Rat Trigeminal Ganglion Neurons Research Articles

Temporomandibular joint inflammation decreases the voltage-gated K + channel subtype 1.4-immunoreactivity of trigeminal ganglion neurons in rats

Voltage-gated K + (Kv) channels are one of the important physiological regulators of the membrane potentials in excitable cells, including sensory ganglion neurons. The aim of the present study was to investigate whether temporomandibular joint (TMJ) inflammation alters expression of Kv channel subtype 1.4 (Kv1.4) of trigeminal ganglion (TRG) neurons innervating TMJ relating allodynia (pain caused by normally innoxious stimulation), by using both behavioral and immunohistochemical techniques. TMJ inflammation was induced by injection of Complete Freund’s Adjuvant (CFA) into the rat TMJ. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ inflamed rats was significantly lower than that in naïve rats. TMJ afferents were identified by fluorogold (FG) labeling. The mean numbers of Kv1.4-/neurofilament (NF) 200(myelinated fiber marker) positive- and negative-immunoreactivities FG-labeled small-/medium-diameter TRG neurons in inflamed rats were significantly decreased when compared with those in the naïve rats. These findings suggest that TMJ inflammation reduces the expression of Kv1.4 subunits in the small-/medium sized (A δ-/C-) TRG neurons and this may contribute to trigeminal inflammatory allodynia in TMJ disorder. These results lead us to suggest that Kv channel openers may be a potential therapeutic agents for prevention of mechanical allodynia.

Effects of capsaicin on VGSCs in TRPV1 −/− mice

Two different mechanisms by which capsaicin blocks voltage-gated sodium channels (VGSCs) were found by using knockout mice for the transient receptor potential V1 (TRPV1 −/−). Similar with cultured rat trigeminal ganglion (TG) neurons, the amplitude of tetrodotoxin-resistant (TTX-R) sodium current was reduced 85% by 1 μM capsaicin in capsaicin sensitive neurons, while only 6% was blocked in capsaicin insensitive neurons of TRPV1 +/+ mice. The selective effect of low concentration capsaicin on VGSCs was reversed in TRPV1 −/− mice, which suggested that this effect was dependent on TRPV1 receptor. The blockage effect of high concentration capsaicin on VGSCs in TRPV1 −/− mice was the same as that in capsaicin insensitive neurons of rats and TRPV1 +/+ mice. It is noted that non-selective effect of capsaicin on VGSCs shares many similarities with local anesthetics. That is, firstly, both blockages are concentration-dependent and revisable. Secondly, being accompanied with the reduction of amplitude, voltage-dependent inactivation curve shifts to hyperpolarizing direction without a shift of activation curve. Thirdly, use-dependent blocks are induced at high stimulus frequency.

The functional difference between transient and sustained K + currents on the action potentials in tetrodotoxin-resistant adult rat trigeminal ganglion neurons

To determine whether there is a difference between a transient K + current ( I A) and a sustained K + current ( I K) regarding the neuronal function in small-diameter adult rat trigeminal ganglion (TG) neurons, which were insensitive to tetrodotoxin (TTX, 1 μM), we performed two different types of experiments. Primary cultures of dissociated TG neurons were prepared, and electrophysiological recordings were performed with the whole-cell configuration using the patch-clamp technique. In the voltage-clamp mode, two distinct K + current components, ( I A) and ( I K), were identified, and two different components (59.5% and 96.3%) of I K to the total K + current were observed at a + 50 mV step-pulse. The IC 50 value for 4-aminoprydine (4-AP, 0.05–50 mM), which inhibited the I A by 50%, was 0.7 mM. That for tetraethylammonium (TEA, 0.02–20 mM) to inhibit 50% of I K was 1.5 mM. In the current-clamp mode, we used 0.5 mM 4-AP and 2 mM TEA at each concentration nearly equal to the IC 50 value. Irrespective of the absence or presence of TEA (2 mM), 0.5 mM 4-AP application increased the number of action potentials due to the decreased duration of the depolarization phase (DDP). TEA in the presence and absence of 4-AP prolonged the duration of action potentials as well as the duration of repolarization phase (DRP). These results suggested that I A and I K had independent effects regulating the intrinsic firing properties of the action potential number and timing, respectively, in adult rat TTX-R TG neurons.

Changes in Osmolality Sensitize the Response to Capsaicin in Trigeminal Sensory Neurons

Changes in tonicity in the peripheral nervous system can activate nociceptors and produce pain. Under local inflammatory conditions the peripheral terminals of nociceptors are subject to deviations from isotonicity. Previously it was shown that several members of the TRP(V) family of ion channels are responsive to changes in tonicity. Here we explore how changes in tonicity affect TRPV1 receptor-mediated responses to capsaicin in dissociated rat trigeminal ganglion (TG) neurons. Using whole cell patch-clamp and calcium imaging, we found that mild anisotonicity (260 and 348 mOsm/kg for hypotonicity and hypertonicity, respectively) strikingly sensitized the capsaicin-evoked current, I(caps). Confocal immunolocalization studies also revealed a modest anisotonicity-mediated redistribution of TRPV1 toward the plasma membrane of TG neurons. With respect to downstream signaling pathways, tonicity-induced sensitization of I(caps) was dependent on whether hypo- or hypertonic stimuli were applied. Specifically, antagonism of PKA- and PI3K-activated pathways appreciably reduced the hypertonicity-induced sensitization of I(caps), whereas inhibition of PKC-mediated pathways selectively reduced the sensitization produced by hypotonic solutions. In summary, whereas the overall effects of hypo- and hypertonicity resulted in a similar pattern of potentiation of I(caps), intracellular signaling pathways were selective for hypo- versus hypertonicity-induced tuning of capsaicin-activated currents.

Molecular Basis of Cav2.3 Calcium Channels in Rat Nociceptive Neurons

Ca(v)2.3 calcium channels play an important role in pain transmission in peripheral sensory neurons. Six Ca(v)2.3 isoforms resulting from different combinations of three inserts (inserts I and II in the II-III loop and insert III in the carboxyl-terminal region) have been identified in different mammalian tissues. To date, however, Ca(v)2.3 isoforms unique to primary sensory neurons have not been identified. In this study, we determined Ca(v)2.3 isoforms expressed in the rat trigeminal ganglion neurons. Whole tissue reverse transcription (RT)-PCR analyses revealed that only two isoforms, Ca(v)2.3a and Ca(v)2.3e, are present in TG neurons. Using single cell RT-PCR, we found that Ca(v)2.3e is the major isoform, whereas Ca(v)2.3e expression is highly restricted to small (<16 mum) isolectin B4-negative and tyrosine kinase A-positive neurons. Ca(v)2.3e was also preferentially detected in neurons expressing the nociceptive marker, transient receptor potential vanilloid 1. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recordings provided evidence of an association between an R-type calcium channel component and Ca(v)2.3e expression. Our results suggest that Ca(v)2.3e in sensory neurons may be a potential target for the treatment of pain.

Effects of phorbol-12,13-dibuterate on sodium currents and potassium currents in rat trigeminal ganglion neurons

The effects of phorbol-12,13-dibuterate (PDBu) on total sodium current (I(Na)-total), tetrodotoxin-resistant sodium current (I(Na)-TTXr), 4-AP-sensitive potassium current (I(A)) and TEA-sensitive potassium current (I(K)) in trigeminal ganglion (TG) neurons were investigated. Whole-cell patch clamp techniques were used to record ion currents in cultured TG neurons of rats. Results revealed that 0.5 micromol/L PDBu reduced the amplitude of I(Na)-total by (38.3+/-4.5)% (n=6, P<0.05), but neither the G-V curve (control: V (0.5)=-17.1+/-4.3 mV, k=7.4+/-1.3; PDBu: V (0.5)=-15.9+/-5.9 mV, k=5.9+/-1.4; n=6, P>0.05) nor the inactivation rate constant (control: 3.6+/-0.9 ms; PDBu: 3.6+/-0.8 ms; n=6, P>0.05) was altered. 0.5 micromol/L PDBu could significantly increase the amplitude of I(Na)-TTXr by (37.2+/-3.2)% (n=9, P<0.05) without affecting the G-V curve (control: V (0.5)=-14.7+/-6.0 mV, k=6.9+/- 1.4; PDBu: V (0.5)=-11.1+/-5.3 mV, k=8.1+/-1.5; n=5, P>0.05) or the inactivation rate constant (control: 4.6+/-0.6 ms; PDBu: 4.2+/-0.5 ms; n=5, P>0.05). 0.5 mumol/L PDBu inhibited I(K) by (15.6+/-5.0) % (n=16, P<0.05), and V (0.5) was significantly altered from - 4.7+/-1.4 mV to -7.9 +/-1.8 mV (n=16, P<0.05). I(A) was not significantly affected by PDBu, 0.5 mumol/L PDBu decreased I(A) by only (0.3+/-3.2)% (n=5, P>0.05). It was concluded that PDBu inhibited I(Na)-total but enhanced I(Na)-TTXr, and inhibited I(K) without affecting I(A). These data suggested that the activation of PKC pathway could exert the actions.

Effects of static magnetic fields on the voltage-gated potassium channel currents in trigeminal root ganglion neurons

To evaluated the effects of moderate-intensity static magnetic fields (SMF) on two types of voltage-gated potassium channel (VGPC) currents: I K,A and I K,V, whole-cell patch-clamp experiments were conducted on acute dissociated rat trigeminal root ganglion (TRG) neurons. The results demonstrated that 125 mT SMF could influence the inactivation kinetics of these two VGPC currents by altering the inactivation rate and velocity. No significant change was observed in the activation properties. These findings supported the hypothesis that biological membrane would be deformed in moderate-intensity SMF and the physiological characteristics of ion channels on the membrane would be influenced. The mechanism underlying the different effects of SMF on the I K,A and I K,V inactivation was also discussed.

Integrins regulate opioid receptor signaling in trigeminal ganglion neurons

The binding of integrins to the extracellular matrix results in focal organization of the cytoskeleton and the genesis of intracellular signals that regulate vital neuronal functions. Recent evidence suggests that integrins modulate G-protein-coupled receptor (GPCR) signaling in hippocampal neurons. In this study we evaluated the hypothesis that integrins regulate the mu opioid receptor in rat trigeminal ganglion neurons. For these studies, primary cultures of adult rat trigeminal ganglion neurons were used to demonstrate the colocalization of β1 and β3 integrins with mu opioid receptor in caveolin-1-rich membrane fractions, and at focal adhesions sites generated by integrin ligand binding. Furthermore, we show that the mu opioid receptor agonist, DAMGO ([D-Ala 2,N-MePhe 4,Gly-ol 5]enkephalin), inhibits cyclic AMP (cAMP) accumulation in response to prostaglandin E2 (PGE 2) stimulation in bradykinin-primed, but not unprimed, cultured trigeminal ganglia neurons. Application of soluble GRGDS (Gly-Arg-Gly-Asp-Ser) peptides that bind specific integrins (i.e. RGD-binding integrins) completely abolished the DAMGO effect in bradykinin-primed trigeminal ganglia neurons, but did not alter bradykinin-mediated hydrolysis of phosphatidylinositol. Likewise, monospecific anti-β1 and anti-β3 integrin subunit antibodies blocked this DAMGO effect in bradykinin-primed trigeminal ganglia neurons. Indeed, application of anti-β1 integrin subunit actually reversed DAMGO signaling, resulting in increased cAMP accumulation in these cells. This suggests that the relative amounts of specific activated integrins at focal adhesions may govern signaling by the mu opioid receptor, perhaps by altering interactions with G proteins (e.g. Gαi vs. Gαs). Collectively, these data provide the first evidence that specific integrins regulate opioid receptor signaling in sensory neurons.

Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and ATP-mediated currents in sensory neurons have been shown to be mediated by both P2X 3 and P2X 2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X 2 and P2X 3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalization with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, non-peptidergic fibers ( Bandeiraea simplicifolia isolectin B4; IB4). Forty percent of P2X 2 and 64% of P2X 3 receptor-expressing cells were IB4 positive, and 33% of P2X 2 and 31% of P2X 3 receptor-expressing cells were NF200 positive. Approximately 40% of cells expressing P2X 2 receptors also expressed P2X 3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labeled and 52% of these neurons expressed either P2X 2 or P2X 3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems.

Mechanisms involved in modulation of trigeminal primary afferent activity in rats with peripheral mononeuropathy

In order to clarify the mechanisms underlying the changes in primary afferent neurons in trigeminal neuropathic pain, a chronic constriction nerve injury model of the infraorbital nerve (ION-CCI) was developed in rats. Mechanical allodynia was observed at 3 days after ION-CCI and lasted more than 14 days. Single-unit activities were recorded from the ION of anesthetized rats. C-, Abeta- and Adelta-units were identified on the basis of their conduction velocity. Adelta-units were frequently encountered at a later period after ION-CCI. The highest Adelta-spontaneous activity was recorded at 3 days after ION-CCI and progressively decreased after that, but spontaneous activity was still higher at 14 days after ION-CCI than that of naïve rats. Mechanical-evoked responses of Adelta-units were also highest at 3 days after ION-CCI and then gradually decreased. In consideration of these data, patch-clamp recordings were performed on medium to large size neurons of the dissociated trigeminal ganglion (TRG). Patch-clamp recordings revealed that the IK (sustained) and IA (transient) in rats with ION-CCI were significantly smaller than those of naïve rats, and correlated with an increase in duration of repolarization phase and a decrease in duration of depolarization phase, respectively. The hyperpolarization-activated current (Ih) was significantly larger in TRG neurons of rats with ION-CCI as compared with those of naïve rats. The present results suggest that Ih, IK and IA in Adelta-afferent neurons in TRG are significantly involved in the changes in afferent spontaneous activity and mechanically evoked activity that accompany mechanical allodynia produced by trigeminal nerve injury.

Characterization of three types of ATP-activated current in relation to P2X subunits in rat trigeminal ganglion neurons

In the present study, ATP-activated currents (I ATPs) recorded from rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique are classified into three types (F, I and S) based on the characteristics of their activation and desensitization. The time of rising phase (R 10–90) of types F, I and S of I ATP is measured to be 33.6 ± 4.5, 62.2 ± 9.9 and 302.1 ± 62.0 ms respectively, and positively correlated to cell size. The time of decaying phases (D 10–90) of types F and S is 399.4 ± 58.2 and > 1500 ms, respectively. The dose–response curves for the three types of I ATP show that their EC 50 values are close (3.44 × 10 − 5 , 4.89 × 10 − 5 and 4.14 × 10 − 5 M for types F, I and S respectively, P > 0.05). Their reversal potentials are basically the same, varying from + 4 to + 10 mV. In addition, using whole-cell patch clamp technique in combination with single cell immunohistochemical staining for P2X receptor subunits, our results suggest that the type distinction of ATP-activated current was associated with cell size and P2X receptor subunits: small-sized cells with type F of I ATP express only P2X1 and/or P2X3 subunits, while cells with types S and I of I ATP express P2X2 or P2X4 in addition to P2X1 and P2X3.

Calcitonin gene‐related peptide enhances release of native brain‐derived neurotrophic factor from trigeminal ganglion neurons

Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is coexpressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1-1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose-dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8-37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity.

Targeting of CTCF to the nucleolus inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism

Multiple functions have been reported for the transcription factor and candidate tumour suppressor, CTCF. Among others, they include regulation of cell growth, differentiation and apoptosis, enhancer-blocking activity and control of imprinted genes. CTCF is usually localized in the nucleus and its subcellular distribution during the cell cycle is dynamic; CTCF was found associated with mitotic chromosomes and the midbody, suggesting different roles for CTCF at different stages of the cell cycle. Here we report the nucleolar localization of CTCF in several experimental model systems. Translocation of CTCF from nucleoplasm to the nucleolus was observed after differentiation of K562 myeloid cells and induction of apoptosis in MCF7 breast cancer cells. CTCF was also found in the nucleoli in terminally differentiated rat trigeminal ganglion neurons. Thus our data show that nucleolar localization of CTCF is associated with growth arrest. Interestingly, the 180 kDa poly(ADP-ribosyl)ated isoform of CTCF was predominantly found in the nucleoli fractions. By transfecting different CTCF deletion constructs into cell lines of different origin we demonstrate that the central zinc-finger domain of CTCF is the region responsible for nucleolar targeting. Analysis of subnucleolar localization of CTCF revealed that it is distributed homogeneously in both dense fibrillar and granular components of the nucleolus, but is not associated with fibrillar centres. RNA polymerase I transcription and protein synthesis were required to sustain nucleolar localization of CTCF. Notably, the labelling of active transcription sites by in situ run-on assays demonstrated that CTCF inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism.

Nitric oxide regulation of calcitonin gene‐related peptide gene expression in rat trigeminal ganglia neurons

Calcitonin gene-related peptide (CGRP) and nitric oxide are involved in the underlying pathophysiology of migraine and other diseases involving neurogenic inflammation. We have tested the hypothesis that nitric oxide might trigger signaling mechanisms within the trigeminal ganglia neurons that would coordinately stimulate CGRP synthesis and release. Treatment of primary trigeminal ganglia cultures with nitric oxide donors caused a greater than four-fold increase in CGRP release compared with unstimulated cultures. Similarly, CGRP promoter activity was also stimulated by nitric oxide donors and overexpression of inducible nitric oxide synthase (iNOS). Cotreatment with the antimigraine drug sumatriptan greatly repressed nitric oxide stimulation of CGRP promoter activity and secretion. Somewhat surprisingly, the mechanisms of nitric oxide stimulation of CGRP secretion did not require cGMP or PI3-kinase signaling pathways, but rather, nitric oxide action required extracellular calcium and likely involves T-type calcium channels. Furthermore, nitric oxide was shown to increase expression of the active forms of the mitogen-activated protein kinases Jun amino-terminal kinase and p38 but not extracellular signal-related kinase in trigeminal neurons. In summary, our results provide new insight into the cellular mechanisms by which nitric oxide induces CGRP synthesis and secretion from trigeminal neurons.

Mechanosensitivity of voltage‐gated K+ currents in rat trigeminal ganglion neurons

We investigated the mechanosensitivity of voltage-gated K+ channel (VGPC) currents by using whole-cell patch clamp recording in rat trigeminal ganglion (TG) neurons. On the basis of biophysical and pharmacological properties, two types of VGPC currents were isolated. One was transient (I(K,A)), the other sustained (I(K,V)). Hypotonic stimulation (200 mOsm) markedly increased both I(K,A) and I(K,V) without affecting their activation and inactivation kinetics. Gadolinium, a well-known blocker of mechanosensitive channels, failed to block the enhancement of I(K,A) and I(K,V) induced by hypotonic stimulation. During hypotonic stimulation, cytochalasin D, an actin-based cytoskeletal disruptor, further increased I(K,A) and I(K,V), whereas phalloidin, an actin-based cytoskeletal stabilizer, reduced I(K,A) and I(K,V). Confocal imaging with Texas red-phalloidin showed that actin-based cytoskeleton was disrupted by hypotonic stimulation, which was similar to the effect of cytochalasin D. Our results suggest that both I(K,A) and I(K,V) are mechanosensitive and that actin-based cytoskeleton is likely to regulate the mechanosensitivity of VGPC currents in TG neurons.

Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5′-fluorouridine into nascent RNA

In this study we have used the transcription assay with 5′-fluorouridine incorporation into nascent RNA to analyze the nuclear organization and dynamics of transcription sites in rat trigeminal ganglia neurons. The 5′-FU administrated by i.p. injection was successfully incorporated into nuclear domains containing actively transcribing genes of trigeminal neurons. 5′-Fluorouridine RNA-labeling was detected with immunocytochemistry at light and electron microscopy levels. The 5′-fluorouridine incorporation sites were detected in the nucleolus, particularly on the dense fibrillar component, and in numerous transcription foci spread throughout the euchromatin regions, without preferential positioning at the nuclear periphery or in the nuclear interior. Double labeling experiments to combine 5′-fluorouridine incorporation with molecular markers of nuclear compartments showed the absence of transcription sites in Cajal bodies and nuclear speckles of splicing factors. Similarly, no 5′-fluorouridine labeling was detected in well-characterized chromatin silencing domain, the telomeric heterochromatin. The specificity and sensitivity of the run-on transcription assay in trigeminal ganglia neurons was verified by the i.p. administration of the transcription inhibitor actinomycin D. The dramatic reduction in RNA synthesis upon actinomycin D treatment was associated with two important cellular events, heterochromatin silencing and formation of DNA damage/repair nuclear foci, demonstrated by the expression of tri-methylated histone H4 and phosphorylated H2AX, respectively. 5′-Fluorouridine incorporation in animal models provides a useful tool to investigate the organization of gene expression in mammalian neurons in both normal physiology and experimental pathology systems.

Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation

The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund’s adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (V h=−60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 μm), voltage-dependent transient K + current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltage-dependent transient K + current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at two-fold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund’s adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient K + current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder.

Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with aδ/c‐fibers and colocalization with trk receptors

The transient receptor potential (TRP) superfamily of cation channels contains four temperature-sensitive channels, named TRPV1-4, that are activated by heat stimuli from warm to that in the noxious range. Recently, two other members of this superfamily, TRPA1 and TRPM8, have been cloned and characterized as possible candidates for cold transducers in primary afferent neurons. Using in situ hybridization histochemistry and immunohistochemistry, we characterized the precise distribution of TRPA1, TRPM8, and TRPV1 mRNAs in the rat dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons. In the DRG, TRPM8 mRNA was not expressed in the TRPV1-expressing neuronal population, whereas TRPA1 mRNA was only seen in some neurons in this population. Both A-fiber and C-fiber neurons expressed TRPM8, whereas TRPV1 was almost exclusively seen in C-fiber neurons. All TRPM8-expressing neurons also expressed TrkA, whereas the expression of TRPV1 and TRPA1 was independent of TrkA expression. None of these three TRP channels were coexpressed with TrkB or TrkC. The TRPM8-expressing neurons were more abundant in the TG compared with the DRG, especially in the mandibular nerve region innervating the tongue. Our data suggest heterogeneity of TRPM8 and TRPA1 expression by subpopulations of primary afferent neurons, which may result in the difference of cold-sensitive primary afferent neurons in sensitivity to chemicals such as menthol and capsaicin and nerve growth factor.

Tumor necrosis factor‐α stimulation of calcitonin gene‐related peptide expression and secretion from rat trigeminal ganglion neurons

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.

Pharmacological interactions between calcium/calmodulin-dependent kinase II α and TRPV1 receptors in rat trigeminal sensory neurons

Multiple lines of evidence suggest that calcium/calmodulin-dependent kinase II α (CaMKIIα) plays an important role in the spinal dorsal horn in nociceptive models of chemical, inflammatory and nerve injury. Moreover, CaMKIIα phosphorylates the vanilloid receptor type 1 (TRPV1), thereby regulating vanilloid agonist binding to the receptor. Herein, we have explored a possible interaction of CaMKIIα activity with the TRPV1 receptor in rat trigeminal ganglion (TG) neurons in vitro. Inhibition of CaMKIIα with KN-93 (5 μM) inhibited capsaicin (CAP)- and n-arachidonoyl-dopamine (NADA)-evoked calcitonin gene-related peptide (CGRP) release effectively decreasing the E max for both compounds. This effect was not mimicked by the inactive compound KN-92 (5 μM), indicating that the effect was mediated by CaMKIIα inhibition. CAP also stimulated a significant ∼50% increase in autophosphorylation of CaMKIIα at Thr 286/287. Immunocytochemistry for phospho-CaMKIIα indicated that this effect specifically occurred in TRPV1-positive TG neurons. These findings indicate that phopho-CaMKIIα is likely to play a role in presynaptic primary afferents in animal models of nociceptive hypersensitivity and provide support for CaMKIIα modulation of TRPV1 activity in sensory neurons.