SENP1 in the retrosplenial agranular cortex regulates core autistic-like symptoms in mice.

Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder, causing defects of social interaction and repetitive behaviors. Here, we identify a de novo heterozygous gene-truncating mutation of the Sentrin-specific peptidase1 (SENP1) gene in people with ASD without neurodevelopmental delay. We find that Senp1+/- mice exhibit core autistic-like symptoms such as social deficits and repetitive behaviors but normal learning and memory ability. Moreover, we find that inhibitory and excitatory synaptic functions are severely affected in the retrosplenial agranular (RSA) cortex of Senp1+/- mice. Lack of Senp1 leads to increased SUMOylation and degradation of fragile X mental retardation protein (FMRP), also implicated in syndromic ASD. Importantly, re-introducing SENP1 or FMRP specifically in RSA fully rescues the defects of synaptic function and autistic-like symptoms of Senp1+/- mice. Together, these results demonstrate that disruption of the SENP1-FMRP regulatory axis in the RSA causes autistic symptoms, providing a candidate region for ASD pathophysiology.

CXCR3 contributes to neuropathic pain via ERK activation in the anterior cingulate cortex.

The anterior cingulate cortex (ACC) is activated by noxious stimuli and is involved in the affective component of pain processing; but its role in the sensory component of pain remains largely unknown. Studies have verified that Chemokine (C-X-C motif) receptor 3 (CXCR3) is involved in nociceptive sensitization in the spinal cord after peripheral nerve injury; however, the expression of CXCR3 in the ACC and its role in neuropathic pain has not been reported. Here, we showed that CXCR3 co-localized with neurons in the ACC and the upregulation of CXCR3 corresponded with hypersensitive behaviors after a chronic constriction injury of the sciatic nerve. Pharmacological blockade of CXCR3 using local injection of its inhibitor, AMG487, into the ACC significantly attenuated hyperalgesia induced by chronic constriction injury and suppressed the phosphorylation of extracellular signal-regulated kinase (ERK). Collectively, these results suggest that CXCR3 in the ACC is involved in hyperalgesia induced by peripheral nerve injury and ERK may be a downstream target.

Adenosine Kinase Expression in the Frontal Cortex in Schizophrenia.

The adenosine hypothesis of schizophrenia posits that reduced availability of the neuromodulator adenosine contributes to dysregulation of dopamine and glutamate transmission and the symptoms associated with schizophrenia. It has been proposed that increased expression of the enzyme adenosine kinase (ADK) may drive hypofunction of the adenosine system. While animal models of ADK overexpression support such a role for altered ADK, the expression of ADK in schizophrenia has yet to be examined. In this study, we assayed ADK gene and protein expression in frontocortical tissue from schizophrenia subjects. In the dorsolateral prefrontal cortex (DLPFC), ADK-long and -short splice variant expression was not significantly altered in schizophrenia compared to controls. There was also no significant difference in ADK splice variant expression in the frontal cortex of rats treated chronically with haloperidol-decanoate, in a study to identify the effect of antipsychotics on ADK gene expression. ADK protein expression was not significantly altered in the DLPFC or anterior cingulate cortex (ACC). There was no significant effect of antipsychotic medication on ADK protein expression in the DLPFC or ACC. Overall, our results suggest that increased ADK expression does not contribute to hypofunction of the adenosine system in schizophrenia and that alternative mechanisms are involved in dysregulation of this system in schizophrenia.

Chewing augments stress-induced increase of pERK-immunoreactive cells in the rat cingulate cortex.

The eff ;ects of chewing during restraint stress on the anterior, mid- and posterior cingulate cortices were investigated in rats using immunohistochemistry to detect the expression of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2), a marker of responding cells. The rats were divided into three groups: control (no immobilization), stress-only (immobilized), and stress-with-chewing (immobilized and allowed to chew a wooden stick). Significant increases in the number of pERK1/2-immunoreactive cells in the anterior, mid- and posterior cingulate cortices were noted in the stress-only group when compared with the control group (p < 0.05). Furthermore, the number of pERK1/2-immunoreactive cells in the anterior, mid- and posterior cingulate cortices in the stress-with-chewing group was also significantly higher than that in the stress-only group (p < 0.05). These findings indicate that the cingulate cortex plays a role in the negative-feedback effect and might be an essential part of the brain where the ameliorating effects of chewing against stress are produced.

Transcription-independent expression of PKMζ in the anterior cingulate cortex contributes to chronically maintained neuropathic pain.

Protein kinase M ζ is well known for its role in maintaining memory and pain. Previously, we revealed that the activation of protein kinase M ζ in the anterior cingulate cortex plays a role in sustaining neuropathic pain. However, the mechanism by which protein kinase M ζ is expressed in the anterior cingulate cortex by peripheral nerve injury, and whether blocking of protein kinase M ζ using its inhibitor, zeta inhibitory peptide, produces analgesic effects in neuropathic pain maintained chronically after injury, have not previously been resolved. In this study, we show that protein kinase M ζ expression in the anterior cingulate cortex is enhanced by peripheral nerve injury in a transcription-independent manner. We also reveal that the inhibition of protein kinase M ζ through zeta inhibitory peptide treatment is enough to reduce mechanical allodynia responses in mice with one-month-old nerve injuries. However, the zeta inhibitory peptide treatment was only effective for a limited time.

Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase.

Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.

[Comparative study of glutamate dehydrogenase in the brain of patients with schizophrenia and mentally healthy people]. / Glutamatdegidrogenaza v mozge bol'nykh shizofreniei i psikhicheski zdorovykh lits.

AIM: To compare the glutamate dehydrogenase (GDH) activity and amounts of GDHI, GDHII, and GDHIII immunoreactive forms in prefrontal, anterior and posterior cingulate cortex and cerebellar cortex of patients with schizophrenia and control subjects. MATERIAL AND METHODS: GDH enzymatic activity was measured and levels of GDH immunoreactive forms were determined in extracts of autopsied samples of prefrontal, anterior and posterior cingulate cortex (areas 10, 24, and 23 by Brodmann), and cerebellar cortex of patients with schizophrenia (n=8) and controls (n=9). RESULTS AND CONCLUSION: GDH enzymatic activity was significantly increased in the prefrontal cortex (area 10) (p<0.004), the posterior cingulate cortex (area 23) (p<0.05) and the cerebellar cortex (p<0.002) and was unchanged in the anterior cingulate cortex (area 24) in patients with schizophrenia compared to controls. The levels of immunoreactive GDH I, GDH II and GDH III were significantly higher in the prefrontal cortex of patients with schizophrenia than in controls (p<0.008, p<0.003, and p<0.0001, respectively). Levels of all three immunoreactive GDH forms were unchanged in the anterior cingulate cortex (area 24), but they were increased in the posterior cingulate cortex (area 23) (p<0.004, p<0.001 and p<0.02, respectively). The levels of immunoreactive GDH II and GDH III, but not GDH I, were significantly increased in the cerebellar cortex of patients with schizophrenia compared with the control group (p<0.02 and p<0.001, respectively). The alteration in the levels of GDH immunoreactive forms in the brain of patients with schizophrenia is one of the causes of impaired brain glutamate metabolism and an important aspect of schizophrenia pathogenesis.

Cingulate Overexpression of Mitogen-Activated Protein Kinase Phosphatase-1 as a Key Factor for Depression.

BACKGROUND: Depression is frequently associated with chronic pain or chronic stress. Among cortical areas, the anterior cingulate cortex (ACC, areas 24a and 24b) appears to be important for mood disorders and constitutes a neuroanatomical substrate for investigating the underlying molecular mechanisms. The current work aimed at identifying ACC molecular factors subserving depression. METHODS: Anxiodepressive-like behaviors in C57BL/6J male mice were induced by neuropathic pain, unpredictable chronic mild stress, and optogenetic ACC stimulation and were evaluated using novelty suppressed feeding, splash, and forced swim tests. ACC molecular changes in chronic pain-induced depression were uncovered through whole-genome expression analysis. Further mechanistic insights were provided by chromatin immunoprecipitation, Western blot, and immunostaining. The causal link between molecular changes and depression was studied using knockout, pharmacological antagonism, and local viral-mediated gene knockdown. RESULTS: Under chronic pain-induced depression, gene expression changes in the ACC highlighted the overexpression of a regulator of the mitogen-activated protein kinase pathway, mitogen-activated protein kinase phosphatase-1 (MKP-1). This upregulation is associated with the presence of transcriptionally active chromatin marks (acetylation) at its proximal promoter region as well as increased cyclic adenosine monophosphate response element-mediated transcriptional activity and phosphorylation of cyclic adenosine monophosphate response element binding protein and activating transcription factor. MKP-1 overexpression is also observed with unpredictable chronic mild stress and repeated ACC optogenetic stimulation and is reversed by fluoxetine. A knockout, an antagonist, or a local silencing of MKP-1 attenuates depressive-like behaviors, pointing to an important role of this phosphatase in depression. CONCLUSIONS: These data point to ACC MKP-1 as a key factor in the pathophysiology of depression and a potential target for treatment development.

Pre-LTP requires extracellular signal-regulated kinase in the ACC.

The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders.

Short-term anesthesia inhibits formalin-induced extracellular signal-regulated kinase (ERK) activation in the rostral anterior cingulate cortex but not in the spinal cord.

BACKGROUND: The rostral anterior cingulate cortex (rACC) has been implicated in the negative affective response to injury, and importantly, it has been shown that activation of extracellular signal-regulated kinase (ERK) signaling in the rACC contributes to the full expression of the affective component of pain in rodents. In this study, we investigated whether administration of anesthesia at the time of injury could reduce phosphorylated-ERK (PERK) expression in the rACC, which might eliminate the negative affective component of noxious stimulation. Intraplantar hindpaw formalin stimulation, an aversive event in the awake animal, was given with or without general isoflurane anesthesia, and PERK expression was subsequently quantified in the rACC using immunohistochemistry. Furthermore, as numerous studies have demonstrated the importance of spinal ERK signaling in the regulation of nociceptive behaviour, we also examined PERK in the superficial dorsal horn of the spinal cord. FINDINGS: Formalin injection with and without short-term (<10 min) general isoflurane anesthesia induced the same level of PERK expression in spinal cord laminae I-II. However, PERK expression was significantly inhibited across all laminae of the rACC in animals anesthetized during formalin injection. The effect of anesthesia was such that levels of PERK were the same in formalin and sham treated anesthesized animals. CONCLUSIONS: This study is the first to demonstrate that isoflurane anesthesia can inhibit formalin-induced PERK in the rACC and therefore might eliminate the unpleasantness of restraint associated with awake hindpaw injection.

Relationship of monoamine oxidase-A distribution volume to postpartum depression and postpartum crying.

Postpartum depression (PPD) has a prevalence rate of 13% and a similarly high proportion of women report a subclinical state of one or more major depressive episode symptoms. The aim was to investigate whether monoamine oxidase-A (MAO-A) VT, an index of MAO-A density, is increased in the prefrontal and anterior cingulate cortex (PFC and ACC), during PPD or when a PPD spectrum symptom, greater predisposition to crying, is present. MAO-A is an enzyme that increases in density after estrogen decline, and has several functions including creating oxidative stress, influencing apoptosis and monoamine metabolism. Fifty-seven women were recruited including 15 first-onset, antidepressant naive, PPD subjects, 12 postpartum healthy who cry due to sad mood, 15 asymptomatic postpartum healthy women, and 15 healthy women not recently pregnant. Each underwent [(11)C]-harmine positron emission tomography scanning to measure MAO-A VT. Both PPD and greater predisposition to crying were associated with greater MAO-A VT in the PFC and ACC (multivariate analysis of variance (MANOVA), group effect, F21,135=1.856; p=0.019; mean combined region elevation 21% and 14% in PPD and crying groups, respectively, relative to postpartum asymptomatic). Greater MAO-A VT in the PFC and ACC represents a new biomarker in PPD, and the PPD symptom of predisposition to crying. Novel strategies for preventing PPD (and some PPD symptoms) may be possible by avoiding environmental conditions that elevate MAO-A level and enhancing conditions that normalize MAO-A level. These findings also argue for clinical trials in PPD with the newer, well-tolerated MAO-A inhibitor antidepressants.

Infralimbic cortex Rho-kinase inhibition causes antidepressant-like activity in rats.

Depression is one of the most common psychiatric disorders in the world; however, its mechanisms remain unclear. Recently, a new signal-transduction pathway, namely Rho/Rho-kinase signalling, has been suggested to be involved in diverse cellular events in the central nervous system; such as epilepsy, anxiety-related behaviors, regulation of dendritic and axonal morphology, antinociception, subarachnoid haemorrhage, spinal cord injury and amyotrophic lateral sclerosis. However there is no evidence showing the involvement of Rho-kinase pathway in depression. In addition, the infralimbic cortex, rodent equivalent to subgenual cingulate cortex has been shown to be responsible for emotional responses. Thus, in the present study, intracranial guide cannulae were stereotaxically implanted bilaterally into the infralimbic cortex, and the effects of repeated microinjections of a Rho-kinase (ROCK) inhibitor Y-27632 (10 nmol) were investigated in rats. Y-27632 significantly decreased immobility time and increased swimming and climbing behaviors when compared to fluoxetine (10 µg) and saline groups in the forced swim test. In addition, Y-27632 treatment did not affect spontaneous locomotor activity and forelimb use in the open-field and cylinder tests respectively; but it enhanced limb placing accuracy in the ladder rung walking test. Our results suggest that Y-27632 could be a potentially active antidepressant agent.

Inhibition of p38 mitogen-activated protein kinase activation in the rostral anterior cingulate cortex attenuates pain-related negative emotion in rats.

The emotional components of pain are far less studied than the sensory components. Previous studies have indicated that the rostral anterior cingulate cortex (rACC) is implicated in the affective response to noxious stimuli. Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been documented to play an important role in diverse kinds of pathological pain states. We used formalin-induced conditioned place aversion (F-CPA) in rats, an animal model believed to reflect the emotional response to pain, to investigate the involvement of p38 MAPK in the rACC after the induction of affective pain. Intraplantar formalin injection produced a significant activation of p38 MAPK, as well as mitogen-activated kinase kinase (MKK) 3 and MKK6, its upstream activators, in the bilateral rACC. p38 MAPK was elevated in both NeuN-positive neurons and Iba1-positive microglia in the rACC, but not GFAP-positive cells. Blocking p38 MAPK activation in the bilateral rACC using its specific inhibitor SB203580 or SB239063 dose-dependently suppressed the formation of F-CPA. Inhibiting p38 MAPK activation did not affect formalin-induced two-phase spontaneous nociceptive response and low intensity electric foot-shock induced CPA. The present study demonstrated that p38 MAPK signaling pathway in the rACC contributes to pain-related negative emotion. Thus, a new pharmacological strategy targeted at the p38 MAPK cascade may be useful in treating pain-related emotional disorders.

Up-regulation of PKMζ expression in the anterior cingulate cortex following experimental tooth movement in rats.

OBJECTIVE: To explore the involvement of synaptic plasticity in pain induced by experimental tooth movement, we evaluated the expression of protein kinase M zeta (PKMζ), an enzyme necessary for maintaining long-term potentiation (LTP) in the anterior cingulate cortex (ACC). METHODS: Male Sprague-Dawley rats weighing 250-300g were used. The change of the expression of PKMζ in the ACC was measured by western blot, and the mRNA of PKMζ was detected by quantitative real-time PCR 1, 3, 7 days after experimental tooth movement. The average time spent on mouth-wiping behaviour of rats involved in pain perception was detected. After that a selective PKMζ inhibitor, called myristoylated ζ-pseudosubstrate inhibitory peptide (ZIP) was injected into ACC, and the effects of ZIP were evaluated. RESULTS: The mouth-wiping behaviour of rats was significantly increased 1, 3, and 7 days after experimental tooth movement. Changes in PKMζ levels were not detected on day 1 but were found to be increased 3 days following the tooth movement, and then declined to the baseline 7 days after tooth movement in the ACC. PKMζ mRNA levels were not significantly different between the experimental and sham-treated groups at the three time points. Time spent on mouth-wiping behaviour was reduced after ZIP was injected into ACC 3 days after tooth movement, and the analgesic effect last for at least 24h. CONCLUSION: PKMζ in the ACC acts to maintain the pain induced by experimental tooth movement. Increased expression of PKMζ protein is attributed to persistent translation of PKMζ mRNA. Synaptic plasticity may be involved in the development of tooth movement pain.

[Changes of protein kinases Mζ expression in the anterior cingulate cortex after applying three different magnitude of orthodontic force].

OBJECTIVE: To investigate the regulatory effect of central synaptic plasticity on pain induced by experimental tooth movement and to analyzethe expression of protein kinases Mζ (PKMζ) in the anterior cingulate cortex (ACC) after applying different magnitude of orthodontic force. METHODS: One hundred and thirty-six male Sprague-Dawley (SD) rats (200-250 g) were used in this study. Orthodontic tooth movement devices were placed on the teeth in the experimental group, and different orthodontic forces (0.39, 0.78, 1.17 N) were applied to move the maxillary first molars, respectively. The same mechanical devices were placed on the teeth in sham-treated group and no orthodontic force was applied. No orthodontic procedure was applied in blank control group. The average time spent on mouth- wiping behavior in each group was recorded after experimental tooth movement. Brain tissue of the anterior cingulate cortex was isolated on day 3 after experiment, and the expression level of PKMζ was analyzed with the method of immunohistochemistry and enzyme-linked immune sorbent assay. ζ-pseudosubstrate inhibitory peptide (ZIP), a selective inhibitor for PKMζ, was injected into ACC on day 3 after experimental tooth movement, and the effects of ZIP on mouth-wiping behavior were evaluated. RESULTS: No statistical difference was found between the blank control group and the sham- treated group in the average time spent on mouth-wiping, value of A and expression level of PKMζ (P > 0.05). Compared with the sham-treated group and blank control group, the average time of mouth-wiping behavior [(58.6±6.9), (66.3±7.8), (78.9±8.7) s], value of A (4 569±454, 6 850±365, 8 294±558) and expression level of PKMζ [(0.32±0.02), (0.34±0.02), (0.36±0.02) mg/L] in 0.39, 0.78, 1.17 N force group were found to be up-regulated with the increase of orthodontic force (P < 0.05). LSD test in three experimental sub-group showed statistical difference (P < 0.05). After microinjection of ZIP, the average time spent on mouth-wiping behavior significantly decreased (P < 0.01), while microinjecting saline did not change rats' mouth-wiping behavior (P > 0.05). CONCLUSIONS: More pain caused by increased orthodontic force maybe due to the up-regulation of PKMζ in the anterior cingulate cortex.

Copper pathology in vulnerable brain regions in Parkinson's disease.

Synchrotron-based x-ray fluorescence microscopy, immunofluorescence, and Western blotting were used to investigate changes in copper (Cu) and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and in nondegenerating brain regions in cases of Parkinson's disease (PD) and appropriate healthy and disease controls. In PD and incidental Lewy body disease, levels of Cu and Cu transporter protein 1, were significantly reduced in surviving neurons in the SN and LC. Specific activity of the cuproprotein superoxide dismutase 1 was unchanged in the SN in PD but was enhanced in the parkinsonian anterior cingulate cortex, a region with α-synuclein pathology, normal Cu, and limited cell loss. These data suggest that regions affected by α-synuclein pathology may display enhanced vulnerability and cell loss if Cu-dependent protective mechanisms are compromised. Additional investigation of copper pathology in PD may identify novel targets for the development of protective therapies for this disorder.

Decreased NOS1 expression in the anterior cingulate cortex in depression.

Decreased function of the anterior cingulate cortex (ACC) is crucially involved in the pathogenesis of depression. A key role of nitric oxide (NO) has also been proposed. We aimed to determine the NO content in the cerebrospinal fluid (CSF) and the expression of NO synthase (NOS) isoforms, that is, NOS1, NOS2, and NOS3 in the ACC in depression. In depressive patients, CSF-NOx levels (the levels of the NO metabolites nitrite and nitrate) were significantly decreased (P = 0.007), indicating a more general decrease of NO production in this disorder. This agreed with a trend toward lower NOS1-mRNA levels (P = 0.083) and a significant decrease of NOS1-immunoreactivity (ir) (P = 0.043) in ACC. In controls, there was a significant positive correlation between ACC-NOS1-ir cell densities and their CSF-NOx levels. Furthermore, both localization of NOS1 in pyramidal neurons that are known to be glutamatergic and co-localization between NOS1 and GABAergic neurons were observed in human ACC. The diminished ACC-NOS1 expression and decreased CSF-NOx levels may be involved in the alterations of ACC activity in depression, possibly by affecting glutamatergic and GABAergic neurotransmission.

Contribution of PKMζ-dependent and independent amplification to components of experimental neuropathic pain.

Injuries can induce adaptations in pain processing that result in amplification of signaling. One mechanism may be analogous to long-term potentiation and involve the atypical protein kinase C, PKMζ. The possible contribution of PKMζ-dependent and independent amplification mechanisms to experimental neuropathic pain was explored in rats with spinal nerve ligation (SNL) injury. SNL increased p-PKMζ in the rostral anterior cingulate cortex (rACC), a site that mediates, in part, the unpleasant aspects of pain. Inhibition of PKMζ within the rACC by a single administration of ζ-pseudosubstrate inhibitory peptide (ZIP) reversed SNL-induced aversiveness within 24 hours, whereas N-methyl-d-aspartate receptor blockade with MK-801 had no effects. The SNL-induced aversive state (reflecting "spontaneous" pain), was re-established in a time-dependent manner, with full recovery observed 7 days post-ZIP administration. Neither rACC ZIP nor MK-801 altered evoked responses. In contrast, spinal ZIP or MK-801, but not scrambled peptide, transiently reversed evoked hypersensitivity, but had no effect on nerve injury-induced spontaneous pain. PKMζ phosphorylation was not altered by SNL in the spinal dorsal horn. These data suggest that amplification mechanisms contribute to different aspects of neuropathic pain at different levels of the neuraxis. Thus, PKMζ-dependent amplification contributes to nerve injury-induced aversiveness within the rACC. Moreover, unlike mechanisms maintaining memory, the consequences of PKMζ inhibition within the rACC are not permanent in neuropathic pain, possibly reflecting the re-establishment of amplification mechanisms by ongoing activity of injured nerves. In the spinal cord, however, both PKMζ-dependent and independent mechanisms contribute to amplification of evoked responses, but apparently not spontaneous pain.

Activation of glycine site and GluN2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats: implications for affective pain.

OBJECTIVE: The rostral anterior cingulate cortex (rACC) is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors (NMDARs) are highly expressed in the rACC and mediate pain-related affect by activating a signaling pathway that involves cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and/or extracellular regulated kinase (ERK)/cAMP-response element-binding protein (CREB). The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC. METHODS: Immunohistochemistry and Western blot analysis were used to separately assess the expression of phospho-ERK (pERK) and phospho-CREB (pCREB) in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB. RESULTS: Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antagonist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine-binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up-regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB. CONCLUSION: Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.

Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia.

Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia.