Adult Rat Trigeminal Ganglion Neurons Research Articles

Tooth pulp inflammation increases brain-derived neurotrophic factor expression in rodent trigeminal ganglion neurons

Nociceptive pathways with first-order neurons located in the trigeminal ganglion (TG) provide sensory innervation to the head, and are responsible for a number of common chronic pain conditions, including migraines, temporomandibular disorders and trigeminal neuralgias. Many of those conditions are associated with inflammation. Yet, the mechanisms of chronic inflammatory pain remain poorly understood. Our previous studies show that the neurotrophin brain-derived neurotrophic factor (BDNF) is expressed by adult rat TG neurons, and released from cultured newborn rat TG neurons by electrical stimulation and calcitonin gene-related peptide (CGRP), a well-established mediator of trigeminal inflammatory pain. These data suggest that BDNF plays a role in activity-dependent plasticity at first-order trigeminal synapses, including functional changes that take place in trigeminal nociceptive pathways during chronic inflammation. The present study was designed to determine the effects of peripheral inflammation, using tooth pulp inflammation as a model, on regulation of BDNF expression in TG neurons of juvenile rats and mice. Cavities were prepared in right-side maxillary first and second molars of 4-week-old animals, and left open to oral microflora. BDNF expression in right TG was compared with contralateral TG of the same animal, and with right TG of sham-operated controls, 7 and 28 days after cavity preparation. Our ELISA data indicate that exposing the tooth pulp for 28 days, with confirmed inflammation, leads to a significant upregulation of BDNF in the TG ipsilateral to the affected teeth. Double-immunohistochemistry with antibodies against BDNF combined with one of nociceptor markers, CGRP or transient receptor potential vanilloid type 1 (TRPV1), revealed that BDNF is significantly upregulated in TRPV1-immunoreactive (IR) neurons in both rats and mice, and CGRP-IR neurons in mice, but not rats. Overall, the inflammation-induced upregulation of BDNF is stronger in mice compared to rats. Thus, mouse TG provides a suitable model to study molecular mechanisms of inflammation-dependent regulation of BDNF expression in vivo.

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.

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.

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.

Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons.

Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 microM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed approximately 60% of the LVA ICa, whereas approximately 10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega-conotoxin GVIA (omega-CgTx), an N-type ICa blocker, suppressed approximately 65% of the whole cell HVA ICa at the concentration of 1 microM. The omega-CgTx-resistant HVA ICa was sensitive to nifedipine (10 microM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by approximately 25% of the drug-free control ( approximately 70% of the omega-CgTx-resistant ICa). The combination of 10 microM nifedipine and 1 microM omega-CgTx left approximately 13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 microM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.

Ginseng root extract inhibits calcium channels in rat sensory neurons through a similar path, but different receptor, as μ-type opioids

The effect of Panax ginseng root extract on Ca 2+ current of adult rat trigeminal ganglion neurons was investigated using whole-cell patch-clamp methods. The application of P. ginseng root extract (100 μg/ml) produced rapid, reversible reduction of the Ca 2+ current by 22 ± 4%. Treatment with pertussis toxin (250 ng/ml) for 16 h reduced the inhibition to 4 ± 1%. The continual presence of 1 μM DAGO, a selective μ-opioid agonist that inhibits Ca 2+ channels, occluded further inhibition of Ca 2+ current by P. ginseng root extract. Yohimbine, phaclofen, atropine, and naloxone — antagonists of α 2-adrenergic, GABA B, muscarinic, and opiate receptors, respectively — did not block the inhibitory effect on Ca 2+ current of P. ginseng root extract. Thus, P. ginseng root extract acts on sensory neurons through a similar pathway as μ-type opioids: both inhibit Ca 2+ channels through pertussis toxin-sensitive GTP-binding proteins. However, the receptor for P. ginseng root extract is not an α 2-adrenergic, GABA B, muscarinic, or opioid receptor.

Identification of acetylcholine receptors in adult rat trigeminal ganglion neurons

Nicotinic acetylcholine receptors (nAChRs) were identified in a subpopulation of cultured adult rat trigeminal ganglia (TG) neurons by whole-cell patch-clamp recordings. Dimethylphenylpiperazinium (DMPP), a nAChR agonist, induced inward currents in 2168 of TG neurons having soma diameters greater than 28 μm. These currents were inhibited by hexamethonium, mecamylamine and atropine, indicating the presence of neuronal ganglionic-type nAChRs. This interpretation is consistent with the finding that the nicotine- or DMPP-induced currents were not inhibited by α-bungarotoxin (α-Bng) in 5 of the 9 cells tested with this compound. However, in 2 of the 9 cells tested, the DMPP-induced currents were completely inhibited by α-Bng, and in the remaining two cells tested, the currents were partially inhibited by α-Bng. About 22% of the cells having diameters ≧28μm were specifically labeled with FITC-labeled α-Bng, whereas only 2% of the cells with soma diameters <28μm were labeled. These data taken together suggest that more than one subtype of nAChR is present in TG.