A novel affective-motivational-based Overground System for detecting spinal cord injury-associated thermal and mechanical hypersensitivity in rats.

BACKGROUND: Preclinical research for neuropathic pain has depended primarily on the use of behavioural nociceptive testing that is sensory-discriminatory-based and reflexive in nature. This can be particularly problematic in spinal cord injury (SCI)-associated neuropathic pain research where hyperreflexia may develop thus confounding interpretation of reflexive responses as pain symptoms. To address this, we have designed an affective-motivational-based Overground System that has interchangeable floors to allow examination of nociceptive behaviours in response to mechanical and cold stimuli prior to and following spinal cord injury. METHODS: The animals were pretrained for 3-4 weeks to cross three Overground System surfaces (cold, rough and neutral) at a consistent pace in order to obtain a food reward (Froot Loops™ ). RESULTS: Following SCI, crossing times were significantly decreased due to more rapid crossing over the cold surface despite forgoing of the food reward, in contrast with the sham control animals. This was correlated with the development of cold hypersensitivity as assessed by standard acetone droplet testing. In response to the rough surface, slower paced crossings (increased time to cross) were observed post-SCI (also resulting in forgoing of the food reward), compared to pre-SCI times and sham control animals. This was correlated with the development of mechanical hypersensitivity as assessed by standard von Frey testing. CONCLUSIONS: Different responses in crossing behaviour for the cold and rough surfaces suggest a precise adaptive change in decision-making behaviour depending on the stimulus presented; thus, outcome measures may be easily tailored for different types of hypersensitivity symptoms. SIGNIFICANCE: We have designed an Overground System that is easy to establish and addresses a major concern in preclinical pain research by providing a cognitive- and motivational-based system for hypersensitivity detection. The affective-motivational-based Overground System allows examination of pain-like behaviours in response to cold (thermal) and rough (mechanical) stimulation prior to and following spinal cord injury. This system provides a more holistic and comprehensive assessment of nociceptive responses following SCI and helps overcome concerns of hyperreflexia confounding-evoked behavioural outcome measures in SCI models. Further, the incorporation of cognitive and motivational components brings preclinical research closer to replicating the clinical experience of a patient's motivation to participate in rewarding lifestyle activities in relation to their pain.

The HNF1A mutant Ala180Val: Clinical challenges in determining causality of a rare HNF1A variant in familial diabetes.

AIMS: Heterozygous mutations in hepatocyte nuclear factor-1A (HNF1A) cause maturity-onset diabetes of the young type 3 (MODY3). Our aim was to compare two families with suspected dominantly inherited diabetes and a new HNF1A variant of unknown clinical significance. METHODS: The HNF1A gene was sequenced in two independently recruited families from the Norwegian MODY Registry. Both familes were phenotyped clinically and biochemically. Microsatellite markers around and within the HNF1A locus were used for haplotyping. Chromosomal linkage analysis was performed in one family, and whole-exome sequencing was undertaken in two affected family members from each family. Transactivation activity, DNA binding and nuclear localization of wild type and mutant HNF-1A were assessed. RESULTS: The novel HNF1A variant c.539C>T (p.Ala180Val) was found in both families. The variant fully co-segregated with diabetes in one family. In the other family, two subjects with diabetes mellitus and one with normal glucose levels were homozygous variant carriers. Chromosomal linkage of diabetes to the HNF1A locus or to other genomic regions could not be established. The protein functional studies did not reveal significant differences between wild type and variant HNF-1A. In each family, whole-exome sequencing failed to identify any other variant that could explain the disease. CONCLUSIONS: The HNF1A variant p.Ala180Val does not seem to cause MODY3, although it may confer risk for type 2 diabetes mellitus. Our data demonstrate challenges in causality evaluation of rare variants detected in known diabetes genes.

Clinical reappraisal of SHORT syndrome with PIK3R1 mutations: toward recommendation for molecular testing and management.

SHORT syndrome has historically been defined by its acronym: short stature (S), hyperextensibility of joints and/or inguinal hernia (H), ocular depression (O), Rieger abnormality (R) and teething delay (T). More recently several research groups have identified PIK3R1 mutations as responsible for SHORT syndrome. Knowledge of the molecular etiology of SHORT syndrome has permitted a reassessment of the clinical phenotype. The detailed phenotypes of 32 individuals with SHORT syndrome and PIK3R1 mutation, including eight newly ascertained individuals, were studied to fully define the syndrome and the indications for PIK3R1 testing. The major features described in the SHORT acronym were not universally seen and only half (52%) had four or more of the classic features. The commonly observed clinical features of SHORT syndrome seen in the cohort included intrauterine growth restriction (IUGR) <10th percentile, postnatal growth restriction, lipoatrophy and the characteristic facial gestalt. Anterior chamber defects and insulin resistance or diabetes were also observed but were not as prevalent. The less specific, or minor features of SHORT syndrome include teething delay, thin wrinkled skin, speech delay, sensorineural deafness, hyperextensibility of joints and inguinal hernia. Given the high risk of diabetes mellitus, regular monitoring of glucose metabolism is warranted. An echocardiogram, ophthalmological and hearing assessments are also recommended.

Weight cycling promotes fat gain and altered clock gene expression in adipose tissue in C57BL/6J mice.

Repeated attempts to lose weight by temporary dieting may result in weight cycling, eventually further gain of body fat, and possible metabolic adaptation. We tested this with a controlled experiment in C57BL/6J mice subjected to four weight cycles (WC), continuous hypercaloric feeding (HF), or low-fat feeding (LF). To search for genes involved in an adaptive mechanism to former weight cycling and avoid acute effects of the last cycle, the last hypercaloric feeding period was prolonged by an additional 2 wk before euthanization. Total energy intake was identical in WC and HF. However, compared with HF, the WC mice gained significantly more total body mass and fat mass and showed increased levels of circulating leptin and lipids in liver. Both the HF and WC groups showed increased adipocyte size and insulin resistance. Despite these effects, we also observed an interesting maintenance of circulating adiponectin and free fatty acid levels after WC, whereas changes in these parameters were observed in HF mice. Global gene expression was analyzed by microarrays. Weight-cycled mice were characterized by a downregulation of several clock genes (Dbp, Tef, Per1, Per2, Per3, and Nr1d2) in adipose tissues, which was confirmed by quantitative PCR. In 3T3-L1 cells, we found reduced expression of Dbp and Tef early in adipogenic differentiation, which was mediated via cAMP-dependent signaling. Our data suggest that clock genes in adipose tissue may play a role in metabolic adaptation to weight cycling.

The nuclear receptors NUR77, NURR1 and NOR1 in obesity and during fat loss.

BACKGROUND: Adipose tissue is critical for systemic metabolic health. Identifying key factors regulating adipose tissue function is a research priority. The NR4A subfamily of nuclear receptors (NRs) (NR4A1/NUR77, NR4A2/NURR1 and NR4A3/NOR1) has emerged as important proteins in different disease states and in the regulation of metabolic tissues, particularly in liver and muscle. However, the expression of the NR4A members in human adipose tissue has not previously been described, and their target genes are largely unknown. OBJECTIVE: To determine whether the NR4As are differentially expressed in human adipose tissue in obesity, and identify potential NR4A target genes. DESIGN: Prospective analysis of s.c. adipose tissue before and 1 year after fat loss, and during in vitro differentiation of primary human preadipocytes. Case-control comparison of omental (OM) adipose tissue. SUBJECTS: A total of 13 extremely obese patients undergoing biliopancreatic diversion with duodenal switch for fat loss, 12 extremely obese patients undergoing laparoscopic sleeve gastrectomy and 37 lean individuals undergoing hernia repair or laparotomy were included in the study. Measurements were done by quantitative PCR gene expression analysis of the NR4A members and in silico promoter analysis based on microarray data. RESULTS: There was a strong upregulation of the NR4As in extreme obesity and normalization after fat loss. The NR4As were expressed at the highest level in stromal-vascular fraction compared with adipocytes, but were downregulated in both fractions after fat loss. Their expression levels were also significantly higher in OM compared with s.c. adipocytes in obesity. The NR4As were downregulated during differentiation of primary human preadipocytes. Moreover, the NR4As were strongly induced within 30 min of tissue incubation. Finally, promoter analysis revealed potential NR4A target genes involved in stress response, immune response, development and other functions. Our data show altered adipose tissue expression of the NR4As in obesity, suggesting that these stress responsive nuclear receptors may modulate pathogenic potential in humans.

Diagnostic screening of NEUROD1 (MODY6) in subjects with MODY or gestational diabetes mellitus.

AIMS: Diagnostic screening of NEUROD1 in patients with maturity-onset diabetes of the young (MODY) without mutations in the known MODY-genes (MODYX) and in subjects diagnosed with gestational diabetes mellitus. METHODS: Direct sequencing of NEUROD1 was performed in (i) 73 probands with clinical MODY without mutations in hepatocyte nuclear factor (HNF)-4alpha (MODY1), glucokinase (MODY2) and hepatocyte nuclear factor (HNF)-1alpha (MODY3), and (ii) 51 subjects diagnosed with gestational diabetes. Control material consisted of 105 anonymous blood donors. RESULTS: Mean age at diagnosis of diabetes was 22 and 30 years in the MODYX patients and gestational diabetes mellitus subjects, respectively. Mean fasting blood glucose (9.6 +/- 4.3 vs. 5.7 +/- 1.0 mml/l) as well as glycosylated haemoglobin (8.2 +/- 2.4 vs. 6.0 +/- 0.6%) were higher in the MODYX patients than subjects with gestational diabetes. NEUROD1 mutations were not detected in our two study groups. Three previously reported polymorphisms were found: Ala45Thr, Pro197His and IVS1 -32 nt C>T. The amino acid substitution serine to cysteine in codon 29 (designated Ser29Cys) was detected in one out of 105 control subjects. As the control material consisted of anonymous blood donors, we were prevented from investigation of possible co-segregation between the sequence variant Ser29Cys and diabetes mellitus. CONCLUSIONS: As we found no NEUROD1 mutations, diagnostic screening for this gene is not warranted in Norwegian MODYX patients. Our study also suggests that NEUROD1 is not a candidate gene in gestational diabetes mellitus (GDM). The sequence variant Ser29Cys was identified in one anonymous DNA sample, but we were prevented from studying possible co-segregation with diabetes mellitus.

Preserved insulin response to tolbutamide in hepatocyte nuclear factor-1alpha mutation carriers.

AIMS: Diabetic subjects with mutations in the gene encoding hepatocyte nuclear factor (HNF)-1alpha (MODY3) are prone to develop hypoglycaemia at low doses of glibenclamide, interpreted as sulphonylurea hypersensitivity. The present study was undertaken to compare the plasma insulin responses to glucose and tolbutamide in HNF-1alpha mutation carriers with those of healthy control subjects. METHODS: Seven mutation carriers; three normoglycaemic, two with impaired glucose tolerance, and two with newly detected diabetes, underwent an oral glucose tolerance test and a tolbutamide-modified intravenous glucose tolerance test with measurements of plasma insulin. Twenty-two healthy subjects served as controls. RESULTS: The plasma insulin response to intravenous glucose was reduced in the HNF-1alpha mutation carriers compared to the control subjects, with an area under the curve (median (interquartile range)) of 812 min pmol/l (421, 1647) and 1933 min pmol/l (1521, 2908), respectively (P = 0.03). In striking contrast, the plasma insulin response to tolbutamide was preserved, with an area under the curve of 2109 min pmol/l (1126, 3172) and 2250 min pmol/l (1614, 3276) in the mutation carriers and control subjects, respectively. CONCLUSIONS: HNF-1alpha mutation carriers are characterized by preserved tolbutamide-induced insulin secretion. Compared to healthy subjects, our MODY3 individuals did not show any increased serum insulin response to tolbutamide, suggesting that HNF-1alpha mutation carriers are not characterized by sulphonylurea hypersensitivity.

A gel electrophoresis method for detection of mitochondrial DNA mutation (3243 tRNA(Leu (UUR))) applied to a Norwegian family with diabetes mellitus and hearing loss.

Blood cells of selected patients from a large Norwegian family with maternally transmitted diabetes mellitus, hearing loss and muscular dysfunction were screened for possible A3243G mutation tRNA(Leu (UUR)) in mitochondrial DNA. We selected 7 patients from 3 of the 4 generations of the family and 10 unrelated healthy control subjects for mutation analysis using denaturing gradient gel electrophoresis (DGGE) and both manual and automated DNA sequencing. The A3243G mutation was found in peripheral blood cells of all 7 patients, but in none of the controls. The mutation was in the form of heteroplasmy and the amount of mutant DNA was found to be between 10% and 35% of total mtDNA in individual patients. This is the first report of a Norwegian family with maternally inherited diabetes and hearing loss carrying the A3243G mutation in mitochondrial DNA.

Peripheral nerve exposure to HIV viral envelope protein gp120 induces neuropathic pain and spinal gliosis.

Painful sensory neuropathy is a common and debilitating consequence of human immunodeficiency virus (HIV). The underlying causes of neuropathic pain are most likely not due to direct infection of the nervous system by active virus. The goal of this study was to determine whether epineural exposure to the HIV-1 envelope protein gp120 could lead to chronic painful peripheral neuropathy. Two doses of gp120 or BSA control were transiently delivered epineurally via oxidized cellulose wrapped around the rat sciatic nerve. Animals were assessed for neuropathic pain behaviors at several intervals from 1-30 days following nerve surgery. Allodynia and hyperalgesia were observed within 1-3 days following gp120 and sustained throughout the testing period. The gp120-exposed sciatic nerve exhibited early but transient pathology, notably axonal swelling and increased tumor necrosis factor alpha (TNF-alpha) within the nerve trunk. In contrast, intense astrocytic and microglial activation was observed in the spinal cord, and this gliosis persisted for at least 30 days following epineural gp120, in parallel with neuropathic pain behaviors. These findings demonstrate that limited peripheral nerve exposure to HIV protein can induce persistent painful sensory neuropathy that may be sustained and magnified by long-term spinal neuropathology.

Spinal allografts of adrenal medulla block nociceptive facilitation in the dorsal horn.

Transplantation of chromaffin cells into the lumbar subarachnoid space has been found to produce analgesia, most conspicuously against chronic neuropathic pain. To ascertain the neurophysiological mechanism, we recorded electrical activity from wide-dynamic-range dorsal horn neurons in vivo, measuring the short-lasting homosynaptic facilitatory effect known as windup, which is induced by repetitive C-fiber input. Rats were given adrenal medulla allografts, or, as controls, striated-muscle allografts. The adrenal-transplanted rats showed analgesia 3--4 wk after transplantation, measured as a reduction in flinching reflexes 30--55 min after subcutaneous formalin injection. Recordings were made under halothane anesthesia, 3--7 days following the behavioral testing. The average C-fiber response and subsequent afterdischarge were facilitated severalfold in control rats by 1-Hz cutaneous electrical stimulation. Such facilitation was essentially absent in adrenal-transplanted animals and also in the A-fiber response of both preparations. Extirpation of transplanted tissue several hours prior to recording did not significantly affect this difference. In conclusion, the adrenal transplants block short-term spinal nociceptive facilitation, probably by stimulating some persistent cellular process that may be an important determinant, but not the only one, of their analgesic effect.

Automated method for isolation of adrenal medullary chromaffin cells from neonatal porcine glands.

An automated method for the isolation of neonatal porcine adrenal chromaffin cells is described. Adrenal chromaffin cells are potentially useful for therapeutic transplantation, but current isolation methodology suffers from labor intensiveness and variability in yield and viability due to imprecision of manual techniques, enzyme purity, and gland age and species. The described approach utilizes an adaptation of an automated procedure previously described for isolation of pancreatic islets. Results from neonatal porcine adrenal glands revealed consistent cell yields with high (approximately 99%) viability. Catecholamine assays showed that the resultant cultures continue to produce and secrete norepinephrine and epinephrine. Immunocytochemical analysis indicated that the majority of cells in the preparation are chromaffin cells and adrenal cortical cells. The procedure meets the following requirements: 1) minimal traumatic action on the adrenal chromaffin cells, 2) continuous digestion in which the adrenal cells that are progressively liberated can be saved from further mechanical action, 3) minimal human intervention in the digestion process, and 4) high yield and viability of the isolated adrenal chromaffin cells.

Alterations in endogenous brain beta-endorphin release by adrenal medullary transplants in the spinal cord.

While transplants of adrenal medullary cells into the spinal subarachnoid space may produce antinociception via inhibition of spinal pain transmission pathways, alterations at higher central nervous system (CNS) centers have not been addressed. Recent findings suggest that prolonged noxious stimulation results in release of endogenous beta-endorphin in the brain, possibly as a compensatory mechanism to reduce nociception. The goal of this study was to determine whether adrenal medullary transplants in the spinal subarachnoid space alter endogenous beta-endorphin secretion in the hypothalamic arcuate nucleus, its principal CNS source. Pain behaviors and arcuate beta-endorphin secretion by microdialysis were monitored during the formalin pain test in animals with spinal adrenal medullary or control transplants. Basal levels of extracellular beta-endorphin were 3-fold higher in adrenal medullary-implanted than in controls. In control animals, formalin induced robust pain behaviors and a marked transient increase in beta-endorphin release 30-60 min following injection. In contrast, pain behaviors were attenuated and the formalin-induced increase in beta-endorphin was completely blocked in adrenal medullary implanted animals. Findings from these studies indicate that adrenal medullary transplants in the spinal subarachnoid space can alter beta-endorphin release in the arcuate nucleus both basally and in response to noxious stimuli. Thus, spinally placed adrenal medullary transplants not only alter local spinal cord pharmacology, but can alter endogenous neurochemistry at higher pain processing centers as well.

Generation and initial characterization of conditionally immortalized chromaffin cells.

Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.

Adrenal medullary explants as an efficient tool for pain control: adhesive biomolecular components are involved in graft function ex vivo.

Adrenal medullary (AM) tissue transplantation into the central nervous system has been reported as a potential source of opioid peptides and catecholamines, which have analgesic effects useful in the control of chronic pain. Clinical trials, involving allogeneic graft of whole tissue explants into the subarachnoid space of the lumbar spinal cord, have already been reported. The aim of the present study was to determine whether adhesion and function of AM explants were related in some extent and how this relationship could account for improvement of AM tissue in terms of analgesic activity before grafting. Our experiments demonstrated a significant correlation between the adherent state of AM organoids during culture and a sustained secretion of Met-enkephalin and catecholamines by chromaffin cells (CC). These findings suggest that optimal culture condition for AM organoid adhesion can be defined for maintenance of tissue, prior to transplantation. Using immunocytochemistry, flow cytometry, and ELISA assays we showed that different cell adhesion molecules (CAMs) and extracellular matrix ECM proteins were expressed and released by AM cells during culture. Adherent AM organoids expressed increased levels of specific neural CAMs (NCAM and HNK-1 epitope) and integrin chains (beta1, alpha1, alpha2, alpha4, alpha5) and deposited markedly higher levels of fibronectin, but also laminin and collagen IV. Those molecules and probably adhesion processes they control might be involved in the maintenance of the CC-secreting neuroendocrine phenotype through cellular signaling pathways.

Spinal CSF from rats with painful peripheral neuropathy evokes catecholamine release from chromaffin cells in vitro.

The environment presented by host tissue may influence cellular transplants in the CNS depending on injury or disease. Here we examined whether chronic pain alters cerebrospinal fluid (CSF), thereby enhancing the analgesic effect of transplanted adrenal cells. CSF samples were taken intracisternally from rats with neuropathic pain induced by chronic constriction injury of the sciatic nerve. The samples were applied to cultured bovine chromaffin-cell clusters while catecholamine release was measured by fast cyclic voltammetry. This caused marked and sustained elevations in catecholamine levels, compared to CSF from sham-operated controls, which were reversible by the nicotinic antagonist mecamylamine. These results suggest that chronic neuropathic pain produces increased CSF levels of secretogogues for chromaffin cells, and illustrates the importance of host microenvironmental factors in determining graft function.

Initial characterization of the transplant of immortalized chromaffin cells for the attenuation of chronic neuropathic pain.

Cultures of embryonic day 17 (E17) rat adrenal and neonatal bovine adrenal cells were conditionally immortalized with the temperature-sensitive allele of SV40 large T antigen (tsTag) and chromaffin cell lines established. Indicative of the adrenal chromaffin phenotype, these cells expressed immunoreactivity (ir) for tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines. At permissive temperature in vitro (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffin-like morphology, and contain detectable TH-ir. At nonpermissive temperature in vitro (39 degrees C), these cells stop proliferating and express increased TH-ir. When these immortalized chromaffin cells were transplanted in the lumbar subarachnoid space of the spinal cord I week after a unilateral chronic constriction injury (CCI) of the rat sciatic nerve, they survived longer than 7 weeks on the pia mater around the spinal cord and continued to express TH-ir. Conversely, grafted chromaffin cells lost Tag-ir after transplant and Tag-ir was undetectible in the grafts after 7 weeks in the subarachnoid space. At no time did the grafts form tumors after transplant into the host animals. These grafted chromaffin cells also expressed immunoreactivities for the other catecholamine-synthesizing enzymes 7 weeks after grafting, including: dopamine-beta-hydroxylase (DbetaH) and phenylethanolamine-N-methyltransferase (PNMT). The grafted cells also expressed detectable immunoreactivities for the opioid met-enkephalin (ENK), the peptide galanin (GAL), and the neurotransmitters y-aminobutyric acid (GABA) and serotonin (5-HT). Furthermore, after transplantation, tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI were significantly reduced during a 2-8-week period, related to the chromaffin cell transplants. The maximal antinociceptive effect occurred 1-3 weeks after grafting. Control adrenal fibroblasts, similarly immortalized and similarly transplanted after CCI, did not express any of the chromaffin antigenic markers, and fibroblast grafts had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that embryonic and neonatal chromaffin cells can be conditionally immortalized and will continue to express the phenotype of primary chromaffin cells in vitro and in vivo; grafted cells will ameliorate neuropathic pain after nerve injury and can be used as a homogeneous source to examine the mechanisms by which chromaffin transplants reverse chronic pain. The use of such chromaffin cell lines that are able to deliver antinociceptive molecules in models of chronic pain after nerve and spinal cord injury (SCI) offers a novel approach to pain management.

NMDA-induced spinal hypersensitivity is reduced by naturally derived peptide analog [Ser1]histogranin.

N-methyl-D-aspartate (NMDA) receptor activation is thought to initiate a cellular cascade of events in the spinal cord that leads to neuronal hyperactivation and exaggerated persistent pain behaviors. Previous studies have demonstrated that implantation of adrenal medullary tissue into the spinal subarachnoid space reduces abnormal pain behaviors such as hyperalgesia and allodynia, possibly by intervening in the NMDA hyperexcitability cascade. Histogranin is a 15-amino acid peptide possessing NMDA receptor antagonist activity that has been isolated from adrenal medullary tissue. The present study examined the ability of stable analog [Ser1]histogranin to reduce abnormal pain-related behaviors induced in rats by direct activation of spinal NMDA receptors. The intrathecal injection of NMDA (5.0, 10.0, 20.0 nmol) produced significant thermal and mechanical hyperalgesia and tactile allodynia in a dose-related fashion. [Ser1]histogranin injected intrathecally prior to NMDA injections dose dependently attenuated or completely blocked hyperalgesia and allodynia. In addition, [Ser1]histogranin administration following NMDA-induction of abnormal pain behaviors reversed these effects. These results demonstrate that a naturally derived adrenal medullary neuropeptide can prevent and reverse NMDA-mediated spinal hyperexcitability. The distinct profile and robust activity of [Ser1]histogranin suggest novel alternative approaches in the management of pain and other CNS disorders involving abnormal excitatory neurotransmission.