The neuropeptide calcitonin gene-related peptide (CGRP) from the trigeminal ganglion has been established as a key player in the pathogenesis of migraine. In this study, we provide evidence that the responsiveness of neuronal CGRP receptors is strongly enhanced in vitro and in vivo by expression of human receptor activity-modifying protein-1 (hRAMP1), an obligatory subunit of the CGRP receptor. We first demonstrated that activation of CGRP receptors on cultured trigeminal ganglion neurons increased endogenous CGRP mRNA levels and promoter activity. To establish whether RAMP1 is limiting in vivo as indicated from the culture studies, a transgenic mouse expressing hRAMP1 in the nervous system was generated. After CGRP injection into the whiskerpad, the hRAMP1 transgenic mice displayed 2.2 Ϯ 0.2-fold greater plasma extravasation, which is a measure of neurogenic inflammation. These results demonstrate that RAMP1 is functionally rate limiting for CGRP receptor activity in the trigeminal ganglion, which raises the possibility that elevated RAMP1 might sensitize some individuals to CGRP actions in migraine.

Using microdialysis, we investigated the circadian rhythms of the extracellular concentrations of dopamine, glutamate and γ‐aminobutyric acid (GABA) in the striatum and nucleus accumbens of the awake rat. Wistar rats were maintained in a 12 hr dark:12 hr light (12:12) cycle for 2 wk before the experiment began. The neurotransmitter levels were measured every 30 min for 30 hr in control (maintaining the 12:12 cycle) or in experimental conditions under a 24‐h light period (continuous light) or under a 24‐h dark interval (continuous dark). The dopamine metabolites, DOPAC and HVA, and the main serotonin metabolite, 5‐HIAA, were measured along with arginine and glutamine under all conditions. In 12:12 conditions, a circadian rhythm of dopamine, glutamate and GABA was found in both the striatum and nucleus accumbens. Again under 12:12 conditions, DOPAC, HVA, 5‐HIAA, and arginine, but not glutamine, fluctuated in a circadian rhythm. In the striatum under constant light conditions, there was a circadian rhythm of dopamine, glutamate, GABA, DOPAC and HVA, but not 5‐HIAA. By contrast, when the rats were kept under continuous dark, dopamine and its metabolites, DOPAC and HVA (but not glutamate and GABA), did not fluctuate in a circadian rhythm. In the nucleus accumbens, under both constant light or dark conditions, no changes were found in the circadian rhythm in any of the neurotransmitters and metabolites studied. These findings show that in the striatum, dopamine but not glutamate and GABA, seem to be influenced by light. In the nucleus accumbens, however, the three neurotransmitters had a circadian rhythm, which was independent of light.

Calcitonin gene-related peptide (CGRP) is a key player in migraine. To address the role of CGRP in mechanical allodynia, which is a common feature of migraine, we used CGRP-sensitized transgenic mice. These mice have elevated nervous system expression of the human receptor activity-modifying protein-1 (hRAMP1) subunit of the CGRP receptor. Under baseline conditions, the nestin/hRAMP1 mice and control littermates had similar hindpaw withdrawal thresholds to von Frey filaments. The effect of CGRP was tested using a filament that elicited a withdrawal response on 20% of its presentations. Following intrathecal injection of 1 nmol CGRP in the nestin/hRAMP1 mice, the response frequency was 80% within 30 min. The antagonist CGRP(8-37) blocked the increased response. In control littermates, a five-fold higher dose of CGRP was required to elicit a similar response. In contrast to intrathecal injection, peripheral CGRP did not increase the mechanical responses. Intraplantar injection of capsaicin was used to test the efficacy of endogenous CGRP. Capsaicin increased mechanical responses in the nestin/hRAMP1 and control mice, although a higher dose was required in controls. In contrast to control mice, there was also a contralateral paw response in nestin/hRAMP1 mice, which is consistent with central sensitization. PERSPECTIVE: In this study we show central CGRP-induced mechanical allodynia that is enhanced by overexpression of RAMP1 in nervous system. These data suggest that hypersensitivity to CGRP could be a potential mechanism underlying central sensitization in migraine and point to CGRP receptor antagonists as a possible therapy for other pain disorders.

The purpose of this study was to investigate possible circadian changes in extracellular concentrations of glutamate (GLU) and gamma-aminobutyric acid (GABA). and the influence of melatonin on the levels of these neurotransmitters in the neostriatum of awake rats using in vivo microdialysis. At the same time, the concentrations of the amino acids taurine (TAU), glutamine (GLN) and arginine (ARG), as well as dopamine (DA) and its metabolites 3, 4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), were measured in the extracellular fluid. When dialysates were collected over a 24-hr period (6 hr dark, 12 hr light, 6 hr dark), both GLU and GABA, without the infusion of melatonin, exhibited statistically significant rhythms, with higher levels of these constituents during the dark and lower levels during the day. Perfusion with melatonin (for 19 consecutive hours) prevented the daytime reductions in both GLU and GABA. Of the amino acids measured in the dialysates collected from the neostriatum of non-perfused rats, only ARG exhibited a significant change during the light:dark cycle; again, lowest concentrations were measured during the day. While melatonin perfusion did not statistically significantly influence neostriatal levels of TAU and ARG, GLN levels continued to drop during the infusion of the indoleamine. Dialysate concentrations of DA, DOPAC and HVA exhibited circadian rhythms which were not influenced by melatonin perfusion. The findings indicate there are differential effects of melatonin on extracellular neurotransmitter concentrations in the neostriatum of the awake rat. The results also suggest that the day:night variations in GLU and GABA may relate to daily changes in endogenous melatonin production, while DA and its metabolites are minimally influenced by this secretory product.

BackgroundReal-time quantitative PCR (qPCR) is a technique frequently used to measure changes in mRNA expression. To ensure validity of experimental findings, it is important to normalize the qPCR data to reference genes that are stable and unaffected by the experimental treatment to correct for variability among samples. Unlike in some models of neuropathic pain, reference genes for models of inflammatory injury have not been validated. This study examined four candidate reference genes in an effort to identify and validate optimal genes for normalization of transcriptional changes occurring in the dorsal horn of the spinal cord and the rostral ventromedial medulla (RVM) following intraplantar injection of complete Freund’s adjuvant (CFA).ResultsThe expression of hypoxanthine phosphoribosyltransferase 1 (Hprt1), beta-actin (Actb), mitogen-activated protein kinase 6 (Mapk6), and beta-2-microglobulin (B2m) was quantified in the dorsal horn and RVM of rats four days or two weeks after intraplantar injection of CFA or saline. The range of expression levels among these four genes differed by as much as 16-fold within the dorsal horn and the RVM. All four of these reference genes were stably expressed in both tissues and did not differ between saline and CFA-treated animals. Analyses using the statistical algorithms in geNorm and NormFinder programs determined that Mapk6 was the most stable gene and recommended the combination of Mapk6 and Actb, or Mapk6 and Hprt1, in such experimental conditions.ConclusionsThis study validated the four genes Hprt1, Actb, Mapk6 or B2m and showed that any one or combination of two of them are good reference genes for normalization of mRNA expression in qPCR experiments in the spinal cord and RVM in the CFA model of inflammatory injury.

This study examined whether mice with a deficiency of neurofibromin, a Ras GTPase activating protein, exhibit a nociceptive phenotype and probed a possible contribution by calcitonin gene-related peptide. In the absence of inflammation, Nf1+/− mice (B6.129S6 Nf1/J) and wild type littermates responded comparably to heat or mechanical stimuli, except for a subtle enhanced mechanical sensitivity in female Nf1+/− mice. Nociceptive phenotype was also examined after inflammation induced by capsaicin and formalin, which release endogenous calcitonin gene-related peptide. Intraplantar injection of capsaicin evoked comparable heat hyperalgesia and mechanical hypersensitivity in Nf1+/− and wild type mice of both genders. Formalin injection caused a similar duration of licking in male Nf1+/− and wild type mice. Female Nf1+/− mice licked less than wild type mice, but displayed other nociceptive behaviors. In contrast, intraplantar injection of CGRP caused greater heat hyperalgesia in Nf1+/− mice of both genders compared to wild type mice. Male Nf1+/− mice also exhibited greater mechanical hypersensitivity; however, female Nf1+/− mice exhibited less mechanical hypersensitivity than their wild type littermates. Transcripts for calcitonin gene-related peptide were similar in the dorsal root ganglia of both genotypes and genders. Transcripts for receptor activity-modifying protein-1, which is rate-limiting for the calcitonin gene-related peptide receptor, in the spinal cord were comparable for both genotypes and genders. The increased responsiveness to intraplantar calcitonin gene-related peptide suggests that the peripheral actions of calcitonin gene-related peptide are enhanced as a result of the neurofibromin deficit. The analgesic efficacy of calcitonin gene-related peptide receptor antagonists may therefore merit investigation in neurofibromatosis patients.

This study examined the distribution of γ-aminobutyric acid (GABA)B receptors on immunohistochemically identified neurons, and levels of GABAB(1) and GABAB(2) mRNA, in the L4 and L5 dorsal root ganglia (DRG) of the rat in the absence of injury and 2 weeks after L5 spinal nerve ligation. In uninjured DRG, GABAB(1) immunoreactivity colocalized exclusively with the neuronal marker (NeuN) and did not colocalize with the satellite cell marker S-100. The GABAB(1) subunit colocalized to >97% of DRG neurons immunoreactive (IR) for neurofilament 200 (N52) or calcitonin gene-related peptide (CGRP), or labeled by isolectin B4 (IB4). Immunoreactivity for GABAB(2) was not detectable. L5 spinal nerve ligation did not alter the number of GABAB(1)-IR neurons or its colocalization pattern in the L4 DRG. However, ligation reduced the number of GABAB(1)-IR neurons in the L5 DRG by ≈38% compared with sham-operated and naïve rats. Specifically, ligation decreased the number of CGRP-IR neurons in the L5 DRG by 75%, but did not decrease the percent colocalization of GABAB(1) in those that remained. In the few IB4-positive neurons that remained in the L5 DRG, colocalization of GABAB(1)-IR decreased to 75%. Ligation also decreased levels of GABAB(1) and GABAB(2) mRNA in the L5, but not the L4 DRG compared with sham-operated or naïve rats. These findings indicate that the GABAB receptor is positioned to presynaptically modulate afferent transmission by myelinated, unmyelinated, and peptidergic afferents in the dorsal horn. Loss of GABAB receptors on primary afferent neurons may contribute to the development of mechanical allodynia after L5 spinal nerve ligation.

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