Quantitative X-ray microanalysis of adrenal medullary cells of young adult and aged rats after glutaraldehyde fixation and potassium dichromate treatment

X-ray microanalysis has been used to detect chromium in the histochemical reaction product resulting from the reaction of noradrenaline with glutaraldehyde during fixation of the rat adrenal medulla and subsequent treatment with potassium dichromate. In unstained ultrathin sections, noradrenaline cells can be identified by their content of highly electron-dense storage granules, which enables individual granules to be analysed quantitatively to assess the amount of bound chromium within them. In young adult (4-month-old) rats the mean chromium content of noradrenaline-containing adrenal medullary granules was 443.6±50.7 mM/kg dry weight. In aged (24-month-old) animals the mean chromium content was 267.0±64.0 mM/kg dry weight which was significantly (P<0.01) lower then the value for the young adult rats. Some noradrenaline cells contained granule populations, which were markedly less electron dense than those in the young adults and this is reflected in the ranges of chromium values recorded between individual cells in the 24-month-old animals. There were also noradrenaline cells in the medulla of the aged animals, which contained highly electron-dense granules but these did not contain as much bound chromium as the highest values recorded in the young adult animals. The results are discussed in the context of the growth of the rat adrenal medulla throughout the lifespan and with respect to the effects of age on the integrity of storage granules.     

Changes in the development of central noradrenaline neurones following neonatal administration of 6-hydroxydopamine

Abstract— The effects of the neurotoxic compound 6-hydroxydopamine on central noradrenaline (NA) neurones have been investigated in the adult rat after systemic administration of the drug at birth. This treatment produced a permanent and selective reduction in endogenous noradrenaline, [³H]noradrenaline uptake in vitro and the number of histochemically demonstrable noradrenaline nerve terminals in the forebrain, certainly related to neuroneal degeneration. The fluorescence morphology of the noradrenaline perikarya in the locus coeruleus was not notably affected. In the pons-medulla region, the 6-hydroxydopamine treatment led to an almost two-fold increase in endogenous noradrenaline with a similar increase in [³H]noradrenaline uptake and formation of ³H-catecholamines from [³H]tyrosine. Fluorescence histochemistry revealed an increased number of noradrenaline nerve terminals which in addition showed an increased fluorescence intensity. Subcellular distribution studies of endogenous noradrenaline in pons—medulla disclosed the highest relative noradrenaline increase in the microsomal fraction after 6-hydroxydopamine at birth. Sucrose gradient centrifugations disclosed that the pons-medulla synaptosomes from 6-OH-DA treated rats sedimented at a higher sucrose concentration than those from untreated controls. It is concluded that treatment of neonate rats with 6-hydroxydopamine produces a selective degeneration of noradrenaline nerve terminals in the forebrain, especially in the cerebral cortex, whereas in the pons-medulla this treatment leads to an increased intraneuronal noradrenaline concentration due to accumulation of noradrenaline in collateral systems not affected by 6-hydroxydopamine and probably also to an increased outgrowth of noradrenaline nerve terminals.     

Immunohistochemical localization of neurokinin-l receptor in the lumbar spinal cord of young rats: morphology and distribution

The type and distribution of neurokinin-1 (NK-1) receptor-expressing neurones were studied in young (14-day-old) rats' lumbar spinal cord using pre-embedding immunohistochemistry. The heaviest immunoreactivity was observed in the middle part and lateral fourth of lamina I where the great majority of immunoreactive perikarya represented fusiform and multipolar cells. In lamina II the middle and medial part showed moderate immunoreactivity, most of the cells resembled stalked cells. In lamina III the labelled perikarya were evenly distributed, while those in lamina IV accumulated mainly in the lateral part. In both laminae most of the labelled neurones represented central cells, the rest of them belonged to the antenna-type cells with long dorsally directed dendrites penetrating the superficial laminae. The immunoreactivity in laminae V-VII was uniform and relatively weak. In lamina VIII the immunopositive perikarya were encountered only rarely while in lamina IX virtually all motoneurones showed weak immunoreactivity. Lamina X contained small, multipolar and fusiform labelled perikarya. In conclusion, we found that the general appearance of the NK-1 receptor immunostaining and the major type of NK-I receptor-expressing neurones were similar to that found previously in adult spinal cord. Using the same method as Brown and colleagues the number of labelled NK- 1 receptor immunoreactive cells was similar in young and adult animals except lamina I where the number of immunoreactive neurones was twice that in adults.     

Influence of extracellular matrix proteins on the expression of neuronal polarity

The influence of laminin (LN) and fibronectin (FN) on the differentiation of individual neurones from the embryonic rat central nervous system was studied in vitro. In control cultures or in the presence of soluble FN, most neurones had several dendrite-like and one axon-like processes. On substratum-bound LN, multipolar and unipolar cells were present. Soluble LN and bound FN induced a very simple neuronal morphology, most neurones having only one axon-like neurite as defined by morphological and immunocytochemical characteristics. The significant reduction of neuronal adhesion and spreading in conditions leading to the growth inhibition of dendrite-like processes suggests that, contrary to that of axons, dendrite growth strongly depends on neuronal adhesion. We propose a model in which the different dependency of axonal and dendritic outgrowth towards adhesion and spreading is explained by the respective physical properties of the two types of neurites.     

Effect of repeated exercise on postnatal development of the noradrenaline content of the albino rat heart, spleen and skeletal muscle

The authors studied the effect of repeated elevation of sympathetic activity on the postnatal development of the noradrenaline content of tissues of the albino rat. Between the ages of 15 and 29 days, young rats were forced to swim in water heated to 25 degrees C, 3 X 30 min on weekdays and 1 X 30 min on Saturdays and Sundays. At 30, 45 and 65 days, the noradrenaline content of the tissues was determined spectrofluorometricaLly by the trihydroxyindole method. The noradrenaline content of the heart of trained rats was higher than in the controls in all the given age groups and the size of the absolute difference rose with advancing age. The noradrenaline content of the spleen developed similarly. Repeated exercise did not lead to an increase in the noradrenaline content of skeletal muscle. The results show that the repeated elevation of the activity of sympathetic adrenergic neurones which occurs in young rats during exercise is a long-term factor stimulating the development of sympathetic innervation of the heart and spleen. The development of the neurones innervating skeletal muscle was not stimulated, probably because the activity of these neurones is not increased by stress.     

Dependence of the cytotoxic effect of guanethidine on the degree of sympathetic activity

Young rats aged 15-29 days received a subcutaneous injection of guanethidine sulphate (5 mg/kg body weight) every day. Owing to damage to the postganglionic sympathetic neurones, on about the 60th day of life we observed a significant decrease in the noradrenaline concentration in these animals' hearts compared with the controls. If every guanethidine injection was followed immediately by intensive physical exercise, there was no drop in the heart noradrenaline concentration. Physical exercise of the same intensity performed a few hours before injecting guanethidine did not prevent the drop in the noradrenaline concentration in the heart. The results show that an exercise-induced increase in sympathetic activity, at a time when guanethidine is circulating in the blood and accumulating in the adrenergic neurones, inhibits the cytotoxic effect of guanethidine. Isolated physical exercise performed between the 15th and 29th day of life leads to an increase in the noradrenaline content of the heart of rats aged 60 days.     

Postnatal androgenization induces premature aging of rat ovaries

In the present paper, we report that ovaries of adult rats treated with testosterone propionate (TP) on a critical postnatal Day 5 exhibit histologic and immunohistochemical findings which resemble those of the anovulatory ovaries in middle-aged female rats. The sterile rat model has been long known whereas ovarian failure seems to be a reason for anovulation with normal hypothalamo-pituitary-gonadotropin background. Appropriate function of ovarian steroidogenic cells is also regulated by mesenchymal cells. To characterize the ovarian failure, we studied the histology, luteinizing hormone receptor (LHR) expression, and characterized changes of vascular pericytes, T cells, and dendritic cells in ovarian steroidogenic compartments consisting of interstitial cells (ISC) of ovarian interstitial glands, and granulosa and theca interna cells of ovarian follicles. Normal adult ovaries contained 63% of mature interstitial glands. The mature ISC exhibited moderate cytoplasmic and strong surface LHR expression and fine (<5 micrometer) cytoplasmic vacuoles (ISC of 'luteal type'). They originated from young ISC of 'thecal type,' which exhibited strong cytoplasmic LHR expression. Remaining 37% were aged interstitial glands, which consisted of aged ISC (increased cytoplasmic vacuolization, nuclear pyknosis, and reduced surface LHR expression) and regressing ISC (weak cytoplasmic and no surface LHR expression). However, no mature ISC of 'luteal type' were detected in anovulatory ovaries of adult rats (45- and 60-day-old) injected with TP (100 or 500 microgram) on postnatal Day 5 (TP rats). Their ovaries contained 96% of aged interstitial glands with aged and regressing ISC. Remaining 4% were abnormal interstitial glands with direct transition of young ISC of 'thecal type' into aged ISC (young/aged glands). Lack of mature ISC, and similar amount of aged (96%) and young/aged interstitial glands (4%) was also detected in anovulatory ovaries of untreated persistently estrous middle-aged (10-month-old) females (aging PE rats). The aging process in TP and aging PE rats was accompanied by regression of vascular pericytes, T cells, and dendritic cells within the interstitial glands. In addition, anovulatory ovaries of TP rats and aging PE females contained mature follicles exhibiting LHR overexpression by granulosa cells, and aged (cystic) follicles with reduced layers of granulosa cells lacking LHR expression. In contrast, when the rats were injected with 500 microgram of TP later, on postnatal Day 10, the adult females exhibited estrous cycles and normal ovaries with corpora lutea. These results show that injection of TP during the critical postnatal period causes a lack of mature and preponderance of aged ISC in adult ovaries, accompanied by degeneration of mesenchymal cells. We suggest that mesenchymal cells regulate qualitative aspects of tissue-specific cells, and this function of mesenchymal cells is programmed during the critical period of development.     

The effect of ageing on the SIF-cells in the hypogastric (main pelvic) ganglion of the rat

Summary The effect of ageing on SIF-cells was studied by the formaldehyde-induced fluorescence (FIF) method and by electron microscopy (EM). Microspectrofluorimetry was used to record emission spectra in FIF preparations. In newborn and in young adult (8–12 weeks) rats a single type of SIF-cell emitting greenish-yellow FIF was found while in aged rats a second type of SIF-cell emitting yellowish-brown granular FIF was also present. The intensity of the yellowish-brown FIF was lower than that of the greenish-yellow FIF. Also a few bright cytoplasmic fluorescent areas were occasionally found in some SIF-cells. The distribution of the SIF-cells through the ganglion did not change remarkably with age.In EM after glutaraldehyde-fixation in newborn and in young adult rats two types of small granule-containing (SGC) cells were distinguished according to the size of the dense cored vesicles, 1) 50–150 nm and 2) 50–250 nm. In aged rats, a third type of SGC-cells containing 50×250 nm elongated dense core vesicles could also be distinguished. After KMnO₄-fixation in newborn and in young adult rats the classification was identical with glutaraldehyde-fixation. In aged rats three types of storage granules were found after KMnO₄, 1) 100–300 nm empty vesicles and 2) 100–300 nm vesicles with small dense core, 3) 100–500 nm irregular in shape and filled with electron opaque material with a more dense core.     

Aging Differentially Affects the Loss of Neuronal Dendritic Spine,Neuroinflammation and Memory Impairment at Rats after Surgery

It is known that age is an important factor for postoperative cognitive dysfunction (POCD) and the patients with POCD suffer from the impairment of multiple brain regions and multiple brain functions. However currently animal studies of POCD mainly focus on hippocampus region, therefore in this study we performed partial hepatectomy in young adult and aged rats to test the questions (1) whether POCD in animals involves other brain areas besides hippocampus; (2) how age influences POCD of young adult and aged animals. We found that (1) in young adult rats, the memory was not significantly affected (P>0.05) 1d, 3d and 7d after partial hepatectomy, but was significantly impaired (p<0.001) in aged rats 1d and 3d post-surgery; (2) in young adult rats, the surgery did not significantly affect the densities of dendritic spines of neurons at CA1, dentate gyrus (DG) and cingulate cortex (P>0.05, respectively) 1d and 3d post-surgery, but the spine densities at CA1 and DG of aged rats were significant reduced 1d and 3d post-surgery (p<0.001, respectively), however this didn’t happen at cingulate cortex (P>0.05); (3) In young adult rats, surgery didn’t affect the activation of microglia and levels of TNF-α and IL-1β at hippocampus (P>0.05), but significantly activated microglia and increased levels of TNF-α and IL-1β at hippocampus of aged rats (P<0.05). Our data suggest that (1) partial hepatectomy-induced POCD mainly involves hippocampus impairments, and (2) differential loss of neuronal dendritic spines and neuroinflammation at hippocampus are most likely the mechanism for the formation of POCD in aged rats.     

Golgi-Cox studies on the central nervous system of a gastropod mollusc

Summary Complete neurones were impregnated in the brain of the pulmonate gastropod pond snail, Lymnaea stagnalis L. using the Golgi-Cox method. Mapping of small to medium sized neurones identified in living preparations by the position of the perikarya was possible. Simple monopolar and bifurcating monopolar neurones with varying lateral patterns of short fine fibres were common in the pond snail brain. Larger neurones have more complex and numerous branches originating from axons close to the perikarya than smaller ones. Stem processes originating on the cell body were observed on neurones above 30 in somal diameter. Possible sites for the location of chemical synapses were suggested. Functional types of neurones were difficult to separate on morphological grounds. Giant or very large neurones are small in number in pond snail ganglia, compared with medium or small neurones.The authors wish to thank Mr. Colin Atherton for photographic assistance and the U.K. Science Research Council for a grant to P. R. B.     

Sympathetic and sensory innervation of the urinary tract in young adult and aged rats: a semi-quantitative histochemical and immunohistochemical study

Summary The sympathetic innervation of the urinary tract of young adult (4 months) and aged (24+ months) rats has been examined by glyoxylic acid-induced fluorescence for the detection of noradrenaline and by immunofluorescence using antisera against tyrosine hydroxylase (TH) and neuropeptide Y (NPY). Immunostaining for calcitonin gene-related peptide (CGRP), known to be present in pelvic sensory nerves, was also performed. Semi-quantitative estimations of nerve densities were made of noradrenergic and peptidergic fibres innervating the smooth musculature of the ureter, bladder and urethra, and of the urinary tract vasculature. In the aged rats the overall patterns of innervation remained unchanged. However, with the exception of the vesical vasculature, the density of noradrenergic innervation decreased as did the intensity of histofluorescence. A similar pattern of results was observed by TH and NPY immunofluorescence. The results present evidence for a diminution in the sympathetic control of the urinary tract in aged rats. The pattern and density of CGRP-immunoreactive nerves was unchanged in the aged animals suggesting that pelvic visceral sensory innervation is more resistant to the effects of advancing age.     

Electron microscopical study of synaptic glomeruli in cerebellum transplanted to the anterior eye chamber

Fetal cerebellar anlage from rat fetuses of 15-16 operational days were grafted into the anterior chamber of the eye of adult female albino rat recipients. Survival time of the transplants--containing both cerebellar cortex and cerebellar nuclei--was 2 to 2 1/2 months. Electron microscopical (EM) studies of the thin, under-developed granular layer of the laminated cerebellar cortex revealed the presence of well differentiated cerebellar glomeruli, surrounded by granule cell perikarya. As in the normal cerebellar cortex, the central profile of the glomerular complex was the large mossy terminal, containing spheroid synaptic vesicles, and forming synaptic contacts with dendrites and dendritic digits of the granule cells. Golgi cell axonal varicosities, containing ovoid or pleomorphic synaptic vesicles were found also on the periphery of the glomeruli. In addition, in several synaptic glomeruli, a third neuronal element was also observed, containing flat, discoidal vesicles and receiving synaptic contacts from mossy and Golgi axons, but being also presynaptic to granule cell dendrites. It is suggested that all mossy terminals in the cerebellar transplant originate from the cerebellar nucleus. Morphological evidence is also provided that the presynaptic dendrite-like processes--never found in normal cerebellar cortex--are also processes of nuclear neurons.     

Size and degeneration increase in herring bodies during aging in hamsters.

The hypothalamo-neurohypophysial tract of young, adult and aged male hamsters was studied at lateral and ventral regions of hypothalamus by means of electron microscopy. Neurosecretory swelling axons (Herring bodies) were usually found as classically described containing abundant neurosecretory granules, mitochondria, few microtubules and profiles of smooth endoplasmic reticulum in all groups of age. However, in aged hamsters, starting at 18-month-old subjects, we observed that the size of some neurosecretory axons was highly increased. Autophagic and degenerative features were seen in the larger ones. These data could suggest abnormal axonal storage or axonal transport blocked during aging. The implications in the role of hypothalamus-neurohypophysial system during aging are discussed.     

Microfluorimetric quantification of catecholamine fluorescence in rat sympathetic ganglia

Summary The formaldehyde-induced fluorescence (FIF) technique was used to generate catecholamine fluorphores in the perikarya of the sympathetic neurons in the superior cervical ganglion of adult rats. During microfluorimetric quantification, the photodecomposition was eliminated by a rapid measuring procedure with a small excitation field and by using only visible light between the measurements.The catecholamine fluorescence, induced in protein microdroplets with increasing noradrenaline concentrations, was linear up to 2×10^–2 M which exceeds the noradrenaline content of even the most intensively fluorescent neurons. Thus, the differences in fluorescence intensities directly reflect the physiological state of each neuron with respect to their catecholamine content. The mean histograms reveal the changes which can only occur in certain neurons, and which can disappear if the mean only is assessed. The microfluorimetric method was sensitive enough to detect even minute changes induced by reserpine treatment in the catecholamine content of the sympathetic ganglion cells.     

Photometric and fluorimetric studies of protomitochondria from liver of young and adult rats

Protomitochondria (PRM) — young organelles — were isolated from liver cells of young (1 month) and adult (9 months) rats. PRM were characterized by photometric and fluorimetric methods. It was found that the number of PRM of various diameters in young and adult rats differs. PRM and mitochondria (MCH) from young and adult rats differ significantly. It was detected by fluorescence of 1-anilinonaphtalene-8-sulfonate (ANS), indicating a different number of membrane binding sites for this probe. An increase of the cytochrome content was observed during ripening of PRM to MCH, more pronounced in young animals. In contrast, the amount of cytochromes in MCH decreases during maturation of young rats to adult rats. The obtained data suggest that massive transformation of PRM to MCH in the cells of young animals takes place, similar to the transformation in the cells of adult animals observed earlier.     

Effect of aging on the rate of axonal transport of choline-phosphoglycerides

The anterograde axonal transport of choline-phosphoglycerides was studied in sciatic nerve motoneurons of adult (3-month-old) and aged (24-month-old) rats. After the spinal cord injection of [2-³H]glycerol, choline-phosphoglycerides; the major phospholipid class was transported along the nerve. The axonal transport rate was determined by plotting the distance covered by the front of transported radioactivity as a function of the time employed. In aged animals the rate of the choline-phosphoglyceride anterograde axonal transport was about 68% lower than that of adults; furthermore, the rate slowed down along the nerve in the proximal-distal direction. This alterated axonal transport mechanism might contribute to the degenerative processes observed in distal regions of peripheral nerve fibers of aged animals.     

Development and Ageing of the Rat Nigrostriatal Dopamine System Studied with Fast Cyclic Voltammetry

Fast cyclic voltammetry at carbon fibre microelectrodes was used to measure electrically stimulated dopamine release in the striatum of anaesthetised young, adult, and senescent Wistar rats. By alteration of stimulus parameters and by use of nomifensine, investigation of dopamine release, uptake, and compartmentalisation within the striatum was possible. The rate of dopamine release was highest in adult rats. No difference was observed between young and old animals. The size of the releasable (newly synthesised) dopamine pool was also largest in the adult group, again with no significant difference occurring between young and aged rats. The rate of dopamine uptake was highest in adult rats, although, when expressed as a function of dopamine release, young and aged rats showed proportionally greater uptake. Nomifensine (10 mg/kg i.p.) increased dopamine release by mobilising the inert storage pool to a greater extent in young than in adult rats, whereas the effect of the drug on uptake was similar in all age groups. The functional significance and possible explanation of these results are discussed.     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Postnatal development of vascularity in the inferior colliculus of the young rat

The inferior colliculus in the rat midbrain is an auditory relay center whose functional maturation occurs postnatally. We examined by morphometry the vascularity and the nuclear profile density of the inferior colliculus in normal young rats at different ages (before and after the onset of auditory input). We also compared this region with a frontal region of the cerebral cortex in 24-day-old rats. The inferior colliculus from aldehyde-perfused Sprague-Dawley rats aged 5, 9, 14, and 24 days was analyzed by light microscopy of semithin plastic sections. The central region (mostly the central nucleus) was sampled at 5 levels representing its entire rostrocaudal extent. Patent-blood-vessel profiles were counted and classified according to their size and profile orientation. Counts of nuclear profiles in the same sections were also made. In the inferior colliculus of rats between 5 and 24 days of age, the small (less than 10-microns diameter) cross-sectioned vessel profiles increased over 6-fold in number per unit area. Correspondingly the vascular volume density, estimated by differential point counting, increased between these ages. However, there was a decrease in the number of neuronal and glial nuclear profiles per unit area, probably because of growth in the volume of the neuronal perikarya and processes, along with cell emigration reported to occur at early postnatal ages. This study has shown that an increase in vascularity in the central region of the rat inferior colliculus continues for up to 2 weeks after the onset of hearing.(ABSTRACT TRUNCATED AT 250 WORDS)