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The epigenetic clock, defined as the DNA methylome age (DNAmAge), is a candidate biomarker of ageing. In this study, we aimed to characterize the behaviour of this marker during the human lifespan in more detail using two follow-up cohorts (the Young Finns study, calendar age i.e. cAge range at baseline 15-24 years, 25-year-follow-up, N = 183; The Vitality 90+ study, cAge range at baseline 19-90 years, 4-year-follow-up, N = 48). We also aimed to assess the relationship between DNAmAge estimate and the blood cell distributions, as both of these measures are known to change as a function of age. The subjects' DNAmAges were determined using Horvath's calculator of epigenetic cAge. The estimate of the DNA methylome age acceleration (Δ-cAge-DNAmAge) demonstrated remarkable stability in both cohorts: the individual rank orders of the DNAmAges remained largely unchanged during the follow-ups. The blood cell distributions also demonstrated significant intra-individual correlation between the baseline and follow-up time points. Interestingly, the immunosenescence-associated features (CD8+CD28- and CD4+CD28- cell proportions and the CD4/CD8 cell ratio) were tightly associated with the estimate of the DNA methylome age. In summary, our data demonstrate that the general level of Δ-cAge-DNAmAge is fixed before adulthood and appears to be quite stationary thereafter, even in the oldest-old ages. Moreover, the blood DNAmAge estimate seems to be tightly associated with ageing-associated shifts in blood cell composition, especially with those that are the hallmarks of immunosenescence. Overall, these observations contribute to the understanding of the longitudinal aspects of the DNAmAge estimate.

Background and aims: Taking medical history from a very old patient for either clinical or research purposes raises the question of the reliability of the information obtained. We ascertained whether the self-reported medical history and self-rated health of 90-year-olds would be in agreement with their medical records. Methods: Information on chronic diseases and self-rated health was collected using medical records and a mailed questionnaire in a population sample of community-living 90-year-old subjects in Tampere, Finland. The results were compared with earlier studies on self-reported medical history. Results: The inter-source agreement of the reported diseases was relatively good. As expected, many of the diseases diagnosed were under-reported in the mailed questionnaire. However, dementia, depression and arthritis were reported more often than doctors had recorded them. Of the respondents, 78% reported their current health as good or average. Subjects with heart disease, stroke, rheumatoid arthritis, Parkinson’s disease or depression reported poor health more often than those not having these diseases. Conclusions: The agreement between medical records and self-reported medical history obtained by questionnaire showed a similar pattern to earlier studies in younger old populations, but in the 90-year olds, the differences became larger. In future studies, special attention should be paid to the oldest old who under-report certain diagnosed medical conditions.

Background Several studies have focused on predictors of mobility limitations and disabilities. Yet little is known about the pace and patterns of mobility changes among very old people. This study examined changes in functional mobility among individuals aged 90 years and older during a 2-9-year follow-up. In addition, we were interested in the patterns of mobility changes. Methods Data were collected through a mailed questionnaire in the years 2001, 2003, 2007 and 2010. The study population (n = 948) consisted of individuals from three cohorts (2001, 2003, 2007) who participated in at least two survey rounds and answered the mobility questions. The length of the follow-up varied from 2–9 years between individuals as well as according to how many times an individual took part in the survey. Multilevel ordinal logistic regression analysis was used to evaluate the effects of time, age, gender, cohort and chronic conditions on changes in mobility. Results At the baseline, “younger” old people, men and individuals in the cohorts for 2003 and 2007 had significantly better mobility compared with women, older individuals and individuals in the 2001 cohort. In addition, individuals with fewer chronic conditions had better mobility than those with more diseases. Mobility declined for most of the participants during the follow-up. The difference in the change in mobility over time for gender, age or chronic conditions was not statistically significant. The analyses were performed with a subgroup of participants aged 90–91 years at the baseline, and results did not differ substantially from the results for the entire study sample. However, the effect of chronic conditions on the change in mobility was statistically significant among participants aged 90-91years. Conclusions No differences were observed in the rate of mobility decline over time between age or gender. The effect of chronic conditions on the change in mobility was significant only among individuals aged 90–91 years. The prevention efforts are important and should focus even more, also among the oldest-old, on additional modifiable risk factors such as maintaining muscle strength. BioMed Central open access

Mitochondrial DNA (mtDNA) coding region polymorphisms, as well as the 150T polymorphism in the noncoding region, have been associated with longevity. We have studied here the association of 150T with longevity further and assessed differences in this association between various mtDNA haplogroups. We analysed a sample of 321 very old subjects and 489 middle-aged controls from Finland and Japan. 150T was more frequent among the very old than among the controls in both the Finnish and Japanese subjects. Interestingly, the association was not similar in all haplogroups, and a stratified analysis revealed that two additional common polymorphisms, 489C and 10398G, modified the association between 150T and longevity. These findings suggest that longevity is partly determined by epistatic interactions involving these three mtDNA loci.

Data on how body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) are associated with body fat in the oldest-old people are scarce. The purpose of this study was to examine if BMI, WC or WHR are associated with leptin, a biological surrogate measure of body fat in 90-year-old people. The data comes from the Vitality 90+ Study, a prospective population-based study of people living in Tampere, Finland. BMI, WC, WHR and plasma concentration of leptin were available for 160 women and 54 men aged 90 years. BMI and WC had a strong significant positive association with leptin both in women and in men, but WHR was associated with leptin only in men. In conclusion, based on the circulating level of leptin, BMI and WC, and WHR in men, reflect body fat in 90-year-old people, but WHR seems to be a poor indicator of body fat in 90-year-old women.

Background and aims: The oldest-old population is expanding rapidly. There is a new need for clinical information about this group, which is actively using social and health care. We studied the population of people born in 1907–1910 and living in the city of Tampere (Finland) at the age 90 (N=916, 79.4% women, 20.6% men); 71.7% of the population lived in the community and 28.3% in institutions. Methods: Medical records of 832 (90.8%) nonagenarians were obtained. We registered diagnoses of chronic diseases or diseases that required hospitalization at any time of their lives, as well as physicians’ notes on their memory and mobility. Diseases were coded and grouped according to ICD-10. Results: The most common diagnosis groups were cardiovascular diseases (78.3%), gastrointestinal diseases (58.6%), infections (53.6%) and trauma (49.6%). There was an average of 8 chronic or severe diseases mentioned in patient records. The diagnosis of dementia was mentioned in 26.7% of cases, most of them living in institutions; a problem with memory — from for-getfulness to dementia — was mentioned in 35.9% of cases; 37.5% were able to move using no or a light support, 8.3% were bedridden. Conclusions: We conclude that this age group suffers from numerous chronic diseases influencing mobility and cognition. Dementia seems to be the most important symptom leading to institutionalization.

Sequence variation in mitochondrial DNA (mtDNA) may cause slight differences both in the functioning of the respiratory chain and in free radical production, and an association between certain mtDNA haplogroups and longevity has been suggested. In order to determine further the role of mtDNA in longevity, we studied the frequencies of mtDNA haplogroups and haplogroup clusters among elderly subjects and controls in a Finnish population. Samples were obtained from 225 persons aged 90-91 years (Vitality 90+) and from 400 middle-aged controls and 257 infants. MtDNA haplogroups were determined by restriction fragment length polymorphism. The haplogroup frequencies of the Vitality 90+ group differed from both those of the middle-aged controls ( P=0.01) and the infants ( P=0.00005), haplogroup H being less frequent than among the middle-aged subjects ( P=0.001) and infants ( P=0.00001), whereas haplogroups U and J were more frequent. Haplogroup clusters also differed between Vitality 90+ and both the middle-aged subjects ( P=0.002) and infants ( P=0.00001), the frequency of haplogroup cluster HV being lower in the former and that of UK and WIX being higher. These data suggest an association between certain mtDNA haplogroups or haplogroup clusters and longevity. Furthermore, our data appear to favour the presence of advantageous polymorphisms and support a role for mitochondria and mtDNA in the degenerative processes involved in ageing.

Infection with human cytomegalovirus (CMV) affects the function and composition of the immune system during ageing. In addition to the presence of the pathogen, the strength of the immune response, as measured by the anti-CMV IgG titre, has a significant effect on age-related pathogenesis. High anti-CMV IgG titres have been associated with increased mortality and functional impairment in the elderly. In this study, we were interested in identifying the molecular mechanisms that are associated with the strength of the anti-CMV response by examining the gene expression profiles that are associated with the level of the anti-CMV IgG titre. Results The level of the anti-CMV IgG titre is associated with the expression level of 663 transcripts in nonagenarians. These transcripts and their corresponding pathways are, for the most part, associated with metabolic functions, cell development and proliferation and other basic cellular functions. However, no prominent associations with the immune system were found, and no associated transcripts were found in young controls. Conclusions The lack of defence pathways associated with the strength of the anti-CMV response can indicate that the compromised immune system can no longer defend itself against the CMV infection. Our data imply that the association between high anti-CMV IgG titres and increased mortality and frailty is mediated by basic cellular processes. BioMed Central open access

Selegiline is a selective and irreversible monoamine B inhibitor with the capacity to increase the level of several antioxidative enzymes in rat brain. It can protect adrenergic neurons against injury induced by neurotoxins such as MPTP, DSP-4 and AF64A in animal studies. In addition, the protective action is not limited to catecholaminergic cells, as selegiline can also minimize the loss of developing motoneurons after axotomy. The aim of this study was to determine whether selegiline can protect peripheral catecholaminergic neurons against the neurotoxic effect of 6-OHDA. This kind of protective effect against 6-OHDA neurotoxicity has not been reported before. Wistar albino male rats aged 4 or 24 months were treated with selegiline or saline solution 1 h before 6-OHDA injection. At 2 weeks after the 6-OHDA injection, the superior cervical ganglia (SCG) and submandibular glands (SMG) were studied using catecholamine histofluorescence and immunohistochemistry for tyrosine hydroxylase (TH). The number of TH-positive cells in the SCG and the length and number of adrenergic nerve fibers in the SMG were quantified. Our findings showed that 6-OHDA caused a reduction of TH immunoreactivity and catecholamine histofluorescence in neuronal somata, as well as a decrease in the number and length of adrenergic nerve fibers in the submandibular gland. Selegiline pretreatment protected SCG neurons and their postganglionic nerve fibers in SMG against these changes in a dose-dependent manner. The mechanism through which selegiline exerts its neuroprotective effect is as yet unknown.

Sympathetic ganglion tissue of 3-months- and 18-months-old Fischer-344 rats was autotransplanted into the adrenal gland in order to determine the effect of aging on the survival of grafted neurons. Adrenal cortex was chosen as the host tissue because it is well vascularized and has a high concentration of glucocorticoids, which stimulate the synthesis of catecholamines. At 4 weeks following the transplantation, the density of neurons was decreased in all transplants, but approximately the same proportion of remaining neurons showed tyrosine hydroxylase immunoreactivity as in intact ganglia. At 8 weeks, a subpopulation of large neurons showed an increased accumulation of age pigment. The heavily pigmented neurons were usually devoid of catecholamines, whereas small non-pigmented neurons frequently showed strong catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. There was no marked difference between old and young animals in the survival of transplanted neurons. The results show that the sympathetic neurons from both 3-months-and 18-months-old animals survived the autotransplantation procedure. The humoral environment of the adrenal cortex may be beneficial for the restoration of the integrity of sympathetic neurons.

Enkephalin (ENK)- and neurotensin (NT)-immunoreactivities (IR) were localized in cat adrenal medulla using immunocytochemistry. ENK was localized mainly in adrenaline cells, 70%-80% of which were heavily labelled. About 5%-10% of the noradrenaline cells were ENK-immunoreactive (IR). A dense network of ENK-IR nerves was localized among adrenaline cells. NT was localized only in the noradrenaline cells, 60%-70% of which were immunoreactive. NT- and ENK-IR were localized in separate noradrenaline cells. A dense network of NT-IR nerves was restricted among noradrenaline cells. The present findings confirm that ENK- and NT-like peptides are localized in separate adrenal medullary cells and demonstrate that also the preganglionic nerves are divided in subpopulations according to their neuropeptide content. These nerves have a selective localization among adrenaline and noradrenaline cells and may have physiological significance in the control of secretion of catecholamines and neuropeptides from adrenal medulla.

The formaldehyde-induced fluorescence (FIF) of the cytoplasm of individual developing neurons of the main pelvic ganglion was recorded microspectrofluorimetrically in order to follow changes in catecholamine (noradrenaline) content during development. For each ganglion, the fluorescence intensity profile was estimated and shown graphically as columns expressing percentage distribution of relative intensities in different intensity classes. During development, the number of weakly fluorescent neurons increases. Treatment with testosterone shifts the profile towards higher intensities in four- and six-week-old animals. Testosterone affected the main pelvic ganglion but not the superior cervical ganglion. The intensity of the cytoplasmic FIF, which correlates with the catecholamine (noradrenaline) content of the object tissue, showed a tendency to decrease during development. This change was not obvious vy visual observation because of the increase in cell size and the toal bulk of the ganglion. Other possible factors affecting visual observation are discussed.

According to the earlier concept, the paraganglia of man are believed to degenerate during the first postnatal years after their dominance during the fetal period. Clinical case reports on persisting paraganglia led us to extensive exploration of surgical material obtained from urological and gynecological surgery. The formaldehyde induced fluorescence (FIF) was used for tracing the catecholamine containing tissues. The fluorescence intensities were recorded with a Leitz MPV 2 microspectrophotometer. Solitary, small paraganglia were found in all patients studied. They were especially frequent in the walls of the urinary bladder and in the connective tissue surrounding the urogenital organs. The intensity of the fluorescence was comparable to pharmacological standard of 10(-2) M noradrenaline and at the same level as the FIF of human fetal paraganglia. All cells of the paraganglionic clusters exhibited FIF and no signs of degeneration could be observed. It is suggested that the paraganglia of man do not degenerate postnatally but persist as a remarcable catecholamine reservoir, which might be of physiological importance.

Enkephalin-like immunoreactivity was demonstrated using the peroxide-antiperoxidase method. Only the adrenaline-storing cells of the hamster adrenal medulla were labelled.

The effect of ageing on adrenergic neurons was studied in the hypogastric ganglion of the male rat using the formaldehyde-induced fluorescence method. With age, two changes were obvious. Firstly, the fluorescence intensity of the neurons decreased throughout postnatal life and the number of completely non-fluorescent adrenergic neurons increased. Secondly, the amount of non-specific fluorescence due to lipofuscin pigment increased. The pigment fluorescence was also found around the neurons in satellite cells.

Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeks in vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor. In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlier in vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of synaptic vesicles were regularly observed, occasionally in synapse-like contact with other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensity fluorescent (SIF) cells were observed. Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.

Synaptosomes from one day old and adult rat brain were studied. Specific cytochemical methods for demonstrating monoaminergic (MA) nerve endings were used. Permanganate fixation after preincubation with 5-OHDA or alpha-methyl-NA demonstrated MA synaptosomes. Their number was small in the adult (less than 1 %) and still smaller in the one day old rat brain. The MA synaptosomes from developing rats were different from the adult ones. The large amount of endoplasmic reticulum in developing synaptosomes suggests that granular vesicles are formed from endoplasmic reticulum in nerve endings.

The development of the hypogastric ganglion of normal and testosterone-treated rats was studied using formaldehyde-induced fluorescence (FIF) method. The fluorescence intensities were recorded microspectrofluorimetrically. In normally developing rats cytoplasmic FIF decreases and cell size increases with age. In normally developing rats cytoplasmic FIF decreases and cell size increases with age. In testosterone-treated animals FIF increases during 2--6 weeks compared to the controls. The differences between control and experimental rats were significant. The diameters were significantly longer in treated animals in three and four week old groups. Vacuolated neurons were seen earlier in testosterone-treated rats. No changes in FIF or in cell size were noticed in the superior cervical ganglion. The male sex steroid, testosterone evidently influences the catecholamine turnover and cellular growth during development in the male pelvic ganglion.

The peroxidase-antiperoxidase method was applied to demonstrate enkephalin-like immunoreactivity in the human sympathetic ganglia. A network of immunoreactive fibres was found in light microscopy between the nonreactive neurons. Electron microscopy showed immunostaining to be restricted to axonal profiles containing both large and small vesicles. The strongest reaction was associated with large vesicles.

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.

The sympathetic ganglia of adult and aged humans were obtained during vascular, gynaecological and urological surgery, and studied using the formaldehyde-induced fluorescence (FIF) method for histochemical demonstration of catecholamines. Microspectrofluorimetry was applied to characterize the emission spectra of the flurophores. The sympathetic ganglia contained two types of cells exhibiting FIF: the adrenergic neurons and the small, intensely fluorescent (SIF) cells. The adrenergic neurons contained pigment granules exhibiting autofluorescence with emission maximum at 530---540 nm. The number of these granules increased with age while the FIF of the neurons decreased. In the oldest patients, most of the neurons in the sympathetic ganglia showed only pigment fluorescence but not FIF. The decrease of the perikaryonal catecholamine stores is interpreted as a degenerative change leading to impaired function of the neurons. The patterns of AChE did not change with age. The SIF cells were sparse and were mostly observed near the ganglia, forming paraganglia of varying size. Because of the rarity of the SIF cells, the effect of ageing could not be judged reliably.

The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.

The distribution of nerves containing immunoreactivity for the VIP and enkephalins has been demonstrated in the human prostate and seminal vesicles using the immunoperoxidase bridge. VIP-containing nerves were detected in both organs studied mainly in association with the epithelium, while nerves containing ELI seemed to be related to smooth muscle. Compared with the distribution of adrenergic and cholinergic nerves in the prostate marked differences in the density of the innervation were detected. The possible nature of these peptide-containing nerves is discussed.

Carotid bodies of adult albino rats were examined using the formaldehyde-induced fluorescence (FIF) method for the demonstration of fluorogenic monaomines and staining with I% Toluidine Blue for morphological observations.

The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 A) and large granular vesicles (diameter 700–1200 A) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 A) and large granular vesicles (diameter 700–1200 A). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 A was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.

The effect of hydrocortisone, prednisolone, estrogen, progesterone and testosteron on the differentiation of the paracervical ganglion of the rat uterus was examined by the formaldehyde induced fluorescence (FIF) method. Prednisolone and hydrocortisone treatment caused about a tenfold increase in the number of small, intensively fluorescent (SIF) cells. The SIF-cells were larger and organized in larger groups than in untreated animals. The SIF-cells were also arranged in a satellite cell position around the ganglion cells, which were non-fluorescent or showed a weak to moderate FIF. The amount of solitary SIF-cells was increased. No effects detectable by the FIF-method were found with certainty after administration of estrogen, progesterone or testosterone.

The effects of 17-B-estradiol on the fine structure of the autonomic nerves of the rabbit oviduct and myometrium were studied by means of KMnO4-fixation. The main changes due to prolonged estrogen treatment were the following: (1) the dimensions of the axons increased, (2) the amount of smooth endoplasmic reticulum in the axons increased, (3) the amounts of both large granular vesicles (LGV) and large agranular vesicles (LAV) in the axons increased and (4) electron dense grains were found within the endoplasmic tubuli of the axons. Furthermore, it seems probable that the amount of small granular vesicles was also considerably higher after two weeks of estrogen treatment.

The autonomic nerves of the myometrium of the rabbit were studied in order to demonstrate simultaneously the adrenergic nature of an axon and the localization of acetylcholinesterase (AChE) in the same axons. The synaptic vesicles of the adrenergic axons and nerve terminals remained partially filled with the electron dense material typical for them after formaldehyde fixation and short incubation time for AChE. AChE stain was localized regularly on the axons which contained agranular synaptic vesicles and also on axons which contained dense cored synaptic vesicles beeing probably adrenergic. The role of AChE on the adrenergic axons is discussed.

The fine structure of mitochondrial accumulations in axonal swellings of human sympathetic ganglia is described. A typical swelling contained, in addition to regularly organized mitochondria, bundles of neurofilaments and vesicles as well as large dense-cored vesicles and myelin figures. Synaptic contacts between axonal swellings with mitochondrial accumulations and ganglion cells were not found. A three-dimensional model of the mitochondrial accumulation based on serial sectioning is presented. The possible degenerative and regenerative features of these accumulations are discussed. It is possible that mitochondrial accumulations are functionally active energy producers rather than results of degenerative processes.

The distribution of enkephalin-like immunoreactivity in paraganglia of the urinary bladder of adult humans was studied by use of immuno-electron microscopy. All paraganglionic cells were positively stained. Enkephalin-like immunoreactivity was located in chromaffin granules. Chromaffin cells in the paraganglia showed only few degenerative features, suggesting undisturbed function of the cells.

The ultrastructure and protein content of the five subfractions of the crude mitochondrial fraction from the brain stem of the 1-day old and adult rat was examined. The morphological composition of the subfractions after fixation in glutaraldehyde and osmiumtetroxide in the adult rat brain stem resembled that previously reported for the whole brain; synaptosomes sedimented in a sucrose gradient in subfractions C and D. In the 1-day old rat, mature synaptosomes were found in subfractions A, B, C and D; E contained mainly free mitochondria. 80–95% of the processes in the adult and 10–30% in the 1-day old rat contained synaptic vesicles which were of four types: (1) small agranular vesicles (2) large dense core vesicles (3) large agranular vesicles (4) coated vesicles. Pre- and postsynaptic membrane thickenings were demonstrated in many nerve-ending particles. In the subfractions of the 1-day old rat the protein content was one half and the distribution resembled that in the adult. Evidently nerve endings develop faster in the brain stem than in cortical areas; a serotoninor adrenergic origin of the early synaptosomes is suggested.

Sympathetic post-ganglionic neurones in the coeliac-superior mesenteric ganglion (CSMG) complex of aged (24 month) rats have been studied by glyoxylic acid-induced fluorescence and electron miscroscopy. Comparisons have been made with the CSMG of young adult (4 month) rats. In the aged rats the noradrenaline fluorescence of the majority of neuronal perikarya was very low or absent and few intraganglionic fluorescent varicosities were seen. Lipofuscin pigment was very prominent at the nuclear pole region of neurones and also in dendrites and axonal processes. Ultrastructural studies revealed large accumulations of residual bodies at the nuclear poles and in axons and dendritic profiles. Within the perikarya many mitochondria were distorted or swollen, the rough endoplasmic reticulum was disarranged and much dilated as were Golgi cisternae. Primary lysosomes were encountered throughout the neurone perikaryon and its axonal or dendritic processes. In contrast to the young adult CSMG, no evidence for loading of transmitter storage vesicles with an identical dose level of 5-hydroxydopamine was detected in any part of the neurones of aged rats. This might reflect an impairment of the uptake mechanisms and/or storage of noradrenaline in aged sympathetic neurones and their axonal and dendritic processes.