Cytometric investigations in histochemically identified neurons — Changes of fluorescence intensities and nuclear diameters in dopamine neurons of the arcuate nucleus in the cycling rat

Summary The relative formaldehyde-induced fluorescence intensities and the diameter of cell nuclei of fluorescent perikarya of the arcuate nucleus were recorded in serial cross-sectioned hypothalamic arcuate nuclei of regularly cycling rats (5 estrous and 4 proestrous animals). Stress-induced changes of cytometric parameters were avoided by preadaptation of animals to handling procedures. Dopamine neurons in a 75 m thick periventricular layer of the arcuate nucleus exhibited significantly smaller nerve cell nuclei and significantly reduced relative fluorescence intensities in proestrous rats. Both of these cytometric parameters indicate a decrease in the activity of periventricular dopamine neurons of the arcuate nucleus. The reported findings might support the hypothesis that dopamine inhibits the release of LH-RH.In partial fulfillment of requirements for an academic degree (Dr. med.)Supported by a grant from the Deutsche Forschungsgemeinschaft (-Ha 726/5-) and the Stiftung Volkswagenwerk (I/35 582) to H.G.H.     

高脂血症大鼠弓状核超微结构及神经肽Y神经元的变化

目的:观察高脂血症大鼠弓状核超微结构及神经肽Y(neuropeptide Y,NPY)免疫阳性神经元的变化。方法:实验组大鼠高脂饲料饲养6周测血脂,透射电镜观察弓状核超微结构,免疫组化染色观察测量NPY免疫阳性神经元光密度、数量及截面积。结果:实验组血清总胆固醇及低密度脂蛋白胆固醇明显升高;弓状核超微结构出现明显病理学改变。NPY神经元平均光密度、细胞数、截面积减少。结论:高脂血症可诱导弓状核神经元超微结构和NPY神经元改变,为进一步探讨血脂代谢异常与中枢神经的内分泌关系提供形态学依据。     

Dendritic arborization and axon trajectory of neurons in the hypothalamic arcuate nucleus of the rat--updated

Neurons in the hypothalamic arcuate nucleus (arcuate neurons) were traced on Golgi-impregnated sections. Dendrites of arcuate neurons showed characteristic orientation patterns. Dendrites along the lateral side follow the convex border of the nucleus by running parallel to the tanycyte processes. Neurons located in the ventrolateral portion of the nucleus have dendrites running parallel to the basal surface of the hypothalamus. Fine, beaded axons of arcuate neurons project mostly ventrally, and less frequently dorsally and dorsolaterally. Ventrally projecting axons converge towards the tuberoinfundibular sulcus which emerges into the ventral portion of the arcuate nucleus from below.     

Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons

The distribution and coexistence of neuropeptide Y (NPY) and somatostatin (SOM) were evaluated in rat and human cerebral cortex and in the rat hypothalamic arcuate nucleus (n) using double immunofluorescent staining in which primary antisera were raised in different species. The results of the study indicate extensive coexistence of NPY and SOM in both rat and human cortex but only occasional coexistence in the rat arcuate n.     

Morphological evidence for synaptic junctions between substance P-containing neurons in the arcuate nucleus of the rat

The presence of synaptic junctions between substance P-containing neurons (SP neurons) was shown in the hypothalamic arcuate nucleus of rats by pre-embedding electron microscopic immunohistochemistry. The immunoreactive perikarya were synapsed by immunoreactive fiber terminals as well as immunonegative fiber terminals. The functional implication of the synapses between SP neurons is discussed in relation to autoregulatory mechanisms of the neurons.     

Dendritic arborization and axon trajectory of neurons in the hypothalamic arcuate nucleus of the rat

Summary Dendritic arborization patterns of neurons in the hypothalamic arcuate nucleus and the course of their axons outside the median eminence were studied using Golgi material, electron microscopic detection of degenerated axon terminals following surgical interference, and horseradish peroxidase technique.Two kinds of neurons can be distinguished in the arcuate nucleus: small fusiform and somewhat larger polygonal cells. Fusiform neurons having two, sparsely arborizing stem dendrites are localized mainly in the medial and dorsal parts of the nucleus. A variant of the fusiform neurons (pear shaped cells) has only one dendritic stem, and the axon originates at the other pole of the neuron. These latter cells are found along the ependymal wall of the third ventricle. Polygonal neurons which occupy the ventral and lateral portions of the nucleus have 4–5 repeatedly branching stem dendrites. In addition to the well known course of the axons of the arcuate neurons towards the median eminence, there are axons of both fusiform and polygonal cells which leave the nucleus in lateral or dorsolateral direction. Axons having this course frequently issue a collateral branch along the first 100–150 m of their trajectory.A knife-cut through the arcuate nucleus or along its lateral border resulted in degeneration of axon terminals in the area between the arcuate and ventromedial nuclei, and in a ventromedial sector of the ventromedial nucleus itself. A cut located in the area between the arcuate and ventromedial nuclei caused degenerated axon terminals over a large part of the ventromedial nucleus with a considerable increase in the ventromedial portion.Labeled fusiform and polygonal neurons can be seen in the arcuate nucleus following the injection of horseradish peroxidase into the lateral hypothalamus.The results suggest unequivocally that the neurons in the arcuate nucleus have efferent connections not only towards the median eminence but also to the hypothalamic ventromedial nucleus and lateral hypothalamic area.     

Estrogen stimulates neuronal plasticity in the deafferented hypothalamic arcuate nucleus in aged female rats

The hypothalamic arcuate nucleus (ARCN) was examined ultrastructurally 3 weeks after the complete deafferentation of the medial basal hypothalamus (MBH) with the island isolation technique in ovariectomized aged female rats (720-930 days of age). The mean numbers of axodendritic and axosomatic synapses in the ARCN decreased to about one-third of those in the intact controls. However, the treatment with estradiol benzoate (2 micrograms/day) during the 3 weeks following the day of brain surgery brought about a marked increase in the numbers of these synapses. The data suggest that the ARCN neurons of aged female rats still retain plasticity to react to deafferentation under influences of estrogen.     

Effects of intracerebroventricular administration of neuropeptide W30 on neurons in the hypothalamic paraventricular nucleus in the conscious rat

We demonstrated that intracerebroventricular (i.c.v.) administration of NPW30 increases the arterial blood pressure (ABP), heart rate (HR), and plasma catecholamine concentrations in conscious rats. NPW has been reported to be an important stress mediator in the central nervous system that modulates the hypothalamus-pituitary-adrenal (HPA) axis and sympathetic outflow. To examine the effects of NPW30 on the neural activity of the hypothalamic paraventricular nucleus (PVN), which is an integrative center of the autonomic and endocrine functions relevant to stress responses, we simultaneously recorded the single-unit activity in the PVN, ABP, and HR in conscious freely moving rats. Of the non-phasic (irregular) PVN neurons (n=35) examined, NPW30 (i.c.v. 3 nmol) elicited excitation in 22 neurons, inhibition in 7 neurons, and no response in 6 neurons, accompanied with increases in ABP and HR, whereas low-dose NPW30 (i.c.v. 0.3 nmol) did not affect the unit activity, ABP, or HR. Neurons that were affected by NPW30 were then further examined for their responses to perturbation in ABP and systemic administration of cholecystokinin-8 (CCK). The majority of neurons also showed responses to CCK, phenylephrine (PE), or nitroprusside (SNP). Our data suggest that central NPW30 modulates PVN neuronal activities, which might be involved in the regulation of cardiovascular function and energy balance through the autonomic nervous system, particularly, under stress-related conditions.     

Neuronostatin is co-expressed with somatostatin and mobilizes calcium in cultured rat hypothalamic neurons

Neuronostatin (NST) is a newly identified peptide of 13-amino acids encoded by the somatostatin (SST) gene. Using a rabbit polyclonal antiserum against the human NST, neuronostatin-immunoreactive (irNST) cells comparable in number and intensity to somatostatin immunoreactive (irSST) cells were detected in the hypothalamic periventricular nucleus. Fewer and/or less intensely labeled irNST cells were noted in other regions such as the hippocampus, cortex, amygdala, and cerebellum. Double-labeling hypothalamic sections with NST- and SST-antiserum revealed an extensive overlapping of irNST and irSST cells in the periventricular nucleus. Pre-absorption of the NST-antiserum with NST (1 μg/ml) but not with SST (1 μg/ml) abrogated irNST and vice versa. The activity of NST on dissociated and cultured hypothalamic neurons was assessed by the Ca²⁺ imaging method. NST (10, 100, 1000 nM) concentration-dependently elevated intracellular Ca²⁺ concentrations [Ca²⁺]_i in a population of hypothalamic neurons with two distinct profiles: (1) a fast and transitory increase in [Ca²⁺]_i, and (2) an oscillatory response. Whereas, SST (100 nM) reduced the basal [Ca²⁺]_i in 21 of 61 hypothalamic neurons examined; an increase was not observed in any of the cells. Optical imaging with a slow-responding voltage sensitive dye DiBAC₄(3) showed that NST (100 nM) depolarized or hyperpolarized; whereas, SST (100 nM) hyperpolarized a population of hypothalamic neurons. The result shows that NST and SST, though derived from the same precursor protein, exert different calcium mobilizing effects on cultured rat hypothalamic neurons, resulting in diverse cellular activities.     

不同时程应激对大鼠下丘脑弓状核和室周核神经细胞的影响

目的:观察不同时程应激对大鼠下丘脑弓状核和室周核神经细胞的影响及酪氨酸羟化酶(TH)的表达变化,为应激性损伤的机制研究提供病理形态学依据。方法:建立每日束缚固定8 h加冰水游泳5 min的应激大鼠模型,分为1、3、7、14、21d组及各时间点正常对照组,采用硫堇染色观察弓状核、室周核内神经细胞尼氏体变化及细胞形态学改变;TH免疫组织化学标记观察大鼠下丘脑弓状核和室周核内多巴胺能神经细胞阳性表达变化,采用全景组织细胞定量分析系统,进行统计分析。结果:较长时程反复的应激刺激导致了大鼠弓状核和室周核神经细胞细胞质内尼氏体消失及部分细胞固缩。TH免疫组织化学标记显示随着应激时程的延长,下丘脑弓状核和室周核内多巴胺能神经细胞阳性表达数目减少。结论:较长时程的反复应激刺激可导致大鼠下丘脑弓状核和室周核神经细胞损伤及多巴胺能神经细胞缺失。     

Changes in the female arcuate nucleus morphology and neurochemistry after chronic ethanol consumption and long-term withdrawal

Ethanol is a macronutrient whose intake is a form of ingestive behavior, sharing physiological mechanisms with food intake. Chronic ethanol consumption is detrimental to the brain, inducing gender-dependent neuronal damage. The hypothalamic arcuate nucleus (ARN) is a modulator of food intake that expresses feeding-regulatory neuropeptides, such as alpha melanocyte-stimulating hormone (α-MSH) and neuropeptide Y (NPY). Despite its involvement in pathways associated with eating disorders and ethanol abuse, the impact of ethanol consumption and withdrawal in the ARN structure and neurochemistry in females is unknown. We used female rat models of 20% ethanol consumption for six months and of subsequent ethanol withdrawal for two months. Food intake and body weights were measured. ARN morphology was stereologically analyzed to estimate its volume, total number of neurons and total number of neurons expressing NPY, α-MSH, tyrosine hydroxylase (TH) and estrogen receptor alpha (ERα). Ethanol decreased energy intake and body weights. However, it did not change the ARN morphology or the expression of NPY, α-MSH and TH, while increasing ERα expression. Withdrawal induced a significant volume and neuron loss that was accompanied by an increase in NPY expression without affecting α-MSH and TH expression. These findings indicate that the female ARN is more vulnerable to withdrawal than to excess alcohol. The data also support the hypothesis that the same pathways that regulate the expression of NPY and α-MSH in long-term ethanol intake may regulate food intake. The present model of long-term ethanol intake and withdrawal induces new physiological conditions with adaptive responses.     

Descending input from the hypothalamic paraventricular nucleus to sympathetic preganglionic neurons in the rat

Summary The descending projection of the hypothalamic paraventricular nucleus (PVN) to the sympathetic preganglionic neurons (SPNs) in the upper thoracic cord of the rat was studied. PVN-fibers were labeled by anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), while SPNs were retrogradely labeled with cholera toxin subunit B (CTb) which was injected into the superior cervical ganglion. SPNs labeled with CTb were mainly observed in the nucleus intermediolateralis (IML) pars principalis and pars funicularis, and a small number of them were in the nucleus intercalatus (IC) and central autonomic nucleus (CA). SPNs found in the IML had dendrites that projected in various directions. Five types of dendritic projections were noted: medial, rostral, caudal, lateral (including dorsolateral) and ventral. Longitudinal dendritic bundles interconnected each cell cluster in the IML. Medial dendrites of the IML, together with dendrites of the IC and CA, formed transverse dendritic bundles extending from the IML to the central canal. The transverse dendritic bundles disentangled near the midline and formed a loose dendritic plexus in the region just dorsal to the central canal. PVN-fibers labeled with PHA-L were observed primarily in lamina I and intermediate gray (lamina VII). Although varicose PVN-fibers and SPNs coexisted in the IML, the tight packing of the dendritic bundles prevented any clear demonstration of direct contacts between them. On the other hand, PVN-fibers were occasionally found to appose and wind around the primary or secondary dendrites of some SPNs of the CA and IC. These dendrites were studded with varicosities of PVN-fibers for a short length, and terminal boutons of PVN-fibers were also seen to make contact directly with the dendrites. The results of this study substantiated a direct connection between the PVN and SPNs, using a combination of immunohistochemical techniques for PHA-L and CTb. The possible involvement of a direct pathway from the PVN to SPNs in cardiovascular regulation is discussed.Abbreviations AF anterior funiculus - CA central autonomic nucleus - CC central canal - CTb cholera toxin subunit B - HRP horseradish peroxidase - IC nucleus intercalatus - IMf nucleus intermediolateralis pars funicularis - IML nucleus intermediolateralis - IMp nucleus intermediolateralis pars principalis - LDB longitudinal dendritic bundle - LF lateral funiculus - PF posterior funiculus - PHA-L Phaseolus vulgaris leucoagglutinin - PVN hypothalamic paraventricular nucleus - SPNs sympathetic preganglionic neurons - TDB transverse dendritic bundle     

Tachykinin-induced changes in β-endorphin gene expression in the rat arcuate nucleus

Previous neuroanatomical data have indicated the presence of synaptic connections between tachykinergic terminals and proopiomelanocortin (POMC) neurons in the arcuate nucleus. Consequently, tachykinins may regulate the activity of POMC neurons. To evaluate the functional signification of this regulation, the effect of intracerebroventricular injections of neurokinin A (NKA) on POMC mRNA levels was studied by using in situ hybridization. Repeated injection of NKA (40 μg/animal per day during 3 days) induced a 48% increase in POMC mRNA expression as compared to NaCl injected control animals. In conclusion the results of this study show an excitatory effect of tachykinin on POMC neurons and suggest a direct and/or indirect excitatory control of POMC neuronal activity by endogenous tachykinins.     

Descending projections from the hypothalamic paraventricular nucleus to the A5 area,including the superior salivatory nucleus,in the rat

Summary The descending projection of the hypothalamic paraventricular nucleus (PVN) to the A5 area was elucidated using a technique that combines retrograde labeling with horseradish peroxidase (HRP), anterograde labeling with PHA-L (Phaseolus vulgaris) leucoagglutinin and immunohistochemistry for dopamine-hydroxylase (DBH). Following an iontophoretic injection of PHA-L into the PVN, HRP was applied to the greater petrosal nerve. Frozen sections of the hypothalamus and the caudal pons were first treated according to a protocol for HRP histochemistry using tetramethylbenzidine with cobalt-enhanced diaminobenzidine, and then they were processed for displaying PHA-L, and then for DBH immunohistochemistry. PHA-L labeled fibers from the PVN were observed in a ventrolateral part of the pontine reticular formation corresponding to the A5 area, where they give rise to a dense network around the cells of origin of the greater petrosal nerve (GPN cells) and DBH-positive cells. Terminals or varicosities labeled with PHA-L were preferentially observed around the somata of GPN cells, suggesting direct contact. However, apparent contact between both elements was hardly ever observed. On the other hand, terminals or varicosities were occasionally observed in close relation to DBH positive cells. These results suggest that descending fibers of the PVN project more strongly to GPN cells than to DBH-positive cells. The relationship of this fiber pathway to control of the secretomotor or cardiovascular systems is discussed.     

Synaptic relationships between induced neuropeptide Y-like immunoreactive terminals and cuneothalamic projection neurons in the rat cuneate nucleus following median nerve transection

Previous studies have demonstrated that following complete median nerve transection (CMNT), neuropeptide Y-like immunoreactive (NPY-LI) fibers were dramatically increased and predominantly expressed in the ventral portion of the middle cuneate nucleus (CN), reaching maximum numbers at four weeks. Ultrastructurally, NPY-LI terminals made axodendritic synapses, but the postsynaptic elements are unknown. In the present study, using retrograde tract-tracing of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and NPY immunocytochemistry we examined the synaptic relationships between cuneothalamic projection neurons (CTNs) and NPY-LI terminals in the rat CN following CMNT. The injury-induced NPY-LI fibers were distributed throughout the rostrocaudal extent of the CN. Further, the greatest number of HRP-labeled CTNs was observed in the ventral portion of the middle CN. Ultrastructurally, the NPY-LI terminals made asymmetric axodendritic synaptic contact with the HRP-labeling CTN dendrites. These data suggest that injury-induced NPY may modulate the excitability of CTNs, and thus, play a role in the transmission of neuropathic sensation following median nerve injury.     

Messenger RNA for neuropeptide Y in the arcuate nucleus increases in parallel with plasma adrenocorticotropin during sepsis in the rat

Loss of appetite occurs in the cecal ligation and puncture (CLP) model of sepsis in conjunction with the activation of central neural stress pathways. Neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus is upregulated by several stressors and is stimulatory to feeding. To examine the response of NPY messenger RNA in the arcuate nucleus to sepsis, we used biotinylated RNA probes and a quantitative non-isotopic in situ hybridization approach in cryo-preserved sections from rats made septic by CLP. The mRNA in arcuate neurons was upregulated from the first day after CLP. By the afternoon of the third day through the morning of the fourth day, the average grey level of NPY mRNA clusters was 30% greater after CLP than after sham surgery (P < 0.05), and the integrated optical density based on both the grey level and the amount of area with detectable mRNA was 60% greater after CLP than after sham surgery (P < 0.03). Both the average grey level and area with detectable staining were positively correlated to plasma ACTH (r = 0.953 and 0.917, respectively, n = 10 and P < 0.01 in each case). Thus sepsis increases the expression of the mRNA for NPY in the arcuate nucleus in proportion to the magnitude of the stress response. However, the suppression of feeding behavior in the CLP model suggests that sepsis activates additional mechanisms that negate the orexigenic contribution of the neuronal increase in NPY mRNA.     

Expression and colocalization patterns of calbindin-D28k,calretinin and parvalbumin in the rat hypothalamic arcuate nucleus

Calcium binding proteins (CaBPs) form a diverse group of molecules that function as signal transducers or as intracellular buffers of Ca²⁺ concentration. They have been extensively used to histochemically categorize cell types throughout the brain. One region which has not yet been characterized with regard to CaBP expression is the hypothalamic arcuate nucleus, which plays a vital role in neuroendocrine control and the central regulation of energy metabolism. Using in situ hybridization and immunofluorescence, we have investigated the cellular distribution of the three CaBPs, calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) in the rat arcuate nucleus. Both mRNA and immunoreactivity was detected in the arcuate nucleus for CB – located in the medial aspects – and CR – located ventrolaterally. No PV mRNA was detected in the arcuate nucleus. Immunofluorescence results for PV were ambiguous; while one antibody detected a group of cell somata, a different antibody failed to visualize any arcuate nucleus cell profiles. Using double-labeling, neither of the examined CaBPs were observed in cells immunoreactive for the signaling molecules agouti gene-related protein, tyrosine hydroxylase, neurotensin, growth hormone-releasing hormone, somatostatin, enkephalin, dynorphin or galanin. We did, however, observe CB- and CR-immunoreactivity, in two distinct populations of neurons immunoreactive for the melanocortin peptide α-melanocyte-stimulating hormone. These data identify distinct subpopulations of arcuate neurons defined by their expression of CaBPs and provide further support for differentiation between subpopulations of anorexigenic melanocortin neurons.     

Direct neural connection from the medial preoptic area to the hypothalamic arcuate nucleus of the rat

Summary Direct neural connections from the medial preoptic area (MPOA) to the arcuate nucleus were studied on light and electron microscope level by a multistep experimental procedure. The hypothalamic deafferentation technique of Halász and Pupp (1965) was used in itself or was combined with electrolytic lesions. In order to eliminate all fibers of more rostral source traversing the medial preoptic area an extended rostral preoptic deafferentation was made, and a survival time of at least 3 weeks was chosen to allow for the complete disappearance of the degenerated fragments from the arcuate nucleus. In the main experimental group the medial preoptic area was destroyed by an electrolytic lesion 3 or more weeks following such rostral preoptic deafferentations. In these animals degenerated fibers and terminals certainly of preoptic origin were found distributed bilaterally, with a predominance on the side of the lesion. — These data suggest that axons originating from medial preoptic neurons terminate in the arcuate nucleus, thus constituting a preoptico-tuberal pathway.