Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

 Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility. Accepted: 20 April 1998     

Light and electron microscopic immunocytochemical localization of vasoactive intestinal polypeptide VIP-like activity in the rat small intestine

Vasoactive intestinal polypeptide nerve processes and cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the inner circular smooth muscle coat and mainly in the mucosa, but were absent in the longitudinal muscle layer. Submucosal blood vessels were often surrounded by immunoreactive vasoactive intestinal polypeptide positive nerves, in close associations (distance less than 40 mn) to blood vessel basement membranes and to smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labeled fibers surrounded unstained neural cell bodies. The synaptic vesicles of vasoactive intestinal polypeptide positive terminals were 35-40 nm in diameter and some dense core vesicles (80-120 nm in diameter) were also observed in the same profiles. These observations suggest that vasoactive intestinal polypeptide nerves may participate in regulating smooth muscle activity and local blood flow in the small intestine.     

Somatostatin immunopositive neurons in the small intestine of the bullfrog (Rana catesbeiana).

Somatostatin immunopositive neurons in the small intestine of the bullfrog (Rana catesbeiana) were studied using immunohistochemistry and surgical denervation of the mesenteric nerve. Immunopositive nerve elements were distributed throughout the small intestine, including nerve fibers in the myenteric plexus, circular muscle layer, submucosal layer, and mucosa. Somatostatin immunopositive nerve cell bodies occurred in the myenteric plexus but not in the submucosal layer. These cell bodies were surrounded by immunopositive nerve fibers forming basket-like terminals, and thus some of these cells may be interneurons. After denervation of the mesenteric nerve, adrenaline immunopositive nerve fibers disappeared almost completely from the small intestine, but no changes occurred in the distribution of somatostatin immunopositive neurons. Neurons in the coeliac ganglion projecting into the small intestine were adrenaline immunopositive but somatostatin immunonegative. The results indicate that somatostatin immunopositive neurons in the small intestine of the bullfrog are primarily intrinsic in origin.     

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold–silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62 ± 0.07 μm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20–80 nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99 ± 0.07 μm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.     

Direct synaptic projections to the myenteric ganglion of the rat subdiaphragmatic esophagus from the dorsal motor nucleus of the vagus

We have examined the ultrastructure of the myenteric ganglion of the subdiaphragmatic esophagus and determined whether the ganglion neurons receive direct projections from the dorsal motor nucleus of the vagus (DMV) using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) as an anterograde tracer. The neurons (22.2 microm x 13.3 microm) of myenteric ganglion in the esophagus contained dark cytoplasm having many free ribosomes, mitochondria, and an oval nucleus, and received only a few axon terminals contacting somata. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Approximately 85% of the axon terminals contacting dendrites and about 50% of the axon terminals contacting somata contained pleomorphic vesicles, while the rest contained round synaptic vesicles. When WGA-HRP was injected into the DMV, anterogradely labeled fibers and terminals were found in the myenteric ganglia. The WGA-HRP labeled terminals were large (1.97 microm) and contained round clear vesicles and small granular vesicles. These labeled terminals contacted exclusively the small dendrites, but not the somata. These results suggest that the DMV neurons project directly to the myenteric ganglion neurons and regulate the esophageal muscles via the ganglion neurons.     

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold–silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62 ± 0.07 μm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20–80 nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99 ± 0.07 μm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.     

The ultrastructure of somatostatin-immunoreactive cell bodies, nerve fibers and terminals in the dorsal horn of rat spinal cord

The distribution and ultrastructure of somatostatin-immunoreactive neurons, nerve fibers and axon terminals in the dorsal horn of rat thoracic spinal cord were studied by immunohistochemistry at both the light and electron microscopic levels. Somatostatin-immuno reactive neurons were predominantly observed in Rexed laminae I and II of the dorsal horn of spinal cord. Somatostatin-immunoreactive electron dense peroxidase material was concentrated in the Golgi apparatus and rough endoplasmic reticulum of the somatostatin-immunoreactive neurons, and was characteristically sparse in other regions of the cytoplasm. In the somatostatin-immunoreactive fibers and terminals, immunoreactive electron dense material was concentrated in the microtubules and large synaptic vesicles.     

Termination pattern and fine structural characteristics of GABA- and [Met]enkephalin-containing nerve fibers and synapses in the superior cervical ganglion of adult rat.

Morphological features of nerve fibers and synapses containing GABA and [Met]enkephalin were studied at the light and electron microscopic levels in the superior cervical ganglia of rats by pre- and postembedding immunohistochemistry. Both GABA and [Met]enkephalin immunoreactivities were found in varicose nerve fibers, forming diffuse networks which were denser in the rostral than in the caudal part of each ganglion. For both antigens rich and basket-like innervation was observed around some of the principal neurons. The GABA-immunoreactive fibers were evenly stained, while in case of [Met]enkephalin-positive nerve fibers the varicosities showed intensive immunopositivity only. Postembedding immunochemistry revealed that both inhibitory substances were located in axon varicosities which established asymmetric synapses of Gray I type. Fine structural investigation revealed that GABA-like immunoreactivity was confined in the nerve endings to the clear synaptic vesicles of 40 nm diameter, whereas the immunogold particles, indicating the occurrence of [Met]enkephalin, were located over the large dense-cored vesicles of 120 nm diameter. The clear and dense-cored vesicles were, however, mixed in the nerve endings labeled by either neurotransmitter substance. Interestingly, the [Met]enkephalin-immunopositive axon terminals were found, consequently, in synaptic contacts with dendrites containing dense bodies in a row underlying the postsynaptic membrane thickening. Since nerve terminals with GABA-like immunoreactivity established synapses of Gray I type without such subjunctional bodies, one can reasonably assume that, in spite of similarities in termination pattern, there is no co-existence of GABA and enkephalin in the axons in the superior cervical ganglion.     

大鼠延髓孤束核吻侧段内脑啡肽阳性终末与γ-氨基丁酸阳性神经元联系的形态学研究

目的 观察大鼠孤束核吻侧段(rNTS)内脑啡肽阳性(ENK-ir)终末与γ-氨基丁酸(GABA-ir)阳性神经元之间的联系.方法 免疫荧光双重标记技术,包埋前免疫组织化学方法染色结合免疫金颗粒标记的电镜双标技术.结果 激光扫描共焦显微镜下可见rNTS内有大量的ENK-ir纤维和终末及散在的GABA-ir神经元.ENK-ir终末与GABA-ir神经元之间形成密切接触.在电镜下可见ENK-ir阳性产物主要定位于圆形清亮突触小泡及大颗粒囊泡表面.ENK-ir轴突终末与GABA-ir及阴性神经元胞体及树突之间形成对称性和非对称性的突触联系,以对称性突触为主.结论 rNTS内的ENK-ir终末可能通过抑制或增强GABA能神经元活性,或直接抑制味觉感受神经元活性的方式,参与NTS内味觉信息的感受和调节.     

Neuronal location of 5-hydroxytryptamine3 receptor-like immunoreactivity in the rat colon

The presence of 5-hydroxytryptamine(3) receptors on enteric neurons is known from pharmacological data that date back more than 40 years. However, an adequate account of which neurons bear these receptors has not been made because suitable antisera have not been available. We have found that the majority of antisera that have been raised against sequences from the 5-hydroxytryptamine(3) receptor also recognize pre-prosomatostatin. We report that this source of false labeling can be eliminated by pre-incubating the antisera with a peptide designed for this purpose. We have used the pre-absorbed antiserum to localize 5-hydroxytryptamine(3) receptors in the rat colon. Immunoreactive nerve cell bodies occurred in the myenteric and submucosal ganglia. The majority had smooth cell bodies and long, smooth processes, that is, Dogiel type II morphology. The initial segments of the long processes of the Dogiel type II neurons were strongly immunoreactive. About 12% of immunoreactive myenteric nerve cells were of the same or smaller size, and had multiple short filamentous processes. Some of the immunoreactive Dogiel type II neurons were also immunoreactive for calretinin in both plexuses, and the majority were immunoreactive for calbindin in submucosal ganglia. Specific immunoreactivity occurred in non-varicose, but not in varicose, fibers in the myenteric and submucosal ganglia, and in fiber bundles that traversed the longitudinal and circular muscle layers. Immunoreactive varicose fibers were observed only in the mucosa. It is concluded that 5-hydroxytryptamine(3) receptors occur on intrinsic sensory neurons in the rat colon, and on extrinsic sensory nerve fibers that innervate the colon.     

Ultrastructural identification of noradrenergic axons and their distribution within the enteric plexuses of the guinea-pig small intestine

Summary Noradrenergic axons in the enteric plexuses of the guinea-pig ileum have been identified at the ultrastructural level using three techniques: the chromaffin reaction, localization of dopamine--hydroxylase (DBH) with horseradish peroxidase-conjugated antibody, andin vivo andin vitro loading with 5-hydroxydopamine (5-OHDA).In the myenteric (Auerbach's) plexus from normal ileum all of these methods produced electron-dense deposits in a distinctive population of axonal varicosities that contained many flattened vesicles (usually more than 30% of the total number of vesicles), as well as oval or irregularly shaped vesicles. When noradrenergic axons to the small intestine had degenerated after surgical denervation, no profiles containing vesicles with electron-dense deposits were observed with the chromaffin reaction, DBH localization or loading with 5-OHDA. Pretreatment with 6-hydroxydopamine (6-OHDA) substantially reduced the number of noradrenergic axons identified by these three techniques. Axons with many flattened vesicles of similar dimensions but without dense cores were found in myenteric plexus from conventionally fixed intestine. These axons had the same distribution within the ganglia as cytochemically labelled noradrenergic terminals and disappeared after extrinsic denervation.In the normal submucous (Meissner's) plexus, both 5-OHDA loading and the chromaffin reaction produced electron-dense granules in small and large vesicles in some axon terminals. In ganglia labelled by these techniques, reactive terminals contained many small round vesicles and few flattened and large round vesicles as did a population of nonreactive terminals. In axon terminals of submucous plexus labelled with anti-DBH, flattened vesicles were found to be more numerous than with the other treatments. As in the myenteric plexus, all reactive axons disappeared from the submucous plexus after extrinsic denervation. In conventionally processed submucous ganglia, noradrenergic axon profiles could not be distinguished from some non-noradrenergic profiles on the basis of types and proportions of vesicles.In the myenteric plexus noradrenergic axon terminals were seen most often near the edges of ganglia. Noradrenergic varicosities also occurred near nerve cell bodies but were rarely found in internodal strands. In the submucous plexus noradrenergic terminals appeared to be randomly distributed throughout submucous ganglia. No axosomatic synapses formed by noradrenergic axons were found in either plexus, but synapses on nerve processes were occasionally encountered in submucous ganglia.     

Ultrastructural immunocytochemical localization of substance P in the cat small intestine

The immunocytochemical peroxidase-antiperoxidase technique was used to identify substance P (SP) in the cat small intestine at the ultrastructural level in the nerve plexuses. In the myenteric plexus the SP containing fibres form synapses with the other nerve terminals and run parallel to the circular muscle. The SP in these fibres is present in the vesicles of 80 to 120 nm or 150 to 250 nm in diameter. The results support the contention that SP containing nerve terminals are present in the enteric nervous system.     

In vivo monitoring of age-related changes in rat brain using quantitative diffusion magnetic resonance imaging and magnetic resonance relaxometry

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.     

Ultrastructure of the pineal gland of the monkey, Macaca fascicularis, with special reference to the presence of synaptic junctions on pinealocytes

Summary The present study described the normal ultrastructure of the monkey pineal gland. The gland was composed of the principal pinealocytes, intramural neurons and glial cells. The nucleus of the pinealocytes was deeply infolded with evenly distributed chromatin materials. The abundant cytoplasm was rich in organelles including the well-developed Golgi apparatuses, multivesicular bodies, dense-cored vesicles and widely scattered free and polyribosomes. A variety of axon terminals was observed and the majority of them contained pleomorphic agranular vesicles with a few large dense-cored vesicles. A few terminals showed flattened vesicles or small dense cored vesicles. Some of the axon terminals formed synaptic contacts with the cell bodies of pinealocytes. These synapses were mainly concentrated in the posterior third of the gland. The occasional intramural neurons observed were postsynaptic to axon terminals containing round agranular vesicles. The sources of the nerve fibres and terminals forming synaptic junctions with pinealocytes and intramural neurons were discussed.     

Fine structures of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat lateral septum

The fine structures of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the rat lateral septum were investigated by means preembedding immunoelectron microscopy. A number of CRF axon terminals formed synapses with cell bodies of non-immunoreactive septal neurons. They occasionally had broad terminal bulges whose subregions showed little or no immunoreactivity for CRF. CRF axon terminals were also in synaptic contact with non-immunoreactive dendrites or dendritic spines. Some dendrites with CRF were postsynaptic to non-immunoreactive axon terminals.     

The tight junction component protein, claudin-4, is expressed by enteric neurons in the rat distal colon

The expression of a tight junction (TJ) component protein, claudin-4, in the enteric neurons was investigated in the rat distal colon by immunohistochemistry and RT-PCR. Claudin-4 immunoreactivity was detected in almost all neurofilament-positive enteric neurons both of the submucosal and the myenteric plexuses, and both of the cell bodies and the neurofibers. The immunoreactivity of enteric neurons for claudin-4 was divided into two types: strongly and weakly positive neurons. Especially in the myenteric plexus, the stained neurons were classified by Dogiel's morphological classification of enteric neurons. The strongly stained claudin-4 positive neurons show Dogiel type II morphology, while the weakly stained claudin-4 positive neurons show Dogiel type I morphology. These immunohistochemical data were supported by mRNA expression in the muscle plus submucosa preparation containing the submucosal and myenteric plexuses, as well as mucosa preparation. The physiological function of claudin-4 expressed on enteric neurons is unclear up to now. It is however suggested that claudin-4 expressed on enteric neurons might play roles for the neural activity, for example as insulation between neurofibers. In conclusion, the present study clearly shows that claudin-4 is expressed by enteric neurons. This is the first evidence that the neuron itself expresses the TJ component protein, claudin-4, in the nervous system.     

Distribution of the high-affinity choline transporter in the central nervous system of the rat

In cholinergic nerve terminals, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake is thought to be the rate-limiting step in acetylcholine synthesis. The high-affinity choline transporter cDNA responsible for the activity was recently cloned. Here we report production of a highly specific antibody to the high-affinity choline transporter and distribution of the protein in the CNS of the rat. The antibody stained almost all known cholinergic neurons and their terminal fields. High-affinity choline transporter-immunoreactive cell bodies were demonstrated in the olfactory tubercle, basal forebrain complex, striatum, mesopontine complex, medial habenula, cranial nerve motor nuclei, and ventral horn and intermediate zone of the spinal cord. Noticeably, high densities of high-affinity choline transporter-positive axonal fibers and puncta were encountered in many brain regions such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem. Transection of the hypoglossal nerve resulted in a loss of high-affinity choline transporter immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus, which paralleled a loss of immunoreactivity to choline acetyltransferase. The antibody also stained brain sections from human and mouse, suggesting cross-reactivity.These results confirm that the high-affinity choline transporter is uniquely expressed in cholinergic neurons and is efficiently transported to axon terminals. The antibody will be useful to investigate possible changes in cholinergic cell bodies and axon terminals in human and rodents under various pathological conditions.     

Dual axonal terminations from the retrosplenial and visual association cortices in the laterodorsal thalamic nucleus of the rat

Light and electron microscopic tracing studies were conducted to assess the synaptic organization in the laterodorsal thalamic nucleus (LD) of the rat and the laminar origins of corticothalamic terminals from the retrosplenial and visual association cortices to LD. A survey of the general ultrastructure of LD revealed at least three types of presynaptic terminals identified on the basis of size, synaptic vesicle morphology, and synaptic membrane specializations: (1) small axon terminals with round synaptic vesicles (SR), which accounted for the majority of terminal profiles and made asymmetric synaptic contacts predominantly with small dendritic shafts and spines; (2) large axon terminals with round synaptic vesicles (LR), which formed asymmetric synaptic contacts mainly with large dendritic shafts; and (3) small to medium-size axon terminals with pleomorphic synaptic vesicles (SMP), which symmetrically synapsed with a wide range of postsynaptic structures from cell bodies to small dendrites. Synaptic glomeruli were identified, whereas no presynaptic dendrites were found. To characterize and identify corticothalamic terminals arising from the retrosplenial and visual association cortices that project to LD, wheat germ agglutinin conjugated to horseradish peroxidase (WGA–HRP) was injected into these cortices. Axons anterogradely labeled with WGA–HRP ended in both SR and LR terminals. On the other hand, dextran-tetramethylrhodamine injected into LD as a retrograde fluorescent tracer labeled large pyramidal cells of layer V as well as small round or multiform cells of layer VI in the retrosplenial and visual association cortices. These findings provide the possibility that corticothalamic terminations from cortical neurons in layer V end as LR terminals, while those from neurons in layer VI end as SR boutons.     

Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal polypeptide have synaptic contacts with axons immunoreactive for neuropeptide Y: an immunoelectron microscopic study in the rat

An electron microscopic study showed by using a dual immunolabeling technique that in the suprachiasmatic nucleus of the rat, axon terminals immunoreactive for neuropeptide Y (NPY) made synaptic contacts upon neurons immunoreactive for vasoactive intestinal polypeptide (VIP). Diaminobenzidine (DAB)-labeled NPY axon terminals made synaptic contacts on silver-gold-labeled VIP perikarya and dendritic processes. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles labeled with DAB chromogen. At the synaptic portion, a symmetrical thickening of the pre- and post-synaptic membranes was evident.     

大鼠三叉神经本体感觉中枢通路二、三级核团内Parvalbumin样阳性神经元突触联系的电镜研究

本教研室以往的研究证实 Parvalbum in样免疫阳性细胞广泛分布于三叉神经本体感觉中枢四级通路的各级中继核团 ,其中有 30 %～ 5 0 %为投射神经元。本研究应用电镜免疫组织化学技术进一步对此通路第二、三级神经元所在地的 Parvalbumin样阳性神经元及其纤维和终末的突触联系进行了观察。结果显示 Parvalbum in样阳性结构主要形成以下几种突触联系 :( 1) Par-valbumin样阳性轴突与 Parvalbumin样阳性胞体或树突形成轴 -体或轴 -树突触 ,其中以非对称性突触为主 ,对称性突触较少 ;( 2 )Parvalbumin样阳性轴突分别与 Parvalbumin样阴性神经元的胞体或树突形成轴 -体或轴 -树突触 ,这些突触联系以对称性为主 ,非对称性大约占 30 %左右 ;( 3) Parvalbumin样阴性终末与 Parvalbumin样阳性树突形成以对称性为主的轴 -树突触 ,这种突触大约占所有突触联系的 5 0 %。以上结果表明 :面口部本体感觉信息由三叉神经中脑核神经元向丘脑腹后内侧核传递的过程中 ,Par-valbumin样阳性轴突终末可通过突触传导机制而兴奋或抑制二、三级核团内的投射或中间神经元而发挥其重要作用