Phillip E. Kunkler

TRPA1 receptors mediate environmental irritant-induced meningeal vasodilatation

&NA; The TRPA1 receptor is a member of the transient receptor potential (TRP) family of ion channels expressed in nociceptive neurons. TRPA1 receptors are targeted by pungent compounds from mustard and garlic and environmental irritants such as formaldehyde and acrolein. Ingestion or inhalation of these chemical agents causes irritation and burning in the nasal and oral mucosa and respiratory lining. Headaches have been widely reported to be induced by inhalation of environmental irritants, but it is unclear how these agents produce headache. Stimulation of trigeminal neurons releases CGRP and substance P and induces neurogenic inflammation associated with the pain of migraine. Here we test the hypothesis that activation of TRPA1 receptors is the mechanistic link between environmental irritants and peptide‐mediated neurogenic inflammation. Known TRPA1 agonists and environmental irritants stimulate CGRP release from dissociated rat trigeminal ganglia neurons and this release is blocked by a selective TRPA1 antagonist, HC‐030031. Further, TRPA1 agonists and environmental irritants increase meningeal blood flow following intranasal administration. Prior dural application of the CGRP antagonist, CGRP8–37, or intranasal or dural administration of HC‐030031, blocks the increases in blood flow elicited by environmental irritants. Together these results demonstrate that TRPA1 receptor activation by environmental irritants stimulates CGRP release and increases cerebral blood flow. We suggest that these events contribute to headache associated with environmental irritants.

Lower GABAA receptor binding in the amygdala and hypothalamus of spontaneously hypertensive rats

The central GABAergic system is associated with normal blood pressure regulation, but the role of GABA receptors in genetic hypertension remains unclear. This study was conducted to investigate GABAA receptor binding in several brain regions of spontaneously hypertensive (SHR) rats during development of hypertension. GABAA receptor binding was labeled with [35S]TBPS and was assessed by quantitative autoradiography with the aid of a computer-assisted image analysis system. Densities of GABAA receptor binding sites were significantly lower in all hypothalamic and amygdaloid nuclei evaluated in 4-week-old SHR rats, when compared with their age-matched normotensive Wistar-Kyoto rats. At 12 weeks of age, GABAA receptor binding remained significantly lower in the central amygdaloid nucleus and paraventricular hypothalamic nucleus of SHR rats. Collectively, the results suggest that GABAA receptors in these nuclei are likely to be involved in the initiation and maintenance of hypertension. In conclusion, this study supports a notion that downregulation of GABAA receptor binding occurs in the hypothalamus and amygdala of SHR rats and may play a role in genetic hypertension.

Galanin-containing neurons in the solitary nucleus and locus coeruleus of spontaneously hypertensive rats are associated with genetic hypertension ☆

Spontaneously hypertensive (SHR) rats contained more galanin (GAL) content and GAL mRNA in locus coeruleus (LC) at the prehypertensive, but not at the well-established hypertensive stage, than did age-matched Wistar-Kyoto (WKY) rats. However, there was also more GAL content, but not GAL mRNA, in the nucleus tractus solitarii (NTS) of SHR rats than WKY rats at both stages. This study suggests that galaninergic neurons in the LC and NTS may participate in the pathogenesis of genetic hypertension.

Sensitization of the trigeminovascular system following environmental irritant exposure.

Background Air pollution is linked to increased emergency room visits for headache, and migraine patients frequently cite chemicals or odors as headache triggers, but the association between air pollutants and headache is not well understood. We previously reported that nasal administration of environmental irritants acutely increases meningeal blood flow via a TRPA1-dependent mechanism involving the trigeminovascular system. Here, we examine whether chronic environmental irritant exposure sensitizes the trigeminovascular system. Methods Male rats were exposed to acrolein, a TRPA1 agonist, or room air by inhalation for four days prior to meningeal blood flow measurements. Some animals were injected daily with a TRPA1 antagonist, AP-18, or vehicle prior to inhalation exposure. Trigeminal ganglia were isolated following blood flow measurements for immunocytochemistry and/or qPCR determination of TRPV1, TRPA1 and CGRP levels. Results Acrolein inhalation exposure potentiated blood flow responses both to TRPA1 and TRPV1 agonists compared to room air. Acrolein exposure did not alter TRPV1 or TRPA1 mRNA levels or TRPV1 or CGRP immunoreactive cell counts in the trigeminal ganglion. Acrolein sensitization of trigeminovascular responses to a TRPA1 agonist was attenuated by pre-treatment with AP-18. Interpretation These results suggest trigeminovascular sensitization as a mechanism for enhanced headache susceptibility after chemical exposure.

Calcitonin gene-related peptide (CGRP) content and CGRP receptor binding sites in discrete forebrain regions of alcohol-preferring vs. -nonpreferring rats, and high alcohol-drinking vs. low alcohol-drinking rats

This study showed that alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats possess fewer calcitonin gene-related peptide (CGRP) receptor binding sites than their respective controls in the central amygdaloid nucleus (CeA) which is known to be related to anxiety. Since P and HAD rats are selectively bred for high alcohol preference, and alcohol can produce anxiolytic effect, one can postulate that P and HAD rats preferentially drink alcohol in order to obtain its anxiolytic effect. This study supports a hypothesis that deficit of CGRP receptors in the CeA of P and HAD rats may contribute to alcohol preference.

Intraganglionic signaling as a novel nasal-meningeal pathway for TRPA1-dependent trigeminovascular activation by inhaled environmental irritants

Headache is the most common symptom associated with air pollution, but little is understood about the underlying mechanism. Nasal administration of environmental irritants activates the trigeminovascular system by a TRPA1-dependent process. This report addresses questions about the anatomical pathway involved and the function of TRP channels in this pathway. TRPV1 and TRPA1 are frequently co-localized and interact to modulate function in sensory neurons. We demonstrate here that resiniferatoxin ablation of TRPV1 expressing neurons significantly reduces meningeal blood flow responses to nasal administration of both TRPV1 and TRPA1 agonists. Accordingly resiniferatoxin also significantly reduces TRPV1 and CGRP immunostaining and TRPV1 and TRPA1 message levels in trigeminal ganglia. Sensory neurons of the trigeminal ganglia innervate the nasal epithelium and the meninges, but the mechanism and anatomical route by which nasal administration evokes meningeal vasodilatation is unclear. Double retrograde labeling from the nose and meninges reveals no co-localization of fluorescent label, however nasal and meningeal labeled cells are located in close proximity to each other within the trigeminal ganglion. Our data demonstrate that TRPV1 expressing neurons are important for TRPA1 responses in the nasal-meningeal pathway. Our data also suggest that the nasal-meningeal pathway is not primarily by axon reflex, but may instead result from intraganglionic transmission.

Noradrenergic Innervation of Ectopic Granule Cells following Low-Level X-Irradiation

The distribution pattern of the noradrenergic system within the cerebellar cortex following low-level X-irradiation was studied using immunocytochemistry. Following one X-irradiation exposure on postnatal day 1, the laminar distribution of tyrosine hydroxylase-like immunoreactive (THIR) fibers was similar to controls at postnatal day 30, but the orientation of the fibers in the molecular layer (ML) was slightly changed. Successive daily doses, however, produced alterations in both the pattern and concentration of THIR fibers within the ML of the cerebellar cortex. In addition, THIR fibers were found among ectopic cell clusters within the ML. This relationship suggests a potential role for the noradrenergic system in the development and/or maintenance of the ectopic cell clusters.

Noradrenergic Innervation of the External Granular Layer of the Rat following Low-Level X-Irradiation

The distribution of the noradrenergic system within the rat cerebellar cortex following low-level X-irradiation was studied using tyrosine hydroxylase immunocytochemistry. X-Irradiation treatments consisted of 1, 3, or 5 successive daily dose beginning on postnatal day 1. When studied 24 h after the last exposure, a dense plexus of tyrosine hydroxylase-like immunoreactive (THIR) fibers, not found in age-matched controls, was observed in the reduced external granular layer (EGL) in each treatment group. At 10 days of age, the THIR fibers were found in association with the reconstituted cells of the premigratory zone of the EGL. The rapid and abnormal innervation of the EGL by the THIR fibers suggests that the noradrenergic system could play a role in the regeneration of this layer.

Quantitative autoradiography on [35S]TBPS binding sites of gamma-aminobutyric acidA receptors in discrete brain regions of high-alcohol-drinking and low-alcohol-drinking rats selectively bred for high- and low-alcohol preference

It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA(A) receptors are involved in alcohol preference in HAD rats.

Environmental toxin acrolein alters levels of endogenous lipids, including TRP agonists: A potential mechanism for headache driven by TRPA1 activation

Exposure to airborne toxins can trigger headaches, but the mechanisms are not well understood. Some environmental toxins, such as acrolein, activate transient receptor potential ankyrin 1 (TRPA1), a receptor involved in pain sensation that is highly expressed in the trigeminovascular system. It has been shown in rat models that repeated exposure to acrolein induces trigeminovascular sensitization to both TRPA1 and TRP vanilloid 1 (TRPV1) agonists, a phenomenon linked to headache. In this study, we test the hypothesis that the sensitization of trigeminovascular responses in rats after acrolein exposure via inhalation is associated with changes in levels of endogenous lipids, including TRPV1 agonists, in the trigeminal ganglia, trigeminal nucleus, and cerebellum. Lipidomics analysis of 80 lipids was performed on each tissue after acute acrolein, chronic acrolein, or room air control. Both acute and chronic acrolein exposure drove widespread alterations in lipid levels. After chronic acrolein exposure, levels of all 6 N-acyl ethanolamines in the screening library, including the endogenous cannabinoid and TRPV1 agonist, N-arachidonoyl ethanolamine, were elevated in trigeminal tissue and in the cerebellum. This increase in TRPV1 ligands by acrolein exposure may indicate further downstream signaling, in that we also show here that a combination of these TRPV1 endogenous agonists increases the potency of the individual ligands in TRPV1-HEK cells. In addition to these TRPV1 agonists, 3 TRPV3 antagonists, 4 TRPV4 agonists, and 25 orphan lipids were up and down regulated after acrolein exposure. These data support the hypothesis that lipid signaling may represent a mechanism by which repeated exposure to the TRPA1 agonist and environmental toxin, acrolein, drives trigeminovascular sensitization.