Silver staining of the neuronal nucleus in electron microscopy: Nucleolar organization in supraoptic nuclei of the rat hypothalamus

Summary The present paper describes a simple, efficient method for silver impregnation of supraoptic nuclei of the rat hypothalamus using a modification of the ammoniacal silver technique of Cajal (1903). This procedure, involving a silver-developer sequence in tissue blocks prior to plastic embedding, permits the simultaneous study of Ag-impregnated supraoptic neurons at both light and electron microscopic levels.Visualization of secretory magnocellular neurons impregnated by this technique using the electron microscope reveals a good preservation of nuclear structures. A selective accumulation of silver grains was observed over heterochromatin clumps and nucleoli, which allows the identification of the nucleolar fibrillar centers and also the dense fibrillar component as the main areas involved in the silver reaction. The meaning of such a silver-distribution pattern is discussed in the light of recent ultrastructural and biochemical data.     

Plasticity of secretory neurons in the supraoptic and paraventricular nuclei of the hypothalamus on exposure to light

Light and electron microscopic methods were used to analyze changes in secretory neurons in the supraoptic (SON) and paraventricular (PVN) nuclei in the hypothalamus in 100 adult male rats at time points from the first minutes to 180 days after 48 hours of full-time exposure to bright light. At the early time points after exposure, the cellular formulae of the SON and PVN shifted towards functionally active neurons with minimal quantities of secretory granules, large nuclei and nucleoli, low RNA contents, small numbers of rough endoplasmic reticulum cisterns, vacuoles, and lysosomes in the perikarya. The number of cells depositing secretion was greater than in controls at 24 h in the SON and PVN and at 10 days in the SON. Normalization of the cellular formula and the structural organization of the protein-synthesizing apparatus of PVN neurons occurred at 10–30 days, with normalization in the SON at 30–180 days. These data provide evidence that the range of plasticity of neurons in the PVN on exposure to full-time bright light was more significant than that in the SON. __________ Translated from Morfologiya, Vol. 127, No. 2, pp. 24–27, March–April, 2005.     

Projections from the raphe nuclei to the suprachiasmatic nucleus of the rat

The presence of serotonergic afferents in the hypothalamic suprachiasmatic nucleus (SCN) is well documented and several functional roles of serotonin (5-HT) in circadian function are well established. However, there is some controversy about the precise location of the serotonergic neurones from where this input arises. Discrete injection of the tracer Cholera toxin, subunit B, (ChB) was centred in the rat SCN, and a few retrograde labelled neurones were distributed in the dorsal and median raphe nuclei (MnR) and in the rostral part of the raphe magnus (RMg), but no neurones were found in the raphe pallidus or raphe obscurus. In addition, a group of neurones was consistently found in the medial part of the pontine supra lemniscal nucleus but not including the serotonergic B₉ region. A combination of retrograde tracing with Fluoro-Gold together with 5-HT-immunolabelling, showed few double-labelled neurones in the dorsal, MnR and B₉. However, the majority of projecting neurones were not co-storing 5-HT immunoreactivity. Phaseolus vulgaris-leucoagglutinin (PHA-L) injections in the dorsal raphe resulted in faint labelling, whereas the MnR gave rise to several labelled fibres in the SCN. Individual delicate PHA-L nerve fibres were found in all compartments of the SCN both in terms of rostrocaudal, ventromedial and dorsomedial extent, without any sign of a topographical organisation of the MnR input to the SCN. PHA-L injections into RMg gave rise to labelling of a few processes within the SCN. In conclusion, the main serotonergic input to the rat SCN originates from a few neurones in the MnR.     

Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits

Serotonin 5-HT₃ receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT₃ receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT₃A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT₃A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT₃A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres. Electronic Publication     

Electron microscopic observations on the possible retinohypothalamic projection in the rat

Summary Degenerating nerve fibres and boutons were searched with the aid of the electron microscope in the arcuate nucleus of rats 2–7 days after bilateral destruction of the retina.In the arcuate nucleus of the control animals as well as in the operated animals, 4 types of boutons were distinguished on the basis of vesicular contents and glial ensheathment. In the operated animals changes interpreted as degenerating were found in small myelinated axons and boutons of type II (boutons containing both synaptic and granular vesicles). The changes were similar to those described in the literature as the dark type of degeneration in experimentally interrupted axons and boutons. Similar changes were not found in the unoperated animals. The conclusion is reached, that a small number of fibres of the optic tract reach the arcuate nucleus to terminate here.Financial support for this work was given by the Calouste Gulbenkian Foundation and by the 3rd Development Plan of the Portuguese Ministry of Education.     

大鼠下丘脑大细胞分泌系统内一氧化氮合酶与催产素的共存

目的　为进一步阐明一氧化氮 (NO)与催产素 (OT)在下丘脑神经内分泌调节中的作用提供形态学资料。方法　用NADPH d组织化学方法 ,并结合免疫组织化学相结合技术 ,观察大鼠下丘脑大细胞分泌系统内一氧化氮合酶 (NOS)与OT免疫阳性神经元的分布与共存关系。结果　NOS与OT在下丘脑室旁核、视上核等下丘脑各大神经分泌核区内的分布基本类似 ,并呈部分共存关系。并且初次在第三脑室室周区内观察到NOS/OT双重反应阳性触液神经元。结论　研究结果提示NO与OT在有关下丘脑生殖与性行为的神经内分泌调节活动中起着重要的介质作用。     

Noradrenergic innervation of vasopression-containing neurons in the rat hypothalamic supraoptic nucleus

The noradrenergic innervation of vasopressin (VP)-containing neurons in the supraoptic nucleus (SON) of the rat hypothalamus was studied electron microscopically by using double-labeling immunocytochemistry combining the pre-embedding peroxidase-anti-peroxidase method with post-embedding immunocolloidal gold staining. Noradrenaline-like immunoreactive axon terminals were found to make synaptic contacts with neurophysin II-like immunoreactive neurons in the SON. This study provides morphological evidence for noradrenergic control of neuronal activity of VP-containing neurons at the SON level.     

Age-associated alteration of serotonergic synaptic neurochemistry in rat rostral hypothalamus

Levels of monoamines and metabolites in rostral and caudal hypothalamic fragments of young (3-4 months) and old (25-26 months) Fischer 344-rats were compared. There were no differences between fragments of young animals. In senescent rats, 5-hydroxyindoleacetic acid (5HIAA) increased in both rostral and caudal hypothalamus. The ratio 5HIAA/5HT (serotonin), an index of serotonin turnover, increased only rostrally. This age-related increase of 5HIAA/5HT in rostral hypothalamus was associated with a decrease in [3H]-spiperone binding. There were no age-related changes of MAO-A activity in the hypothalamus or of serum tyrosine or tryptophan levels.     

Site-specific regulation of gene expression by estrogen in the hypothalamus of adult female rats

Estrogen plays critical roles in the neuroendocrine system of adult female rats through separate actions, respectively, in the preoptic area (POA) and the ventromedial nucleus of the hypothalamus (VMH). Seven-week-old rats were treated with/without estrogen after they were ovariectomized, and four estrogen-responsive, neuronal system-related genes, encoding alpha4 neuronal nicotinic acetylcholine receptor (Chrna4), GABA_A receptor delta (Gabrd), serotonin receptor 6 (Htr6), and GABA transporter 2 (Slc6a13), were investigated by real-time RT-PCR and Western blot analyses to examine their differential regulation by estrogen between the anterior part containing POA and the posterior part containing VMH. We further examined Bax, Bcl2, and Prkce, the former two genes to be involved in the gene expression network of Chrna4 and the latter gene, that of Gabrd. The regulation of Bax and Bcl2 by estrogen differed between the anterior and posterior parts. The results demonstrated differential regulation of these neuronal system-related genes by estrogen between the anterior and posterior parts of the hypothalamus and suggested the roles of gene expression networks for the respective genes in the neuroendocrine system of adult female rats.     

Circadian rhythm in metabolic activity of suprachiasmatic, supraoptic and raphe nuclei

Previous studies using 2-deoxyglucose (2-DG) autoradiography have demonstrated that the suprachiasmatic nucleus (SCN) of the hypothalamus, a putative neural circadian pacemaker, displays circadian rhythmicity in its metabolic activity. In the present study, we show that distinct circadian variations in 2-DG uptake occur not only in the suprachiasmatic, but also in the supraoptic and median raphe nuclei of the rat brain. On the other hand, several other brain areas failed to display systematic circadian variations in 2-DG uptake. These results indicate that circadian metabolic rhythms are not unique to the SCN. Further studies are required to precisely define the extent of such phenomena.     

The fine structure of serotonin and tyrosine hydroxylase immunoreactive terminals in the ventral posterior thalamic nucleus of cat and monkey

Summary Immunocytochemical methods have been combined with serial thin section analysis to study the synaptic organization of serotonin (5-HT) and tyrosine hydroxylase (TH) immunoreactive terminals in the ventral posterior nucleus of the cat and monkey thalamus. One hundred 5-HT immunoreactive terminals from the cat and approximately forty 5-HT and TH immunoreactive terminals from the monkey were selected for analysis in serial thin sections. Only 7–10% of the immunoreactive terminals could be revealed to form conventional synaptic contacts. Most of these could be identified as the asymmetrical type. Dendritic shafts belonging to relay neurons were the major targets of these monoamine immunoreactive terminals. The remainder made intimate membrane associations with relay cell dendrites and somata or with presynaptic dendrites of interneurons, but no overt membrane specializations could be detected. The present results suggest that the modulation of thalamocortical relay function by brainstem monoamine pathways in the somatosensory thalamus may occur by release of transmitters at atypical contact sites.     

Effects of electrochemical stimulation of the hypothalamus on serum prolactin and LH in rats

Summary Plasma levels of prolactin and LH were measured by radioimmunoassay following electrochemical stimulation of the medial preoptic area (MPO) or the arcuate nucleus (Arc.N.) in pentobarbital anesthetized proestrous rats. Differences in the secretion pattern of prolactin and LH were observed when stimulated by means of acutely or chronically implanted electrodes. Acute implantation and stimulation of the MPO resulted in no change in serum prolactin levels, whereas stimulation by means of chronically implanted electrodes evoked a marked increase in serum prolactin. The general observation was that electrostimulation in the acute experiments causes a less sharp but more prolonged prolactin and LH release from pituitary than stimulation through chronically implanted electrodes.Aided in part by NIH research grants CA 10771 and AM 4784.     

Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat

The first binaural integration within the auditory system responsible for sound localization depends upon commissural neurons that connect the two symmetrical cochlear nuclei. These cells in the deep polymorphic layer of the rat dorsal cochlear nucleus were identified with the electron microscope after injection of the retrograde tracer, Wheat Germ Agglutinin conjugated to Horseradish Peroxydase, into the contralateral cochlear nucleus. Commissural neurons are multipolar or bipolar with an oval to fusiform shape. Few commissural neurons, most inhibitory but also excitatory, connect most of the divisions of the rat cochlear nuclei. The most common type is a glycinergic, sometimes GABAergic, moderately large cell. Its ergastoplasm is organized into peripheral stacks of cisternae, and few axo-somatic synaptic boutons are present. Another type of commissural neuron is a medium-sized, spindle-shaped cell, glycine and GABA-negative, with sparse ergastoplasm and synaptic coverage. A giant, rare type of commissural neuron is glycine-positive and GABA-negative, with short peripheral stacks of ergastoplasmic cisternae. It is covered with synaptic boutons, many of which contain round synaptic vesicles. Another rare type of commissural neuron is a moderately large cell, oval to fusiform in shape, immunonegative for both glycine and GABA, and contacted by many axo-somatic boutons. It contains large dense mitochondria and numerous dense core vesicles of peptidergic type. Some labelled boutons, mostly inhibitory and probably derived from commissural neurons, contact pyramidal, cartwheel, giant and tuberculo-ventral neurons. The prevalent inhibition of electrical activity in a cochlear nucleus observed after stimulation of the contralateral cochlear nucleus may be due to commissural inhibitory terminals which contact excitatory neurons such as pyramidal and giant cells. Other inhibitory commissural terminals which contact inhibitory neurons such as cartwheel and tuberculo-ventral neurons, may explain the stimulation of electrical activity in the DCN after contralateral stimulation.     

Transplantation of fetal lateral geniculate nucleus to the occipital cortex: connectivity with host's area 17

Summary The developing lateral geniculate complex was excised from fetal albino rats at 18 days of gestation and implanted into the occipital cortex of host animals at 5 days of postnatal age. Groups of host animals were sacrificed at 10, 20 and 30 days following this procedure. The transplant tissue of selected animals was stereotaxically lesioned 2 days prior to scheduled sacrifice and their brains subjected to either Fink-Heimer or electron microscopic analysis of the distribution and density of degenerating efferents from the transplant. The remaining animals were analysed by means of Bodian, Golgi-Cox or electron microscopic techniques. Transplanted neurons displayed typical dendritic branching patterns of geniculate relay neurons by 20 days following implantation. Intrinsic neurons, characterized by a small ovoid soma and two main stem dendrites, only became evident in transplant tissue by 30 days and were much reduced in number. Synapses developed by 10 days and rapidly increased in number by 20 and 30 days. Most complexes were simple axo-dendritic, asymmetric junctions. Multiple serial and reciprocal complexes, as well as the characteristic glomerular complex, failed to appear. Analysis of Bodian stained material revealed a dense network of fibers coursing about the transplant. Distinct bundles of these fibers were observed extending from the medial edge of the transplant into area 17 by 20 days following implantation. A Fink-Heimer analysis of animals whose transplants were stereotaxically lesioned revealed degeneration in Layers II–VI of the primary visual cortex but the majority of these fibers terminated within the lateral two-thirds of Layer IV. Few degenerated fibers could be found in the underlying white matter indicating that efferents from the transplant found their way to their correct target zone by growing through a complex neuropil which provided minimal physical substrates to guide such growth. Most of the contacts formed by these fibers were simple junctions along the shafts of dendrites with a wide range in diameter. It is concluded that the nearby host visual neurons, which are the correct target cells for the afferents arising in the transplant, induced a directed growth of these fibers.     

Coiled bodies in supraoptic nuclei of the rat hypothalamus during the postnatal period

In electron microscopic studies of the supraoptic nuclei of the rat hypothalamus, structures identified as “coiled bodies” were found in magnocellular neurons. Although they could be seen elsewhere in mature neurosecretory cells, coiled bodies were commonly encountered in developing neurons during the postnatal period in both sexes. They appeared as distinctive nuclear inclusions consisting of round-to-oval networks of short electron-dense strands embedded in a less dense, fibrillar matrix, and lacking a limiting membrane. In fine structure and stain-affinity, they bore a resemblance to the fibrillar component of the nucleolus. Coiled bodies were located either in close association with the nucleolus or free within the nucleoplasm, showing no specific relationships with the perinucleolar chromatin or with the nuclear envelope. Their origin and functional meaning is discussed in the light of recent ultrastructural and biochemical data on cellular differentiation and nucleolar behavior.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactive neuronal elements in rat hypothalamus and median eminence with special reference to morphological background of its effect on anterior pituitary — light and electron microscopic immunocytochemistry

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellular portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.     

Distribution of histaminergic neuronal cluster in the rat and mouse hypothalamus

Histidine decarboxylase (HDC) catalyzes the biosynthesis of histamine from l-histidine and is expressed throughout the mammalian nervous system by histaminergic neurons. Histaminergic neurons arise in the posterior mesencephalon during the early embryonic period and gradually develop into two histaminergic substreams around the lateral area of the posterior hypothalamus and the more anterior peri-cerebral aqueduct area before finally forming an adult-like pattern comprising five neuronal clusters, E1, E2, E3, E4, and E5, at the postnatal stage. This distribution of histaminergic neuronal clusters in the rat hypothalamus appears to be a consequence of neuronal development and reflects the functional differentiation within each neuronal cluster. However, the close linkage between the locations of histaminergic neuronal clusters and their physiological functions has yet to be fully elucidated because of the sparse information regarding the location and orientation of each histaminergic neuronal clusters in the hypothalamus of rats and mice. To clarify the distribution of the five-histaminergic neuronal clusters more clearly, we performed an immunohistochemical study using the anti-HDC antibody on serial sections of the rat hypothalamus according to the brain maps of rat and mouse. Our results confirmed that the HDC-immunoreactive (HDCi) neuronal clusters in the hypothalamus of rats and mice are observed in the ventrolateral part of the most posterior hypothalamus (E1), ventrolateral part of the posterior hypothalamus (E2), ventromedial part from the medial to the posterior hypothalamus (E3), periventricular part from the anterior to the medial hypothalamus (E4), and diffusely extended part of the more dorsal and almost entire hypothalamus (E5). The stereological estimation of the total number of HDCi neurons of each clusters revealed the larger amount of the rat than the mouse. The characterization of histaminergic neuronal clusters in the hypothalamus of rats and mice may provide useful information for further investigations.     

Subcellular fractionation of hypothalamus and pituitary stalk of the bull. Morphologic study and serotonin-norepinephrine distribution

Summary Hypothalamus and pituitary stalk of the bull were fractionated in a sucrose density gradient according to the Hebb and Whittaker (1958) procedure. The crude mitochondrial-synaptosomal fraction was layered on a discontinuous sucrose density gradient (0.6; 0.8; 1; 1.2 M) and, after 63,000 g (average) centrifugation for 2 hours, 4 bands and one pellet were obtained. The fraction layered on 0.8 M sucrose contained mainly myelin and some rare synaptosomes. The fractions layered on 0.8 M, 1.0 M, and 1.2 M sucrose were very rich in synaptosomes. Different populations of the latter were present: one type contained only clear synaptic vesicles; the second type, in addition to clear synaptic vesicles, had a population of vesicles with homogeneous dense core (about 1000 Å diameter). There was no significant difference between the light fractions of hypothalamus and of pituitary stalk. However, in the heavier fractions derived from the pituitary stalk a considerable number of large profiles containing dense core granules of 2000 Å diameter were present.In the primary fractions norepinephrine is recovered mainly in the crude synaptosomal fraction, while serotonin is present in higher concentration in the supernatant. In the density gradient serotonin and norepinephrine distribute principally in the three synaptosome-rich fractions, serotonin being more concentrated in the lightest synaptosomal fraction, while norepinephrine is mainly recovered in the heaviest fraction. The differences in the amine distribution in the fractions derived from hypothalamus and pituitary stalk are described and discussed in relation to the different morphology of those fractions.Supported in part by a CNR research grant.Presented in part at the 2nd International Meeting of the International Society for Neurochemistry, Milano 1969.     

Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host neostriatum

Summary In adult rats with a unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopamine pathway, grafts of embryonic ventral mesencephalon can establish extensive efferent connections with the previously denervated host neostriatum and can compensate for motor and sensorimotor asymmetries induced by the lesion. The object of this study was to examine the afferent synaptic inputs to grafted dopaminergic neurons, implanted into a cortical cavity overlying the previously denervated caudate-putamen, using electron microscopic immunocytochemistry. The dopaminergic neurons of the grafts in the same animals had previously been shown to re-innervate the host neostriatum, to form synaptic connections therein and to attenuate the lesion-induced motor asymmetry that occured in response to amphetamine (Freund et al. 1985). In the light microscope, the grafts were found to contain numerous tyrosine hydroxylase-immunoreactive perikarya, dendrites, axons and axonal swellings which had distinct distributions. In addition axons and axonal swellings that were immunoreactive for either substance P or glutamate decarboxylase were present. Electron microscopic analysis of the boutons contacting tyrosine hydroxylase-immunoreactive neurons in the grafts revealed the presence of at least five distinct types of afferent synaptic boutons based on their immunochemistry, morphology, or types of membrane specialization. One type was itself immunoreactive for tyrosine hydroxylase; such synapses are extremely rare in the intact substantia nigra, none were found in the contralateral substantia nigrae or the substantia nigra of a control rat. Three of the remaining types had ultrastructural features that were similar to synaptic terminals that were immunoreactive for substance P or glutamate decarboxylase. These synapses were similar to the types of synapses found contacting dopaminergic neurons in the substantia nigra contralateral to the graft or the substantia nigra of a control rat. The results demonstrate that, in the absence of the normal extrinsic afferent inputs, the intracortical mesencephalic grafts have a well-developed local synaptic circuitry. It is suggested that local circuit regulation of dopaminergic neurons within the graft may, at least in part, be responsible for the maintenance of a normal or close to normal functional activity.     

Distribution and origins of substance P-immunoreactive projections to the paraventricular and supraoptic nuclei: Partial overlap with ascending catecholaminergic projections

Anatomical and pharmacological evidence suggests a role for substance P (SP) in the control of vasopressin secretion, but the origins of SP-immunoreactive (IR) projections to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus have not yet been identified. Combined axonal transport, immunohistochemical, and ablation approaches were used to characterize the organization of SP-IR projections to the PVH. The results may be summarized as follows: (1) SP-IR projections are broadly and prominently distributed throughout the SO and both the magnocellular and parvicellular divisions of the PVH. The distribution within the PVH is quite uniform. (2) Combined retrograde transport-immunohistochemical analyses identified multiple potential sources of SP-IR inputs to the PVH. These included a number of hypothalamic cell groups, the laterodorsal and peduculopontine tegmental nuclei, and the rostral and caudal aspects of the ventrolateral medulla. Portions of the tegmental and medullary SP-IR neurons that were retrogradely labelled following tracer deposits in the PVH also stained positively for choline acetyltransferase or tyrosine hydroxylase, respectively. (3) To evaluate the distribution and prominence of medullary SP-IR projections to the PVH and SO, staining for SP and catecholamine-synthesizing enzymes was carried out in animals that had previously received knife cuts at the level of the pontomedullary border. Pronounced, and roughly parallel decrements in staining for peptide and amines were seen in the magnocellular division of the PVH and in the SO; less marked reductions in SP-IR varicosities are in a position to influence multiple visceral regulatory cell types in the PVH and SO. Inputs to the magnocellular neurosecretory system arise in large measure from medullary neurons in which SP coexists with catecholamines. SP-IR projections to the parvicellular division of the PVH appear to originate from a number of sources.