Evidence for involvement of the neural pathway containing the peripheral vagus nerve, medullary visceral zone and central amygdaloid nucleus in neuroimmunomodulation

It is now evident that a bidirectional communication network exists between the central nervous system (CNS) and immune system (IS). However, the way in which the IS passes inform to the brain is not quite clear.In the present study, one of the neural pathways involved in the cytokine-to-brain communication was investigated in the rat. This pathway starts at the vagal nerve projecting to the medullary visceral zone (MVZ), an arc-shape band from the dorsomedial to ventrolateral area in the middle-caudal segment of the medulla oblongata, and terminates at the central amygdaloid nucleus (Ce) which receives projections from large catecholaminergic neurons in the MVZ. Animals were randomly divided into two experimental groups. Triple-labeling was used in Group I animals to combine wheat germ aggulutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunostaining. WGA-RP was stereotaxically injected into the unilateral Ce of the animals and, after a survival period of 48 h, intraperitoneal (IP) injection of lipopolysaccharide (LPS) was performed. Seven kinds of labeled neurons were observed in the MVZ, namely, HRP-, Fos- or TH-singly-labeled neurons; Fos/HRP-, Fos/TH- or HRP/TH-doubly-labeled neurons; and Fos/HRP/TH-triply-labeled neurons. As for Group II animals, bilateral subdiaphragmatic vagotomy (SDV) or sham operation was performed, followed 4 weeks later by IP injection of LPS. The number of Fos-positive neurons within the Ce and MVZ was significantly lower (P<0.01) in rats having SDV when compared with those receiving sham operation. Our results suggest that part of the peripheral immune information can be conveyed through the vagus to the catecholaminergic neurons in the MVZ, where it is transported to the Ce. The MVZ is a neural relay station in the immune-to-brain communication and might play a significant role in neuroimmuno-modulation via the vagus-MVZ-Ce pathway.     

Visceral noxious stimulation induced expression of Fos protein in medullary catecholaminergic neurons projecting to nucleus accumbens in the rat: a study with triple labeling method of HRP tracing combined with Fos and TH immunohistochemistry

Horseradish peroxidase (HRP) was stereotaxically injected into the nucleus accumbens (Acb), and visceral noxious stimulation given by injecting formalin into the stomach. Sections of the medulla were subjected to HRP reaction combined with immunohistochemical reactions for Fos protein (ABC method) and tyrosine hydroxylase (TH, PAP method). The catecholaminergic neurons of the medulla (including vagal complex, ventrolateral medulla and reticular formation between them) which expressed Fos protein and projected to Acb were studied. The results showed that HRP retrogradely labeled cells were seen in the medulla bilaterally with apparent ipsilateral predominance and TH-LI and Fos-LI single labeled cells were bilaterally distributed; HRP/TH,TH/Fos double labeled neurons were more numerous than HRP/Fos double-labeled neurons. HRP/TH/Fos triple-labeled neurons were small in number and were mainly distributed in the nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM), but only a few labeled cells were located in RF between nTS and VLM. It is concluded that TH-LI neurons in the medulla projected to Acb and some of them expressed Fos protein after noxious stimulation of the stomach.     

延髓内脏带至杏仁核投射通路参与迷走神经刺激抑制癫痫发作的研究

目的 观察迷走神经→延髓内脏带(MVZ)→杏仁中央核的儿茶酚胺能通路是否参与了迷走神经刺激(vagus nerve stimulation,VNS)抑制癫痫的调节；是否存在由迷走神经→延髓内脏带→海马的直接投射参与抑痫。方法 将逆行追踪剂WGA—HRP注入大鼠—侧杏仁中央核或腹侧海马，48h后，给予迷走神经刺激，观察MVZ内WGA—HRP逆行标记的细胞、Fos蛋白、TH阳性神经元的表达及分布。结果 杏仁核注射组大鼠MVZ内可见HPR／Fos／TH二重标记的细胞；海马注射组MVZ内未见HRP逆标神经元，但HRP逆行标记与Fos阳性双重标记细胞出现存隔区和下丘脑室旁该。结论 提示迷走神经→延髓内脏带→杏仁中央核的投射通路直接参与VNS抑痫过程，而且与儿茶酚胺能神经元有关；迷走神经→延髓内脏带→隔区、下丘脑室旁核中继至海马的间接通路也参与了抑痫。     

大鼠延髓内脏带与下丘脑室旁核、视上核往返投射通路参与高渗调节反应的研究

目的探讨延髓内脏带(MVZ)与下丘脑室旁核(PVN)和视上核(SON)之间是否存在往返渗透压投射通路。方法通过给予大鼠饮用3%氯化钠的方法制作高渗刺激模型,并用WGA-HRP逆行追踪、抗Fos、抗酪氨酸羟化酶(TH)或加压素(VP)及胶质纤维酸性蛋白(GFAP)免疫组织化学相结合的四重标记方法,观察MVZ、PVN和SON中WGA-HRP、Fos、TH、VP和GFAP阳性分布及表达状况。结果高渗刺激后MVZ、PVN和SON内Fos阳性细胞明显增多;GFAP阳性结构也明显增多,其分布与Fos阳性细胞分布基本一致,表现为胞体肥大、突起粗长。星形胶质细胞(AST)紧密包绕在神经元周围形成神经元-AST复合体(N-ASC)。结论神经元和AST以N-ASC的形式共同参与渗透压调节反应,体内存在MVZ和SON或PVN之间往返的渗透压调节通路。     

Tyrosine hydroxylase protein in Lewy bodies of parkinsonian and senile brains

Immunohistochemistry with the antisera against tyrosine hydroxylase (TH) was performed on the neurons with Lewy bodies (LBs) in the sections at the level of the basal ganglia, midbrain, pons, medulla oblongata and spinal cord. These were obtained from 3 autopsy cases with Parkinson's disease and 2 senile brains without parkinsonism. In catecholamine neurons, stained positively with the antisera to TH, specific immunoreactions were found in the LBs. These specific stainings were accentuated and/or restricted to the peripheral, less dense, zone of the LBs in the neurons of catecholaminergic regions. LBs in the neurons of the innominate substance, raphe nuclei of the brain stem and spinal cord did not stain positively with TH antisera. This suggests that the production of LBs is complicated, but that it is also closely correlated to the biochemical properties of each neuron in the various regions. In catecholamine neurons in which LBs frequently occurred, TH enzyme protein might play an important part in the productions of LBs.     

The Role of Ascending Neuronal Pathways in Stress-Induced Release of Noradrenaline in the Hypothalamic Paraventricular Nucleus of Rats

Central catecholaminergic pathways carrying pain-related signals to the hypothalamic paraventricular nucleus (PVN) were investigated in laboratory rats. Four per cent formalin injected subcutaneously was employed as a stressful stimulus. Neuronal activity in brainstem catecholaminergic and paraventricular neurones was assessed by Fos immunohistochemistry. Stress-induced noradrenaline (NE) release from nerve terminals in the PVN was measured in extracellular fluid by in-vivo microdialysis. Within 30 min, formalin elicited a four- to sixfold increase in plasma ACTH and corticosterone concentrations and intense Fos-like activity was seen in the superficial zones of the lumbar spinal cord ipsilateral to the side of the formalin injection. In brainstem catecholaminergic neurones, the PVN, and midline thalamic nuclei, formalin-induced Fos-immunopositivity was equally present in the ipsi- and contralateral sides of the injection. An immediate elevation (4-5 times higher than baseline levels) of NE levels was measured in both the right and left PVN after a formalin injection into the right paw. Unilateral surgical transections at the medulla-spinal cord junction failed to affect formalin-induced elevations in NE levels in the PVN independently of the side of the formalin injection or the knife cut. Thus, this observation clearly shows that fibres carrying pain-evoked signals ascend bilaterally from the spinal cord to the brainstem and forebrain. Hemisections of the medulla oblongata between the level of A1-A2 NE cell groups and the locus coeruleus reduced but did not eliminate formalin-induced NE release from the PVN ipsilateral to the knife cut. This effect was independent of the side of the formalin injection. In the contralateral PVN, high and similar NE levels were measured in response to a formalin injection into the right or the left leg. The present study indicates that formalin-induced pain signals are carried by sensory fibres to the ipsilateral spinal cord. From there, axons of different dorsal horn neurones reach noradrenergic cells on both sides of the medulla oblongata. The majority of noradrenergic fibers ascend on the same side and innervate the ipsilateral PVN. Since formalin administration resulted in a moderate elevation of NE levels in the PVN on the operated side, the role of other ascending noradrenergic (from the locus coeruleus) or noncatecholaminergic fibres that could modulate NE release from the PVN should be considered.     

Flurothyl-induced seizures in rats activate Fos in brainstem catecholaminergic neurons

Autonomic changes accompany seizures in both animals and humans. While ictal autonomic dysfunction can be life-threatening, the participating neural networks involved are poorly understood. In this study we examined the activation of Fos following generalized seizures in brainstem structures known to mediate autonomic function. Adult female rats were sacrificed 2 h after flurothyl-induced seizures. Double-immunostaining for c-Fos and dopamine-beta-hydroxylase (DBH), and c-Fos and phenylethanol-N-methyl-transferase (PNMT) were performed in brainstem slices. Numbers of DBH-labeled neurons expressing Fos-like immunoreactivity (FLI) (DBH/Fos) and PNMT labeled neurons expressing FLI (PNMT/Fos) were counted in the noradrenergic (A1, A2, A5, A7) and adrenergic (C1, C2) cell groups localized in pons and medulla oblongata. Among the experimental animals, the highest degree of co-localization of DBH/Fos neurons was observed in the locus coeruleus (A6; 87.7%), and in the A1(72.8%) cell group located in the caudal ventrolateral medulla (VLM). No co-localization of DBH/Fos neurons was observed in control animals. The highest degree of co-localization of PNMT/Fos neurons was observed in the C1 adrenergic cell group (84.2%) located in the rostral VLM. Control animals showed very few (5.5%) PNMT/Fos co-localized neurons in the C1 adrenergic cell group. Our results indicate that flurothyl-induced generalized seizures in rats activate catecholaminergic neurons in the pons and medulla oblongata. Further studies are necessary to determine whether activation of brainstem catecholaminergic neurons contribute to the autonomic manifestations that frequently accompany epileptic seizures.     

Distribution of glutamic acid decarboxylase mRNA-containing neurons in rat medulla projecting to thoracic spinal cord in relation to monoaminergic brainstem neurons.

Recent neurophysiological work has suggested the existence of monosynaptic gamma-aminobutyric acidergic (GABAergic) projections from the medulla oblongata to sympathetic preganglionic neurons. The purpose of the present study was to identify the possible anatomical location of these neurons. The location of GABAergic neurons with projection to the thoracic spinal cord was studied by using in situ hybridization for both 65-kD and 67-kD isoforms of glutamic acid decarboxylase (GAD) mRNA (GAD-65 and GAD-67, respectively) combined with midthoracic spinal cord injections of the tracer Fast Blue. Tyrosine hydroxylase (TH) or tryptophan hydroxylase immunohistochemistry was combined with GAD mRNA detection and Fast Blue to determine whether any bulbospinal catecholaminergic or serotonergic cell groups in the medulla also are GABAergic. GAD-67 and GAD-65 mRNA-containing neurons had similar distribution patterns in the medulla oblongata, with some areas exhibiting lighter labeling for GAD-65 mRNA. GABAergic bulbospinal neurons were located in the caudal part of the solitary nucleus, the parasolitary nucleus, the vestibular nuclei, the ventral medial medulla, the raphe nuclei, and parapyramidal areas. TH-immunoreactive neurons in the A1, A2, C1, and C2 areas or the area postrema did not contain either GAD-67 or GAD-65 mRNA. GAD mRNA-positive bulbospinal cells were present medial to theA1 and C1 catecholaminergic cell groups, with little or no overlap. Serotonergic neurons positive for GAD mRNAwere found in the parapyramidal area and just dorsal to the pyramidal tract in the raphe magnus. This population included bulbospinal neurons. In conclusion, GABAergic neurons with projections to the thoracic spinal cord exist in a restricted number of medullary nuclei from which inhibitory sympathetic control may originate.     

Colocalization of fixative-modified glutamate and glutaminase but not GAD in rubrospinal neurons

In an attempt to identify putative neurotransmitters of rubrospinal neurons, immunocytochemical procedures were utilized in combination with retrograde tracing techniques in 15 adult male rats. Following injections of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) into the spinal cord, midbrain sections were processed with a combined procedure that allowed visualization of both the retrograde tracer and one or more antigens including glutamate, glutaminase, and glutamatic acid decarboxylase (GAD). Initial colocalization studies demonstrated that glutamatelike and glutaminaselike immunoreactivities were cocontained within the same neurons. Following injections of HRP or WGA-HRP into the spinal cord approximately 53% of retrogradely labeled neurons contained glutamate immunoreactivity. Triple-labeling experiments indicated that glutamatelike immunoreactivity was colocalized with glutaminase immunoreactivity in retrogradely labeled rubrospinal neurons. Retrogradely labeled neurons did not contain GAD immunoreactivity. Moreover, triple labeling experiments verified that glutamatelike immunoreactive retrogradely labeled cells did not cocontain GAD immunoreactivity. These studies demonstrate that glutamate and its synthesizing enzyme, glutaminase, are present in some rubrospinal neurons and raise the possibility that a component of the rubrospinal projection may be glutamatergic. GAD, on the other hand, is not present in rubrospinal neurons. This finding supports the hypothesis that GABAergic neurons play a role as interneurons in the red nucleus.     

Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes

The immunocytochemical localization of the biosynthetic enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)--was used to determine the cytological features and precise neuroanatomical location of catecholaminergic neurons in the medulla oblongata of rat. Perikarya labeled with TH were detected in two bilaterally symmetrical columns located in the ventrolateral and dorsomedial medulla. The distribution and the number of neuronal perikarya containing TH were the same as those containing DBH, except in the dorsal motor nucleus of the vagus at the level of the area postrema where the number of neurons immunocytochemically labeled for TH was considerably greater than those labeled for DBH. The detection of perikarya which show immunoreactivity for TH, used in the biosynthesis of dopamine, noradrenaline, and adrenaline, but not DBH, which converts dopamine to noradrenaline, suggests the existence of dopamine-synthesizing neurons in the medulla. Perikarya labeled with PNMT, used in the biosynthesis of adrenaline, were localized in more restricted regions corresponding to rostral subsets of the dorsal and ventral groups labeled for TH and DBH. Counts of neurons immunocytochemically labeled for TH or PNMT were obtained in order to determine the relative ratio of neurons which contain the enzymes necessary for the synthesis of dopamine, noradrenaline, or adrenaline at various levels of the medulla. At the most caudal levels no PNMT labeled neurons were detected. Further rostral, PNMT-labeled neurons were first detected in the ventrolateral medulla. At the level of the area postrema, the number of PNMT-labeled neurons in the ventrolateral medulla was approximately half of the number of cells showing immunoreactivity for TH. In contrast, few PNMT-labeled cells were detected in the dorsomedial medulla at the level of the area postrema compared to many neurons labeled for TH. At rostral medullary levels, in both the ventrolateral and the dorsomedial regions, the number of neurons labeled for TH and PNMT was essentially the same. Thus most, if not all, of the catecholaminergic neurons in the rostral medulla have PNMT, necessary for the synthesis of adrenaline.     

Bulbospinal respiratory neurons are a source of double synapses onto phrenic motoneurons following cervical spinal cord hemisection in adult rats

The purpose of this study was to determine if the medullary neurons that provide the primary excitatiry drive to phrenic motoneurons (i.e., rostral ventral respiratory group, rVRG) are a source of double synapse formation in the phrenic nucleus after spinal cord hemisection. The axons of rVRG neurons either ipsilateral or contralateral to the hemisection were labeled by injection of a mixture of HRP and WGA-HRP into the rostral ventral respiratory group. Phrenic motoneurons ipsilateral and caudal to the hemisection were labeled by the retrograde transport of HRP. The ultrastructural results indicated that after hemisection, rVRG neurons from both sides of the medulla formed labelled double synapses in the phrenic nucleus.     

Involvement of medullary A2 noradrenergic neurons in the activation of oxytocin neurons after conditioned fear stimuli

Fear-related stimuli activate oxytocin neurons in the hypothalamus and facilitate oxytocin release from the pituitary. Oxytocin neurons in the supraoptic nucleus receive direct noradrenergic innervations from the A1 and A2 cell groups in the medulla oblongata. In the present study, we investigated the role of hypothalamic-projecting noradrenergic neurons in controlling oxytocin cell activity following fear-related stimuli in rats. An unconditioned fear stimulus (intermittently applied footshock) or conditioned fear stimulus induced expression of Fos protein, a protein product of an immediate-early gene, in magnocellular oxytocin neurons in the supraoptic or paraventricular nucleus. A neurotoxin, 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine, microinjected into the vicinity of the supraoptic nucleus, selectively depleted the noradrenaline contents of the nucleus and blocked the Fos expression in the supraoptic nucleus after the unconditioned or conditioned fear stimulus. In the medulla oblongata, the unconditioned fear stimulus induced expression of Fos protein in both A2/C2 and A1/C1 catecholaminergic neurons. On the other hand, the conditioned fear stimulus induced expression of Fos protein preferentially in the A2/C2 neurons. Furthermore, the unconditioned fear stimulus induced Fos expression in the A1/C1 and A2/C2 catecholaminergic neurons labelled with retrograde tracers previously injected into the supraoptic nucleus. The conditioned fear stimulus induced Fos expression preferentially in the A2/C2 catecholaminergic neurons labelled with the retrograde tracers. These data suggest that the conditioned fear-induced oxytocin cell activity is mediated by the A2 noradrenergic neurons projecting to oxytocin neurons, while the unconditioned fear response is mediated by both A2 and A1 noradrenergic neurons.     

Localization of Barrington's nucleus in the pontine dorsolateral tegmentum of the rabbit.

The localization of Barrington's nucleus in the dorsolateral pons of the rabbit and its projections to the sacral spinal cord were examined by using retrograde and anterograde labeling methods combined with immunohistochemistry. After injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or a fluorescence tracer, tetramethylrhodamine-dextran amine (TMR), into the sacral spinal cord segments, a cluster of neurons labeled with WGA-HRP or TMR were seen in the pontine dorsolateral tegmentum. To identify whether the retrogradely labeled neurons were situated within the locus coeruleus, the sections containing TMR-labeled neurons through the pons were incubated with anti-tyrosine hydroxylase (TH) antibody and observed under epifluorescence microscope. It was shown that the cluster of TMR-labeled neurons in the dorsolateral tegmentum were surrounded by TH-positive neurons, but they were negatively immunostained with TH-like immunoreactivity. In anterograde experiment, injection of WGA-HRP into the dorsolateral tegmentum resulted in many anterogradely labeled nerve fibers and terminals in the sacral spinal cord, including the sacral parasympathetic nucleus. The present results suggest that the cluster of neurons in the dorsolateral tegmentum of the rabbit may correspond to Barrington's nucleus revealed in the rat and cat, and thus may be involved in micturtion reflex of the rabbit.     

c-Fos expression in the myenteric plexus, spinal cord and brainstem following injection of formalin in the rat colonic wall

Fos expression induced by injection of dilute formalin (50 microl, 5% in physiological saline) into the colonic wall was examined in the myenteric plexus, lumbosacral spinal cord and brainstem of the rat. The aims of this study were (i) to determine whether neurons in these regions express Fos in response to the injection of formalin into the colon and (ii) to examine whether administration of an alpha 2 adrenoceptor agonist modulates Fos expression. Tissues were removed 2 h after the injection of saline or formalin. Saline injected in the colon induced Fos in enteric glia in the myenteric plexus. The number of Fos immunoreactive nuclei significantly increased in both myenteric neurons and enteric glia after the injection of formalin. Similarly, Fos immunoreactive neuronal nuclei were significantly increased in the spinal cord, area postrema and nucleus of the solitary tract after the injection of formalin. Pretreatment of rats with the alpha 2 adrenoceptor agonist xylazine (2, 4 and 8 mg/kg) 15 min before the injection of formalin, dose-dependently reduced the number of Fos immunoreactive neuronal and glial nuclei in the myenteric plexus, and neuronal nuclei in the spinal cord and brainstem. Simultaneous administration of xylazine (8 mg/kg) and the alpha 2 adrenoceptor antagonist yohimbine (1 mg/kg) reversed the effects of xylazine in the spinal cord and brainstem, but not in the myenteric plexus. These data show that injection of formalin in the colonic wall results in Fos expression in myenteric neurons and enteric glia, and neurons in the spinal cord and brainstem. This may be due to the direct chemical stimulation of the innervation of the colon and/or the subsequent acute colitis. The observed neuronal Fos expression can be modulated by an alpha 2 adrenoceptor agonist through noradrenergic pathways and/or reduction of the excitability of the enteric neural circuitry.     

家兔脑桥被盖背外侧部向骶髓的直接投射

为研究家兔脑桥排尿反射通路的构成,本实验采用顺、逆行追踪技术结合免疫组化方法对家兔脑桥被盖背外侧部与骶髓的纤维联系进行了研究。将麦芽凝集素辣根过氧化物酶（ＷＧＡ－ＨＲＰ）或荧光素四甲基罗达明葡聚糖胺（ＴＭＲ）注射到骶髓后,在脑桥被盖背外侧部发现一团ＷＧＡ－ＨＲＰ或ＴＭＲ标记的神经元。为确定这些逆标细胞是否属于蓝斑核内的神经元,将ＴＭＲ注射例的脑桥切片用抗酪氨酸羟化酶（ＴＨ）抗体孵育并进行免疫荧光组化染色。结果显示,ＴＨ阳性细胞环绕于ＴＭＲ标记神经元周围,但未见ＴＭＲ／ＴＨ双标细胞。将ＷＧＡ－ＨＲＰ注射于脑桥被盖背外侧部后,在骶髓节段的骶髓副交感核、中间带灰质和后连合核等处发现有大量顺行标记的纤维和终末。结合在其他种属动物上的研究资料,推测位于家兔脑桥被盖背外侧部的这团神经元可能相当于在大鼠和猫相应部位发现的与脑桥排尿反射密切相关的Ｂａｒｒｉｎｇｔｏｎ核     

The overlap of spinothalamic and dorsal column nuclei projections in the ventrobasal complex of the rat thalamus: a double anterograde labeling study using light microscopy analysis

Projections from the spinal cord and the dorsal column nuclei (DCN) to the ventrobasal complex of the thalamus (VB) were studied in the rat by using double anterograde labeling strategy. This strategy was based on the injection of 3H-leucine into the DCN and of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the spinal cord and their subsequent transport. Adjacent 30-micron-thick sections were then processed differentially for autoradiography or for HRP by using tetramethyl benzidine (TMB) as a chromogen. Similar areas of the ventrobasal complex were labeled, in adjacent sections, after a large injection of 3H-leucine into the DCN and when wheat germ agglutinin-HRP had been injected in any part of the spinal cord. If, however, a small injection of the radioactive tracer was centered in the gracile nucleus and compared with an injection of WGA-HRP placed in the lumbar enlargement of the cord, the rostral and dorsal portions of the lateral VB were labeled from both sources. On the other hand, if tritiated leucine was injected into the cuneate nucleus, and WGA-HRP placed in the cervical enlargement, then the caudal and ventral portions of the lateral VB demonstrated overlap of both labels. The present results show that, in the rat, areas of termination of both the spinothalamic tract and the lemniscal pathway originating from the DCN overlap in the lateral VB. This overlap is somatotopically organized, thus indicating that the same area of the VB receives somatic inputs from one particular part of the body through both pathways. These results are discussed in comparison to those of comparable studies performed in the cat and in the monkey and with reference to the electrophysiological data that have demonstrated that, in the rat VB, neurons responding to noxious stimulation are intermingled with neurons exclusively responding to non-noxious stimulation.     

Distribution of norepinephrine-containing neurons projecting to the parietal associative cortex and spinal cord in the cat locus coeruleus

The distribution of neurons projecting to the parietal associative cortex and spinal cord in the locus coeruleus (LC) of the cat was examined by the retrograde transport of the horseradish peroxidase (HRP) and catecholamine histofluorescence technique. It was demonstrated that neurons projecting to the parietal cortex were localized predominantly in the dorsal LC and the most dense labeling was observed at the coronal section corresponding to frontal plane P-1. Neurons projecting to the spinal cord were concentrated in the ventral LC and the largest quantity of labeled neurons was found in the frontal plane P-3. Microinjections of HRP into the parietal cortex and spinal cord also caused intense retrograde labeling of neurons in the reticular formations of mesencephalon, pons varolii and medulla oblongata. Coeruleo-cortical and coeruleo-spinal projection neurons formed in the LC two partially overlapping cellular populations which could not be differentiated according to morphologic characteristics of their cells. It was concluded that the parietal associative cortex of cat as well as spinal cord had direct afferent inputs both from the LC and from the reticular formation.     

Monosynaptic projections from the lateral periaqueductal gray to the nucleus retroambiguus in the rhesus monkey: implications for vocalization and reproductive behavior

The periaqueductal gray (PAG) is known to be essential for vocalization and reproductive behavior. The PAG controls components of these behaviors by means of projections to the nucleus retroambiguus (NRA), a group of premotor neurons in the caudal medulla oblongata. In the accompanying study (VanderHorst et al., 2000 [accompanying study]), the NRA and its lumbosacral projections have been identified in the rhesus monkey. The present light and electron microscopical tracing study describes the PAG-NRA pathway in primates. To locate midbrain neurons projecting to the NRA, wheat germ agglutinin horseradish peroxidase (WGA-HRP) was injected into the NRA in six monkeys. To determine the distribution pattern of PAG axons in the medulla oblongata, WGA-HRP was injected into the PAG and adjacent tegmentum in three additional monkeys. In one of these three monkeys, biotinylated dextran amine and cholera toxin subunit b were injected into the lumbosacral cord to retrogradely identify NRA neurons. The results show that a compact group of neurons in the medial part of the lateral PAG at the intercollicular level sends a dense projection to the NRA. The projection is bilateral with a clear ipsilateral predominance. At the ultrastructural level, there are monosynaptic contacts between PAG fibers and NRA neurons, including NRA neurons that project to the lumbosacral cord. The synaptic contacts were primarily asymmetrical and the labeled terminal profiles contained spherical and dense core vesicles. It is concluded that there exists a strong and direct PAG-NRA pathway in the rhesus monkey. Because NRA neurons projecting to the lower lumbar cord are included, the PAG-NRA projection is likely to be involved not only in vocalization but also in other behaviors, such as receptive posture.     

Projection from the ventrolateral medullary neurons containing tyrosine hydroxylase to the central amygdaloid nucleus in the rat.

By using a double-labeling produce of retrograde horseradish peroxidase (HRP) tracing and the immunocytochemical localization of tyrosine hydroxylase (TH), the present study ascertained that the axonal fibers of catecholaminergic neurons in the caudal ventrolateral medulla projected to the central amygdaloid nucleus in the rat. The majority of double-labeled cells were observed primarily around the level of the obex. 92% of HRP retrogradely labeled cells contained TH-like immunoreactive (TH-IR), but only 31% of TH-IR cells was labeled with HRP.     

Effects of baclofen on colon inflammation-induced Fos, CGRP and SP expression in spinal cord and brainstem

The present study demonstrates sites of expression for Fos protein in the brainstem and lumbosacral spinal cord of rats subjected to mustard oil irritation of the colon. The protective effect of baclofen, a selective GABA(B) receptor agonist, on the induced Fos protein increases was determined. Mustard oil injected into the lumen of the colon produces an acute site-specific inflammation. Immunocytochemical localization of Fos protein in neuronal nuclei was evident after 1 h, was greatest at 2 h and was still evident but declining at 8 h. In the spinal cord the majority of Fos labeled neurons were localized in the superficial laminae of lumbar (L6) cord with more found in the sacral (S1) cord. Some labeled neurons were also found in the deeper spinal laminae, intermediolateral nucleus and around lamina X. Brainstem sites expressing Fos included the nucleus of the solitary tract in the medulla, parabrachial, locus coeruleus, pontine and caudal dorsal raphe nuclei and periaqueductal gray. Weak Fos protein labeling existed in a few cells in vehicle control animals. Systemic administration of the GABA(B) receptor agonist, baclofen (10 mg/kg, i.p.), significantly reduced Fos expression in the spinal cord after mustard oil treatment but significantly increased the relative number of nuclei labeled in the nucleus of the solitary tract. Baclofen also significantly decreases dorsal horn CGRP immunoreactivity relative to the increased levels seen after inflammation of the colon. The SP content increases observed after inflammation of the colon were not altered by baclofen. These data suggest that: (1) neurons in regions important for nociceptive transmission, descending inhibitory control and autonomic control are activated by noxious stimulation of the colon, and (2) baclofen specifically reduces Fos expression in the superficial dorsal horn of the spinal cord induced by nociceptive afferent input.