adolescent nicotine exposure on synaptic transmission, plasticity and intrinsic excitability in layer V pyramidal neurons (PNs) of the mice insular cortex five weeks after the treatment. We found that excitatory synaptic transmission and potentiation were enhanced in these neurons. Following adolescent nicotine exposure, insular layer V PNs displayed enhanced intrinsic excitability, which was reflected in changes in relationship between current strength and spike number, inter-spike interval, spike current threshold and refractory period. In addition, spike-timing precision evaluated by standard deviation of spike timing was decreased following nicotine exposure. Our data indicate that adolescent nicotine exposure enhances synaptic transmission, plasticity and intrinsic excitability in layer V PNs of the mice insular cortex at later life, which might contribute to severe nicotine dependence in adulthood.

L-bupivacaine Inhibition of Nociceptive Transmission in Rat Peripheral and Dorsal Horn Neurons.

BACKGROUND: Although the widely used single L-enantiomers of local anesthetics have less toxic effects on the cardiovascular and central nervous systems, the mechanisms mediating their antinociceptive actions are not well understood. The authors hypothesized that significant differences in the ion channel blocking abilities of the enantiomers of bupivacaine would be identified. METHODS: The authors performed electrophysiologic analysis on rat dorsal root ganglion neurons in vitro and on spinal transmissions in vivo. RESULTS: In the dorsal root ganglion, these anesthetics decreased the amplitudes of action potentials. The half-maximum inhibitory concentrations of D-enantiomer D-bupivacaine were almost equal for Aß (29.5 µM), Aδ (29.7µM), and C (29.8 µM) neurons. However, the half-maximum inhibitory concentrations of L-bupivacaine was lower for Aδ (19.35 µM) and C (19.5 µM) neurons than for A ß (79.4 µM) neurons. Moreover, D-bupivacaine almost equally inhibited tetrodotoxin-resistant (mean ± SD: 15.8 ± 10.9% of the control, n = 14, P < 0.001) and tetrodotoxin-sensitive (15.4 ± 15.6% of the control, n = 11, P = 0.004) sodium currents. In contrast, L-bupivacaine suppressed tetrodotoxin-resistant sodium currents (26.1 ± 19.5% of the control, n = 18, P < 0.001) but not tetrodotoxin-sensitive sodium currents (74.5 ± 18.2% of the control, n = 11, P = 0.477). In the spinal dorsal horn, L-bupivacaine decreased the area of pinch-evoked excitatory postsynaptic currents (39.4 ± 11.3% of the control, n = 7, P < 0.001) but not touch-evoked responses (84.2 ± 14.5% of the control, n = 6, P = 0.826). In contrast, D-bupivacaine equally decreased pinch- and touch-evoked responses (38.8 ± 9.5% of the control, n = 6, P = 0.001, 42.9 ± 11.8% of the control, n = 6, P = 0.013, respectively). CONCLUSIONS: These results suggest that the L-enantiomer of bupivacaine (L-bupivacaine) effectively inhibits noxious transmission to the spinal dorsal horn by blocking action potential conduction through C and Aδ afferent fibers.

Correction to: Ascending noradrenergic excitation from the locus coeruleus to the anterior cingulate cortex.

An amendment to this paper has been published and can be accessed via the original article.

Anti-Epileptic Effects of FABP3 Ligand MF1 through the Benzodiazepine Recognition Site of the GABAA Receptor.

Recently, we developed the fatty acid-binding protein 3 (FABP3) ligand MF1 (4-(2-(1-(2-chlorophenyl)-5-phenyl-1H-pyrazol-3-yl)phenoxy) butanoic acid) as a therapeutic candidate for α-synucleinopathies. MF1 shows affinity towards γ-aminobutyric acid type-A (GABAA) receptor, but its effect on the receptor remains unclear. Here, we investigate the pharmacological properties of MF1 on the GABAA receptor overexpressed in Neuro2A cells. While MF1 (1-100 µm) alone failed to evoke GABA currents, MF1 (1 µm) promoted GABA currents during GABA exposure (1 and 10 µm). MF1-promoted GABA currents were blocked by flumazenil (10 µm) treatment, suggesting that MF1 enhances receptor function via the benzodiazepine recognition site. Acute and chronic administration of MF1 (0.1, 0.3 and 1.0 mg/kg, p.o.) significantly attenuated status epilepticus (SE) and the mortality rate in pilocarpine (PILO: 300 mg/kg, i.p.)-treated mice, similar to diazepam (DZP: 5.0 mg/kg, i.p.). The anti-epileptic effects of DZP (5.0 mg/kg, i.p.) and MF1 (0.3 mg/kg, p.o.) were completely abolished by flumazenil (25 mg/kg, i.p.) treatment. Pentylenetetrazol (PTZ: 90 mg/kg, i.p.)-induced seizures in mice were suppressed by DZP (5.0 mg/kg, i.p.), but not MF1. Collectively, this suggests that MF1 is a mild enhancer of the GABAA receptor and exercises anti-epileptic effects through the receptor's benzodiazepine recognition site in PILO-induced SE models.

Ascending noradrenergic excitation from the locus coeruleus to the anterior cingulate cortex.

Anterior cingulate cortex (ACC) plays important roles in sensory perception including pain and itch. Neurons in the ACC receive various neuromodulatory inputs from subcortical structures, including locus coeruleus noradrenaline (LC-NA) neurons. Few studies have been reported about synaptic and behavioral functions of LC-NA projections to the ACC. Using viral-genetic method (AAV-DIO-eYFP) on DBH-cre mice, we found that LC-NA formed synaptic connections to ACC pyramidal cells but not interneurons. This is further supported by the electron microscopic study showing NAergic fibers contact the presynaptic inputs and post-synaptic areas of the pyramidal cells. NA application produced both pre- and post-synaptic potentiation effects in ACC excitatory transmission in vivo and in vitro. Activation of LC-NA projection to the ACC by optogenetic method produced enhancement of excitatory transmission in vitro and induced scratching and behavioral sensitization for mechanical stimulation. Our results demonstrate that LC-NA projections enhance or facilitate brain responses to pain and itch by potentiating glutamatergic synaptic transmissions in the ACC.

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2.

It has been reported that neuroinflammation occurs in the central nervous system (CNS) in patients with neuropathic pain, Alzheimer's disease and autism spectrum disorder. The 18-kDa translocator protein TSPO is used as an imaging target in positron emission tomography to detect neuroinflammation, and its expression is correlated with microglial activation. However, the mechanism underlying the transcriptional regulation of Tspo induced by inflammation is not clear. Here, we revealed that lipopolysaccharide (LPS) -induced Tspo expression was activated by the AP-1 complex in a mouse microglial cell line, BV-2. Knockdown of c-Fos and c-Jun, the components of AP-1, reduced LPS-induced Tspo expression. Furthermore, the enrichment of Sp1 in the proximal promoter region of Tspo was increased in the presence of LPS. In addition, the binding of histone deacetylase 1 (HDAC1) to the enhancer region, which contains the AP-1 site, was decreased by LPS treatment, but there were no significant differences in HDAC1 binding to the proximal promoter region with or without LPS. These results indicated that HDAC1 is involved not in the proximal promoter region but in the enhancer region. Our study revealed that inflammatory signals induce the recruitment of AP-1 to the enhancer region and Sp1 to the proximal promoter region of the Tspo gene and that Sp1 may regulate the basal expression of Tspo.

Chronic pain models amplify transient receptor potential vanilloid 1 (TRPV1) receptor responses in adult rat spinal dorsal horn.

Persistent pain is associated with negative affect originating from hypersensitivity and/or allodynia. The spinal cord is a key area for nociception as well as chronic pain processing. Specifically, the dorsal horn neurons in lamina II (substantia gelatinosa: SG) receive nociceptive inputs from primary afferents such as C fibers and/or Aδ fibers. Transient receptor potential vanilloid 1 (TRPV1) is a major receptor to sense heat as well as nociception. TRPV1 are expressed in the periphery and the central axon terminals of C fibers and/or Aδ fibers in the spinal cord. Activating TRPV1 enhances the release of glutamate in the spinal cord from naïve rodents. Here, we studied whether or not chronic pain could alter the response of TRPV1 channels to exogenous, capsaicin through study of synaptic transmission and neural activity in rat SG neurons. Using in vitro whole-cell patch-clamp recording, we found that bath application of capsaicin facilitated both the frequency and amplitude of miniature and spontaneous excitatory postsynaptic currents beyond a nerve injury and a complete Freund's adjuvant injection observed in the naïve group. Strikingly, capsaicin produced larger amplitudes of inward currents in pain models than compared to the naïve group. By contrast, the proportions of neurons that show capsaicin-induced inward currents were similar among naïve and pain groups. Importantly, the capsaicin-induced inward currents were conducted by TRPV1 and required calcium influx that was independent of voltage-gated calcium channels. Our study provides fundamental evidence that chronic inflammation and neuropathic pain models amplify the release of glutamate through the activation of TRPV1 in central axon terminals, and that facilitation of TRPV1 function in rat spinal SG neurons may contribute to enhanced capsaicin-induced inward currents.

Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo.

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem. The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood. Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group. Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats.

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Ethanol-induced enhancement of inhibitory synaptic transmission in the rat spinal substantia gelatinosa.

Recent studies have shown that ethanol produces a widespread modulation of neuronal activity in the central nervous system. It is not fully understood, however, how ethanol changes nociceptive transmission. We investigated acute effects of ethanol on synaptic transmission in the substantia gelatinosa (lamina II of the spinal dorsal horn) and mechanical responses in the spinal dorsal horn. In substantia gelatinosa neurons, bath application of ethanol at low concentration (10 mM) did not change the frequency and amplitude of spontaneous inhibitory postsynaptic currents. At medium to high concentrations (20-100 mM), however, ethanol elicited a barrage of large amplitude spontaneous inhibitory postsynaptic currents. In the presence of tetrodotoxin, such enhancement of spontaneous inhibitory postsynaptic currents was not detected. In addition, ethanol (20-100 mM) increased the frequency of spontaneous discharge of vesicular GABA transporter-Venus-labeled neurons and suppressed the mechanical nociceptive response in wide-dynamic range neurons in the spinal dorsal horn. The present results suggest that ethanol may reduce nociceptive information transfer in the spinal dorsal horn by enhancement of inhibitory GABAergic and glycinergic synaptic transmission.

Reduced synaptic function of Kainate receptors in the insular cortex of Fmr1 Knock-out mice.

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP). Kainate receptor (KAR) is a subfamily of ionotropic glutamate receptors (iGluR) that acts mainly as a neuromodulator of synaptic transmission and neuronal excitability. However, little is known about the changes of synaptic KAR in the cortical area of Fmr1 KO mice. In this study, we performed whole-cell patch-clamp recordings from layer II/III pyramidal neurons in the insular cortex of Fmr1 KO mice. We found that KARs mediated currents were reduced in Fmr1 KO mice. KARs were mainly located in the synaptosomal fraction of the insular cortex. The abundance of KAR subunit GluK1 and GluK2/3 in the synaptosome was reduced in Fmr1 KO mice, whereas the total expressions of these KARs subunits were not changed. Finally, lack of FMRP impairs subsequent internalization of surface GluK2 after KAR activation, while having no effect on the surface GluK2 expression. Our studies provide evidence indicating that loss of FMRP leads to the abnormal function and localization of KARs. This finding implies a new molecular mechanism for Fragile X syndrome.

Involvement of GABAB receptor in the antihypersensitive effect in anterior cingulate cortex of partial sciatic nerve ligation model.

The role of the GABAB receptor in the anterior cingulate cortex (ACC) of neuropathic pain is unclear. Injection of a GABAB receptor antagonist CGP35348 into the ACC induced mechanical hypersensitivity in normal rats. Activation of the GABAB receptor injected by a GABAB receptor agonist baclofen into the ACC attenuated mechanical hypersensitivity in partial sciatic nerve ligation (PSNL) rats. Co-microinjection of CGP35348 with a muscarinic M1 receptor agonist McN-A-343 into the ACC significantly inhibited McN-A-343-induced antihypersensitivity in PSNL rats. These results suggest that the GABAB receptor in the ACC contributes to mechanical hypersensitivity and is involved in muscarinic M1 receptor-mediated antihypersensitivity.

Chronic inflammatory pain induced GABAergic synaptic plasticity in the adult mouse anterior cingulate cortex.

Background Chronic pain is a persistent unpleasant sensation that produces pathological synaptic plasticity in the central nervous system. Both human imaging study and animal studies consistently demonstrate that the anterior cingulate cortex is a critical cortical area for nociceptive and chronic pain processing. Thus far, the mechanisms of excitatory synaptic transmission and plasticity have been well characterized in the anterior cingulate cortex for various models of chronic pain. By contrast, the potential contribution of inhibitory synaptic transmission in the anterior cingulate cortex, in models of chronic pain, is not fully understood. Methods Chronic inflammation was induced by complete Freund adjuvant into the adult mice left hindpaw. We performed in vitro whole-cell patch-clamp recordings from layer II/III pyramidal neurons in two to three days after the complete Freund adjuvant injection and examined if the model could cause plastic changes, including transient and tonic type A γ-aminobutyric acid (GABAA) receptor-mediated inhibitory synaptic transmission, in the anterior cingulate cortex. We analyzed miniature/spontaneous inhibitory postsynaptic currents, GABAA receptor-mediated tonic currents, and evoked inhibitory postsynaptic currents. Finally, we studied if GABAergic transmission-related proteins in the presynapse and postsynapse of the anterior cingulate cortex were altered. Results The complete Freund adjuvant model reduced the frequency of both miniature and spontaneous inhibitory postsynaptic currents compared with control group. By contrast, the average amplitude of these currents was not changed between two groups. Additionally, the complete Freund adjuvant model did not change GABAA receptor-mediated tonic currents nor the set of evoked inhibitory postsynaptic currents when compared with control group. Importantly, protein expression of vesicular GABA transporter was reduced within the presynpase of the anterior cingulate cortex in complete Freund adjuvant model. In contrast, the complete Freund adjuvant model did not change the protein levels of GABAA receptors subunits such as α1, α5, ß2, γ2, and δ. Conclusion Our results suggest that the induction phase of inflammatory pain involves spontaneous GABAergic plasticity at presynaptic terminals of the anterior cingulate cortex.

Top-down descending facilitation of spinal sensory excitatory transmission from the anterior cingulate cortex.

Spinal sensory transmission is under descending biphasic modulation, and descending facilitation is believed to contribute to chronic pain. Descending modulation from the brainstem rostral ventromedial medulla (RVM) has been the most studied, whereas little is known about direct corticospinal modulation. Here, we found that stimulation in the anterior cingulate cortex (ACC) potentiated spinal excitatory synaptic transmission and this modulation is independent of the RVM. Peripheral nerve injury enhanced the spinal synaptic transmission and occluded the ACC-spinal cord facilitation. Inhibition of ACC reduced the enhanced spinal synaptic transmission caused by nerve injury. Finally, using optogenetics, we showed that selective activation of ACC-spinal cord projecting neurons caused behavioral pain sensitization, while inhibiting the projection induced analgesic effects. Our results provide strong evidence that ACC stimulation facilitates spinal sensory excitatory transmission by a RVM-independent manner, and that such top-down facilitation may contribute to the process of chronic neuropathic pain.

Chronic diazepam administration increases the expression of Lcn2 in the CNS.

Benzodiazepines (BZDs), which bind with high affinity to gamma-aminobutyric acid type A receptors (GABAA-Rs) and potentiate the effects of GABA, are widely prescribed for anxiety, insomnia, epileptic discharge, and as anticonvulsants. The long-term use of BZDs is limited due to adverse effects such as tolerance, dependence, withdrawal effects, and impairments in cognition and learning. Additionally, clinical reports have shown that chronic BZD treatment increases the risk of Alzheimer's disease. Unusual GABAA-R subunit expression and GABAA-R phosphorylation are induced by chronic BZD use. However, the gene expression and signaling pathways related to these effects are not completely understood. In this study, we performed a microarray analysis to investigate the mechanisms underlying the effect of chronic BZD administration on gene expression. Diazepam (DZP, a BZD) was chronically administered, and whole transcripts in the brain were analyzed. We found that the mRNA expression levels were significantly affected by chronic DZP administration and that lipocalin 2 (Lcn2) mRNA was the most upregulated gene in the cerebral cortex, hippocampus, and amygdala. Lcn2 is known as an iron homeostasis-associated protein. Immunostained signals of Lcn2 were detected in neuron, astrocyte, microglia, and Lcn2 protein expression levels were consistently upregulated. This upregulation was observed without proinflammatory genes upregulation, and was attenuated by chronic treatment of deferoxamine mesylate (DFO), iron chelator. Our results suggest that chronic DZP administration regulates transcription and upregulates Lcn2 expression levels without an inflammatory response in the mouse brain. Furthermore, the DZP-induced upregulation of Lcn2 expression was influenced by ambient iron.

Propofol Anesthesia Is Reduced in Phospholipase C-Related Inactive Protein Type-1 Knockout Mice.

The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pretreatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phosphorylation of cortical the GABAA-R ß3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABAA-R ß3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital.