ERK potentiates p38 in central sensitization induced by traumatic occlusion.

This study was to investigate the role of p38 activation via ERK1/2 phosphorylation in neurons and microglia of the spinal trigeminal subnucleus caudalis (Vc) in the promotion of orofacial hyperalgesia induced by unilateral anterior crossbite (UAC) traumatic occlusion in adult rats. U0126, a p-ERK1/2 inhibitor, was injected intracisternally before UAC implant. The effects of the U0126 injection were compared to those following the injection of SB203580, a p-p38 inhibitor. Mechanical hyperalgesia was evaluated via pressure pain threshold measurements. Brain stem tissues were processed for a Western blot analysis to evaluate the activation of ERK1/2 and p38. Double immunofluorescence was also performed to observe the expression of p-ERK1/2 and p-p38 in neurons (labeled by NeuN) and microglia (labeled by OX42). The data showed that UAC caused orofacial hyperalgia ipsilaterally on d1 to d7, peaking on d3 (P<0.05). An upregulation of p-ERK1/2 was observed in the ipsilateral Vc on d1 to d3, peaking on d1. An upregulation of p-p38 was also observed on d1 to d7, peaking on d3 (P<0.05). p-ERK1/2 primarily co-localized with NeuN and, to a lesser extent, with OX42, while p-p38 co-localized with both NeuN and OX42. Pretreatment with U0126 prevented the upregulation of both p-ERK1/2 and p-p38. Similarly to an intracisternal injection of SB203580, U0126 pretreatment attenuated the UAC-induced orofacial hyperalgesia. These data indicate that UAC caused orofacial hyperalgesia by inducing central sensitization via the activation of ERK1/2 and p38 in both neurons and microglia in the Vc, potentially impacting the effects of p-ERK1/2 during p38 activation.

The expression of succinate semialdehyde dehydrogenase in the caudal part of the spinal trigeminal nucleus is down-regulated after electrical stimulation of the dura mater surrounding the superior sagittal sinus in conscious rats.

Using two-dimensional gel electrophoresis (2-DE), we examined proteomic changes in the caudal part of the spinal trigeminal nucleus induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus (ES-SSS) in conscious rats. After image analysis of 2-DE gels, nine protein spots of interest were excised from the gels and identified by liquid chromatography-tandem mass spectrometry. Among the nine, succinate semialdehyde dehydrogenase (SSADH) was found to be down-regulated after ES-SSS. This result was validated with Real-time polymerase chain reaction and Western blot analyses. Because SSADH degrades GABA, decreased expression of it increases the local concentration of GABA in the caudal part of the spinal trigeminal nucleus after ES-SSS; this has not been reported before and may participate in the modulation of trigeminovascular headache.

Distribution of heme oxygenase-2 and NADPH-diaphorase in the spinal trigeminal nucleus of the rat.

Heme oxygenase (HO)/carbon monoxide (CO) and nitric oxide synthase (NOS)/nitric oxide (NO) systems are involved in sensory information processing. The present study was undertaken to examine the distribution of HO-2 and NOS in the spinal trigeminal nucleus (STN) of the rat, using histochemistry and immunohistochemistry. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining was found that NADPH-d activity was more prominent in the nucleus caudalis (Vc) and the dorsomedial subdivision of the nucleus oralis (Vo) than in other spinal trigeminal regions. Immunohistochemistry for HO-2 revealed that HO-2 staining neurons distributed extensively, which intensity was higher in the rostral than caudal part of the STN. The colocalization of NADPH-d and HO-2 was mainly confined in the Vc. The expression and distribution of NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and might function in the processing orofacial signal in the STN together.

Oestrogen-modulated increase of calmodulin-dependent protein kinase II (CamKII) in rat spinal trigeminal nucleus after systemic nitroglycerin.

Migraine can be triggered by systemic administration of the nitric oxide (NO) donor nitroglycerin (NTG) and by abrupt falls in plasma oestradiol. Calmodulin-dependent protein kinase II (CamKII) present in superficial dorsal horns is thought to play a role in sensitization of central nociceptors, a phenomen present in migraineurs. We therefore examined in rats the expression of CamKII in the caudal trigeminal nucleus (TNC) after subcutaneous NTG (10 mg/kg) and its modulation by oestrogen. In male rats and in ovariectomized females, after 4 h NTG increased significantly CamKII expression in the superficial layers of TNC, but not in the upper thoracic spinal cord. NTG had no effect on CamKII expression in oestradiol-treated ovariectomized animals. Thus NTG, i.e. NO, selectively enhances CamKII in the rat TNC and oestradiol blocks this effect. These data may help to understand the mechanisms by which NO triggers migraine attacks and oestrogens influence migraine severity.

Nitroglycerin-induced nNOS increase in rat trigeminal nucleus caudalis is inhibited by systemic administration of lysine acetylsalicylate but not of sumatriptan.

Systemic administration of nitroglycerin (NTG), a nitric oxide (NO) donor, in migraineurs triggers after several hours an attack of which the precise mechanisms are unknown. We found previously in rats that nitroglycerin (10 mg/kg s.c.) is able to increase significantly after 4 h the number of neuronal nitric oxide synthase (nNOS)-immunoreactive neurones in the cervical part of trigeminal nucleus caudalis. In the present experiments, we demonstrate that the 5-HT1B/D agonist sumatriptan (0.6 mg/kg s.c.) does not alter this phenomenon when given before NTG. By contrast, pretreatment with lysine acetylsalicylate (50 mg/kg i.m.) attenuates the NTG-induced nNOS expression in the superficial laminae of trigeminal nucleus caudalis. These findings suggest that effect of NTG on nNOS at a high dosage may involve the cycloxygenase pathway and that activation of the peripheral 5-HT1B/D receptors is not able to modify this effect. These data could help to better understand the role of NO in the pathogenesis of headaches and the action of antimigraine drugs.

Nitric oxide synthase inhibition lowers activity of neurons with meningeal input in the rat spinal trigeminal nucleus.

Nitric oxide is thought to control transmitter release and neuronal activity in the spinal dorsal horn and the spinal trigeminal nucleus, where nociceptive information from extra- and intracranial tissues is processed. Extracellular impulse activity was recorded from neurons in the rat spinal trigeminal nucleus with afferent input from the cranial dura mater. In contrast to the inactive isomer D-NAME, infusion of the nitric oxide synthase inhibitor L-NAME (20 mg/kg) significantly reduced neuronal activity and increased systemic blood pressure. It is concluded that nitric oxide production contributes to the ongoing activity of sensitized neurons in the spinal trigeminal nucleus. The results suggest that nitric oxide may be involved in the generation and maintenance of primary headaches such as migraine.

Nitric oxide synthase/nicotinamide adenine dinucleotide phosphate-diaphorase in the brainstem trigeminal nuclei after transection of the masseteric nerve in rats.

In this study, the responses of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS) activities were quantitatively analyzed at different times in both ipsilateral and contralateral sides of trigeminal nuclei, after unilateral trigeminal muscle nerve transection, in Sprague Dawley rats. In the control animals, both NADPH-d- and nNOS-positive neurons were constitutively distributed in the rostrolateral solitary tract nucleus, dorsomedial part of trigeminal nucleus oralis (Vo/Sn), and superficial layers (VcI/II) of the trigeminal nucleus caudalis (Vc). NADPH-d-positive neurons appeared in the trigeminal mesencephalic nucleus ipsilaterally at 5 days (mean +/- SEM: 30.5 +/- 5.6) and were maintained until 8 weeks (33 +/- 10.6) after the denervation. In the trigeminal motor nucleus, NADPH-d-positive neurons appeared transiently and bilaterally, peaking at 1 week (663.5 +/- 156.2, ipsilateral side; 687.5 +/- 118.6, contralateral side) after unilateral denervation of the masseteric nerve. In both Vo/Sn and Vc, the number of NADPH-d-positive neurons in the control animals showed a decrease at 3 days but significantly increased from 5 days to 1 week and gradually fell to the control values by 8 weeks after the denervation. There were no significant differences observed between the two sides in either Vo/Sn or Vc. nNOS-positive neurons were similarly distributed and the numbers of labeled neurons were similar to those of NADPH-d-positive neurons after the denervation, although the changes were delayed by approximately 1 week. In conclusion, after unilateral nerve transection, the peak NADPH-d activity occurs 1 week prior to nNOS activity.

Nitric oxide synthase and glutamate receptor immunoreactivity in the rat spinal trigeminal neurons expressing Fos protein after formalin injection.

Although recent studies implicated glutamate receptors and nitric oxide in nociception, much still needs to be known about their localisation in neurons involved in nociceptive transmission from the orofacial region. In this study, c-fos expression indicated by Fos immunohistochemistry in the caudal spinal trigeminal nucleus induced by subcutaneous injection of formalin into the lateral face of the rat was used as a marker for nociceptive neurons. The study sought to determine whether Fos-positive neurons express nitric oxide synthase, glutamate N-methyl-D-aspartate type receptor subunit 1, and glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type receptor subunit 2/3; and whether they project to the thalamus. After formalin injection, many Fos-positive nuclei appeared in the superficial laminae of the ipsilateral trigeminal nucleus. Confocal laser scanning microscope revealed that almost all neurons with Fos immunofluorescent nuclei were colocalised with N-methyl-D-aspartate receptor 1, 94% with glutamate receptor 2/3 and 14% with nitric oxide synthase. Some of them were closely related to neurons labelled by nitric oxide synthase. Lastly, some of the Fos-positive neurons were labelled by tetramethylrhodamine-dextran injected into the trigeminothalamic tract or the thalamic region. The results suggested that activation of N-methyl-D-aspartate receptor 1 and glutamate receptor 2/3 upon glutamate release in response to noxious stimulation to the orofacial region might mediate c-fos expression in neurons involved in nociception. The expression of Fos in the neurons could also be mediated by nitric oxide produced from the same, as well as neighbouring neurons, when nociceptive stimulation persisted. Fos-positive neurons in the spinal trigeminal nucleus may project to the thalamus, relaying orofacial nociception to the higher sensory centre.

Postnatal development of cytochrome oxidase activity in fiber tracts of the rat brain.

This paper describes postnatal changes in cytochrome oxidase (C.O.) activity in developing fiber tracts. Quantitative histochemistry was used to measure changes in C.O. activity in nine white matter regions at postnatal days (P) 7, 12, 17, 30, and 60 in the rat. At P7, enzyme activity was maximal in the spinal trigeminal tract, medial longitudinal fasciculus, and cerebellar white matter. At P12, maximal levels were measured in the medial lemniscus and cerebral peduncle. C.O. activity increased from low levels at P7 to maximal levels by P17 in the hippocampal commissure, posterior and anterior corpus callosum, and anterior commissure. In all nine regions, C.O. activity decreased by P60. Thus, peaks in C.O. activity shifted as a function of postnatal age in a caudo-rostral direction. The regional heterogeneity in the age of onset in C.O. fluctuations suggests that vulnerability to injury and metabolic dysfunction during the perinatal period will differentially affect white matter structures, depending on the age of onset of such disruptions.

FRAP-positive and capsaicin-sensitive terminals in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis.

FRAP (fluoride-resistant acid phosphatase)-reactivity in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis (STNC) was examined by light and electron microscopy. Degenerated figures of terminals caused by capsaicin were compared with the FRAP-positive terminals. Scalloped (fan-like) or indented, sinuous, slender, and cap-like figures with closely packed agranular synaptic vesicles of various sizes were common to both FRAP-positive and capsaicin-sensitive terminals. These terminals had glomerular or nonglomerular endings. Sometimes FRAP-positive and capsaicin-sensitive glomerular terminals made presynapses with surrounding dendrites. Frequently, both nonglomerular terminals were in direct contact with the neuronal soma. The terminal features of FRAP-positive and capsaicin-sensitive ones in the mouse STNC are the same as those seen in the superficial dorsal horn of the spinal cord. These findings suggest that some of the FRAP-positive terminals are capsaicin-sensitive, thereby indicating their nociceptive primary afferent.

Induction of NADPH-diaphorase/nitric oxide synthase in the brainstem trigeminal system resulting from cerebellar lesions.

Recent evidence indicates that NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) can be induced in cerebellar afferent neurons following mechanical, thermal, or chemical damage to the cerebellar cortex (Saxon and Beitz [1994] Neuroreport 5:809-812). The present study reports on the induction of NADPH-d/NOS in neurons of the brainstem trigeminal complex (BVC). Three groups of rats were used: Group I received a unilateral glass micropipette lesion into the vermal/paravermal region of the cerebellar cortex, group II received a concurrent injection of fluoro-gold along with the pipette lesion, and in group III the cerebellar cortex on one side was aspirated. Following survival times of 7-120 days, animals were processed for NADPH-d histochemistry. All three groups showed projection-specific induction of NADPH-d in different regions of the brainstem trigeminal complex. Induced neurons were distributed throughout the ipsilateral subnucleus interpolaris, principal trigeminal nucleus, and intertrigeminal nucleus. Subnucleus oralis contained a small number of induced neurons localized to the ipsilateral dorsomedial portion of the subnucleus. Projection-specific induction was confirmed by the presence of neurons double-labeled for NADPH-d and Fluoro-Gold. Although the functional consequences of NADPH-d/NOS induction remain to be elucidated, the induction of these enzymes in precerebellar neurons suggests that nitric oxide may play a role in the neuronal response to target specific lesions.

Localization of an isoform of carboxylesterase in rat brain differs from that in human brain.

Liver carboxylesterase (CE) is an enzyme capable of metabolizing drugs, and may also function as a regulator of lipid metabolism. We examined two isoforms of CE (RH1 and RL1) by immunohistochemistry in rat brain. The anti-RL1 antibody did not stain any brain structures. The anti-RH1 antibody, however, stained oligodendrocytes in all brain tissues and tanycytes, as well as some neurons in the deep cingulate gyrus, various hypothalamic nuclei and the spinal trigeminal nucleus. In the central nervous system, rat CE may function as a protective factor against foreign chemicals in these glial and neuronal cells. The distribution differed from that of the homologous human isoform which has been previously found only in endothelial cells in human brain. A possible relation between RH1 positive neurons and the medial pain system is discussed.

Distribution of nitric oxide synthase-immunoreactive interneurons in the spinal trigeminal nucleus.

The spinal trigeminal nucleus is involved in the transmission of orofacial sensory information. Neither the distribution of the neuromessenger, nitric oxide, within the trigeminal system nor the possible relationship of this simple gas with trigeminothalamic neurons has been carefully studied. Using immunocytochemical (against nitric oxide synthase) and histochemical (NADPH-diaphorase staining) techniques, we have found that nitric oxide neurons and processes are more prominent in the nucleus caudalis and the dorsomedial aspect of the nucleus oralis than in other spinal trigeminal regions. To study the relationship of nitric oxide to trigeminothalamic neurons and intertrigeminal interneurons of the spinal trigeminal nucleus, spinal trigeminal neurons were retrogradely labeled with fluorogold by thalamic injections or by injections into the junction of the nucleus interpolaris and nucleus caudalis. Medullary sections were subsequently processed with NADPH-diaphorase histochemistry. None of the diaphorase-stained neurons in the spinal trigeminal nucleus was found to contain fluorogold; however, some diaphorase-stained processes were found in close proximity to trigeminothalamic neurons. Following spinal trigeminal nucleus injections, many diaphorase-stained neurons were found to contain fluorogold, especially in the nucleus caudalis, suggesting that nitric oxide-containing neurons in the spinal trigeminal nucleus are intertrigeminal interneurons. Collectively, these data indicate that nitric oxide is most prominent in interneurons located in nucleus caudalis and that these interneurons give rise to processes that appose trigeminothalamic neurons, raising the possibility that they may indirectly influence orofacial nociceptive processing at the level of the spinal trigeminal nucleus via nitric oxide production.

Ultrastructural changes in acetylcholinesterase activity in the deafferented spinal trigeminal nucleus.

Acetylcholinesterase (AChE) activity was investigated in synaptic areas of the cat spinal trigeminal nucleus (pars interpolaris and pars caudalis) ipsilateral and contralateral to complete retrogasserian rhizotomy. Vibratome sections of tissue taken from animals of 1, 3, 6, 14, and 21 days survival were examined by electron microscopy following a histochemical reaction for AChE activity employing a method based on the Karnovsky-Roots technique for demonstrating reaction product. As degeneration progressed with survival time, enzymatic activity was initially reduced in synaptic clefts of injured afferent terminals and subsequently was enhanced throughout the extracellular space, including within synaptic clefts of possibly reinnervated sites. These changes in enzymatic activity with primary deafferentation are discussed in relation to the process of reinnervation, the development of neuronal hyperactivity, and possible noncholinergic functions of AChE.

Immunoreactive glutamic acid decarboxylase in the trigeminal nucleus caudalis of the cat: a light- and electron-microscopic analysis.

This study used antisera directed against glutamic acid decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), to examine the light- and electron-microscopic distribution of presumed GABA-ergic synapses in the medullary homologue of the cat spinal dorsal horn, the trigeminal nucleus caudalis. At the light-microscopic level, immunoreactive terminals were concentrated in the superficial dorsal horn, laminae I and II. Colchicine was generally ineffective in revealing the distribution of cell bodies. However, in two successful cases, the majority of labeled cells were found in the magnocellular layer, ventral to the substantia gelatinosa, a region that had a lower density of immunoreactive terminals. Other labeled neurons were scattered in laminae I and II. A variety of synaptic arrangements were found at the electron-microscopic level. These derived from two types of labeled terminals. One contained both small round vesicles and large dense-cored vesicles. The second contained small round and pleomorphic vesicles. Some immunoreactive GAD terminals contained a few flat vesicles. Labeled terminals predominantly formed axodendritic synapses, via symmetrical contacts. Several axoaxonic arrangements were also observed. In most cases, the GAD terminal (which did not contain dense-cored vesicles) was presynaptic to another vesicle-containing profile, including the scalloped central terminal thought to derive from primary afferents. Another population of labeled GAD terminals was found postsynaptic to unlabeled vesicle-containing profiles, including central terminals. These data indicate that inhibitory GABA-ergic controls in the trigeminal nucleus caudalis involve both presynaptic and postsynaptic mechanisms and are probably mediated via direct contacts onto ascending projection neurons, as well as via synaptic contacts onto nociceptive primary afferent fibers. The transmission of nociceptive messages by neurons of the spinal cord dorsal horn and trigeminal nucleus caudalis is subject to a variety of segmental and supraspinal controls. Pharmacological and electrophysiological studies have implicated the biogenic amines serotonin and norepinephrine, and the endogenous opioid peptides enkephalin and dynorphin, in those controls (Basbaum and Fields, 1978, 1984; Basbaum et al., 1983; Basbaum, 1985).(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of GABA-T-intensive neurons in the rat hindbrain.

The pharmacohistochemical method previously used to identify the distribution in rat brain of gamma-aminobutyric acid transaminase (GABA-T)-intensive neurons has been applied to the rat pons and medulla. The method involves systemic administration of the irreversible GABA-T inhibitor Gabaculine and the detection, 12 to 15 hours after the injection of the newly synthesized GABA-T by histochemical means. GABA-T-intensive neurons were found to be rich in the following hindbrain structures: inferior colliculus, nuclei of the raphe system, nuclei parabrachialis dorsalis and ventralis, nucleus cuneiformis, nucleus vestibularis medialis, nucleus tractus spinalis nervi trigemini, nucleus vagus, nucleus cochlearis, nucleus reticularis lateralis, nucleus ambiguus, nucleus cuneatus lateralis, inferior olive, and reticular formation of the pons and medulla. Neurons of the deep cerebellar nuclei and the rostral portion of the lateral vestibular nucleus were negative for GABA-T but were surrounded by granular staining indicative of impinging GABA-T-rich nerve endings. These results provide further support for the hypothesis that GABA neurons are far more GABA-T-intensive than other neurons in the central nervous system.

Choline acetyltransferase and acetylcholinesterase in centrifugal labyrinthine bundles of rats.

Activities of choline acetyltransferase and acetylcholinesterase were measured for the acetylcholinesterase-positive fiber bundles containing axons projecting from the brainstem to the labyrinth of the rat. These activities were compared to those of a well-established cholinergic tract: the facial motor root. The choline acetyltransferase activities were roughly similar between the tracts, consistent with a conclusion that the centrifugal labyrinthine fibers are all cholinergic. The acetylcholinesterase activities were much higher in the centrifugal labyrinthine bundle than in the facial motor root, probably relating to the smaller diameters of the labyrinthine fibers. Transection of the centrifugal labyrinthine bundle led to virtually total loss of its choline acetyltransferase activity lateral to the cut, consistent with a centrifugal direction of all the fibers, but loss of only half its acetylcholinesterase activity, even after 34 days. These results agree with those for well-established cholinergic pathways, including the facial motor root in the present study, and with previous suggestions that a component of the acetylcholinesterase in cholinergic tracts might be synthesized by cells other than the neurons in the tract.

[Oblongata in the spinal trigeminal nucleus of the adult rat (author's transl)].

The substantia gelationsa has been noted as a region of central pain control, In 1979, we reported that an intense thiamine monophosphatase (TMPase) activity was specifically localized on the plasma membrane of the synaptic glomeruli, especially dense sinusoid axon terminals (DSA). In the present investigation, the ultracytochemical localization of TMPase activity was studied in the spinal trigeminal nucleus of the adult rat. Vibratome sections of aldehyde-fixed specimens were incubated in the incubation medium (veronal-acetate buffer, pH 5. 8, 27 mM, thiamine monophosphoric acid chloride 2mM, lead nitrate 2.7 mM, sucrose 146 mM, final pH 5.4) for 30 min at 37 degrees C, and processed for light as well as electron microscopy. At the light microscopic level, the reaction products, lead sulfide, were observed to be localized in the substantia gelatinosa of the caudal medulla oblongata in the spinal trigeminal nucleus. At the electron microscopic level, the TMPase activity was positive on the plasma membrane of DSA in the region where is particularly concerned with the relay of thermal and noxious stimuli. It has been suggested by us previously that there may be some relationship between the TMPase activity and pain, mechanism in the spinal cord, and the results that the situation seems to be hold true in the spinal trigeminal nucleus, also.