CD73 Controls Extracellular Adenosine Generation in the Trigeminal Nociceptive Nerves.

Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5'-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.

[A COMPARATIVE STUDY OF CATECHOLAMINERGIC AND NITROXIDERGIC VASOMOTOR NEURONS IN THE NUCLEI OF THE CAUDAL PART OF THE BRAINSTEM OF THE RAT].

Immunohistochemical methods for the demonstration of tyrosine hydrolase (TH) and neuronal form of nitric oxide synthase (nNOS) were used to study the distribution of catecholaminergic and nitroxidergic vasomotor neurons respectively, in the nuclei of the medulla oblongata and the pons of 12 Wistar rats. Most often the expression of TG was found in neurons located in the nucleus and several reticular nuclei (gigantocellular, paragigantocellular, caudal pons nucleus), but the proportion of immunoreactive neurons did not exceed 8-14%. In the other nuclei (reticular parvocellular nucleus and oral pons nucleus, spinal nucleus of the trigeminal nerve) the value of this parameter ranged from 1 to 3%. In a large group of nuclei with proven vasomotor function such neurons were constantly not detected. In the structures with high content of catecholaminergic neurons, nNOS-positive cells were found, as a rule, in fewer numbers than in the nuclei with a limited number of TH-positive neurons.

Substance P, tyrosine hydroxylase and serotonin terminals in the rat caudal nucleus ambiguus.

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.

PKCgamma in Vc and C1/C2 is involved in trigeminal neuropathic pain.

The aim of the present study was to clarify the involvement of protein kinase Cγ (PKCγ) in the facial neuropathic pain following infraorbital nerve injury. We analyzed the change in PKCγ expression in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1/C2) following chronic constriction injury of the infraorbital nerve (ION-CCI). We also studied ION-CCI-mediated mechanical nocifensive behavior in rats. The mechanical head-withdrawal threshold significantly decreased 1 to 14 days after ION-CCI compared with that before ION-CCI and in sham rats. The expression of PKCγ was significantly larger in the ipsilateral Vc compared with the contralateral side in ION-CCI rats 3, 7, and 14 days after ION-CCI. Intrathecal (i.t.) administration of the PKCγ inhibitor chelerythrine prevented an increase in the PKCγ expression in the ipsilateral Vc. Moreover, i.t. administration of chelerythrine annulled ION-CCI-mediated reduction in the head-withdrawal threshold. Taken together, these findings suggest that PKCγ expression in the Vc played an important role in the mechanism of orofacial static mechanical allodynia following trigeminal nerve injury.

Neurochemical features of enkephalinergic neurons in the mouse trigeminal subnucleus caudalis.

Enkephalinergic (ENKergic) neurons have been proposed to play crucial roles in pain modulation in the trigeminal subnucleus caudalis (Vc). To assist an advance in the research of ENKergic neurons, here we used preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice, in which all ENKergic neurons were fluorescent. We first performed fluorescent in situ hybridization combined with immunofluorescent histochemistry to confirm the specificity of this transgenic mouse and its advantages in showing ENKergic neurons in the Vc. Then based on this useful transgenic mouse, we examined the phenotypic diversity of PPE-GFP neurons by immunostaining for several markers that characterize ENKergic neuron subtypes. About 25.9±1.9% of GFP-positive neurons were regarded as immunoreactive for glutamic acid decarboxylase (GAD)(67) mRNA and 14.7±1.4% of GFP-positive neurons were positive for γ-aminobutyric acid. The proportions of calbindin-, calretinin-positive cells among the ENKergic neurons were 8.4±1.2% and 7.3±1.7%, respectively. Only 1.1±0.1% of GFP-positive neurons colocalized with parvalbumin and no GFP-positive neurons were found to co-express neuronal nitric oxide synthase. We then injected retrograde tracer into the thalamic regions and observed that a small number of ENKergic neurons in the Vc were retrogradely labeled with the tracer. The present results provide a detailed morphological evidence of the neurochemical features of ENKergic neurons. These results have broad implications for understanding the functional roles of ENKergic neurotransmission in the Vc.

Expression profile of multiple secretory phospholipase A(2) isoforms in the rat CNS: enriched expression of sPLA(2)-IIA in brainstem and spinal cord.

Phospholipases A(2) (PLA(2)) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A(2) (sPLA(2)) isoforms in the normal rat CNS with focus on sPLA(2)-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA(2)-IB expression was low throughout the CNS, sPLA(2)-IIA expression was high in the brainstem and spinal cord, sPLA(2)-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA(2)-V expression was high in the olfactory bulb and cerebellum, and sPLA(2)-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA(2)-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA(2)-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA(2)-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA(2)-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA(2) in nociceptive transmission.

Increased COX2 in the trigeminal nucleus caudalis is involved in orofacial pain induced by experimental tooth movement.

Pain is among the major problems during orthodontic treatment. Recent studies have shown that central Cyclooxygenase2 (COX2) pathway was involved in several pain models. The present study investigated whether inducible COX2 within the trigeminal nucleus caudalis (Vc) contributed to experimental tooth movement pain in freely moving rats. Elastic rubber bands were inserted between the first and second maxillary molars bilaterally to establish tooth movement model. The directed mouth wiping behavior was used to evaluate the pain during tooth movement. COX2 distribution in Vc was studied by immunohistochemistry and the changes of COX2 expression were detected by Western blot at different time point after rubber band insertion. Our results showed that tooth movement significantly increased COX2 expression in Vc and the time spent on mouth wiping, reaching a maximum at 1 day and then decreasing gradually. Furthermore, the rhythm change of COX2 expression in Vc and the mouth wiping behavior were much correlative with each other. All of the COX2-immunoreactive structures in Vc exhibited NeuN-immunopositive staining and most of these COX2-immunoreactive neurons were Fos-immunopositive. Importantly, the mouth wiping behavior could be attenuated by intracisternal injection of NS-398 (a selective COX2 inhibitor) but not by periodontal administration of NS-398. All these results suggested that increased COX2 in Vc was involved in tooth movement pain and thus may be a central target for orthodontic pain treatment.

Kynurenate derivative attenuates the nitroglycerin-induced CamKIIα and CGRP expression changes.

OBJECTIVE: To examine the efficacy of L-kynurenine and a novel kynurenic acid derivative on the nitroglycerin-induced calmodulin-dependent protein kinase II alpha (CamKIIalpha) and calcitonin gene-related peptide (CGRP) expression changes in the rat caudal trigeminal nucleus. BACKGROUND: Systemic administration of the nitric oxide donor nitroglycerin can trigger an attack in migraineurs. In the rat, nitroglycerin activates second-order neurons in the caudal trigeminal nucleus, and increases expression of the CamKIIalpha and decreases that of the CGRP there. As glutamatergic mechanisms may be crucial in trigeminal pain processing, the aim of our study was to examine the effects of L-kynurenine, a metabolic precursor of the N-methyl D-aspartate receptor antagonist kynurenic acid, on the nitroglycerin-induced changes in CamKIIalpha and CGRP immunoreactivity. METHODS: One hour before the nitroglycerin (10 mg/kg bodyweight, s.c.) injection, the animals were pretreated with L-kynurenine (300 mg/kg bodyweight, i.p.) or 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride (300 mg/kg bodyweight, i.p.), a novel kynurenic acid derivative. Four hours later, the rats were perfused transcardially and the cervical spinal cord segments were removed for immunohistochemistry. Results.- L-kynurenine and 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride pretreatment attenuated the nitroglycerin-induced changes in CamKIIalpha and CGRP immunoreactivity in the rat caudal trigeminal nucleus. CONCLUSIONS: These findings suggest a mechanism by which the inhibition of the excitatory amino acid receptors by kynurenic acid and its derivatives can alter trigeminal nociception.

Selective inhibition of cyclooxygenase-2 attenuates nitroglycerin-induced calmodulin-dependent protein kinase II alpha in rat trigeminal nucleus caudalis.

The nitric oxide donor, nitroglycerin (NTG) can trigger a migraine attack, after a delay of several hours in migraineurs, but not in healthy people. This long delay does not favor a pure vasodilatatory action. In rats, subcutaneous administration of NTG (10mg/kg) significantly and selectively increases the number of calmodulin-dependent protein kinase II alpha (CamKIIalpha)-immunoreactive neurons in the trigeminal caudal nucleus (TNC) after 4h. The aim of our study was to determine if any isoforms of the cyclooxygenase (COX) enzyme might have a role in the NTG-induced increase of CamKIIalpha expression. In our experiments, we demonstrated that pretreatment with NS398, the selective COX-2 inhibitor attenuated the NTG-induced CamKIIalpha expression in the TNC at doses of 3 and 5mg/kg. In contrast, SC560, a selective COX-1 inhibitor failed to modulate this phenomenon in any of the dosages used (1, 5 and 10mg/kg). These findings suggest that COX-2, but not COX-1 derived metabolites are important factors in the NTG-induced CamKIIalpha expression. Thus this isoform may play a significant role in the induction of migraine. These data could help in the better understanding of the pathogenesis of headaches and the action of antimigraine drugs.

Involvement of neuronal, inducible and endothelial nitric oxide synthases in capsaicin-induced muscle hypersensitivity.

Nitric oxide, which has been implicated in the development of hyperalgesia in the spinal system, has not been systematically studied in the trigeminal system, especially in the context of inflammatory muscle pain condition. In this study, we investigated the functional role of centrally released nitric oxide in the pathogenesis of orofacial muscle pain. Specifically, we examined the contribution of neuronal, inducible and endothelial nitric oxide synthases, nNOS, iNOS and eNOS, respectively, in mediating masseter hypersensitivity under acute inflammatory condition. Time-dependent changes in nNOS, iNOS and eNOS protein expression in the subnucleus caudalis (Vc) were assessed following capsaicin injection in the masseter muscle of male Sprague Dawley rats. The expression of all three nitric oxide synthases was significantly up-regulated 30-60 min following capsaicin stimulation, which paralleled the time course of the development of capsaicin-induced masseter hypersensitivity. Pretreatment with each NOS inhibitor significantly attenuated the masseter hypersensitivity. These data showed that all three NOS in the Vc are functionally important for the development of craniofacial muscle hyperalgesia and suggest that the three NOS are closely orchestrated to regulate the level of nitric oxide under normal and pathologic conditions.

Mechanisms involved in an increment of multimodal excitability of medullary and upper cervical dorsal horn neurons following cutaneous capsaicin treatment.

BACKGROUND: In order to evaluate mechanisms that may underlie the sensitization of trigeminal spinal subnucleus caudalis (Vc; the medullary dorsal horn) and upper cervical spinal cord (C1-C2) nociceptive neurons to heat, cold and mechanical stimuli following topical capsaicin treatment of the facial skin, nocifensive behaviors as well as phosphorylation of extracellular regulated-kinase (pERK) in Vc and C1-C2 neurons were studied in rats. RESULTS: Compared to vehicle application, capsaicin application to the lateral facial skin produced 1 hour later a flare in the skin, and also induced significantly greater nocifensive behaviors to heat, cold or mechanical stimulus of the lateral facial skin. The intrathecal (i.t.) injection of the MEK inhibitor PD98059 markedly attenuated the nocifensive behaviors to these stimuli in capsaicin-treated rats. Moreover, the number of pERK-like immunoreactive (pERK-LI) cells in Vc and C1-C2 was significantly larger following the heat, cold and mechanical stimuli in capsaicin-treated rats compared with vehicle-treated rats. The number of pERK-LI cells gradually increased following progressive increases in the heat or mechanical stimulus intensity and following progressive decrease in the cold stimulus. The ERK phosphorylation in Vc and C1-C2 neurons was strongly inhibited after subcutaneous injection of the capsaicin antagonist capsazepine in capsaicin-treated rats. CONCLUSION: The present findings revealed that capsaicin treatment of the lateral facial skin causes an enhancement of ERK phosphorylation in Vc and C1-C2 neurons as well as induces nocifensive behavior to heat, cold and mechanical simulation of the capsaicin-treated skin. The findings suggest that TRPV1 receptor mechanisms in rat facial skin influence nociceptive responses to noxious cutaneous thermal and mechanical stimuli by inducing neuroplastic changes in Vc and C1-C2 neurons that involve in the MAP kinase cascade.

Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2-dependent cyclin E expression.

Hyperhomocysteinemia (HHcy)-abnormally elevated plasma levels of homocysteine (Hcy)-has been associated with the development of neurodegenerative dementia and mild cognitive impairment. This association suggests that HHcy might facilitate memory loss in the elderly. As memory loss can occur through a deteriorated neurogenic capacity, we have studied the effects of Hcy on neural progenitor cells (NPCs) both in vitro and in vivo. We show that Hcy exerts an antiproliferative effect on basic fibroblast growth factor (bFGF) -stimulated NPCs isolated from the postnatal subventricular zone (SVZ), accompanied by inactivation of the extracellular signal-regulated kinase (Erk1/2) and inhibition of Erk1/2-dependent expression of cyclin E. Using a mice model we show that, under normal folate conditions, HHcy exerts an inhibitory effect on adult brain neurogenesis. This inhibition occurs in the caudal areas of the dentate gyrus (DG) of the hippocampus, a neurogenic area mainly involved in learning and memory performance, and in the SVZ, recently implicated in olfactory learning performance. In both areas reduced number of proliferative neuroblasts were found. Since neuroblasts are primarily bFGF-responsive progenitors already committed to a neuronal phenotype, our results strongly suggest that excess Hcy inhibits neurogenesis in the DG and SVZ by inhibiting the bFGF-dependent activation of Erk1/2 in these cells.

Role of soluble guanylate cyclase in the trigeminal subnucleus caudalis in capsaicin-induced muscle hypersensitivity.

Nitric oxide (NO) produces its effects by activating soluble guanylate cyclase (sGC). In the present study, we investigated the potential role of sGC in the subnucleus caudalis (Vc) in mediating masseter hypersensitivity under acute inflammatory condition in male Sprague-Dawley rats. First, our Western blot analysis revealed that sGC protein is reliably detected in the Vc. Subsequent immunohistochemical studies demonstrated that neuronal cell bodies in the superficial laminae of the Vc positively stained for sGC. Astrocytes in deeper lamina of the Vc also showed sGC immunoreactivity. We then tested whether intrathecal administration of sGC inhibitors, methylene blue (MB), and ODQ, in the Vc, attenuates masseter hypersensitivity induced by intramuscular injection of capsaicin. Intrathecal MB or ODQ significantly blocked the capsaicin-induced reduction of mechanical threshold to noxious stimulation of the masseter. These data indicate that the NO-sGC pathway in the Vc is involved in mediating orofacial muscle hypersensitivity under acute inflammatory conditions.

Cox-2 inhibitor attenuates NO-induced nNOS in rat caudal trigeminal nucleus.

OBJECTIVE: The aim of the present study was to determine which isoform of the cyclooxygenase (COX) enzyme plays a role in the neuronal nitric oxide synthase (nNOS) activation caused by nitroglycerin (NTG), in the most caudal part of the trigeminal caudal nucleus (TNC) of the rat. BACKGROUND: Nitric oxide donor, NTG, can trigger migraine attack in migraineurs, but not in healthy persons. In rats, subcutaneous administration of NTG (10 mg/kg) increases significantly the number of nNOS-immunoreactive neurons in the TNC after 4 hours, which could be attenuated by acetyl-salicylate (Aspirin), a nonselective COX-inhibitor. METHODS: SPRD rats were divided into 3 groups: (1) control group (no drug administration), (2) NS398 (selective COX-2 inhibitor) administration (1, 3, or 5 mg/kg), and (3) SC560 (selective COX-1 inhibitor) administration (1, 5, or 10 mg/kg). Thirty minutes after drug administration, the animals received NTG (10 mg/kg) or placebo injection. Four hours later the animals were transcardially perfused and the cervical part of the TNC was removed for immunohistochemistry. Results.-The selective COX-2 inhibitor NS398 in contrast to the selective COX-1 inhibitor SC560 attenuates the NTG-induced nNOS expression dose-dependently. CONCLUSION: These findings suggest that metabolites deriving from COX-2 (but not COX-1) may be the most important factors in the NTG-induced nNOS expression. These data could help to better understand the pathogenesis of headaches and the action of antimigraine drugs.

Protein kinase C gamma-like immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat.

We examined protein kinase C gamma-immunoreactivity (PKCgamma-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCgamma-IR in the MDH was most intense in the SG. The number of neurons with PKCgamma-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCgamma-IR, indicating that SG neurons with PKCgamma might receive nociceptive primary afferent fibers. The results support the notion that PKCgamma in the MDH may contribute to the regulation of the nociception.

Protein kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat.

We examined protein kinase C gamma-like immunoreactivity (PKCgamma-LI) of trigeminothalamic neurons in the rat medullary dorsal horn (MDH) after injecting a retrograde tracer, Fluoro-Gold (FG), into the thalamus. Over 90% of FG-labeled neurons in the marginal layer (lamina I) and a few FG-labeled neurons in the superficial part of the magnocellular layer (lamina III) showed PKCgamma-LI. No PKCgamma-neurons in the substantia gelatinosa (lamina II) were labeled with FG. PKCgamma-mediated regulation of trigeminothalamic neurons may contribute to the changes in MDH activity during persistent pain.

Ultrastructural study of NADPH-d positive neurons in laminae I and II of the rat caudal spinal trigeminal nucleus.

The present study investigated the ultrastructure of neurons in the caudal spinal trigeminal nucleus. These neurons which are believed to function as interneurons in the transmission of orofacial nonreflexive nociceptive information, measured 20 microns x 11 microns, and were nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) positive. The reaction product, formazan, was localized in the nuclear envelope, mitochondria, rough endoplasmic reticulum, and multivesicular bodies of these neurons. It was also localized in the membrane of the smooth endoplasmic reticulum at the axon terminal. The neurons were contacted by both axosomatic and axodendritic synapses formed by both NADPH-d positive and NADPH-d negative axon terminals. Two types of NADPH-d positive axon terminals could be recognized. The first was a large terminal containing many stained mitochondria and unstained small round agranular vesicles mixed with some slightly flattened ones. It formed asymmetrical axodendritic synapse. The second type of axon terminals contained pleomorphic synaptic vesicles and formed asymmetrical synapses upon both dendrites and soma. The sources of NADPH-d positive axon terminals were discussed. Most of the unstained axon terminals forming axosomatic and axodendritic synapses with stained cell bodies and dendrites contained flattened vesicles. In addition to the above, complicated synaptic configurations showing NADPH-d positive axoaxonic synapses in relation to NADPH-d negative dendritic spines were also seen in which a NADPH-d negative dendritic spine was completely contacted by a NADPH-d positive bouton which was in turn contacted by another NADPH-d positive bouton.

Adrenalectomy increases reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity in the rat spinal trigeminal subnucleus caudalis.

Neurons exhibiting reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity (NADPHd) were quantified at 500 microns rostrocaudal intervals in spinal trigeminal nucleus (Vsp) of adenalectomized (ADX), ADX + corticosterone, and sham-ADX rats 6-12 days after surgery. NADPHd neurons were found predominantly in Vsp subnucleus caudalis (Vc) and in dorsomedial subnucleus oralis. ADX significantly increased the number of NADPHd neurons in superficial laminae of Vc, an effect reversed by chronic corticosterone replacement. ADX effects on NADPHd in superficial laminae of Vc but not in deep laminae of Vc or in the periobex region of Vsp paralleled previously observed sites of ADX enhancement of noxious stimulus-induced Fos-like immunoreactivity. The results indicate that chronic changes in adrenal steroid status regulate NADPHd, a mechanism that may both derive from changes in nitric oxide synthase expression and influence the processing of nociceptive information by central trigeminal neurons.

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)

[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.