Use of fluorescent carbocyanine dyes in the study of the organization of the pathways in autopsy material of the human brain

Our report provides evidence that fluorescent carbocyanine dyes (diI and diO) can be used in experimental anatomical studies of the fixed autopsy human brain. The dyes transported in both anterograde and retrograde directions, providing labeling of axons with collaterals and neurons including dendrites. To study the retrograde labeling of pyramidal neurons and anterogradely labeling of afferent fibers in human motor cortex, we applied diI and diO to the white matter, I and III layers of cortex. During 2 months there was no evidence of passive diffusion from labeled fibers and neurons to other neurons or glia. This method will be useful for identifying alterations of neuronal connections associated with neurological and psychiatric disorders.     

Distribution pattern in the human pituitary and hypothalamus of a new neuropeptide: The C-terminal glycoprotein-fragment of human pro-pressophysin (CPP)

Summary The distribution pattern of CPP-containing neurons and fibers in the human pituitary and hypothalamus was studied with a specific antiserum to human CPP and the unlabeled antibody technique. Immunoreactive CPP was found in the magnocellular neurons of the supraoptic nucleus (SON), the paraventricular nucleus (PVN) and in neurons scattered in the supraoptic hypophyseal tract. CPP-containing parvocellular neurons were found in the suprachiasmatic nucleus (SCN). The CPP-containing fibers from the magnocellular neurons formed a tract coursing through the median eminence and the pituitary stalk to the posterior lobe of the hypophysis. In contrast, no such fibers from the SCN projected to SON, PVN and the median eminence. This pattern is identical to that of vasopressin and its associated neurophysin-containing neurons and fibers and strongly supports the concept that CPP is a part of the common precursor for vasopressin and neurophysin II. The biological importance of human CPP other than being a precursor fragment remains to be elucidated.To whom requests for reprints should be addressed     

Corticotropin-releasing hormone in the human hypothalamus. Free-floating immunostaining method

We have clearly demonstrated corticotropin-releasing hormone (CRH) immunoreactive cell bodies and nerve fibers in the human hypothalamus by immunocytochemistry using free-floating sections instead of paraffin-embedded sections. Human hypothalami were obtained at autopsy, fixed and cryostat-sectioned at 40 microns. Free-floating sections were immunostained with antibody to CRH using the Vector ABC system. Most of CRH immunoreactive nerve fibers from the paraventricular nucleus pass under the fornix, while some CRH immunoreactive nerve fibers pass beyond the fornix and some through the fornix. Then the CRH immunoreactive nerve fibers run downward, medially to the supraoptic nucleus and toward the pituitary stalk. This method of immunocytochemistry is a very sensitive and suitable means for immunocytochemical studies of neuropeptides in the human brain.     

Neurotransmitters and neuropeptides in the developing human central nervous system. A review

This is a review on the ontogenesis of major neurotransmitters and neuropeptides in the developing human central nervous system. In general, the molecules under study appeared early in development, usually in the first trimester. Cholinergic neurons were found to be present around the time of neuropeptide formation. The newly formed neuropeptidergic fibers extended towards the cholinergic centers where both might interact. In the major centers of the central nervous system, neuropeptides were also noted to colocalize with various neurotransmitters. For example, in the facial nucleus, enkepahlin and substance P fibers coexisted with cholinergic and catecholaminergic neurons, suggesting complex interactions. In the interpeduncular nucleus, peptidergic neurons acting as interneurons clearly modulated the afferent input to this nucleus. In the hippocampus and in sensory organs such as the retina, there were indications that neuropeptides and gamma-amino butyric acid coexisted. We hypothesize that interactions of neurotransmitters and peptides in neurons and fibers early in development play an indispensable role in the morphogenesis of the human central nervous system.     

Histochemical diagnosis of the neurons of cholinergic synaptic transmission

The authors studied neurons of the medulla oblongata of 5 human fetuses (22-27 weeks of development). Cholinacetyltransferase (CAT) activity was examined by the Berth method. Three neuronal types were diagnosed in the nuclei of the medulla oblongata with regard to CAT localization in the cytoplasm and synapses: (a) cholinergic-cholinoceptive neurons having CAT in the cytoplasm and in the innervating afferent fibers; (b) cholinergic-noncholinoceptive neurons with high CAT content, innervated with noncholinergic afferent fibers; (c) noncholinergic-cholinoceptive neurons carrying cholinergic synapses.     

Immunocytochemical localization of somatostatin in human brain

The distribution of somatostatin (SRIF) was examined in normal human forebrain, using thick vibratome cut sections. The unlabeled antibody enzyme method of immunocytochemistry revealed a widespread distribution of SRIF immunoreactive neurons and fibers throughout the septum, diencephalon and corpus striatum. Within the septum SRIF neurons and fibers were observed in the medial and lateral septal nuclei, the nucleus of the diagonal band, the nucleus accumbens and the bed nucleus of the stria terminalis. SRIF neurons and fibers were found in several hypothalamic and anterior thalamic nuclei as well as all regions of the corpus striatum. An interesting collection of SRIF immunoreactive neurons and processes were observed forming a wide band extending anteriorly from the lateral preoptic area through the lateral hypothalamus and substantia innominata posteriorly. This report on the localization of immunoreactive SRIF in the human forebrain extends previous anatomical findings and lends morphological support to recent biochemical studies.     

人胎大肠氮能神经元发育的研究

By using histochemical methed of NADPH-diaphorse, the development of the nitrergic neurons in the large intestine of human fetus were studied. The results showed: At the fifth month of gestation, weak positive reaction of nitric oxide synthase (NOS) appeared in part of the round cells of intermuscular ganglia. The round cells differentiated into the nitrergic nerve cells. At the sixth month, the bodies of nitrergic neurons were obviously enlarged, the processes of which were lengthened. The nitrergic nerve fibers were seen in the muscle layer, the submucosa and the base of the intestinal gland. The growth and development of nitrergic neurons reached its peak at the seventh month. The number of intermuscular ganglionic cells was increased. The density of nitrergic nerve fibers was increased in the inner circular muscle layer, and have bead-like structures. At the eighth to tenth month, the staining intensity of nitrergic neurons was increased. The myenteric plexus was densely distributed with nitrergic nerve cell bodies, whereas the submucosa and the inner circular muscle layers contained only a few neurons. The nitrergic nerve fibers were observed in all layer of large intestine, the density of the distribution of nitrergic nerve fibers was by far the highest in the inner circular muscle layer, less in the submucosa and outer longitudinal muscle layer, and only a few were found in the mucous layer. To our knowledge, it is the first time that the development of nitrergic neurons in the large intestine of human fetus was demonstrated.     

人胎大肠氮能神经元发育的研究

用NADPH-d组织化学法对人胎大肠氮能神经元的发育进行了观察.结果表明第5个月胎龄时,肌间神经节处圆形细胞中部分细胞出现一氧化氮合酶(NOS)阳性反应,并分化成氮能神经细胞.第6个月胎龄时,氮能神经元胞体增大,突起伸长,在肌层、粘膜下层和肠腺基部出现氮能神经纤维分布.第7个月胎龄时,氮能神经元生长发育达到高峰,肌间神经节细胞数目增多,环肌层神经纤维分布密度增加,膨体结构明显.第8-10个月胎龄时,氮能神经元染色强度加深,其胞体分布以肌间神经节最多,粘膜下层和内环肌层较少.氮能神经纤维的分布密度以内环肌层最高,粘膜下层和外纵肌层次之,粘膜层较低.本研究揭示了大肠氮能神经元发育的变化规律.     

On the neurons with dendrites intermingling with the fibers of the human corpus callosum: a Golgi picture

Golgi preparations of the anterior part of the truncus of the corpus callosum from 11 adult human brains were investigated. The vertical plane of section was situated symmetrically between the frontal and sagittal plane. The use of this oblique plane of section enabled easy identification of the neurons with dendrites intermingling with transcallosal fibers, what was not possible in standard frontal sections. 2 types of such neurons (with features of other interstitial neurons) were described: fusiform and multipolar. Both types of neurons were more frequently impregnated in areas adjacent to induseum griseum, cingular cortex, and in the depth of the callosal sulcus. Multipolar neurons were also present in the central core and in ventral parts of the corpus callosum, but fusiform ones were not present in ventral parts of the corpus callosum truncus. The dentrites of both types of neurons usually were perpendicular to, sometimes also parallel to transcallosal fibers. The impregnation of these neurons in groups and pairs suggest their integrative role, and their planar orientation in mentioned oblique plane corresponds to oblique direction of transcallosal cingulostriatal decussating fibers.     

Choline Acetyltransferase Activities in Single Motor Neurons from Vertebrate Spinal Cords

Single cell bodies of spinal motor neurons were isolated from freeze-dried sections of fresh spinal cords from six species of vertebrates. Single human neurons were also isolated from the spinal cords of three autopsy cases without neurological diseases. Choline acetyltransferase activity of these single neurons was determined by measuring acetyl-CoA formation from CoASH and acetylcholine by use of the enzymatic amplification reactions, CoA and NADP cyclings. The enzyme activity was unevenly distributed in the cytosol of spinal motor neurons of all species, but not measurable in rabbit dorsal root ganglion cells. The specific activity on a dry weight basis varied widely among the individual neurons from the species studied. The average activity was highest with rat neurons and lowest with yellowtail neurons. The neurons from cold-blooded animals (bullfrog and yellowtail) had about one-tenth the activity compared with the warm-blooded animals (cat, rabbit, rat, and hen). Human neurons, obtained under different morbid and post-mortem conditions with three autopsy cases, had very low activities corresponding to those of cold-blooded animals. Since the choline acetyltransferase activity lost from mouse brain after 11 h at 38 degrees C was 50%, the activity in human neurons was believed to actually be low in vivo.     

Quantitative studies of superior cervical sympathetic ganglia in a variety of primates including man. I. The ratio of preganglionic fibers to ganglionic neurons

Estimates of the number of ganglionic neurons of superior cervical sympathetic ganglia and the number of preganglionic axons in the trunks just caudal to these ganglia were obtained from a sample of primates that included: man, chimpanzee, baboon, stump-tailed macaque, rhesus monkey, and squirrel monkey. The number of ganglionic neurons ranged from 63,625 in a squirrel monkey ganglion to 1,041,652 neurons in a human ganglion. Estimates of the number of preganglionic fibers varied between 2,285 in a cervical sympathetic trunk of a squirrel monkey and 12,008 in a human specimen. The resulting ratios of preganglionic fibers to ganglionic neurons ranged from 1:28 in a squirrel monkey ganglion to 1:196 in a human ganglion. The data reported in this study reveal considerable variation in the ratio of pre- to post-ganglionic neurons, and as was noted in regard to the number of cells in the ganglion, the ratios of ganglionic to preganglionic neurons appear to increase as a function of body size. In contrast, the number of preganglionic fibers does not increase as strikingly with body size, but varies greatly in the same species. The resulting ratio between the two orders of neurons is, therefore, less predictable than the number of ganglionic neurons in any given ganglion.     

Gastrin-releasing peptide: neuronal distribution and spatial relation to endocrine cells in the human upper gut

By using immunocytochemical techniques, we have studied the distribution of gastrin releasing peptide (GRP)-containing neurons as well as the spatial relationship between these neurons and the endocrine cells in the human stomach and duodenum. Moderate numbers of immunoreactive fibers were distributed in the smooth muscle and submucosa of the stomach; they were more rare in the duodenal wall. Numerous GRP-containing nerve fibers were found in the oxyntic mucosa, the antral mucosa harboured only few GRP immunoreactive nerve fibers. The mucosa of the proximal duodenum was found to be virtually devoid of such fibers. Only occasionally did we observe signs of a direct contact between GRP-containing nerve fibers and gastrin and somatostatin cells in the antral mucosa. In the oxyntic mucosa GRP-containing nerve fibers sometimes seemed to contact endocrine cells, including somatostatin cells as well as individual parietal cells. In conclusion, although GRP-containing nerve fibers were quite numerous in the wall of the human upper gastro-intestinal (GI)-tract, we observed a lack of intimate spatial relationship between these fibers and endocrine cells in the antral mucosa, suggesting additive mechanisms to a direct innervation of gastrin cells and somatostatin cells by GRP nerve fibers explaining the physiological effects on hormonal release.     

Relationship of ACTH1-39-immunostained fibers and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

Dual antigen immunocytochemical staining procedures were used in the same tissue section to determine the distribution of ACTH immunostained fibers and varicosities within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus and elucidate its anatomical relationship to vasopressin (VP) and oxytocin (OXY)-containing neurons. Double immunostained preparations using glucose oxidase-antiglucose oxidase complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue section were employed. ACTH-immunoreactive (ir) fibers were distributed throughout the periventricular stratum and the parvocellular component of the PVN; in the latter area fibers were particularly dense in the ventral medial portion of the medial parvocellular division. Dual immunostained sections revealed a close anatomical association between opiocortin fibers and oxytocin and vasopressin parvocellular neurons. ACTH immunostained fibers were present in the anterior and medial magnocellular component of PVN and in the ventral medial portion of the posterior magnocellular division; these immunoreactive fibers were in intimate proximity to oxytocin-ir perikarya. The very close approximation between the ACTH-ir fibers and oxytocin-containing cell bodies suggests potential cell to cell communication between the two peptidergic systems in PVN. Few ACTH immunostained fibers were seen in the dorsal lateral portion of the posterior magnocellular division in which vasopressinergic neurons predominate. The present anatomical study supports pharmacological and physiological studies which indicate that opioids can influence the activity of magnocellular PV neurons. This study also elucidates an anatomical relationship between opiocortins (ACTH1-39) and parvocellular PV neurons which suggests that the opiocortin system may play a role in the regulation of both the neuroendocrine and autonomic activities of specific PV neurons.     

Neuronal organization of the left celiac ganglion in the cat

Unit responses of the isolated left celiac ganglion to stimulation of various nerves of the solar plexus were studied by intracellular microelectrode recording in cats before and after degeneration of the preganglionic fibers. The resting potential of the ganglionic neurons was ?62.2±2.9 mV and the amplitude of the spike potential 72.4±3.2 mV. The spike was followed by after-hyperpolarization with a mean amplitude of 24% of the spike amplitude and a duration of between 25 and 180 msec. A characteristic feature of the ganglion was the presence of orthodromic unit responses to stimulation of peripheral nerve fibers of the solar plexus. The higher threshold of activation of the neurons by peripheral fibers than by preganglionic fibers and the preservation of orthodromic unit responses to stimulation of peripheral nerves after degeneration of the preganglionic fibers are evidence that the peripheral reflex arc is closed in this ganglion. Neurons of the left celiac ganglion are divided into three groups. Only preganglionic fibers of the splanchnic nerve with different properties converge on the neurons of the first group (the most numerous); only afferent fibers of peripheral nerves converge on the neurons of the third group (the least numerous); both types of fibers terminate on neurons of the second group. This convergence may lie at the basis of the mechanism of the centrifugal and peripheral reflex interaction in the ganglion for coordinated visceral activity.     

Distribution and dilatory effect of vasoactive intestinal polypeptide (VIP) in human cerebral arteries

Vasoactive intestinal polypeptide (VIP)-containing nerve fibers were demonstrated in human pial arteries by immunocytochemistry. Fine varicose fibers were located in the adventitia close to the media layer. Measurements by radioimmunoassay revealed concentrations of VIP between 0.7 and 2.7 pmol/g in the major arteries at the base of the brain, obtained at autopsy. Isolated human pial arteries, obtained in conjunction with neurosurgery, relaxed in a concentration-dependent manner upon administration of VIP. The relaxation of the vessels amounted to 57 +/- 9% of the contraction elicited by prostaglandin F2 alpha (2.5 microM) with an EC50 value of (8.5 +/- 1.2) X 10(-9) M.     

Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.

Corticosteroid-binding globulin, a specific steroid carrier in serum with high binding affinity for glucocorticoids, is expressed in various tissues. In the present study, we describe the immunocytochemical distribution of this protein in neurons and nerve fibers in the human hypothalamus. CBG immunoreactive perikarya and fibers were observed in the paraventricular, supraoptic, and sexual dimorphic nuclei in the perifornical region, as well as in the lateral hypothalamic and medial preoptic areas, the region of the diagonal band, suprachiasmatic and ventromedial nuclei, bed nucleus of the stria terminalis and some epithelial cells from the choroid plexus and ependymal cells. Stained fibers occurred in the median eminence and infundibulum. Double immunostaining revealed a partial co-localization of corticosteroid-binding globulin with oxytocin and, to a lesser extent, with vasopressin in the paraventricular and the supraoptic nuclei. Double immunofluorescence staining showed coexistence of these substances in axonal varicosities in the median eminence. We conclude that neurons of the human hypothalamus are capable of expressing corticosteroid-binding globulin, in part co-localized with the classical neurohypophyseal hormones. The distribution of CBG immunoreactive neurons, which is widespread but limited to specific nuclei, indicates that CBG has many physiological functions that may include neuroendocrine regulation and stress response.     

Study of neuropeptide Y-containing nerve fibers in the human penis

Summary Neuropeptide Y 1–36 (IR-NPY) immunoreactive nerve-fiber processes have been observed in tunicae of veins and arteries and in smooth muscles of the human penis taken at autopsy or during surgery by use of light-and electron-microscopic immunohistochemical techniques. Numerous IR-NPY nerve fibers were mostly concentrated in the inner part of the adventitia close to the media of the arterial and venous vessels and among the intracavernous smooth muscle cells. IR-NPY nerve fibers were less abundant in veins than in arteries. Positive somata were not observed in the penises. At the ultrastructural level, IR-NPY were localized exclusively in large, dense granules of nerve terminals by means of the postembedding immunogold technique. In the deep dorsal vein, IR-NPY nerve fibers were also located in the media formed by an outer circular and an inner longitudinal layer. In the intracavernous and dorsal arteries, they showed the highest density in the inner part of the adventitia. In the corpora cavernosa and in the corpus spongiosum, IR-NPY nerve processes were intermingled between the smooth-muscle fibers around the sinusoid spaces. IR-NPY nerve fibers were present in the cavernous nerves close to the central arteries. The urethra did not show any IR-NPY-positive nerve fibers. This peculiar distribution of IR-NPY nerve fibers suggested that they could participate in regulating arterial and venous blood flow and intracavernous smooth-muscle tone. NPY may therefore be of importance in some of the mechanisms of penile erection especially during detumescence.     

Immunohistochemical investigation of the nitrergic system in the taste organ of the frog,Rana esculenta

We have studied by immunocytochemistry, the taste discs of the frog, Rana esculenta, with the aim of providing morphological and neurochemical data on the nitrergic system and of assessing the eventual presence of intrinsic neurons associated with the gustatory organs. In taste discs, antibodies against neuronal nitric oxide synthase (nNOS) revealed a positive immunoreaction in the taste receptor cell bodies and processes. The basal cells were also stained. All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity; these fiber were mainly located in the basal plexus. Immunoreactive nerve fibers were also visible at the periphery of the papilla-contacting ciliate cells, which form a ring around the taste disc. In conclusion, the findings obtained in this study suggest that the occurrence of nNOS-immunoreactivity in basal cells, taste cells and nerves might reflect a role for nitric oxide in taste mechanisms of Amphibia. The results may also sustain the physiological implication of NO as a molecule involved in the local target function of maintaining the taste bud mucosal integrity and in regulating the blood flow to the epithelium.     

人十二指肠血管活性肠肽能P物质能神经分布的研究

本文采用免疫组织化学ＡＢＣ法研究血管活性肠肽（ＶＩＰ） 能神经和Ｐ物质（ＳＰ） 能神经在人十二指肠壁内的分布。结果显示： ＶＩＰ能和ＳＰ能神经纤维和神经元均呈棕褐色; ＶＩＰ能神经纤维遍布肠壁各层,ＳＰ能神经纤维主要分布于肌层和神经丛; ＶＩＰ能和ＳＰ能神经元见于肌间和粘膜下神经, 尤以后者为多, 但形态特点不同; 在肌间神经丛, ＳＰ能神经元比ＶＩＰ能神经元多。粘膜内可见ＶＩＰ能和ＳＰ能神经元, 多单个分布在粘膜肌层内。结果表明： １ＶＩＰ能和ＳＰ能神经在人十二指肠壁内分布有差异。２粘膜内存在ＶＩＰ能和ＳＰ能神经元