Phenotypic Alterations in Hippocampal NPY- and PV-Expressing Interneurons in a Presymptomatic Transgenic Mouse Model of Alzheimer's Disease.

Interneurons, key regulators of hippocampal neuronal network excitability and synchronization, are lost in advanced stages of Alzheimer's disease (AD). Given that network changes occur at early (presymptomatic) stages, we explored whether alterations of interneurons also occur before amyloid-beta (Aß) accumulation. Numbers of neuropeptide Y (NPY) and parvalbumin (PV) immunoreactive (IR) cells were decreased in the hippocampus of 1 month-old TgCRND8 mouse AD model in a sub-regionally specific manner. The most prominent change observed was a decrease in the number of PV-IR cells that selectively affected CA1/2 and subiculum, with the pyramidal layer (PY) of CA1/2 accounting almost entirely for the reduction in number of hippocampal PV-IR cells. As PV neurons were decreased selectively in CA1/2 and subiculum, and given that they are critically involved in the control of hippocampal theta oscillations, we then assessed intrinsic theta oscillations in these regions after a 4-aminopyridine (4AP) challenge. This revealed increased theta power and population bursts in TgCRND8 mice compared to non-transgenic (nTg) controls, suggesting a hyperexcitability network state. Taken together, our results identify for the first time AD-related alterations in hippocampal interneuron function as early as at 1 month of age. These early functional alterations occurring before amyloid deposition may contribute to cognitive dysfunction in AD.

Regional and sub-regional differences in hippocampal GABAergic neuronal vulnerability in the TgCRND8 mouse model of Alzheimer's disease.

Hippocampal network activity is predominantly coordinated by γ-amino-butyric acid (GABA)ergic neurons. We have previously hypothesized that the altered excitability of hippocampal neurons in Alzheimer's disease (AD), which manifests as increased in vivo susceptibility to seizures in the TgCRND8 mouse model of AD, may be related to disruption of hippocampal GABAergic neurons. In agreement, our previous study in TgCRND8 mice has shown that hippocampal GABAergic neurons are more vulnerable to AD-related neuropathology than other types of neurons. To further explore the mechanisms behind the observed decrease of GABAergic neurons in 6 month-old TgCRND8 mice, we assessed the relative proportion of somatostatin (SOM), neuropeptide Y (NPY) and paravalbumin (PV) sub-types of GABAergic neurons at the regional and sub-regional level of the hippocampus. We found that NPY expressing GABAergic neurons were the most affected, as they were decreased in CA1-CA2 (pyramidal-, stratum oriens, stratum radiatum and molecular layers), CA3 (specifically in the stratum oriens) and dentate gyrus (specifically in the polymorphic layer) in TgCRND8 mice as compared to non-transgenic controls. SOM expressing GABAergic neurons were decreased in CA1-CA2 (specifically in the stratum oriens) and in the stratum radiatum of CA3, whereas PV neurons were significantly altered in stratum oriens sub-region of CA3. Taken together, these data provide new evidence for the relevance of hippocampal GABAergic neuronal network disruption as a mechanism underlying AD sequelae such as aberrant neuronal excitability, and further point to complex hippocampal regional and sub-regional variation in susceptibility to AD-related neuronal loss.

The expression of apoptosis inducing factor (AIF) is associated with aging-related cell death in the cortex but not in the hippocampus in the TgCRND8 mouse model of Alzheimer's disease.

BACKGROUND: Recent evidence has suggested that Alzheimer's disease (AD)-associated neuronal loss may occur via the caspase-independent route of programmed cell death (PCD) in addition to caspase-dependent mechanisms. However, the brain region specificity of caspase-independent PCD in AD-associated neurodegeneration is unknown. We therefore used the transgenic CRND8 (TgCRND8) AD mouse model to explore whether the apoptosis inducing factor (AIF), a key mediator of caspase-independent PCD, contributes to cell loss in selected brain regions in the course of aging. RESULTS: Increased expression of truncated AIF (tAIF), which is directly responsible for cell death induction, was observed at both 4- and 6-months of age in the cortex. Concomitant with the up-regulation of tAIF was an increase in the nuclear translocation of this protein. Heightened tAIF expression or translocation was not observed in the hippocampus or cerebellum, which were used as AD-vulnerable and relatively AD-spared regions, respectively. The cortical alterations in tAIF levels were accompanied by increased Bax expression and mitochondrial translocation. This effect was preceded by a significant reduction in ATP content and an increase in reactive oxygen species (ROS) production, detectable at 2 months of age despite negligible amounts of amyloid-beta peptides (Aß). CONCLUSIONS: Taken together, these data suggest that AIF is likely to play a region-specific role in AD-related caspase-independent PCD, which is consistent with aging-associated mitochondrial impairment and oxidative stress.

Knockdown of prodynorphin gene prevents cognitive decline, reduces anxiety, and rescues loss of group 1 metabotropic glutamate receptor function in aging.

Expression of dynorphin, an endogenous opioid peptide, increases with age and has been associated with memory impairments in rats. In human, prodynorphin (Pdyn) gene polymorphisms might be linked to cognitive function in the elderly. Moreover, elevated dynorphin levels have been reported in postmortem samples from Alzheimer's disease patients. However, the cellular and molecular processes affected by higher dynorphin levels during aging remain unknown. Using Pdyn(-/-) mice, we observed significant changes in the function and expression of Group 1 metabotropic glutamate receptor (mGluR). Compared with age-matched wild-type (WT) littermates, we found increased expression of mGluR1α and mGluR5 in the hippocampus and cortex of old, but not young, Pdyn(-/-) mice. Increased Group 1 mGluR expression in aged Pdyn(-/-) mice was associated with enhanced mGluR-mediated long-term depression, a form of synaptic plasticity. Notably, whereas aged WT mice developed spatial and recognition memory deficits, aged Pdyn(-/-) mice performed similarly as young mice. Pharmacological treatments with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide, a positive modulator of mGlu5 receptors, or norbinaltorphimine, an antagonist for dynorphin-targeted κ-opioid receptor, rescued memory in old WT mice. Conversely, mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride impaired spatial memory of old Pdyn(-/-) mice. Intact cognition in aged Pdyn(-/-) mice paralleled with increased expression of Group 1 mGluR-related genes Homer 1a and Arc. Finally, aged Pdyn(-/-) mice displayed less anxiety-related behaviors than age-matched WT mice. Together, our results suggest that elevated Pdyn expression during normal aging reduces mGluR expression and signaling, which in turn impairs cognitive functions and increases anxiety.

Alterations in hippocampal network oscillations and theta-gamma coupling arise before Aß overproduction in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by memory impairments. Brain oscillatory activity is critical for cognitive function and is altered in AD patients. Recent evidence suggests that accumulation of soluble amyloid-beta (Aß) induces reorganization of hippocampal networks. However, whether fine changes in network activity might be present at very early stages, before Aß overproduction, remains to be determined. We therefore assessed whether theta and gamma oscillations and their cross-frequency coupling, which are known to be essential for normal memory function, were precociously altered in the hippocampus. Electrophysiological field potential recordings were performed using complete hippocampal preparations in vitro from young transgenic CRND8 mice, a transgenic mouse model of AD. Our results indicate that a significant proportion of 1-month-old TgCRND8 mice showed robust alterations of theta-gamma cross-frequency coupling in the principal output region of the hippocampus, the subiculum. In addition we showed that, compared to controls, these mice expressed negligible levels of Aß. Finally, these network alterations were not due to genetic factors as 15-day-old animals did not exhibit theta-gamma coupling alterations. Thus, initial alterations in hippocampal network activity arise before Aß accumulation and may represent an early biomarker for AD.

ßCTF-correlated burst of hippocampal TNFα occurs at a very early, pre-plaque stage in the TgCRND8 mouse model of Alzheimer's disease.

Tumor necrosis factor-alpha (TNFα) regulates neuronal excitability. We investigated whether alterations in the level of TNFα occur at a time point that precedes the reported seizure-associated hyperexcitability of hippocampal networks in pre-plaque models of Alzheimer's disease (AD). Western blot and ELISA experiments indicated a significant increase in hippocampal TNFα expression in 1-month-old TgCRND8 mice that correlated with levels of the ß-C-terminal fragment (ßCTF) of amyloid-ß protein precursor. CD11b labeling indicated changes in microglial morphology toward an activated state, suggesting that these cells may be a putative source of the observed TNFα increase during this pre-symptomatic stage of AD-like pathology.

Involvement of PKA-dependent upregulation of nNOS-CGRP in adrenomedullin-initiated mechanistic pathway underlying CFA-induced response in rats.

We have previously shown that intrathecal administration of the adrenomedullin (AM) receptor antagonist AM(22-52) produces a long-lasting anti-hyperalgesia effect. This study examined the hypothesis that AM recruits other pronociceptive mediators in complete Freund's adjuvant (CFA)-induced inflammation. Injection of CFA in the hindpaw of rat produced an increase in the expression of nNOS in dorsal root ganglion (DRG) and the spinal dorsal horn. An intrathecal administration of AM(22-52), but not the CGRP antagonist BIBN4096BS, abolished the CFA-induced increase of nNOS. Moreover, AM-induced increase of CGRP was inhibited by the nNOS inhibitors L-NAME and 7-nitroindazole in cultured ganglion explants. Addition of AM to ganglion cultures induced an increase in nNOS protein, which was attenuated by the PKA inhibitor H-89. Treatment with AM also concentration-dependently increased cAMP content and pPKA protein level, but not its non-phosphorylated form, in cultured ganglia. In addition, nNOS was shown to be co-localized with the AM receptor components calcitonin receptor-like receptor and receptor activity-modifying protein 2- and 3 in DRG neurons. The present study suggests that the enhanced activity of nitric oxide (NO) mediates the biological action of AM at the spinal level and that AM recruits NO-CGRP via cAMP/PKA signaling in a mechanistic pathway underlying CFA-induced hyperalgesia.

Hippocampal GABAergic neurons are susceptible to amyloid-ß toxicity in vitro and are decreased in number in the Alzheimer's disease TgCRND8 mouse model.

The relevance of γ-amino-butyric acid (GABA)-ergic dysfunctions in the pathology of Alzheimer's disease (AD) remains a matter of debate. In the present study, we characterized the toxicity of amyloid-ß (Aß) on hippocampal GABAergic neurons both in vivo and in vitro. In the TgCRND8 mouse model of AD, we found a significant decrease in the number of hippocampal neurons immunoreactive for glutamate decarboxylase 67 (GAD67), the enzyme synthesizing GABA. This decrease, which was specific for hippocampal CA1-3 fields, was observed at 6 months of age, long after the overproduction of soluble Aß42 (between 2 and 4 months) and accumulation of insoluble Aß into amyloid plaques (between 4 and 6 months). In vitro, neurotoxicity was observed in primary hippocampal cultures 72 h following the addition of Aß42 solutions containing a mixture of soluble oligomers. Taken together, our results suggest that when cultured and exposed to Aß in vitro, GABAergic neurons are susceptible to Aß42 neurotoxicity. However, in TgCRND8 mice, the number of GABAergic neurons is not altered up to 6 months, in spite of the massive Aß load. Combined with the previously reported increased sensitivity to seizures observed in younger (1.5-2 month-old) TgCRND8 mice, it is likely that Aß toxicity leads to GABAergic neuron dysfunction prior to their losses at a later stage.

On the possible role of ERK, p38 and CaMKII in the regulation of CGRP expression in morphine-tolerant rats.

BACKGROUND: The neuropeptide, calcitonin gene-related peptide (CGRP) has been proposed to be a regulator of the development of morphine analgesic tolerance and thereby could be a target to reduce the induction of this phenomenon under clinical conditions. However, the mechanisms of CGRP regulation are unclear. We investigated here the possible role of the extracellular signal-regulated protein kinase (ERK), p38 and calcium/calmodulin-dependent protein kinase II (CaMKII) in CGRP regulation following chronic morphine treatment. RESULTS: A 7-day treatment with morphine (15 µg/day) led to an increase in CGRP contents in the spinal cord dorsal horn (SCDH) and dorsal root ganglion (DRG) and this effect was prevented by the inhibition of the ERK, p38 or CaMKII pathway. The phosphorylation/activation of ERK, p38 and CaMKII was enhanced in the SCDH following chronic morphine while in DRG only the phosphorylation of CaMKII was increased. Moreover, our chronic morphine treatment up-regulated neuronal nitric oxide synthase (nNOS) levels in the SCDH, an effect blocked by the inhibition of the ERK, p38 or CaMKII pathway. The blockade of nNOS activity also suppressed chronic morphine-induced CGRP increases in the DRG and SCDH. Double immunofluorescence studies revealed that nNOS and CaMKII are co-localized in the SCDH and that CaMKII is activated in CGRP-expressing DRG neurons. CONCLUSIONS: The activation of spinal ERK, p38 and CaMKII, alongside nNOS, is involved in chronic morphine-induced CGRP up-regulation in both the DRG and SCDH. Moreover, the stimulation of CaMKII in the DRG likely directly regulates the expression of CGRP associated with morphine analgesic tolerance.

A role for protein kinase C-dependent upregulation of adrenomedullin in the development of morphine tolerance in male rats.

Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and is a pronociceptive mediator. This study investigated whether AM plays a role in the development of tolerance to morphine-induced analgesia. Repetitive intrathecal injection of morphine increased the expression of AM-like immunoreactivity (AM-IR) in the spinal dorsal horn and dorsal root ganglion (DRG) neurons. Ganglion explant culture study showed that this upregulation of AM-IR was µ-opioid receptor dependent through the use of another agonist, fentanyl, and a selective antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)). The coadministration of the selective AM receptor antagonist AM(22-52) markedly attenuated the development of morphine tolerance, associated thermal hyperalgesia, and increase in AM-IR. A likely autocrine mechanism is supported by the finding that AM-IR is colocalized with AM receptor components in DRG neurons. Furthermore, opiate-induced increase in AM content was blocked by protein kinase C (PKC) inhibitors, whereas a PKC activator increased AM synthesis and release. A treatment with AM(22-52) also inhibited increases in the expression of CGRP-IR in the spinal cord and DRGs as well as in culture ganglion explants, whereas exposure to CGRP failed to alter AM content. Together, these results reveal that a sustained opiate treatment induces an upregulation of AM through the activation of µ-opioid receptors and the PKC signaling pathway. This phenomenon contributes to the development of tolerance to the antinociceptive effects of opiates at least partially via the upregulation of CGRP. Targeting AM and its receptors should be considered as a novel approach to preserve the analgesic potency of opiates during their chronic use.

Morphological evidence for the involvement of microglial p38 activation in CGRP-associated development of morphine antinociceptive tolerance.

Calcitonin gene-related peptide (CGRP) exerts an effect on the development of morphine antinociceptive tolerance, which may in part involve the activation of p38 kinase. In the present study, we investigated the temporal expression and spatial distribution of p38 phosphorylation as well as their possible regulation by CGRP receptor signaling following chronic morphine treatment. A 7-day intrathecal treatment with morphine (15 µg/day) produced tolerance to its analgesic effects as well as a rightward shift in the dose-response curve to its antinociceptive effects. This treatment time-dependently increased p38 phosphorylation in the spinal dorsal horn, as shown by phosphorylated p38-immunoreactive (p-p38-ir) cell counts. The increased phosphorylation occurred first in superficial layers of the spinal dorsal horn and then extended to deeper laminae. Interestingly, accompanying the development of morphine tolerance, p-p38-ir cells, identified as microglia, displayed hypertrophy and increased number of branched processes, suggesting their activation. These various behavioral and morphological changes were blocked by the intrathecal treatment with BIBN4096BS, a non-peptide CGRP receptor antagonist. These data provide additional morphological evidence in support of a role for CGRP in the development of morphine antinociceptive tolerance, possibly by regulating the expression and distribution of p38 phosphorylation in microglia.

Calcitonin gene-related peptide as a regulator of neuronal CaMKII-CREB, microglial p38-NFκB and astroglial ERK-Stat1/3 cascades mediating the development of tolerance to morphine-induced analgesia.

Tolerance to morphine-induced analgesia is an intractable phenomenon, often hindering its prolonged applications in the clinics. The enhanced pronociceptive actions of spinal pain-related molecules such as calcitonin gene-related peptide (CGRP) may underlie this phenomenon and could be a promising target for intervention. We demonstrate here how CGRP regulates the development of morphine analgesic tolerance at the spinal level. A 7-day treatment with morphine led to tolerance to its analgesic effects and enhanced expression of CGRP and its receptor subunits calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). Activation of several cell-type-specific kinase transcription factor cascades is required to mediate this tolerance, including calcium/calmodulin-dependent protein kinase II (CaMKII) and cAMP response element-binding protein (CREB) in neurons, p38 and nuclear factor kappa B (NFκB) in microglia and extracellular signal-regulated protein kinase (ERK) and signal transducer and activator of transcription 1 and 3 (Stat1/3) in astrocytes, because inhibitors of CaMKII, p38 and ERK pathways correspondingly reduced the increases in phosphorylated CREB, acetylated-NFκB and phosphorylated Stat1/3 levels and attenuated the development of tolerance. Interestingly, these cascades were linked to the regulation of glutamatergic N-methyl-d-aspartate (NMDA) receptor expression. Chronic morphine-induced behavioural responses and biochemical events were all subjugated to modulation by disrupting CGRP receptor signaling. Together, these data suggest that CGRP contributes to the development of tolerance to morphine-induced analgesia by regulating the activation of the neuronal CaMKII-CREB, microglial p38-NFκB and astroglial ERK-Stat1/3 cascades. Targeting CGRP-associated signaling molecules may prolong or restore morphine's analgesic properties upon a chronic exposure.

Possible involvement of transthyretin in hippocampal beta-amyloid burden and learning behaviors in a mouse model of Alzheimer's disease (TgCRND8).

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss, possibly triggered by the accumulation of beta-amyloid (Abeta) peptides and the hyperphosphorylation of Tau neurofilament protein. Recent findings have shown that transthyretin (TTR) is a potent scavenger of Abeta peptide deposits, suggesting a possible neuroprotective role for TTR in neurodegenerative processes associated with amyloidogenesis, such as AD. METHODS: To investigate the relationship between TTR and Abeta deposition, we crossed mouse carrying a deletion of TTR (TTR(- or -)) with a transgenic mouse model of AD (TgCRND8), and Abeta burden and spatial learning capacities were evaluated at 4 and 6 months of age (exclusion of the 6 month-old TgCRND8/TTR(- or -) group due to low survival rate). RESULTS: Rather surprisingly, Abeta plaque burden was significantly reduced in the hippocampus of 4-month-old TgCRND8/TTR(+ or -), and to a lesser extent in TgCRND8/TTR(- or -), as compared to age-matched TgCRND8/TTR(+ or +). No difference in plaque burden was found between any groups in 6-month-old animals. At 4 and 6 months of age, all populations of these hybrid transgenic mice displayed similar magnitude of spatial memory deficits in the Morris water maze task. CONCLUSION: Since TgCRND8 mice represent an aggressive model of Abeta deposition with plaques developing as early as 3 months of age, along with spatial learning deficits, it may be already too late at 4 and 6 months of age to observe significant changes due to the deletion of the TTR gene.

Injured nerve-derived COX2/PGE2 contributes to the maintenance of neuropathic pain in aged rats.

Neuropathic pain (NeP) is a debilitating disease afflicting mostly the aged population. Inflammatory responses in injured nerves play a pivotal role in the pathogenesis of NeP. Injured nerve derived cyclooxygenase 2/prostaglandin E2 (COX2/PGE2) contributes to the genesis of NeP at the early stage in young rats. Here we show that COX2/PGE2 is involved in the maintenance of NeP at a chronic stage in aged rats. Eighteen months after partial sciatic nerve ligation (PSNL), NeP remained prominent in aged rats. COX2 expressing macrophages and PGE2 levels were increased in injured nerves. PGE2 receptors (EP1 and EP4) and pain-related ion channel transient receptor potential vanilloid-1 (TRPV1) were increased in the ipsilateral dorsal root ganglion (DRG) neurons of aged PSNL rats. Perineural injection of a selective COX2 inhibitor NS-398 relieved NeP, reversed PSNL increased expression of EP1, EP4 and TRPV1 and suppressed the levels of pain-related peptide substance P and calcitonin gene-related peptide in DRG neurons. These data suggest that injured nerve-derived PGE2 contributes to the maintenance of NeP at the chronic stage in aged rats. Chronically facilitating the synthesis of pain-related molecules in nociceptive DRG neurons is a novel mechanism underpinning the contribution of PGE2.

Upregulation of adrenomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats.

Adrenomedullin (AM), a member of calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pronociceptive mediator [28]. This study was undertaken to investigate the role of AM in a model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Injection of CFA, but not of saline, in the unilateral hindpaw produced an increase in the expression of AM-like immunoreactivity (AM-IR) in laminae I-II of the spinal cord as well as in small- and medium-sized dorsal root ganglion (DRG) neurons at 48 h. The content of AM in DRG on the side ipsilateral to CFA injection started to increase at 4 h and remained at high levels at 24 and 48 h. The selective antagonist of AM receptors, AM(22-52), administered intrathecally (i.t.) 24 h after CFA injection inhibited inflammation-associated hyperalgesia in a dose-dependent manner (2, 5 and 10 nmol). Impressively, this anti-hyperalgesic effect lasted for at least 24 h. I.t. administration of AM(22-52) (10 nmol) also reversed CFA-induced increase in AM-IR in the spinal dorsal horn and DRG. Furthermore, blockade of AM receptors abolished CFA-induced changes in the expression and content of CGRP-like immunoreactivity in these regions. Taken together, our results suggest that the upregulation of AM in DRG neurons contributes to the development of inflammatory pain, and this effect is mediated, at least in part, by enhancing the expression and release of CGRP. Blocking AM receptor downstream signaling effects using antagonists has the potential of relieving pain following the induction of inflammation.

Cell-type specific activation of p38 and ERK mediates calcitonin gene-related peptide involvement in tolerance to morphine-induced analgesia.

Tolerance to morphine-induced analgesia is a well-established phenomenon, often limiting its usefulness in the long-term treatment of pain. The mechanisms underlying tolerance are not well understood. We previously suggested a possible role for spinal calcitonin gene-related peptide (CGRP) in the development of tolerance to morphine-induced analgesia. In the present study, we demonstrate that CGRP is involved in morphine tolerance by differentially regulating the ERK-dependent up-regulation of IL-1beta, TNF-alpha, and microsomal prostaglandin E synthase-1 (mPGES-1) in astrocytes and p38-dependent up-regulation of IL-6 in microglia in the rat spinal cord. A 7-d treatment with morphine induced tolerance to the antinociceptive effect and increased phosphorylated ERK localized in astrocytes and phosphorylated p38 enriched in microglia, both effects being inhibited by blocking CGRP receptors. Interestingly, the inhibition of the ERK pathway suppressed the development of tolerance and morphine-induced up-regulation of IL-1beta, TNF-alpha, and mPGES-1. Blockade of p38 activity also inhibited the development of tolerance and morphine-induced IL-6 up-regulation. Taken together, these data suggest that chronic morphine induces the synthesis of CGRP, which in turn acts on CGRP receptors located on astrocytes and microglia to stimulate ERK and p38, respectively, leading to increased synthesis and release of proinflammatory mediators resulting in tolerance to morphine-induced analgesia.

Adrenomedullin-2/intermedin induces cAMP accumulation in dissociated rat spinal cord cells: evidence for the existence of a distinct class of sites of action.

Adrenomedullin-2/intermedin is structurally related to the calcitonin family of peptides, which includes calcitonin gene-related peptide (CGRP), adrenomedullin, and amylin. We recently reported that CGRP and adrenomedullin act through distinct receptors to induce cyclic adenosine monophosphate (cAMP) accumulation in dispersed cells from embryonic rat spinal cord. Here, we investigated the apparent affinity and efficacy of adrenomedullin-2/intermedin for these receptors. Adrenomedullin-2/intermedin competed with [(125)I]-CGRP for binding to specific embryonic spinal cord cells with a pIC(50) of 9.73 +/- 0.06. Interestingly, adrenomedullin-2/intermedin competed for specific [(125)I]-adrenomedullin binding in a biphasic manner with pIC(50) of 9.03 +/- 0.22 and 6.45 +/- 0.24, respectively. Cellular levels of cAMP were increased by adrenomedullin-2/intermedin (pEC(50) 7.84 +/- 0.08) when cells were exposed to this peptide for 10 min at 37 degrees C. This effect was partially inhibited by the non-peptide antagonist BIBN4096BS (pA(2) 6.56 +/- 0.12), the adrenomedullin antagonist hAM(22-52) (pA(2) 6.36 +/- 0.30), and the adrenomedullin/CGRP antagonist CGRP(8-37) (pA(2) 7.24 +/- 0.60). More interestingly, a highly significant effect of adrenomedullin-2/intermedin on cAMP accumulation (pEC(50) 7.3 +/- 0.14) was still observed even in the presence of a mixture of saturating concentrations of BIBN4096BS, hAM(22-52), and the amylin antagonist AC187. Taken together, these data provide evidence for the possible existence of a distinct class of receptor sites for adrenomedullin-2/intermedin in embryonic rat spinal cord cells.

An organelle proteomic method to study neurotransmission-related proteins, applied to a neurodevelopmental model of schizophrenia.

Limited information is currently available on molecular events that underlie schizophrenia-like behaviors in animal models. Accordingly, we developed an organelle proteomic approach enabling the study of neurotransmission-related proteins in the prefrontal cortex (PFC) of postpubertal (postnatal day 60 (PD60)) neonatally ventral hippocampal (nVH) lesioned rats, an extensively used neurodevelopmental model of schizophrenia-like behaviors. The PFC was chosen because of its purported role in the etiology of the disease. Statistical analysis of 392 reproducible spots on 2-D organelle proteomic patterns revealed significant changes in intensity of 18 proteinous spots in plasma membrane-enriched fractions obtained from postpubertal nVH lesioned rats compared to controls. Mass spectrometric analysis and database searching allowed the identification of a single protein in each of the nine differential spots, including proteins of low abundance, such as neurocalcin delta. Most of the identified dysregulated proteins, including clathrin light chain B, syntaxin binding protein 1b and visinin-like protein 1 are known to be linked to various neurotransmitter systems and to play key roles in plasma membrane receptor expression and recycling as well as synaptic vesicle exocytosis/recycling. Organelle proteomic approaches have hence proved to be most useful to identify key proteins linked to a given behavior in animal models of brain diseases.

Papel de la adrenomedulina cerebelosa en la hipertensión arterial / Paper of the adrenomedulina cerebelosa in the arterial hypertension

La adrenomedulina (AM) y el péptido relacionado con el gen de la calcitonina (CGRP) pertenecen a la superfamilia de los péptidos de CGRP. En el SNC, los sitios de unión para la AM y el CGRP se encuentran presentes en áreas hipotalámicas y en la corteza cerebelosa de la rata. La administración central de AM o de CGRP en ratas induce diuresis, natriuresis e incremento de la presión arterial. El papel de la AM en el cerebelo se desconoce. Con el fin de establecer la posible relación de la AM y CGRP cerebelosa y la regulación cardiovascular, en el presente estudio evaluamos la densidad de sitios de unión para la AM y el CGRP en el cerebelo de ratas espontáneamente hipertensas (SHR) y sus controles normotensos Wistar Kyoto (WKY) adultos de 16 semanas, mediante el uso de técnicas autoradiografícas y empleando 125I-hCGRPα y 125I-hAM13-52 como radioligandos. Los cortes coronales de cerebelo fueron incubados con 35 pM de [125I]-hCGRPα o [125I]-hAM13-52, durante 90 y 120 minutos, respectivamente. La unión no específica fue determinada en presencia de 1µM del ligando no marcado. El análisis densitométrico demostró que existe una colocalización de los sitios de unión para el [125I]-hCGRPα y la [125I]-hAM13-52 en la corteza cerebelosa. En el cerebelo la unión de la [125I]-hAM13-52 en las ratas SHR fue significativamente mayor que las WKY, indicando una mayor expresión de los receptores para la AM en el cerebelo de animales hipertensos. En relación a la unión de [125I]-hCGRPα, se observó también un pequeño incremento significativo en las ratas SHR en relación a las WKY. Con el fin de establecer la posible vía de señalización de la AM en la corteza cerebelosa, se evaluó la actividad de la óxido nítrico sintasa inducida por la AM.

A role for adrenomedullin as a pain-related peptide in the rat.

Adrenomedullin (AM) belongs to the calcitonin gene-related peptide (CGRP) family and is a well known potent vasodilator. We show here that AM is a powerful pain-inducing neuropeptide. AM-like immunoreactivity is widely distributed in both CGRP-containing and lectin IB4-binding nociceptors in dorsal root ganglion and axon terminals in the superficial dorsal horn of the rat spinal cord. Specific binding sites for the radioligand, [(125)I]AM13-52 as well as immunoreactivity for receptor markers such as the calcitonin receptor-like receptor and three receptor-activity-modifying proteins are localized in the superficial dorsal horn, demonstrating the existence of AM/CGRP receptors in this region. Intrathecal injection of rat AM1-50, dose- and time-dependently, induced long-lasting heat hyperalgesia and increased the phosphorylation of Akt and GSK3beta in the dorsal horn. Pre- and posttreatments with the AM receptor antagonist AM22-52 and PI3 kinase inhibitors (LY294002 and Wortmannin) significantly blocked or reversed AM-induced heat hyperalgesia. Pre- and posttreatments with AM22-52 and Wortmannin also significantly blocked or reversed intraplantar capsaicin-induced heat hyperalgesia. Taken together, our results demonstrate that AM acts as a pain-inducing peptide in the dorsal horn. By activating specific receptors (likely AM2) and the PI3K/Akt/GSK3beta signaling pathway, AM could play a significant role in long-lasting heat hypersensitivity and inflammatory heat hyperalgesia.