Late-Life Alcohol Exposure Does Not Exacerbate Age-Dependent Reductions in Mouse Spatial Memory and Brain TFEB Activity.

Alcohol consumption is believed to affect Alzheimer's disease (AD) risk, but the contributing mechanisms are not well understood. A potential mediator of the proposed alcohol-AD connection is autophagy, a degradation pathway that maintains organelle and protein homeostasis. Autophagy is in turn regulated through the activity of Transcription factor EB (TFEB), which promotes lysosome and autophagy-related gene expression. To explore the effect of alcohol on brain TFEB and autophagy, we exposed young (3-month old) and aged (23-month old) mice to two alcohol-feeding paradigms and assessed biochemical, transcriptome, histology, and behavioral endpoints. In young mice, alcohol decreased hippocampal nuclear TFEB staining but increased SQSTM1/p62, LC3-II, ubiquitinated proteins, and phosphorylated Tau. Hippocampal TFEB activity was lower in aged mice than it was in young mice, and Gao-binge alcohol feeding did not worsen the age-related reduction in TFEB activity. To better assess the impact of chronic alcohol exposure, we fed young and aged mice alcohol for four weeks before completing Morris Water and Barnes Maze spatial memory testing. The aged mice showed worse spatial memory on both tests. While alcohol feeding slightly impaired spatial memory in the young mice, it had little effect or even slightly improved spatial memory in the aged mice. These findings suggest that aging is a far more important driver of spatial memory impairment and reduced autophagy flux than alcohol consumption.

Models of Inflammation: Carrageenan Air Pouch.

The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this article has been extensively used to identify potential anti-inflammatory drugs. © 2021 Wiley Periodicals LLC. Basic Protocol: Air pouch model in the rat Alternate Protocol: Air pouch model in the mouse.

Models of Inflammation: Carrageenan- or Complete Freund's Adjuvant (CFA)-Induced Edema and Hypersensitivity in the Rat.

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the processing of pain sensation at CNS sites, the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. Detailed in this article are methods to elicit and measure carrageenan- and complete Freund's adjuvant (CFA)-induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections into either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) or by the trigeminal ganglion neurons (vibrissal pad). © 2021 Wiley Periodicals LLC.

Hippocampal mitogen-activated protein kinase phosphatase-1 regulates behavioral and systemic effects of chronic corticosterone administration.

Clinical reports indicate a bidirectional relationship between mental illness and chronic systemic diseases. However, brain mechanisms linking chronic stress and development of mood disorders to accompanying peripheral organ dysfunction are still not well characterized in animal models. In the current study, we investigated whether activation of hippocampal mitogen-activated protein kinase phosphatase-1 (MKP-1), a key factor in depression pathophysiology, also acts as a mediator of systemic effects of stress. First, we demonstrated that treatment with the glucocorticoid receptor (GR) agonist dexamethasone or acute restraint stress (ARS) significantly increased Mkp-1 mRNA levels within the rat hippocampus. Conversely, administration of the GR antagonist mifepristone 30 min before ARS produced a partial blockade of Mkp-1 upregulation, suggesting that stress activates MKP-1, at least in part, through upstream GR signaling. Chronic corticosterone (CORT) administration evoked comparable increases in hippocampal MKP-1 protein levels and produced a robust increase in behavioral emotionality. In addition to behavioral deficits, chronic CORT treatment also produced systemic pathophysiological effects. Elevated levels of renal inflammation protein markers (NGAL and IL18) were observed suggesting tissue damage and early kidney impairment. In a rescue experiment, the effects of CORT on development of depressive-like behaviors and increased NGAL and IL18 protein levels in the kidney were blocked by CRISPR-mediated knockdown of hippocampal Mkp-1 prior to CORT exposure. In sum, these findings further demonstrate that MKP-1 is necessary for development of enhanced behavioral emotionality, while also suggesting a role in stress mechanisms linking brain dysfunction and systemic illness such as kidney disease.

Strategies for Behaviorally Phenotyping the Transgenic Mouse.

The techniques and protocols to modify the mouse genome described in this volume allow researchers to produce genetic models of a remarkable number and breadth of human disease. The generation of gene-modified mice offers profoundly powerful approaches for bringing known or purported human gene disruptions into mouse models, but the degree to which the resultant mutant mouse recapitulates the complex physiological and behavioral features of the human disease state is a key variable in the ultimate usefulness of the mouse model organism. Accordingly, the behavioral characterization of mice with novel targeted gene mutations is an important initial step in determining the potential impact of a novel mouse model. This chapter addresses strategies useful in the initial observations of the animal that assist in directing the choice of secondary tests to assess more detailed aspects of potentially disrupted behaviors that may be relevant to the disease being modeled. An initial standardized, comprehensive screen that assesses general health, reflexes, and sensorimotor functions is the first step in characterizing behavioral phenotype, and results often suggest areas where more complex complementary behavioral assays may reveal more detailed disruption of normal behavior. This sequential, standardized approach reduces variability between subjects; this chapter also addresses approaches to reducing experimental artifacts due to handling, test order, testing facility environment, and other sources. This brief overview of behavioral phenotyping approaches is intended to provide practical information to streamline initial characterization of new mouse models and maximize the usefulness of efforts to use these models to study human health and disease.

Assessing complex movement behaviors in rodent models of neurological disorders.

Behavioral phenotyping is a crucial step in validating animal models of human disease. Most traditional behavioral analyses rely on investigator observation of animal subjects, which can be confounded by inter-observer variability, scoring consistency, and the ability to observe extremely rapid, small, or repetitive movements. Force-Plate Actimeter (FPA)-based assessments can quantify locomotor activity and detailed motor activity with an incredibly rich data stream that can reveal details of movement unobservable by the naked eye. This report describes four specific examples of FPA analysis of behavior that have been useful in specific rat or mouse models of human neurological disease, which show how FPA analysis can be used to capture and quantify specific features of the complex behavioral phenotypes of these animal models. The first example quantifies nociceptive behavior of the rat following injection of formalin into the footpad as a common model of persistent inflammatory pain. The second uses actimetry to quantify intense, rapid circling behaviors in a transgenic mouse that overexpresses human laminin α5, a basement membrane protein. The third example assesses place preference behaviors in a rat model of migraine headache modeling phonophobia and photophobia. In the fourth example, FPA analysis revealed a unique movement signature emerged with age in a digenic mutant mouse model of Tourette Syndrome. Taken together, these approaches demonstrate the power and usefulness of the FPA in the examination and quantification of minute details of motor behaviors, greatly expanding the scope and detail of behavioral phenotyping of preclinical models of human disease.

NCB5OR Deficiency in the Cerebellum and Midbrain Leads to Dehydration and Alterations in Thirst Response, Fasted Feeding Behavior, and Voluntary Exercise in Mice.

Cytosolic NADH-cytochrome-b5-oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues. We have previously reported that global ablation of NCB5OR in mice results in early-onset lean diabetes with decreased serum leptin levels and increased metabolic and feeding activities. The conditional deletion of NCB5OR in the mouse cerebellum and midbrain (conditional knock out, CKO mice) results in local iron dyshomeostasis and altered locomotor activity. It has been established that lesion to or removal of the cerebellum leads to changes in nutrient organization, visceral response, feeding behavior, and body weight. This study assessed whether loss of NCB5OR in the cerebellum and midbrain altered feeding or metabolic activity and had an effect on serum T3, cortisol, prolactin, and leptin levels. Metabolic cage data revealed that 16 week old male CKO mice had elevated respiratory quotients and decreased respiratory water expulsion, decreased voluntary exercise, and altered feeding and drinking behavior compared to wild-type littermate controls. Most notably, male CKO mice displayed higher consumption of food during refeeding after a 48-h fast. Echo MRI revealed normal body composition but decreased total water content and hydration ratios in CKO mice. Increased serum osmolality measurements confirmed the dehydration status of male CKO mice. Serum leptin levels were significantly elevated in male CKO mice while prolactin, T3, and cortisol levels remain unchanged relative to wild-type controls, consistent with elevated transcript levels for leptin receptors (short form) in the male CKO mouse cerebellum. Taken together, these findings suggest altered feeding response post starvation as a result of NCB5OR deficiency in the cerebellum.

Rats bred for low and high running capacity display alterations in peripheral tissues and nerves relevant to neuropathy and pain.

INTRODUCTION: Diet and activity are recognized as modulators of nervous system disease, including pain. Studies of exercise consistently reveal a benefit on pain. This study focused on female rats to understand differences related to metabolic status and peripheral nerve function in females. METHODS: Here, we investigated parameters of peripheral nerve function relevant to pain in rats selectively bred for high (high-capacity runners; HCR) or low endurance exercise capacity (low-capacity runners; LCR) resulting in divergent intrinsic aerobic capacities and susceptibility for metabolic conditions. RESULTS: LCR female rats have reduced mechanical sensitivity, higher intraepidermal nerve fiber density and TrkA-positive epidermal axons, increased numbers of Langerhans and mast cells in cutaneous tissues, and a higher fat content despite similar overall body weights compared to female HCR rats. Sensory and motor nerve conduction velocities, thermal sensitivity, and mRNA expression of selected genes relevant to peripheral sensation were not different. CONCLUSIONS: These results suggest that aerobic capacity and metabolic status influence sensory sensitivity and aspects of inflammation in peripheral tissues that could lead to poor responses to tissue damage and painful stimuli. The LCR and HCR rats should prove useful as models to assess how the metabolic status impacts pain.

Loss of NCB5OR in the cerebellum disturbs iron pathways, potentiates behavioral abnormalities, and exacerbates harmaline-induced tremor in mice.

Iron dyshomeostasis has been implicated in many diseases, including a number of neurological conditions. Cytosolic NADH cytochrome b5 oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues and is capable of reducing ferric iron in vitro. We previously reported that global gene ablation of NCB5OR resulted in early-onset diabetes and altered iron homeostasis in mice. To further investigate the specific effects of NCB5OR deficiency on neural tissue without contributions from known phenotypes, we generated a conditional knockout (CKO) mouse that lacks NCB5OR only in the cerebellum and midbrain. Assessment of molecular markers in the cerebellum of CKO mice revealed changes in pathways associated with cellular and mitochondrial iron homeostasis. (59)Fe pulse-feeding experiments revealed cerebellum-specific increased or decreased uptake of iron by 7 and 16 weeks of age, respectively. Additionally, we characterized behavioral changes associated with loss of NCB5OR in the cerebellum and midbrain in the context of dietary iron deprivation-evoked generalized iron deficiency. Locomotor activity was reduced and complex motor task execution was altered in CKO mice treated with an iron deficient diet. A sucrose preference test revealed that the reward response was intact in CKO mice, but that iron deficient diet consumption altered sucrose preference in all mice. Detailed gait analysis revealed locomotor changes in CKO mice associated with dysfunctional proprioception and locomotor activation independent of dietary iron deficiency. Finally, we demonstrate that loss of NCB5OR in the cerebellum and midbrain exacerbated harmaline-induced tremor activity. Our findings suggest an essential role for NCB5OR in maintaining both iron homeostasis and the proper functioning of various locomotor pathways in the mouse cerebellum and midbrain.

Models of Inflammation: Carrageenan- or Complete Freund's Adjuvant (CFA)-Induced Edema and Hypersensitivity in the Rat.

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the processing of pain sensation at CNS sites, the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. Detailed in this unit are methods to elicit and measure carrageenan- and complete Freund's adjuvant (CFA)-induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections into either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) or by the trigeminal ganglion neurons (vibrissal pad).

Behavioral effects and mechanisms of migraine pathogenesis following estradiol exposure in a multibehavioral model of migraine in rat.

Migraine is one of the most common neurological disorders, leading to more than 1% of total disability reported and over 68 million visits to emergency rooms or physician's offices each year in the United States. Three times as many women as men have migraine, and while the mechanism behind this is not well understood, 17ß-estradiol (estradiol) has been implicated to play a role. Studies have demonstrated that exposure to estrogen can lead to activation of inflammatory pathways, changes in sodium gated channel activity, as well as enhanced vasodilation and allodynia. Estradiol receptors are found in trigeminal nociceptors, which are involved in signaling during a migraine attack. The purpose of this study was to investigate the role of estradiol in migraine pathogenesis utilizing a multibehavioral model of migraine in rat. Animals were surgically implanted with a cannula system to induce migraine and behavior was assessed following exposure to a proestrus level of estradiol for total locomotor activity, light and noise sensitivity, evoked grooming patterns, and enhanced acoustic startle response. Results demonstrated decreased locomotor activity, increased light and noise sensitivity, altered facial grooming indicative of allodynia and enhanced acoustic startle. Further examination of tissue samples revealed increased expression of genes associated with inflammation and vasodilation. Overall, this study demonstrates exacerbation of migraine-like behaviors following exposure to estradiol and helps further explain the underlying mechanisms behind sex differences found in this common neurological disorder.

Acute estrogen surge enhances inflammatory nociception without altering spinal Fos expression.

Chronic pain is a major neurological disorder that can manifest differently between genders or sexes. The complex actions of sex hormones may underlie these differences; previous studies have suggested that elevated estrogen levels can enhance pain perception. The purpose of this study was to investigate the hypothesis that acute, activational effects of estradiol (E2) increase persistent inflammatory nociception, and anatomically where this modulation occurs. Spinal expression of Fos is widely used as a marker of nociceptive activation. This study used formalin-evoked nociception in ovariectomized (OVX) adult female rats and measured late-phase hindlimb flinching and Fos expression in the spinal cord, and their modification by acute estrogen supplementation similar to a proestrus surge. Six days after ovariectomy, female rats were injected subcutaneously (s.c.) with 10µg/kg E2 or vehicle. Twenty-four hours later, 50µL of 1.25% or 100µL of 5% formalin was injected into the right hindpaw; hindlimb flinches were counted, and spinal cords removed 2h after formalin injection. The numbers of Fos-expressing neurons in sections of the lumbar spinal cord were analyzed using immunohistochemistry. Formalin-induced inflammation produced a dose-dependent increase in late-phase hindlimb flinching, and E2 pretreatment increased flinching following 5%, but not 1.25% formalin injection. Despite the modification of behavior by E2, the number of spinal Fos-positive neurons was not altered by E2 pretreatment. These findings demonstrate that an acute proestrus-like surge in serum estrogen can produce a stimulus-intensity-dependent increase in inflammation-evoked nociceptive behavior. However, the lack of effect on spinal Fos expression suggests that this enhancement of nociceptive signaling by estrogen is independent of changes in peripheral activation of, expression of the immediate early gene Fos by, or signal throughput of spinal nociceptive neurons.

GABA pharmacology: the search for analgesics.

Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABAA and GABAB receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.

Gait analysis at multiple speeds reveals differential functional and structural outcomes in response to graded spinal cord injury.

Open-field behavioral scoring is widely used to assess spinal cord injury (SCI) outcomes, but has limited usefulness in describing subtle changes important for posture and locomotion. Additional quantitative methods are needed to increase the resolution of locomotor outcome assessment. This study used gait analysis at multiple speeds (GAMS) across a range of mild-to-severe intensities of thoracic SCI in the rat. Overall, Basso, Beattie, and Bresnahan (BBB) scores and subscores were assessed, and detailed automated gait analysis was performed at three fixed walking speeds (3.5, 6.0, and 8.5 cm/sec). Variability in hindpaw brake, propel, and stance times were analyzed further by integrating across the stance phase of stepping cycles. Myelin staining of spinal cord sections was used to quantify white matter loss at the injury site. Varied SCI intensity produced graded deficits in BBB score, BBB subscores, and spinal cord white matter and total volume loss. GAMS measures of posture revealed decreased paw area, increased limb extension, altered stance width, and decreased values for integrated brake, propel, and stance. Measures of coordination revealed increased stride frequency concomitant with decreased stride length, resulting in deviation from consistent forelimb/hindlimb coordination. Alterations in posture and coordination were correlated to impact severity. GAMS results correlated highly with functional and histological measures and revealed differential relationships between sets of GAMS dynamics and cord total volume loss versus epicenter myelin loss. Automated gait analysis at multiple speeds is therefore a useful tool for quantifying nuanced changes in gait as an extension of histological and observational methods in assessing SCI outcomes.

Exposure to bisphenol A exacerbates migraine-like behaviors in a multibehavior model of rat migraine.

Migraine is a common and debilitating neurological disorder suffered worldwide. Women experience this condition 3 times more frequently than men, with estrogen strongly implicated to play a role. Bisphenol A (BPA), a highly prevalent xenoestrogen, is known to have estrogenic activity and may have an effect in migraine onset, intensity, and duration through estrogen receptor signaling. It was hypothesized that BPA exposure exacerbates migraine symptoms through estrogen signaling and downstream activation of nociception related pathways. Utilizing a multibehavior model of migraine in ovariectomized female rats, changes in locomotion, light and sound sensitivity, grooming, and acoustic startle were examined. Furthermore, changes in the expression of genes related to estrogen (ERα, GPR30), and nociception (extracellular signal regulated kinase, ERK, sodium gated channel, Nav1.8, and fatty acid amide hydrolase, FAAH) were studied following behavioral experiments. The following results were obtained: BPA treatment significantly exacerbated migraine-like behaviors in rats. Rats exposed to BPA demonstrated decreased locomotion, exacerbated light and sound aversion, altered grooming habits, and enhanced startle reflexes. Furthermore, BPA exposure increased mRNA expression of estrogen receptors, total ERK mRNA and ERK activation, as well as Nav1.8, and FAAH mRNA, indicative of altered estrogen signaling and altered nociception. These results show that BPA, an environmentally pervasive xenoestrogen, exacerbates migraine-like behavior in a rat model and alters expression of estrogen and nociception-related genes.

Characterization of GABA receptors.

Described in this unit are ligand-binding assays for GABAA , GABAB , and the homomeric ρ GABAA (formerly GABAC ) receptor recognition sites in brain tissue. Although GABA binding sites are present in peripheral organs, most research is directed toward examining these receptors in the CNS. These assays may also be used to determine the affinity of an unlabeled compound for the GABA binding sites. Excluded from the unit are ligand-binding assays for other components of the GABAA receptor complex, such as the benzodiazepine or ion-channel binding sites.

Neurochemical analysis of primary motor cortex in chronic low back pain.

The involvement of the primary motor cortex (M1) in chronic low back pain (LBP) is a relatively new concept. Decreased M1 excitability and an analgesic effect after M1 stimulation have been recently reported. However, the neurochemical changes underlying these functional M1 changes are unknown. The current study investigated whether neurochemicals specific to neurons and glial cells in both right and left M1 are altered. N-Acetylaspartate (NAA) and myo-inositol (mI) were measured with proton magnetic resonance spectroscopy in 19 subjects with chronic LBP and 14 healthy controls. We also examined correlations among neurochemicals within and between M1 and relationships between neurochemical concentrations and clinical features of pain. Right M1 NAA was lower in subjects with LBP compared to controls (p = 0.008). Left M1 NAA and mI were not significantly different between LBP and control groups. Correlations between neurochemical concentrations across M1s were different between groups (p = 0.008). There were no significant correlations between M1 neurochemicals and pain characteristics. These findings provide preliminary evidence of neuronal depression and altered neuronalglial interactions across M1 in chronic LBP.

Vitamin D deficiency promotes skeletal muscle hypersensitivity and sensory hyperinnervation.

Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors ("pain-sensing" nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2-4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.

Mechanisms of pain modulation by sex hormones in migraine.

A number of pain conditions, acute as well as chronic, are much more prevalent in women, such as temporomandibular disorder (TMD), irritable bowel syndrome, fibromyalgia, and migraine. The association of female sex steroids with these nociceptive conditions is well known, but the mechanisms of their effects on pain signaling are yet to be deciphered. We reviewed the mechanisms through which female sex steroids might influence the trigeminal nociceptive pathways with a focus on migraine. Sex steroid receptors are located in trigeminal circuits, providing the molecular substrate for direct effects. In addition to classical genomic effects, sex steroids exert rapid nongenomic actions to modulate nociceptive signaling. Although there are only a handful of studies that have directly addressed the effect of sex hormones in animal models of migraine, the putative mechanisms can be extrapolated from observations in animal models of other trigeminal pain disorders, like TMD. Sex hormones may regulate sensitization of trigeminal neurons by modulating expression of nociceptive mediator such as calcitonin gene-related peptide. Its expression is mostly positively regulated by estrogen, although a few studies also report an inverse relationship. Serotonin (5-Hydroxytryptamine [5-HT]) is a neurotransmitter implicated in migraine; its synthesis is enhanced in most parts of brain by estrogen, which increases expression of the rate-limiting enzyme tryptophan hydroxylase and decreases expression of the serotonin re-uptake transporter. Downstream signaling, including extracellular signal-regulated kinase activation, calcium-dependent mechanisms, and cAMP response element-binding activation, are thought to be the major signaling events affected by sex hormones. These findings need to be confirmed in migraine-specific animal models that may also provide clues to additional ion channels, neuropeptides, and intracellular signaling cascades that contribute to the increased prevalence of migraine in women.

Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine.

OBJECTIVE: The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene-related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. BACKGROUND: Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate-buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. METHODS: Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP. RESULTS: Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP-related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP-related genes were upregulated by both IS and PBS applications. CONCLUSIONS: This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation.