A robust evaluation of TDP-43, poly GP, cellular pathology and behavior in a AAV-C9ORF72 (G 4 C 2 ) 66 mouse model.

The G 4 C 2 hexanucleotide repeat expansion in C9ORF72 is the major genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Despite considerable efforts, the development of mouse models of C9-ALS/FTD useful for therapeutic development has proven challenging due to the intricate interplay of genetic and molecular factors underlying this neurodegenerative disorder, in addition to species differences. This study presents a robust investigation of the cellular pathophysiology and behavioral outcomes in a previously described AAV mouse model of C9-ALS expressing 66 G 4 C 2 hexanucleotide repeats. Despite displaying key molecular ALS pathological markers including RNA foci, dipeptide repeat (DPR) protein aggregation, p62 positive stress granule formation as well as mild gliosis, the AAV-(G 4 C 2 ) 66 mouse model in this study exhibits negligible neuronal loss, no motor deficits, and functionally unimpaired TAR DNA-binding protein-43 (TDP-43). While our findings indicate and support that this is a robust and pharmacologically tractable model for investigating the molecular mechanisms and cellular consequences of (G 4 C 2 ) repeat driven DPR pathology, it is not suitable for investigating the development of disease associated neurodegeneration, TDP-43 dysfunction, gliosis, and motor performance. Our findings underscore the complexity of ALS pathogenesis involving genetic mutations and protein dysregulation and highlight the need for more comprehensive model systems that reliably replicate the multifaceted cellular and behavioral aspects of C9-ALS.

Mechanisms of amyloid-ß34 generation indicate a pivotal role for BACE1 in amyloid homeostasis.

The beta­site amyloid precursor protein (APP) cleaving enzyme (BACE1) was discovered due to its "amyloidogenic" activity which contributes to the production of amyloid-beta (Aß) peptides. However, BACE1 also possesses an "amyloidolytic" activity, whereby it degrades longer Aß peptides into a non­toxic Aß34 intermediate. Here, we examine conditions that shift the equilibrium between BACE1 amyloidogenic and amyloidolytic activities by altering BACE1/APP ratios. In Alzheimer disease brain tissue, we found an association between elevated levels of BACE1 and Aß34. In mice, the deletion of one BACE1 gene copy reduced BACE1 amyloidolytic activity by ~ 50%. In cells, a stepwise increase of BACE1 but not APP expression promoted amyloidolytic cleavage resulting in dose-dependently increased Aß34 levels. At the cellular level, a mislocalization of surplus BACE1 caused a reduction in Aß34 levels. To align the role of γ-secretase in this pathway, we silenced Presenilin (PS) expression and identified PS2-γ-secretase as the main γ-secretase that generates Aß40 and Aß42 peptides serving as substrates for BACE1's amyloidolytic cleavage to generate Aß34.

Neurodegenerative Disease-Related Proteins within the Epidermal Layer of the Human Skin.

There is increasing evidence suggesting that amyloidogenic proteins might form deposits in non-neuronal tissues in neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. However, the detection of these aggregation-prone proteins within the human skin has been controversial. Using immunohistochemistry (IHC) and mass spectrometry tissue imaging (MALDI-MSI), fresh frozen human skin samples were analyzed for the expression and localization of neurodegenerative disease-related proteins. While α-synuclein was detected throughout the epidermal layer of the auricular samples (IHC and MALDI-MSI), tau and Aß34 were also localized to the epidermal layer (IHC). In addition to Aß peptides of varying length (e.g., Aß40, Aß42, Aß34), we also were able to detect inflammatory markers within the same sample sets (e.g., thymosin ß-4, psoriasin). While previous literature has described α-synuclein in the nucleus of neurons (e.g., Parkinson's disease), our current detection of α-synuclein in the nucleus of skin cells is novel. Imaging of α-synuclein or tau revealed that their presence was similar between the young and old samples in our present study. Future work may reveal differences relevant for diagnosis between these proteins at the molecular level (e.g., age-dependent post-translational modifications). Our novel detection of Aß34 in human skin suggests that, just like in the brain, it may represent a stable intermediate of the Aß40 and Aß42 degradation pathway.

Patients' experience of partial tooth loss and expectations to treatment: a qualitative study in Danish and Swedish patients.

Knowledge of impairments, wishes and expectations is essential to make correct decisions regarding oral rehabilitation. The purpose of this study was to investigate discomforts, wishes and expectations in patients' with partial edentulism before entering oral rehabilitation. In Copenhagen, Denmark, and Malmö, Sweden, respectively, 20 patients with partial edentulism seeking rehabilitation were interviewed in a semistructured qualitative manner. The interviews were transcribed and analysed yielding overall domains. Six themes appeared as overall domains: (i) experienced impairments, (ii) experienced social awareness, (iii) expectation to treatment, (iv) expectation to durability/survival, (v) coping strategies dealing with the tooth loss including explanations of the tooth loss and (vi) modifications to experienced impairment. The impairments were mostly experienced as problems in social settings. Most participants expressed a simple wish to function normally; a fixed solution was preferred. Many Danish participants accepted a removable solution whereas only few Swedish participants did so. The domains 'coping strategies' and 'modifications' were not part of the chosen topics of interest, indicating a high wish of the participants to explain their tooth loss and how they coped with it. In conclusion, a large degree of social impairment was found in the patient group along with several coping strategies. The impairments were modified by a number of factors indicating that highly individualised care and treatment is needed. A state of normality was described as the primary treatment wish with a higher acceptance of removable solutions in Denmark than in Sweden. For final decision-making, surrounding factors seemed to influence the patients' choices.

An in vivo role for Rho kinase activation in the tumour vascular disrupting activity of combretastatin A-4 3-O-phosphate.

BACKGROUND AND PURPOSE: Combretastatin A-4 3-O-phosphate (CA4P) is in clinical trial as a tumour vascular disrupting agent (VDA) but the cause of blood flow disruption is unclear. We tested the hypothesis that activation of Rho/Rho kinase (ROCK) is fundamental to the effects of this drug in vivo. EXPERIMENTAL APPROACH: Mouse models of human colorectal carcinoma (SW1222 and LS174T) were used. Effects of the ROCK inhibitor, Y27632, alone or in combination with CA4P, on ROCK activity, vascular function, necrosis and immune cell infiltration in solid tumours were determined. Mean arterial BP (MABP) was measured to monitor systemic interactions and the vasodilator, hydralazine, was used to control for the hypotensive effects of Y27632. KEY RESULTS: Y27632 caused a rapid drop in blood flow in SW1222 tumours, with recovery by around 3 h, which was paralleled by MABP changes. Y27632 pretreatment reduced CA4P-induced ROCK activation and partially blocked CA4P-induced tumour vascular effects, in both tumour types. Y27632 also partially inhibited CA4P-induced tumour necrosis and was associated with reduced immune cell infiltration in SW1222 tumours. Hydralazine caused a similar hypotensive effect as Y27632 but had no protective effect against CA4P treatment. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that ROCK activity is critical for full manifestation of the vascular activity of CA4P in vivo, providing the evidence for pharmacological intervention to enhance the anti-tumour efficacy of CA4P and related VDAs.

Analysis of the perceived oral treatment need using Andersen's behavioural model.

OBJECTIVES: The aim of this study was to investigate the influence of specific components of Andersen's behavioural model on adult individuals' perceived oral treatment need. METHODS: A questionnaire was sent to a randomly selected sample of 9,690 individuals, 20 to 89 years old, living in Skåne, Sweden. The 58 questions, some with follow-up questions, were answered by 6,123 individuals; a 63% response rate. Selected for inclusion in the multivariate logistic regression analysis were those questions relating to Andersen's behavioural model, phase five. Responses to "How do you rate your oral treatment need today?" were used as a dependent variable. The 62 questions chosen as independent variables represented the components: individual characteristics, health behaviour and outcomes in the model. RESULTS: Of the independent variables, 24 were significant at the p< or =0.05 level. Low educational level, previously unmet perceived oral TREATMENT need, frequent visiting pattern, perception of worse oral health than one's peers, an external locus of control, and to have received information from one's dental caregiver about a need for oral treatment were all highly significant (p<0.001) variables correlating with high self-perceived oral treatment need. CONCLUSION: The Andersen behavioural model can be a useful theoretical tool for the study of perceived oral treatment need.

An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections.

A fully automatic segmentation and morphological analysis algorithm for the analysis of microvessels from CD31 immunostained histological tumour sections is presented. Development of the algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, to provide the seeds for a 'region-growing' method for object segmentation in the 3D hue, saturation, value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing objects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels. The automatic segmentation was validated against a hand-segmented set of 44 images from three different SW1222 human colorectal carcinomas xenografted into mice. 96.3 ± 0.9% of pixels were found to be correctly classified. Automated segmentation was carried out on a further 53 images from three histologically distinct mouse fibrosarcomas (MFs) for morphological comparison with the SW1222 tumours. Four morphometric measurements were calculated for each segmented vessel: vascular area (VA), ratio of lumen area to vascular area (lu/VA), eccentricity (e), and roundness (ro). In addition, the total vascular area relative to tumour tissue area (rVA) was calculated. lu/VA, e and ro were found to be significantly smaller in MF tumours than in SW1222 tumours (p < 0.05; unpaired t-test). The algorithm is available through the website http://www.caiman.org.uk where images can be uploaded, processed and sent back to users. The output from CAIMAN consists of the original image with boundaries of segmented vessels overlaid, the calculated parameters and a Matlab file, which contains the segmentation that the user can use to derive further results.

Tramadol-induced oral dryness and pilocarpine treatment: effects on total protein and IgA.

Pilocarpine induces a profuse flow of saliva, and it may re-establish saliva production in cases of drug-induced oral dryness. The aim of the study (a sub-study to the previous trial investigating the pilocarpine fluid effects in individuals suffering from drug-induced dry mouth) was to search for saliva quality changes induced by the treatments. Sixty-five individuals were enrolled in a randomized, double-blind, placebo-controlled trial. The subjects received tramadol to induce oral dryness. Secretion rate was measured before and after tramadol, and then after pilocarpine, placebo, or no treatment. All saliva was analyzed for its protein and IgA content in the pilocarpine (n=15) and placebo groups (n=12). At baseline, the flow of saliva was 0.47±0.05ml/min, the protein output 0.17±0.2mg/min and the IgA output 0.022±0.002mg/min. After tramadol treatment (50mg 3×/day over two days), the flow was reduced by 64%, protein output by 52% and the IgA output by 38%. While placebo treatment did not affect any of the variables, the flow was 120%, the protein output 193% and the IgA output 83% of the baseline characteristics after pilocarpine treatment (5mg). Thus, the pilocarpine-induced increase in the flow rate in the state of tramadol-induced oral dryness results in saliva with a well preserved protein concentration but with a decrease in IgA concentration. However, compared to baseline, there was neither a decrease in output nor in concentration of IgA.

Cortical spreading depression-associated cerebral blood flow changes induced by mechanical stimulation are modulated by AMPA and GABA receptors.

Migraine is one of the most prevalent neurological disorders with some 30% of patients additionally suffering from focal neurological disturbances: the aura. The underlying mechanism behind the aura is generally considered to be a form of cortical spreading depression (CSD). We used mechanical stimulation to induce hyperaemia associated with CSD in cats and rats, and studied the effect of a glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor, antagonist, and gamma-aminobutyric acid (GABA)(A) and GABA(B) receptor agonists, to understand better the pharmacology of CSD. All three were able to inhibit CSD-associated cerebral blood flow changes in the rat and in a proportion of cats studied; non-responders showed altered speed of propagation and time to induction. The data suggest AMPA and GABA receptors may be targets of migraine therapy in inhibiting CSD and thus may alter the frequency of migraine aura.

Neurobiology of migraine.

Migraine is a complex disorder of the brain whose mechanisms are only now being unraveled. It is common, disabling and economically costly. The pain suggests an important role of the nociceptive activation, or the perception of activation, of trigeminal cranial, particularly intracranial afferents. Moreover, the involvement of a multi-sensory disturbance that includes light, sound and smells, as well as nausea, suggests the problem may involve central modulation of afferent traffic more broadly. Brain imaging studies in migraine point to the importance of sub-cortical structures in the underlying pathophysiology of the disorder. Migraine may thus be considered an inherited dysfunction of sensory modulatory networks with the dominant disturbance affecting abnormal processing of essentially normal neural traffic.

Suitability of Smopex-102 cation-exchange fiber for analytical purposes and drug monitoring.

The present study aimed to evaluate the suitability of Smopex-102 cation-exchange fiber for the separation of acidic and basic model drugs from biological fluids (e.g. serum) prior to chromatographic analysis. In addition, the interactions of the drugs with the fiber were studied. The study found that basic antidepressant model drugs bound to a considerably greater extent than acidic drugs to poly(acrylic acid) (PAA) grafted Smopex-102 cation-exchange fiber from 25 mM HEPES buffer (pH 7.0) and spiked serum. Drug binding from serum decreased except for acidic drugs due to drug distribution between serum proteins and cation-exchange fiber. Electrostatic interactions were possibly the most important factors affecting drug binding to the fiber. Basic drugs were released most effectively from the fiber by using acetic acid (mean released amount 123.7 +/- 36.3% and mean absolute recovery 95.4 +/- 23.8%). Results demonstrated that the cation-exchange fiber evaluated might be a potential material for separating basic drugs from protein-free and proteinaceous (e.g. serum) liquid solutions for subsequent monitoring and evaluation. However, the drug release solution and release time must be optimized more precisely in order to validate described sample preparation method for each basic drug.

Measuring the velocity of fluorescently labelled red blood cells with a keyhole tracking algorithm.

In this paper we propose a tracking algorithm to measure the velocity of fluorescently labelled red blood cells travelling through microvessels of tumours, growing in dorsal skin flap window chambers, implanted on mice. Preprocessing removed noise and artefacts from the images and then segmented cells from background. The tracking algorithm is based on a 'keyhole' model that describes the probable movement of a segmented cell between contiguous frames of a video sequence. When a history of cell movement exists, past, present and a predicted landing position of the cells will define two regions of probability that resemble the shape of a keyhole. This keyhole model was used to determine if cells in contiguous frames should be linked to form tracks and also as a postprocessing tool to join split tracks and discard links that could have been formed due to noise or uncertainty. When there was no history, a circular region around the centroid of the parent cell was used as a region of probability. Outliers were removed based on the distribution of the average velocities of the tracks. Since the position and time of each cell is recorded, a wealth of statistical measures can be obtained from the tracks. The algorithm was tested on two sets of experiments. First, the vasculatures of eight tumours with different geometries were analyzed; average velocities ranged from 86 to 372 microm s(-1), with minimum and maximum track velocities 7 and 1212 microm s(-1), respectively. Second, a longitudinal study of velocities was performed after administering a vascular disrupting agent to two tumours and the time behaviour was analyzed over 24 h. In one of the tumours there is a complete shutdown of the vasculature whereas in the other there is a clear decrease of velocity at 30 min, with subsequent recovery by 6 h. The tracking algorithm enabled the simultaneous measurement of red blood cell velocity in multiple vessels within an intravital video sequence, enabling analysis of heterogeneity of flow and response to treatment in mouse models of cancer.

Dopamine and migraine: biology and clinical implications.

In the last 30 years dopamine has been considered as playing a role in the pathogenesis of migraine. The literature indicates that migraineurs are hypersensitive to dopamine agonists with respect to some of the premonitory symptoms of migraine such as nausea and yawning. There are various non-specific dopamine D(2) receptor antagonists that show good clinical efficacy in migraine, and also a number of polymorphisms of dopaminergic genes related to migraine. Animal studies have also shown that dopamine receptors are present in the trigeminovascular system, the area believed to be involved in headache pain, and neuronal firing here is reduced by dopamine agonists. There appears to be little effect of dopamine on peripheral trigeminal afferents. We assess some of the limitations of the clinical studies with regard to the therapeutics, and those found in the studies that discovered differences in genetic polymorphisms in migraine, and consider the implications of this on a dopaminergic hypothesis of migraine.

Modulation of nociceptive dural input to the trigeminal nucleus caudalis via activation of the orexin 1 receptor in the rat.

Migraine pathophysiology is thought to involve the trigeminal innervation of the dura mater and intracranial blood vessels. Electrical stimulation of dural blood vessels is painful in humans and causes activation of neurons in the caudal-most portion of the trigeminal nucleus in experimental animals. The hypothalamic neuropeptides orexin A and B are selectively synthesized in the lateral and posterior hypothalamus, and recent findings have implicated their involvement in nociceptive processing. To evaluate the potential for orexin receptor modulation of trigeminovascular nociceptive afferents, we examined the effects of intravenous orexin A and B on responses of neurons in the trigeminal nucleus caudalis. To dissect the receptor pharmacology of responses to stimulation we utilized the novel orexin 1 receptor (OX(1)R) antagonist N-(2-methyl-6-benzoxazolyl)-N''-1,5-naphthyridin-4-yl urea (SB-334867). Orexin A 30 microg/kg (F(1.9,9.8) = 21.93, P < 0.001) and 50 microg/kg (F(3.2,16.4) = 3.28, P < 0.045) inhibited the A-fibre responses to dural electrical stimulation over 60 min. Maximum inhibition was achieved at 25 min for both 30 microg/kg (t(5) = 19.83, n = 6, P < 0.001) and 50 microg/kg (t(5) = 7.74, n = 6, P < 0.001). The response with orexin A 30 microg/kg was reversed by pretreatment with the OX(1)R antagonist SB-334867 (F(3.5,17.5) = 0.49, P = 0.73), which had no effect when given alone. Orexin B and control vehicle administration had no significant effect on trigeminal neuronal firing. The current study demonstrates that orexin A is able to inhibit A-fibre responses to dural electrical stimulation via activation of the OX(1)R.

Intravital microscopy on a closed cranial window in mice: a model to study trigeminovascular mechanisms involved in migraine.

The purpose of the study was to develop a mouse model to study trigeminovascular mechanisms using intravital microscopy on a closed cranial window. In addition, we studied exogenous and endogenous calcitonin gene-related peptide (CGRP)-mediated vasodilation in dural arteries. Arteries in C57BL/6Jico mice were constricted with endothelin-1, which reduced the baseline diameter by 65-75%. Subsequently, vasodilation was induced by alpha-CGRP, capsaicin or transcranial electrical stimulation of perivascular trigeminal nerves in the absence or presence of different concentrations of BIBN4096BS or sumatriptan. Both alpha-CGRP and capsaicin induced vasodilation in preconstricted arteries. Transcranial electrical stimulation also induced current-dependent relaxation of dural arteries with 100 microA producing maximal dilation in the control group. BIBN4096BS blocked the responses evoked by alpha-CGRP and capsaicin, as well as electrical stimulation, whereas sumatriptan attenuated only vasodilation induced by electrical stimulation. This model is likely to prove useful in dissecting elements of the trigeminovascular system and for exploring pathophysiological aspects of migraine, especially in future studies using transgenic mice with mutations relevant to those observed in patients with migraine.

Animal models of migraine: looking at the component parts of a complex disorder.

Animal models of human disease have been extremely helpful both in advancing the understanding of brain disorders and in developing new therapeutic approaches. Models for studying headache mechanisms, particularly those directed at migraine, have been developed and exploited efficiently in the last decade, leading to better understanding of the potential mechanisms of the disorder and of the action for antimigraine treatments. Model systems employed have focused on the pain-producing cranial structures, the large vessels and dura mater, in order to provide reproducible physiological measures that could be subject to pharmacological exploration. A wide range of methods using both in vivo and in vitro approaches are now employed; these range from manipulation of the mouse genome in order to produce animals with human disease-producing mutations, through sensitive immunohistochemical methods to vascular, neurovascular and electrophysiological studies. No one model system in experimental animals can explain all the features of migraine; however, the systems available have begun to offer ways to dissect migraine's component parts to allow a better understanding of the problem and the development of new treatment strategies.

Occipital afferent activation of second order neurons in the trigeminocervical complex in rat.

Stimulation of the greater occipital nerve produces excitation of second order neurons in the trigeminocervical complex. Given that neck pain is very common in primary headache disorders, this convergent excitation may play a role in pain referral from cervical structures. While previous studies have demonstrated a physiological model for this convergence, this study sought an anatomical approach to examine the distribution of second order neurons in the trigeminocervical complex receiving greater occipital nerve input. In addition, the role of glutamatergic NMDA receptor activation within the trigeminocervical complex in response to cervical afferents was studied. Noxious stimulation of the occipital muscle in rat using mustard oil and mineral oil produced significantly altered Fos expression in the trigeminocervical complex compared with the surgical control (H(4)=31.3, P<0.001, Kruskal-Wallis). Baseline expression was 11 (median, range 4, 17) fos positive cells in the trigeminocervical complex, occipital muscle treated with mustard oil produced 23 (17, 33) and mineral oil a smaller effect of 19 (15, 25) fos positive cells, respectively (P=0.046). The effects of both mustard and mineral oil were reversed by the NMDA-receptor antagonist MK801. This study introduces a model for examining trigeminocervical complex activity after occipital afferent stimulation in the rat that has good anatomical resolution and demonstrates involvement of glutamatergic NMDA receptors at this important synapse.

Orexin 1 receptor activation attenuates neurogenic dural vasodilation in an animal model of trigeminovascular nociception.

The pathophysiology underlying the pulsating quality of the pain of a migraine attack is not fully understood, although trigeminal vascular afferents containing the sensory neuropeptide calcitonin gene-related peptide (CGRP) must have a role. Antimigraine drugs, such as triptans, serotonin 5-hydroxytryptamine(1B/1D) receptor agonists, reproducibly block neurogenic vasodilation associated with CGRP release. We examined the effects of the hypothalamic neuropeptides orexin A and orexin B on neurogenic dural vasodilation, dissecting out the receptor pharmacology with the novel orexin 1 (OX1) receptor antagonist N-(2-methyl-6-benzoxazolyl)-N''-1,5-naphthyridin-4-yl urea (SB-334867). Electrical stimulation of dural afferents (50-300 microA) resulted in reproducible dural vasodilation of 136 +/- 9%. Orexin A 30 microg kg(-1), but not 3 and 10 microg kg(-1), inhibited the dilation brought about by electrical stimulation over 60 min and maximally after 15 min by 60% (t7= 7.138; P < 0.001; n = 8). This response was reversed by pretreatment with the OX1 receptor antagonist SB-334867. Addition of CGRP(8-37) at the point of maximal effect of orexin A produced a further significant decrease in neurogenic dural vasodilation compared with orexin A only. CGRP administration (1 microg kg(-1)) produced a reproducible dural blood vessel dilation of 145 +/- 7% that was not inhibited by intravenous administration of orexin A (30 microg kg(-1)). Orexin B had no significant effect even at the highest dose. The current study demonstrates that orexin A is able to inhibit neurogenic dural vasodilation via activation of the OX1 receptor, resulting in inhibition of prejunctional release of CGRP from trigeminal neurons.

Calcium channels modulate nociceptive transmission in the trigeminal nucleus of the cat.

Clinical observations and genetic studies have suggested a role for high-threshold voltage-dependent calcium channels (VDCCs) in the pathogenesis of migraine. This study investigated the role of P/Q-, L- and N-type VDCCs in post-synaptic action potential generation in trigeminovascular nociceptive afferents in the trigeminocervical complex (TCC) of the cat in vivo. Trigeminovascular nociceptive afferents were identified in the TCC by electrical stimulation of the superior sagittal sinus. Forty-six cell bodies were identified by their response to microiontophoresis of l-glutamate and their bipolar action potential shape. Blockade of VDCCs was accomplished by microiontophoresis of omega-agatoxin IVa/TK (P/Q-), omega-conotoxin GVIa (N-) and calciseptine (L-type). Non-selective antagonism was studied using cadmium ions. Non-selective blockade of high threshold VDCC with cadmium resulted in a reduction in l-glutamate-evoked neuronal activity (P=0.01). Blockade of P/Q: TK- (P<0.001), IVA- (P=0.007), L- (P<0.001) and N-type (P<0.001) VDCCs resulted in significant reductions in post-synaptic action potential generation in response to l-glutamate. High threshold VDCCs, including P/Q-, L- and N-type VDCCs, can therefore modulate nociceptive transmission in the trigeminocervical complex in vivo. We discuss the evidence to suggest a role for VDCCs in the pathophysiology of primary headache disorders, and how abnormalities of function may contribute to their pathogenesis.