Calcitonin Gene-related Peptide Expression In Neurons Research Articles

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Activation of Parabrachial Tachykinin 1 Neurons Counteracts Some Behaviors Mediated by Parabrachial Calcitonin Gene-related Peptide Neurons

Many threats activate parabrachial neurons expressing calcitonin gene-related peptide (CGRPPBN) which transmit alarm signals to forebrain regions. Most CGRPPBN neurons also express tachykinin 1 (Tac1), but there are also Tac1-expressing neurons in the PBN that do not express CGRP (Tac1+;CGRP− neurons). Chemogenetic or optogenetic activation of all Tac1PBN neurons in mice elicited many physiological/behavioral responses resembling the activation of CGRPPBN neurons, e.g., anorexia, jumping on a hot plate, avoidance of photostimulation; however, two key responses opposed activation of CGRPPBN neurons. Activating Tac1PBN neurons did not produce conditioned taste aversion and it elicited dynamic escape behaviors rather than freezing. Activating Tac1+;CGRP− neurons, using an intersectional genetic targeting approach, resembles activating all Tac1PBN neurons. These results reveal that activation of Tac1+;CGRP− neurons can suppress some functions attributed to the CGRPPBN neurons, which provides a mechanism to bias behavioral responses to threats.

Grape seed extract suppresses calcitonin gene-related peptide secretion and upregulates expression of GAD 65/67 and GABAB receptor in primary trigeminal ganglion cultures.

The trigeminal ganglion is implicated in the underlying pathology of migraine and temporomandibular joint disorders (TMD), which are orofacial pain conditions involving peripheral and central sensitization. The neuropeptide calcitonin gene-related peptide (CGRP) is synthesized in some trigeminal ganglion neurons, and its release promotes inflammation, peripheral and central sensitization, and pain signaling. Recent studies in preclinical migraine and TMD models provide evidence that dietary supplementation with grape seed extract (GSE) inhibits trigeminal pain signaling. The goal of this study was to investigate the cellular mechanisms by which GSE modulates primary trigeminal ganglion cultures. The effect of GSE on CGRP secretion was determined by radioimmunoassay. To determine if GSE effects involved modulation of CGRP or the GABAergic system, expression of CGRP, GAD 65 and 67, GABAA receptor, and GABAB1 and GABAB2 receptor subunits were investigated by immunocytochemistry. GSE significantly inhibited basal CGRP secretion but did not alter neuronal CGRP expression. GAD 65 and 67 expression levels in neurons were significantly increased in response to GSE. While GSE did not cause a change in the neuronal expression of GABAA, GSE significantly increased GABAB1 expression in neurons, satellite glial cells, and Schwann cells. GABAB2 expression was significantly elevated in satellite glia and Schwann cells. These findings support the notion that GSE inhibition of basal CGRP secretion involves increased neuronal GAD 65 and 67 and GABAB receptor expression. GSE repression of CGRP release coupled with increased GABAB1 and GABAB2 glial cell expression would be neuroprotective by suppressing neuronal and glial excitability in the trigeminal ganglion.

A central alarm system that gates multi-sensory innate threat cues to the amygdala.

SUMMARYPerception of threats is essential for survival. Previous findings suggest that parallel pathways independently relay innate threat signals from different sensory modalities to multiple brain areas, such as the midbrain and hypothalamus, for immediate avoidance. Yet little is known about whether and how multi-sensory innate threat cues are integrated and conveyed from each sensory modality to the amygdala, a critical brain area for threat perception and learning. Here, we report that neurons expressing calcitonin gene-related peptide (CGRP) in the parvocellular subparafascicular nucleus in the thalamus and external lateral parabrachial nucleus in the brainstem respond to multi-sensory threat cues from various sensory modalities and relay negative valence to the lateral and central amygdala, respectively. Both CGRP populations and their amygdala projections are required for multi-sensory threat perception and aversive memory formation. The identification of unified innate threat pathways may provide insights into developing therapeutic candidates for innate fear-related disorders.

Sensory neurons expressing calcitonin gene-related peptide α regulate adaptive thermogenesis and diet-induced obesity

ObjectivesHeat-sensory neurons from the dorsal root ganglia (DRG) play a pivotal role in detecting the cutaneous temperature and transmission of external signals to the brain, ensuring the maintenance of thermoregulation. However, whether these thermoreceptor neurons contribute to adaptive thermogenesis remains elusive. It is also unknown whether these neurons play a role in obesity and energy metabolism. MethodsWe used genetic ablation of heat-sensing neurons expressing calcitonin gene-related peptide α (CGRPα) to assess whole-body energy expenditure, weight gain, glucose tolerance, and insulin sensitivity in normal chow and high-fat diet-fed mice. Exvivo lipolysis and transcriptional characterization were combined with adipose tissue-clearing methods to visualize and probe the role of sensory nerves in adipose tissue. Adaptive thermogenesis was explored using infrared imaging of intrascapular brown adipose tissue (iBAT), tail, and core temperature upon various stimuli including diet, external temperature, and the cooling agent icilin. ResultsIn this report, we show that genetic ablation of heat-sensing CGRPα neurons promotes resistance to weight gain upon high-fat diet (HFD) feeding and increases energy expenditure in mice. Mechanistically, we found that loss of CGRPα-expressing sensory neurons was associated with reduced lipid deposition in adipose tissue, enhanced expression of fatty acid oxidation genes, higher exvivo lipolysis in primary white adipocytes, and increased mitochondrial respiration from iBAT. Remarkably, mice lacking CGRPα sensory neurons manifested increased tail cutaneous vasoconstriction at room temperature. This exacerbated cold perception was not associated with reduced core temperature, suggesting that heat production and heat conservation mechanisms were engaged. Specific denervation of CGRPα neurons in intrascapular BAT did not contribute to the increased metabolic rate observed upon global sensory denervation. ConclusionsTaken together, these findings highlight an important role of cutaneous thermoreceptors in regulating energy metabolism by triggering counter-regulatory responses involving energy dissipation processes including lipid fuel utilization and cutaneous vasodilation.

Elucidating an Affective Pain Circuit that Creates a Threat Memory

Animals learn to avoid harmful situations by associating a neutral stimulus with a painful one, resulting in a stable threat memory. In mammals, this form of learning requires the amygdala. Although pain is the main driver of aversive learning, the mechanism that transmits pain signals to the amygdala is not well resolved. Here, we show that neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus are critical for relaying pain signals to the central nucleus of amygdala and that this pathway may transduce the affective motivational aspects of pain. Genetic silencing of CGRP neurons blocks pain responses and memory formation, whereas their optogenetic stimulation produces defensive responses and a threat memory. The pain-recipient neurons in the central amygdala expressing CGRP receptors are also critical for establishing a threat memory. The identification of the neural circuit conveying affective pain signals may be pertinent for treating pain conditions with psychiatric comorbidities.

Substance P and calcitonin gene related peptide mediate pain in chronic pancreatitis and their expression is driven by nerve growth factor.

Calcitonin gene-related peptide (CGRP), substance P and nerve growth factor play an important role in inflammatory pain in various somatic pain models but their role in chronic pancreatitis has not been well studied. The aim of this study was to investigate the effects of intrathecal administration of calcitonin gene-related peptide antagonist and substance P receptor antagonist on pain behavior in a rat model of chronic pancreatitis and to determine whether nerve growth factor drives the up-regulation of expression of these neuropeptides in sensory neurons. Pancreatitis was induced by retrograde infusion of trinitobenzene sulfonic acid into the pancreatic duct of adult rats. Three weeks post infusion continuous intrathecal infusion of the calcitonin gene-related peptide antagonist alpha CGRP8-37 or neurokinin-1 receptor antagonist CP-96345 or its inactive enantiomer CP-96344 was administered for seven days. The effects of treatment on pancreatic hyperalgesia were assessed by sensitivity of the abdominal wall to von Frey filament probing as well as by the nocifensive response to electrical stimulation of the pancreas. In a separate experiment chronic pancreatitis was induced and pancreas specific dorsal root ganglion neurons labeled with DiI were assessed for calcitonin gene-related peptide and substance P immunoreactivity. Intrathecal infusion of calcitonin gene-related peptide and neurokinin-1 receptor antagonists significantly attenuated behavioral pain responses in rats with chronic pancreatitis. Further, treatment of chronic pancreatitis rats with nerve growth factor antibody significantly reduced pancreas specific neurons expressing calcitonin gene-related peptide and substance P in thoracic dorsal root ganglion. Calcitonin gene-related peptide and substance P mediate pancreatic hyperalgesia in chronic pancreatitis and nerve growth factor in turn sustains the up-regulation of these neuropeptides in pancreatic sensory neurons.

Projections of the sexually dimorphic calcitonin gene-related peptide neurons of the preoptic area determined by retrograde tracing in the female rat.

The medial preoptic area of the rat exhibits morphologic sex differences and is implicated in the control of sexually dimorphic behavior and function. Neurons expressing calcitonin gene-related peptide (CGRP) within the anteroventral periventricular (AVPV) and medial preoptic nucleus (MPN) of the medial preoptic area exhibit female-dominant sex differences in number through organizational and activational effects of gonadal steroids. The present study used retrograde tracing experiments to establish the projections of the AVPV and MPN CGRP neurons in the female rat. After the intraperitoneal administration of Fluoro-Gold to female rats (n = 5), we were unable to detect retrograde tracer in any CGRP-immunoreactive cells of the hypothalamus. Intracerebral injections of 50- to 100-nl volumes of Fluoro-Gold into the mediobasal hypothalamus resulted in up to 70% of CGRP neurons in the AVPV and MPN containing retrograde tracer. Similar large volume tracer depositions in the lateral septum, periaqueductal gray, two likely CGRP projection sites, resulted in no labeling of preoptic CGRP neurons. Experiments using small volume (30-nl) injections of Fluoro-Gold and green fluorescent microspheres at multiple sites in the mediobasal hypothalamus (n = 18) revealed that approximately 60% of AVPV and 30% of MPN neurons expressing CGRP were projecting to the region of the tuberal and ventral premammillary nuclei, with a minor projection to the dorsomedial nucleus. These findings demonstrate a major projection of the preoptic CGRP neurons to the posterior hypothalamus in the female rat and support further a functional role for these neurons in the sexually dimorphic regulation of reproductive functioning.

Role of calcitonin gene-related peptide and substance P in Dahl-salt hypertension.

Calcitonin gene-related peptide (CGRP) and substance P are known to play a counterregulatory role in acquired models of salt-dependent hypertension. In contrast, neuronal production of these peptides is decreased in the spontaneously hypertensive rat, which may contribute to the elevated blood pressure. To determine the role played by CGRP and substance P in Dahl-salt hypertension, 4- to 6-week-old male salt-resistant (DR) and salt-sensitive (DS) rats were divided into 4 groups (n=5/group) and pair-fed low-salt (0.2% NaCl) (DR/LS and DS/LS) and high-salt (8% NaCl) diets (DR/HS and DS/HS) for 3 weeks. After 3 weeks, all the rats had venous (for drug administration) and arterial (for blood pressure monitoring) catheters surgically implanted and were studied in the conscious and unrestrained state. Mean arterial pressure was significantly higher in the DS/HS rats animals (185.8+/-1.6 mm Hg, P<0.001). Intravenous administration of CGRP and SP receptor antagonists was without effect in any of the groups studied. CGRP and SP mRNA content from dorsal root ganglia were not significantly different between the groups. Whereas immunoreactive CGRP was decreased in the DS groups (DS/HS, 9.4+/-0.4 pg/microgram protein; DS/LS, 11.1+/-0.8 pg/microgram protein; P<0.01) compared with the DR groups (DR/HS, 13.9+/-0.6 pg/microgram protein; DR/LS, 14.6+/-0.6 pg/microgram protein), neuronal SP production was similar between all the groups. Thus, CGRP and substance P do not play a counterregulatory role in Dahl-salt hypertension. The decrease in neuronal CGRP expression in DS rats appears to be genetically determined as in SHR, however, and may contribute to the increase in blood pressure following salt-loading.

Expression and regulation of cholecystokinin and cholecystokinin receptors in rat nodose and dorsal root ganglia

Cholecystokinin (CCK) is an important satiety factor, acting via the vagus nerve to influence central feeding centers. CCK binding sites have been demonstrated in the vagal sensory nodose ganglion and within the nerve proper. Using in situ hybridization, expression of the CCK A and B receptors (Rs), as well as of CCK itself, was studied in the normal nodose ganglion (NG), and after vagotomy, starvation and high-fat diet. CCK A-R mRNA expression in dorsal root ganglia (DRGs) was also explored. In the NG, 33% of the neuron profiles (NPs) contained CCK A-R mRNA and in 9% we observed CCK B-R mRNA. CCK mRNA was not found in normal NGs. Peripheral vagotomy decreased the number of CCK A-R mRNA-expressing NPs, dramatically increased the number of CCK B-R mRNA, and induced CCK mRNA and preproCCK-like immunoreactivity in nodose NPs. No significant differences in the number of NPs labelled for either mRNA species were detected following 48 h food deprivation or in rats fed a high-fat content diet. In DRGs, 10% of the NPs expressed CCK A-R mRNA, a number that was not affected by either axotomy or inflammation. This cell population was distinct from neurons expressing calcitonin gene-related peptide mRNA. These results demonstrate that the CCK A-R is expressed by both viscero- and somatosensory primary sensory neurons, supporting a role for this receptor as a mediator both of CCK-induced satiety and in sensory processing at the spinal level. The stimulation of CCK and CCK B-R gene expression following vagotomy suggests a possible involvement in the response to injury for these molecules.

Nerve growth factor enhances calcitonin gene-related peptide expression in the spontaneously hypertensive rat.

Calcitonin gene-related peptide (CGRP) expression is markedly reduced in dorsal root ganglia neurons in the spontaneously hypertensive rat (SHR). This decrease in such a potent vasodilator may contribute to the elevated blood pressure. Therefore, the purpose of this study was to determine whether stimulation of neuronal CGRP expression in SHR, by means of the administration of nerve growth factor, would lower the blood pressure. Nerve growth factor (10 nmol/L per kg/d, IP) was given to 12-week SHR (n=8 to 11/group) once a day for 1, 3, and 7 days. Control SHR received vehicle only. All rats were instrumented for CGRP receptor antagonist (CGRP(8-37)) administration (intravenous) and mean arterial pressure recording. Both the 1- and 3-day NGF treatments lowered the mean arterial pressure to 147+/-5 and 147+/-3 mm Hg, respectively, compared with controls (166+/-3 mm Hg). However, by day 7, the mean arterial pressure had returned to control levels (169+/-5 mm Hg). CGRP(8-37) administration produced a significant mean arterial pressure increase in all 3 nerve growth factor-treated groups (14+/-2, 10+/-2, and 13+/-2 mm Hg). CGRP mRNA levels in dorsal root ganglia were increased in the 3 neurotrophin-treated groups, whereas CGRP peptide content was higher at days 3 and 7. Therefore, nerve growth factor treatment of SHR can enhance neuronal CGRP expression. At days 1 and 3, nerve growth factor produces a depressor response that is primarily mediated by CGRP as evidenced by the pressor effect of CGRP(8-37.) At day 7, CGRP also plays a counterregulatory role, even though the mean arterial pressure has returned to control levels. This finding may result from a nerve growth factor-mediated upregulation of a pressor system that counteracts the hypotensive actions of CGRP. Thus, these data suggest that the decreased production of CGRP in SHR could contribute to the hypertension.

Skin Cell Induction of Calcitonin Gene-Related Peptide in Embryonic Sensory Neurons in Vitro Involves Activin

Target skin cells induce the neuropeptide calcitonin gene-related peptide (CGRP) in naı̈ve embryonic dorsal root ganglion (DRG) neurons in vitro, but the molecular basis of that induction is not known. Recombinant activin or bone morphogenetic proteins (BMPs) dramatically increase the number of sensory neurons with CGRP and substance P in vitro (X. 1). These experiments were designed to test if activin or BMPs accounted for the CGRP-inductive activity by skin cells. To identify factors from skin that induce CGRP, we developed a bioassay in which embryonic DRG neurons isolated before peripheral target contact in vivo are challenged in vitro with specific factors. Conditioned medium from an embryonic rat skin cell line induced neuronal CGRP expression, and induction was blocked by follistatin, implicating transforming growth factor β family members. Immunoblot analysis revealed that the skin cell line medium contained several activin and bone morphogenetic protein moieties. Antibody specific to activin neutralized most of the CGRP-inductive activity in skin conditioned medium. These data indicate that the CGRP-inductive action of skin cells involves activin and establish activin as a candidate regulator of this sensory neuropeptide phenotype during development.

NT-3 modulates NPY expression in primary sensory neurons following peripheral nerve injury.

Peripheral nerve transection induces significant changes in neuropeptide expression and content in injured primary sensory neurons, possibly due to loss of target derived neurotrophic support. This study shows that neurotrophin-3 (NT-3) delivery to the injured nerve influences neuropeptide Y (NPY) expression within dorsal root ganglia (DRG) neurons. NT-3 was delivered by grafting impregnated fibronectin (500 ng/ml; NT group) in the axotomised sciatic nerve. Animals grafted with plain fibronectin mats (FN) or nerve grafts (NG) were used as controls. L4 and L5 DRG from operated and contralateral sides were harvested between 5 and 240 d. Using immunohistochemistry and computerised image analysis the percentage, diameter and optical density of neurons expressing calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and NPY were quantified. Sciatic nerve axotomy resulted in significant reduction in expression of CGRP and SP, and significant upregulation of VIP and NPY (P < 0.05 for ipsilateral vs contralateral DRG). By d 30, exogenous NT-3 and nerve graft attenuated the upregulation of NPY (P < 0.05 for NT and NG vs FN). However, NT-3 administration did not influence the expression of CGRP, SP or VIP. The mean cell diameter of NPY immunoreactive neurons was significantly smaller in the NT-3 group (P < 0.05 for NT vs FN and NG) suggesting a differential influence of NT-3 on larger neurons. The optical densities of NPY immunoreactive neurons of equal size were the same in each group at any time point, indicating that the neurons responding to NT-3 downregulate NPY expression to levels not detectable by immunohistochemistry. These results demonstrate that targeted administration of NT-3 regulates the phenotype of a NPY-immunoreactive neuronal subpopulation in the dorsal root ganglia, a further evidence of the trophic role of neurotrophins on primary sensory neurons.

α2-Adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a potent vasodilator, is produced in dorsal root ganglia (DRG) neurons which extend nerves peripherally to blood vessels and centrally to the spinal cord. We previously reported that neuronal CGRP expression is significantly reduced in the spontaneously hypertensive rat (SHR) which could contribute to the elevated BP. Other studies suggest that the enhanced activity of the sympathetic nervous system in the SHR may mediate, at least in part, this reduction in neuronal CGRP expression via activation of α 2-adrenoreceptors ( α 2-AR) on DRG neurons. To test this hypothesis in vitro we employed primary cultures of adult rat DRG neurons. Neuronal cultures were initially exposed (24 h) to either the α 2-AR agonist UK 14,304 (10 −6 M) or vehicle; however, no changes in CGRP mRNA content or immunoreactive CGRP (iCGRP) release were observed. Using the rationale that in vivo DRG neurons receive a continuous supply of target tissue derived nerve growth factor (NGF), which stimulates CGRP synthesis, the cultured neurons were treated (24 h) with either vehicle, NGF (25 ng/ml) alone, or NGF plus UK. NGF treatment increased CGRP mRNA accumulation 5.5±0.9-fold ( p<0.001) and iCGRP release 2.9±0.4-fold ( p<0.001) over control levels. The stimulatory effects of NGF were markedly attenuated, but not abolished, by UK (NGF+UK vs. control, CGRP mRNA, 2.9±0.4-fold, p<0.05; iCGRP, 1.7±0.2-fold, p<0.05). These values were also significant ( p<0.05) when compared to NGF treatment alone. Experiments performed using the α 2-antagonist yohimbine confirmed that the effects of UK were mediated by the α 2-AR. These results, therefore, demonstrate that α 2-AR activation attenuates the stimulatory effects of NGF on CGRP expression in DRG neurons.

Dexamethasone and activators of the protein kinase A and C signal transduction pathways regulate neuronal calcitonin gene-related peptide expression and release.

Primary cultures of adult rat dorsal root ganglia (DRG) neurons were used to determine if activation of either the protein kinase A or C signal transduction pathways or treatment with the synthetic glucocorticoid dexamethasone modulate neuronal calcitonin gene-related peptide (CGRP) synthesis and release. DRG are the sites of neuronal cell bodies known to produce abundant CGRP levels, and to send axons peripherally to blood vessels and centrally to the spinal cord. Using immunocytochemical techniques, we confirmed that synthesis of immunoreactive CGRP (iCGRP) is restricted to a subpopulation of DRG neurons. Subsequently, we determined that treatment (24 h) of the neurons with either dibutyryl cAMP (1 mM) or phorbol 12-myristate 13-acetate (2 microM) increased CGRP mRNA content 2.2 +/- 0.4 (n = 6, p < 0.03) and 3.0 +/- 0.6-fold (n = 6, P < 0.02) respectively, while secreted iCGRP levels were increased 1.8 +/- 0.2 (n = 14, P < 0.005) and 4.5 +/- 1.0 (n = 14, P < 0.001)-fold over control levels. Treatment of the neurons with dexamethasone alone had no effect on CGRP expression; however, this agent was able to significantly attenuate the stimulatory effects of NGF on both CGRP mRNA accumulation and release of iCGRP. Time course studies demonstrated that in the phorbol ester treated neurons CGRP mRNA levels continued to increase at 48 h, while maximal induction with dibutyryl cAMP occurred at approximately 12 h. These results indicate that local and/or circulating factors which act through the protein kinase A and C signal transduction pathways upregulate both CGRP expression and release, while glucocorticoids attenuate the stimulatory effects of NGF.

Enhanced Neuronal Expression of Calcitonin Gene–Related Peptide in Mineralocorticoid-Salt Hypertension

Dorsal root ganglia neuronal cell bodies synthesize the vasodilator neuropeptide calcitonin gene-related peptide and innervate the blood vessels and spinal cord sites (laminae I and II) involved in blood pressure regulation. We previously demonstrated that calcitonin gene-related peptide mRNA content is significantly decreased in dorsal root ganglia and that immunoreactive calcitonin gene-related peptide levels are reduced in laminae I and II of the dorsal horn of the spinal cord in the spontaneously hypertensive rat compared with Wistar-Kyoto control rats. To determine whether neuronal calcitonin gene-related peptide expression is also altered in mineralocorticoid-salt hypertension, we quantified calcitonin gene-related peptide mRNA levels in dorsal root ganglia and protein content in laminae I and II of the spinal cord in rats with mineralocorticoid-salt-induced hypertension. To control for pellet implantation, saline drinking water, and/or uninephrectomy, four normotensive groups were similarly studied. By Northern hybridization analysis, the ratio of calcitonin gene-related peptide mRNA to 18S rRNA was increased approximately fivefold in hypertensive rats (33 +/- 7) compared with each of the four normotensive control groups (average of the four groups, 6 +/- 0.5; P < .01, mineralocorticoid-salt group versus each group). The density of the peptide, quantified by computer-assisted image analysis, in laminae I and II in the hypertensive rats was also increased (66 +/- 1 versus average of the four groups, 46 +/- 2 arbitrary units; P < .001, mineralocorticoid-salt group versus each group). In conclusion, neuronal levels of calcitonin gene-related peptide mRNA and protein are increased in mineralocorticoid-salt hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of neurons expressing calcitonin gene-related peptide mRNAS in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin generelated peptide mRNA sequences encoding residues 23–30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10–20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.