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Ageing of the human immune system, or immunosenescence, is characterised by distinct changes in the proportion of the various cell types, e.g., increase of the CD14+ monocytic cells, decrease of CD19+ B lymphocytes, and changes in T cell subpopulations, namely increase of CD4+ and CD8+ cells which have lost the costimulatory CD28 antigen. Currently, it is believed that the lifelong antigenic burden may be one of the inducers of immunosenescence. Thus far, only one exogenous stimulus, cytomegalovirus infection, has shown to be a major factor in this respect. To find other possible candidates, we evaluated the role of the evolutionary youngest group of human endogenous retroviruses, namely HERV-K(HML-2), on immunosenescence. HERVs exist in the genome as proviruses, but their activation has been detected in several immunopathologic conditions. The expression of HERV-K(HML-2) env was observed to be lower in the peripheral blood mononuclear cells of nonagenarians (n=61) than in those of young controls (n=37). These mRNA levels did not correlate with the age-associated differences in the proportions of CD14+, CD4+CD28- and CD8+CD28- cells, but in the case of CD19+ B cells a strong positive correlation was observed in the nonagenarians. Thus, these data suggest that HERVs do not function as antigenic drivers of immunosenescence. On the contrary, expression of HERV-K(HML-2) env is associated with more youthful levels of B cells.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35–75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan. published

Plasma cell-free DNA (cf-DNA) has recently emerged as a potential biomarker of aging, reflecting systemic inflammation, and cell death. In addition, it has been suggested that cf-DNA could promote autoinflammation. Because the total cf-DNA pool comprises different cf-DNA species, we quantified the plasma levels of gene-coding cf-DNA, Alu repeat cf-DNA, mitochondrial DNA (mtDNA) copy number, and the amounts of unmethylated and total cf-DNAs. We identified the relationships between these cf-DNA species and age-associated inflammation, immunosenescence, and frailty. Additionally, we determined the cf-DNA species-specific transcriptomic signatures in blood mononuclear cells to elucidate the age-linked leukocyte responses to cf-DNA. The study population consisted of n = 144 nonagenarian participants of the Vitality 90+ Study and n = 30 young controls. In the nonagenarians, higher levels of total and unmethylated cf-DNAs were associated with systemic inflammation and increased frailty. The mtDNA copy number was also directly correlated with increased frailty but not with inflammation. None of the cf-DNA species were associated with immunosenescence. The transcriptomic pathway analysis revealed that higher levels of total and unmethylated cf-DNAs were associated with immunoinflammatory activation in the nonagenarians but not in the young controls. The plasma mtDNA appeared to be inert in terms of inflammatory activation in both the nonagenarians and young controls. These data demonstrate that the plasma levels of total and unmethylated cf-DNA and the mtDNA copy number could serve as biomarkers of frailty. In addition, we suggest that circulating self-DNA, assessed as total or unmethylated cf-DNA, might aggravate immunoinflammatory reactivity in very old individuals.

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.

The effects of aging and lifelong ethanol consumption on astrocytic morphology and glial fibrillary acidic protein-immunoreactivity (GFAP-IR) in the cerebellar vermis obtained from ethanol-preferring Alko, Alcohol (AA) rats were analyzed by using computer-assisted image analysis. The ethanol-consuming animals (both male and female) were given ethanol (10%–12%, vol./vol.) as the only available fluid for 21 months (3–24 months), whereas the young (3 months) and the old (24 months) controls received water. In the male rats, but not in the female rats, an age-related decrease in GFAP-IR was found in folia II, VII, and X of the molecular layer, and in turn, an age-related increase was found in folium X of the granular layer, indicating opposite changes in GFAP-IR for male rats due to aging in adjacent brain regions. In the female rats, 21 months of daily average ethanol consumption of 6.6 g/kg resulted in decreased GFAP-IR in folium VII of the molecular layer, and the decrease in cerebellar GFAP-IR correlated with the average daily ethanol intake ( r =−.886, P =.019) when folia II, IV, VII, and X were analyzed together. No effect of ethanol on GFAP-IR was detected in the granular layer or in the central white matter of the female rats. There was no change in GFAP-IR in any of the three cerebellar layers of the male rats with average daily ethanol consumption of 3.2 g/kg. These results indicate that the Bergmann glial fibers are the GFAP-expressing structures of the cerebellum most sensitive to moderate-to-heavy chronic ethanol exposure and that this effect is dose dependent.

Background: Binge ethanol exposure is known to induce degeneration of central nervous system (CNS) neurons. Sympathetic hyperactivity has been related to ethanol withdrawal symptoms, but the effects of repeated withdrawals on peripheral sympathetic neurons have not been studied previously. Methods: The effects of continuous versus intermittent ethanol consumption on sympathetic neurons of the superior cervical ganglion (SCG) were studied in male Wistar rats. Two-month-old rats were divided into three groups: one group with ethanol (10% v/v) as the drinking fluid throughout the 5½-month experiment (continuous, n=9), one group drinking ethanol on 4 days/week and water on 3 days/week (intermittent, n=9), and a control group (n=9) with water as the only available fluid. All groups had food ad libitum. SCG volume, neuron density, and total number of neurons were measured by using unbiased morphometric methods. Results: As the mean daily ethanol consumption did not differ between the two ethanol-exposed groups (continuous 5.7 g/kg/day versus intermittent 5.8 g/kg/day), the total dose of ethanol consumed was 42% smaller in the intermittent group. The total number of SCG neurons decreased by 28%, and neuron density by 23%, in the intermittent group compared with the control group, whereas no significant neuron loss was observed in the continuous group. The volume of the SCG was similar in all study groups. The results suggest that repeated ethanol withdrawals, rather than ethanol exposure per se, are deleterious to sympathetic neurons. Conclusions: Ethanol-induced degeneration of neurons is not only related to the amount of ethanol consumed, but also to the patterns of drinking.

In the present study, the neuroprotective effects of dexmedetomidine on rat locus coeruleus were studied during a 5-week intermittent ethanol exposure. Male Wistar rats (3 to 4 months old) were given ethanol or isocaloric sucrose by intragastric intubations three times a day for 4 days, which was followed by a 3-day withdrawal period. This 7-day cycle of ethanol exposure and withdrawal was repeated five times. Dexmedetomidine (at a dose decreasing from 30 microg/kg to 10 microg/kg, s.c.) was given to the treatment group during the withdrawal phase. The results showed that, during the 5-week experiment, dexmedetomidine significantly relieved the ethanol withdrawal syndrome, measured as the sum of the three most specific symptoms (rigidity, tremor, and irritability). The total neuron number of locus coeruleus (LC) decreased in the ethanol-treated group by 24%, compared with the nontreated control group and by 11%, compared with the sucrose-treated control group. Interestingly, the LC neuron numbers were found to decrease in the sucrose-intubated rats as well, compared with the nontreated control group. Dexmedetomidine was found to relieve ethanol-induced neuronal loss in the LC. Dexmedetomidine might be a new interesting alternative in the treatment of ethanol withdrawal syndrome, particularly due to its possible neuroprotective effects in the central nervous system.

The aim of this work was to evaluate the effects of long-term ethanol consumption on arterial responses in vitro in young and aged rats. Therefore, Wistar rats (ages 3 and 29 mo, respectively) were allocated to six groups: control-young, sucrose-young, ethanol-young, control-aged, sucrose-aged, and ethanol-aged. The ethanol-fed groups were given 25% ethanol by intragastric gavage three times a day 4 days a week. Responses of mesenteric arterial rings were examined in standard organ chambers after 5 treatment weeks. In norepinephrine-precontracted arterial rings, endothelium-dependent relaxations to acetylcholine, as well as endothelium-independent relaxations to isoproterenol, were attenuated in aged rats when compared with young controls. Relaxation responses to isoproterenol, but not to acetylcholine and nitroprusside, were clearly improved by ethanol treatment in both young and aged rats. The cyclooxygenase inhibitor diclofenac, which reduces the synthesis of dilating and constricting prostanoids, enhanced the relaxation to acetylcholine in all three aged rat groups but was without significant effect in the young rats. In the presence of the nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester the relaxation to acetylcholine in control and sucrose-fed aged rats was markedly reduced compared with control rats, whereas in the young controls and in both young and aged ethanol-exposed groups, distinct relaxations to higher concentrations of acetylcholine were still present. The endothelium-independent relaxations to cromakalim, a hyperpolarizing vasodilator acting via ATP-sensitive potassium channels, were also markedly augmented by ethanol feeding in both young and aged rats. In conclusion, ethanol consumption in both young and aged rats was associated with markedly improved arterial relaxations to isoproterenol and cromakalim, as well as clearly augmented relaxation to acetylcholine during inhibition of cyclooxygenase and nitric oxide synthase. These findings suggest that especially the potassium channel-related component of arterial relaxation was augmented by long-term ethanol exposure.

1. Age-associated deterioration of arterial function may result from long-lasting oxidative stress. Since coenzyme Q (Q10) has been suggested to protect the vascular endothelium from free radical-induced damage, we investigated the effects of long-term dietary Q10 supplementation on arterial function in senescent Wistar rats. 2. At 16 months of age, 18 rats were divided into two groups. The control group was kept on a standard diet while the other group was supplemented with Q10 (10 mg kg(-1) day(-1)). In addition, nine rats (age 2 months) also ingesting a standard diet were used as the young control group. After 8 study weeks the responses of the mesenteric arterial rings in vitro were examined. 3. Endothelium-independent arterial relaxations to isoprenaline and nitroprusside (SNP) were attenuated in aged rats. Increased dietary Q10 clearly enhanced the relaxation to isoprenaline, but did not affect the response to SNP. In addition, vasodilation of noradrenaline-precontracted rings to acetylcholine (ACh), which was also impaired in aged vessels, was improved after Q10 supplementation. Cyclooxygenase inhibition with diclofenac enhanced the relaxation to ACh only in young rats, while it abolished the difference between the old controls and Q10 supplemented rats, suggesting that the improved endothelium-dependent vasodilation observed in Q10 supplemented rats was largely mediated by prostacyclin (PGI2). 4. In conclusion, long-term Q10 supplementation improved endothelium-dependent vasodilation and enhanced beta-adrenoceptor-mediated arterial relaxation in senescent Wistar rats. The mechanisms underlying the improvement of endothelial function may have included augmented endothelial production of PGI2, increased sensitivity of smooth muscle to PGI2, or both.

Aging and gender have a strong influence on the functional capacity of the immune system. In general, the immune response in females is stronger than that in males, but there is scant information about the effect of aging on the gender difference in the immune response. To address this question, we performed a transcriptomic analysis of peripheral blood mononuclear cells derived from elderly individuals (nonagenarians, n = 146) and young controls (aged 19–30 years, n = 30). When compared to young controls, we found 339 and 248 genes that were differentially expressed (p1.5 or

Interleukin-7 is a homeostatic cytokine that contributes to the maintenance of the T cell pool. It also has proinflammatory effects and is involved in the pathogenesis of autoimmune diseases. Due to its homeostatic effects, IL-7 has been proposed as a potential rejuvenation factor for the elderly immune system. We analyzed the correlation of plasma IL-7 concentrations and the proportions of different T cell populations in nonagenarians (n=163) participating in the Vitality 90+ study. Young individuals (n=35, aged 19-30years) were used as controls. The numbers of CD3+, CD14+, CD4+ and CD8+ cells and the expression of the CD28 costimulatory molecule on CD4+ and CD8+ lymphocyte subsets were analyzed using flow cytometry. The plasma IL-7 levels were significantly higher in the nonagenarians compared to the controls (7.86 vs. 5.74pg/ml, p=0.004). In the nonagenarians, plasma IL-7 levels correlated inversely with the proportion of CD3+ T cells and directly with the proportion of CD14+ monocytes and plasma C-reactive protein. No correlation was observed between plasma IL-7 levels and the proportions of CD4+CD28- or CD8+CD28- subsets. These results suggest that the IL-7 levels in nonagenarians do not have an inhibitory effect on the development of immunosenescence; rather they are associated with increased inflammation.

The effects of aging and chronic ethanol administration on the histochemical and morphometric features of rat superior cervical ganglion were studied in a rat strain selected for voluntary alcohol consumption. Ethanol was administered to the experimental group ad libitum (10% v/v in drinking water) from 3 months to 28 months of age, the average ethanol intake being 6.4–5.4 g/kg per day. The sympathetic neurons of the ethanol consuming rats showed several signs of enhanced degeneration, e.g. decreased neuronal packing density, increased amount of age-pigment and decreased intensity of catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. The results may indicate a selective vulnerability of peripheral sympathetic neurons rather than a universal accelerated aging due to chronic ethanol exposure.

Sympathetic ganglion tissue of young (3 months) and aged (24 months) NMRI mice was allotransplanted into the submandibular gland to study the influence of aging on the survival of grafted neurons. The submandibular gland (SMG) was chosen as a host tissue because of its high concentration of NGF and good blood supply. Four weeks postgrafting the viability of transplants was evaluated using the formaldehyde-induced fluorescence technique, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. The density of neurons, catecholamine fluoresence and TH immunoreactivity (THIR) appeared to be almost unchanged when the transplant was completely surrounded by the SMG tissue, whereas transplants located within the interlobular septum and capsule, or even outside the capsule, showed significantly reduced neuronal survival. The shape of most of the transplanted neurons was not different from those in the intact ganglia. The average diameter of the transplanted young neurons was significantly decreased; this was not the case with the aged neurons. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants in both age groups. The transplants in mice treated with 6-OH-dopamine showed considerable regrowth of adrenergic nerve fibers. There seemed to be no marked difference in the survival of transplanted neurons between aged and young animals. The results indicate that the sympathetic neurons from both young adult and aged animals survive the allotransplantation procedure. The neurotrophic factors together with dense vascularization present in the mouse submandibular gland may be beneficial for the restoration of the integrity of mature and aged adrenergic neurons.

The localization of protein kinase C-beta-like immunoreactivity (PKC-beta-LI) was studied in the rat dorsal root ganglion (DRG) using an antibody specific for a peptide sequence common to the beta 1- and beta 2-subtypes. PKC-beta-LI was seen in 45% of neuronal cell bodies and in nerve fibers, which were mostly myelinated. The PKC-beta-LI-containing cell bodies had a diameter significantly larger than the unlabeled cell bodies. The results suggest that PKC-beta is a PKC subtype involved in cell surface signal transduction in the subpopulation of large DRG neurons.

Indoleamine 2,3-dioxygenase (IDO), an enzyme degrading tryptophan (trp) to kynurenine (kyn), suppresses T cell activity. Ageing of the immune system, immunosenescence, includes a decline in T cell function. We therefore sought to establish whether IDO activity is involved in immunosenescence and whether it predicts mortality in aged subjects. We measured kyn/trp, reflecting IDO activity, in 284 nonagenarians and 309 blood donor controls. IDO activity was significantly higher in nonagenarians compared with controls and IDO activity at study entry predicted subsequent mortality in nonagenarians. Thus, increased IDO activity might be a mechanism involved in the decline of T cell responses in immunosenescence.

The effects of chronic, intermittent ethanol exposure on brain cytochrome c oxidase (CO) activity levels were studied in young (3- to 4-month-old) and aged (29- to 30-month-old) male Wistar rats. The rats were given highly intoxicating doses of ethanol three times a day by intragastric intubation for four successive days, followed by a 3-day ethanol-withdrawal period. This 4-day ethanol-exposure with 3-day ethanol-withdrawal cycle was repeated five times to simulate the binge drinking of human alcoholics. The histochemical demonstration of CO showed a markedly decreased activity level in the medial prefrontal cortex (especially layer V pyramids and neuropil) of the ethanol-exposed rats of both age groups compared with findings for the respective controls. In the cerebellar vermis, CO activity level was decreased in the Purkinje neurons of the aged ethanol-exposed rats and in the granule cells of both young and aged ethanol-exposed rats. The CO activity level in the locus coeruleus was decreased in both young and old ethanol-exposed rats, but the decrease was more pronounced in the young ethanol-exposed group. Aging per se did not markedly change CO histochemical findings in either prefrontal or cerebellar cortex, but CO activity levels were increased in the locus coeruleus. In summary, results of the current study support our conclusion that CO activity levels were decreased in the cerebral and cerebellar cortices as well as in the locus coeruleus—CNS regions known to be negatively affected by chronic ethanol exposure. Defective energy metabolism due to decreased CO activity levels might compromise neuronal energy stores and thereby contribute to ethanol-induced brain dysfunction and irreversible CNS degeneration.

Aims: The number of cerebellar microglia after 5 1 ⁄ 2 months of continuous or intermittent ethanol exposure was studied using the optical dissector method. Methods: Male Wistar rats were divided into three groups: an intermittently ethanol-exposed group, a continuously ethanol-exposed group and a control group ( n = 6 in each group). The intermittently treated rats had two ethanol- withdrawal periods per week throughout the experiment. The number of microglia was measured in the anterior (folium II) and the posterior (folium X) cerebellar vermis. Tomato ( Lycopersicon esculentum) lectin was used to stain the cerebellar microglia. Results: The volumes of folia II and X were similar in all the groups. The number of microglia increased in the molecular layer of foliu m II in the intermittently ethanol-exposed group compared with the continuously exposed and control groups. In the granular layer, ther e were no differences between the groups in the number of microglia. Conclusions: The results suggest that the number of cerebellar microglia increases in the anterior vermis before any ethanol-induced cerebellar atrophy is discernible. Repeated ethanol withdrawals see m to be more essential in inducing microgliosis than ethanol intoxication per se.

Acetaldehyde, the first metabolite of ethanol, has been shown to be capable of binding covalently to liver proteins in vivo, which may be responsible for a variety of toxic effects of ethanol. Acetaldehyde-protein adducts have previously been detected in the liver of patients and experimental animals with alcoholic liver disease. Although a role for acetaldehyde as a possible mediato r of ethanol-induced neurotoxicity has also been previously suggested, the formation of protein-acetaldehyde adducts in brain has not been examined. This study was designed to examine the occurrence of acetaldehyde-protein adducts in rat brain after lifelong ethanol exposure. A total of 27 male rats from the alcohol-preferring (AA) and alcohol-avoiding (ANA) lines were used. Four ANA rats and five AA rats were fed 10-12% (v/v) ethanol for 21 months. Both young (n = 10) and old (n = 8) rats receiving water were used as controls. Samples from frontal cortex, cerebellum and liver were processed for immunohistochemical detection of acetaldehyde adducts. In four (tw o ANA, two AA rats) of the nine ethanol-exposed rats, weak or moderate positive reactions for acetaldehyde adducts could be detec ted both in the frontal cortex and cerebellum, whereas no such immunostaining was found in the remaining five ethanol-treated rats or in the control rats. The positive reaction was localized to the white matter and some large neurons in layers 4 and 5 of the front al cortex, and to the molecular layer of the cerebellum. Interestingly, the strongest positive reactions were found among the ANA rats, wh ich are known to display high acetaldehyde levels during ethanol oxidation. We suggest that acetaldehyde may be involved in ethanol-ind uced neurotoxicity in vivo through formation of adducts with brain proteins and macromolecules.

In this experiment we studied the effects of aging and lifelong ethanol consumption on rat peripheral sympathetic neurons. The aim was to find out the possible differences in the vulnerability to ethanol-induced neuronal degeneration between rats of both genders, or between the alcohol-avoiding (ANA) and the alcohol-preferring (AA) lines of rat. The superior cervical ganglia (SCG) of 40 male and 41 female AA and ANA rats were analyzed. The ethanol-exposed groups had 12% ethanol as the only available fluid from 3 to 24 months of age. The young (3 months) and old (24 months) control groups had water instead. SCG neuronal density, volume, and total neuron number were measured by unbiased morphometric methods. No gender difference was seen in either the volume of the SCG or in the SCG neuron number. The volume of the ganglion was significantly increased with age, but the total neuron number did not change. Neuronal density was significantly decreased with age, but lifelong ethanol consumption induced no further decrease. SCG neuron number in the ethanol-exposed groups did not differ from the age-matched or young control groups, but a significant negative correlation (r = -0.70, p

The effects of lifelong ethanol exposure and aging on the morphology of the locus coeruleus (LC) were studied in the AA (Alko, Alcohol) and ANA (Alko, Nonalcohol) rats of both sexes. The ethanol-consuming (EtOH) rats were given 12% (v/v) ethanol as the only drinking fluid from 4 to 22 months of age, whereas the young (3-month-old) and aged (24-month-old) controls had only water available. The total LC neuron numbers were obtained by using the unbiased disector method. In the AA line, as we have previously reported. the EtOH female and male rats displayed a 26-30% loss of LC neurons compared with the controls. In the ANA line, the EtOH females had 30% fewer LC neurons than the controls (EtOH 1579 +/- 377 vs. controls 2264 +/- 269, ANOVA p0.01), whereas the EtOH males showed no neuron loss compared to the controls (EtOH 1848 +/- 525 vs. controls 2216 +/- 152, ANOVA NS). However, taking into account (sex by line ANCOVA) the markedly higher ethanol intake of the female rats in both lines, no gender or line differences in the ethanol-induced LC degeneration were detected. Neither was there any difference in LC neuron numbers between the young and old control rats of either line of rats. In conclusion, chronic alcohol consumption, not aging per se, damages the LC neurons in experimental animals.

The effects of lifelong ethanol consumption and ageing on the morphology of locus coeruleus (LC) were studied in alcohol-preferring AA (Alko Alcohol) rats of both sexes. Ethanol (12% v/v) was the only available liquid for the ethanol-consuming rats from 3 months up to 24 months of age. Young (3-month-old) and old (24-month-old) control groups were included in the measurements. The LC morphometry was performed by an unbiased disector method. In the old control rats, the total neuron number, neuronal density and the volume of the LC proper did not differ from the young controls. In the ethanol-exposed rats, the total neuron number of the LC was decreased by 30% and the LC neuronal density by 22%, compared to the age-matched controls. No gender difference was found in the vulnerability of LC neurons to ethanol-induced degeneration. The results suggest a remarkable sensitivity of the LC neurons to the ethanol-induced degeneration in both male and female rats. The possible mechanisms and functional implications of this neuronal loss are discussed.

Aging and chronic alcohol consumption can cause degenerative changes in the cerebellar cortex. In this study, the effects of aging and lifelong alcohol consumption on cerebellar cortical layer volumes (molecular and granular) and also white matter layer volumes were studied in alcohol-preferring (AA) and nonpreferring (ANA) rats of both sexes. The ethanol-consuming animals (EtOH) had 12% (w/v) ethanol as the only available fluid from 4 to 22 months of age, whereas the young (3 month) and old controls (24 months) had only water to drink. The volumes of molecular, granular, and white matter layers of the cerebellar vermis in folia II, IV, VII, and X were measured by using systematic sampling and a point-counting method. The volumes of the granular and white matter layers showed consistent increase between 3 and 24 months of age, whereas the volume of the molecular layer remained unchanged with increasing age. Individual ethanol intake was measured over a 1-week period at the beginning and at the end of chronic ethanol exposure. Significant (ANOVA, p = 0.000) sex difference was found in the drinking behavior in both lines, with females consuming more alcohol than males (daily ethanol consumption at 22 months of age 3.2 +/- 0.3 vs. 7.1 +/- 0.3 g/kg for AA males and females; 3.2 +/- 0.3 vs. 5.4 +/- 0.4 g/kg for ANA males and females, respectively). The only ethanol-induced effect on the cerebellum was observed in ANA-EtOH females with a 15% reduction in the volumes of the molecular and granular layer in folium II compared with age-matched controls and a significant (p < 0.05, analysis of covariance with ethanol intake as a covariate) line difference in folium II (molecular and granular layers) was observed between ANA-EtOH females and AA-EtOH females. Furthermore, the volume of the molecular layer in folium II was significantly (p < 0.05, analysis of covariance with ethanol intake and body weights as covariates) reduced for ANA-EtOH females, compared with ANA-EtOH males indicating a sex difference in the cerebellar degeneration due to chronic alcohol consumption. Of the three layers studied, the white matter layer was the most resistant layer to the effects caused by chronic alcohol consumption. In view of the fact that AA and ANA rats of both sexes differ regarding the drinking behavior and ethanol metabolism, they provide an important model for further research on ethanol-induced pathological changes in the central nervous system.

The effects of dexmedetomidine, a selective alpha 2-adrenoceptor agonist, on rat sympathetic neurons were studied during a 12-day, heavy ethanol exposure. Adult male Wistar rats were given ethanol or isocaloric sucrose three times a day by intragastric intubation. Both acute (a single dose of 300 micrograms/kg p.o.) and chronic (100 micrograms/kg x 2 P.O. throughout the experiment) effects of dexmedetomidine were tested. The superior cervical ganglia (SCG) of the ethanol-exposed, non-dexmedetomidine-treated rats showed an abnormally high overall level of tyrosine hydroxylase immunoreactivity (TH-IR) and catecholamine histofluorescence. However, a subpopulation of neurons had apparently lost their catecholamine synthetic activity, as they exhibited no TH-IR or catecholamine fluorescence. The ethanol-exposed ganglia also showed structural alterations (e.g., decreased neuronal size and increased occurrence of vacuolated neurons). In the ethanol-exposed, chronically dexmedetomidine-treated group, by contrast, the SCG exhibited TH-IR and catecholamine fluorescence intensities comparable to those seen in the control ganglia. All the structural parameters studied, as well, were at the control level in the chronically dexmedetomidine-treated group. The single dose of dexmedetomidine offered only marginal protection against the ethanol-induced alterations. These results suggest that chronic dexmedetomidine treatment may prevent ethanol-induced overactivity and degeneration of catecholaminergic neurons.

Age-differences in the sensitivity of peripheral sympathetic neurons to chronic ethanol exposure and ethanol withdrawal were studied in male Wistar rats aged 4 months, 12 months, or 24 to 25 months. The superior cervical ganglia (SCG) of the young (4 months) and the 2-year-old rats responded to a 12-day or 4-week ethanol exposure with significantly increased catecholamine turnover, while the ganglia of the middle-aged rats (12 months) showed only a minor increase in the intensity of catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. Extensive neuronal vacuolation was found in the 4 months ethanol-exposed SCG, probably as a reaction of a subpopulation of neurons to increased stimulation. Ethanol-induced neuronal loss was most prominent in the SCG of the oldest age group. Contrary to the marked changes in SCG functional and morphometric parameters, the pelvic sympathetic neurons in the hypogastric ganglion showed no significant changes after ethanol exposure. The pattern of ethanol-induced morphological alterations found in the present study did not provide unambiquous support for either the "accelerated aging" or the "increased vulnerability" concept regarding ethanol-aging interactions in the nervous system.

The myocardial interactions of ageing and lifelong ethanol ingestion were studied in the ethanol-preferring AA (Alko Alcohol) line of rats. Samples of the left ventricle from young control rats (3-month), old control rats (30-month) and rats exposed to ethanol from 3 to 30 months of age, were studied in terms of myocardial ultrastructure and lipopigmentation. Electron microscopic morphometry showed an age-related increase in the volumetric densities of lipofuscin and unspecified sarcoplasm, while the proportions of mitochondria, myofibrils and tubular structures remained unaltered in the left ventricular myocardium. Lifelong ethanol exposure increased the proportion of sarcoplasmic reticulum and transverse tubules, apparently due to dilation of the tubular structures. Mitochondria were significantly larger in the ethanol-exposed rats compared to the control rats of the same age, while the volumetric proportion of mitochondria tended to decrease in the ethanol-exposed group. Fluorescence microscopic image analysis showed that myocardial lipopigmentation (proportion of myocardial area covered by autofluorescent lipopigrnents) was about two-fold in the old ethanol-exposed rats compared to the old control rats, and 13-fold compared to the young controls. It is concluded that ageing and chronic ethanol ingestion produce rather different patterns of alteration in myocardial ultrastructure, which does not support the concept of ethanol-induced accelerated ageing. The enhancement of myocardial lipofuscin accumulation is thought to reflect chronic oxidative stress in the hearts exposed to ethanol.

The effect of chronic (3-4 weeks), heavy ethanol exposure on neuronal vacuolation in rat peripheral nervous system was studied in male Wistar rats. The rats were force-fed with 25% ethanol 3 times a day, which resulted in blood ethanol levels of 53.1 +/- 18.8 mmol/l, i.e., marked intoxication. In the superior cervical ganglia (SCG), the ethanol exposure increased the proportion of vacuolated neurons c. 13-fold (0.2 +/- 0.0% in the control ganglia, 2.7 +/- 0.6% in the EtOH-ganglia, P < 0.001). A considerable population of vacuolated neurons (VN) was seen in the sensory inferior vagal (nodose) ganglia, and occasional neurons with large cytoplasmic vacuoles in the sensory dorsal root ganglia (DRG) of the EtOH-rats. In the hypogastric ganglia, where VN are regularly found in the adult rat, ethanol exposure did not affect the amount or the appearance of the vacuolated neurons. The number of VN in the SCG decreased significantly between 2 days and 1 week after cessation of the exposure, but did not return to control level by 1 month after ethanol withdrawal. In electron microscopy, most of the vacuolated SCG neurons showed normal ultrastructure, apart from the large cytoplasmic vacuoles. Some vacuolated neurons, however, showed neuropathologic changes, e.g., dilated endoplasmic reticulum, mitochondrial alterations and increased numbers of myelin figures. These degenerative changes were more frequent in the vacuolated DRG neurons than in the sympathetic ones. The occurrence of VN in rat peripheral ganglia may represent a reaction to increased stimulation during prolonged ethanol exposure and, especially, during repeated phases of ethanol withdrawal.

The amount and distribution of myocardial lipoptgments ( ‘age pigments’) were studied in alcoholic and control human hearts, to test the hypothesis of ethanol-induced long- term oxidative damage in myocardium. The amount of myocardial lipopigments was measured by image analysis in six men (age 34–60 years) who had a history of chronic alcohol misuse and who died of acute ethanol intoxication, and in their age-matched, non-alcoholic controls. Lipopigmentation in the intoxication cases was 33.5 ± 2.8% (mean ± SEM) higher compared to the controls in the eight myocardial areas studied ( P < 0.001). A linear correlation of myocardial lipopigmentation with age was noticed in both the intoxication group ( R = 0.894) and the controls ( R = 0.927). The amount of lipopigments varied largely from one myocardial area to another, being highest in the most strained areas (left ventricle, interventricular septum). The accumulation of lipopigments is considered a marker of oxidative stress and ageing in the myocardium. The results support the role of free radical-induced oxidative stress in the pathogenesis of ethanol-induced cardiac abnormalities.

Sympathetic ganglion tissue of aged (36 months) Wistar rats was allotransplanted into the submandibular gland (SMG) of young (3 months) animals to study whether sympathetic neurons can outlive the original host. The visbility of the transplants was evaluated one yeat postgrafting, using the formaldehyde-induced fluorescence technique (FIF) for histochemical demonstration of catecholamines, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. One year after transplantation, grafted superior cervical ganglion (SCG) cells demonstrated catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. The transplants consisted of groups of sympathetic neurons dispersed in a fibrous matrix. After long postgrafting time, the sympathetic neurons of aged rats showed several signs of enhanced degeneration; increased autofluoroscent lipopigment, decreased neuronal density and reduced catecholamine fluorescence. The mean diameter of the transplanted aged neurons was significantly decreased. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants. A subpopulation of small and medium-sized grafted neurons sent out fluorescent fibers, which were located in a fibrous scar area but did not extend into submandibular host tissue. The results indicate that a long postgrafting time induced degeneration which is comparable to normal aging changes in grafted very old neurons. Thus, aged sympathetic neurons maintain plasticity to survive as transplants, and under favourable conditions these neurons outlive the original host.

The purpose of this study was to investigate the viability and ultrastructural characteristics of intraocular superior cervical ganglion (SCG) grafts from young (3 months), aged (24 months) and very old (36 months) rats after short-term (1 month) grafting. The formaldehyde-induced fluorescence (FIF) technique for histochemical demonstration of catecholamines was used to indicate the functionality of transplanted neurons. Ultrastructural changes in grafts were demonstrated by electron microscopy. Four weeks after transplantation, catecholamine histofluorescence in young transplants was almost as strong as in the intact ganglia, while aged and very old grafts showed decreased fluorescence and contained a marked accumulation of autofluorescent lipopigment bodies. Catecholamine histofluorescence showed a decrease in neuronal density of 47%, 59% and 68% in young, aged and very old grafted ganglia, respectively. The shape of most of the transplanted neurons did not differ from that in the intact ganglia, but the average diameter of neurons was decreased after grafting. In electron microscopy, both neurons with normal in vivo fine structure and neurons showing some abnormal cytological alterations were seen in each age group of the transplants. The most prominent feature after grafting was the accumulation of different types of lipopigment bodies in the perikarya of neurons. The organization of the rough endoplasmic reticulum was more irregular in transplanted neurons than in intact neurons. In addition, the amount of neurofilament aggregates increased and some mitochondria were swollen in neurons after transplantation. These results suggest that young sympathetic ganglion tissue survives rather well after transplantation into the anterior eye chamber, while in the aged sympathetic ganglion implants the survival rate is poorer. However, aged and very old SCG grafts were shown to contain and continue to produce noradrenaline, indicating that sympathetic neurons maintain their plasticity and regenerative ability in advanced age. Catecholamine histofluorescence and fine structural changes in the cell structure of grafted sympathetic neurons may indicate an accelerated aging process induced by the transplantation procedure.

The dependence of superior cervical ganglion (SCG) adrenergic neurons on their target organ submandibular salivary gland containing high concentrations of nerve growth factor was studied in adult and aged male mice. The submandibular salivary glands were removed (sialectomy) either uni- or bilaterally, and the SCG were studied by fluorescence microscopy and histochemically. Catecholamine fluorescence and tyrosine hydroxylase immunoreactivity decreased after sialectomy, suggesting reduced noradrenaline production. Neuronal density was lower in the aged controls than in the young controls. In both age groups, sialectomy reduced the density of catecholamine-producing neurons. In the mouse SCG, there was remarkable heterogeneity in the size of neuronal somata. In aged control mice there was a greater number of large-size neurons than in young adult control mice. Six weeks postoperatively, no large catecholamine-producing neurons could be observed in the ganglia. Yellow autofluorescent lipopigments accumulated with age in the adrenergic neurons. Sialectomy increased the accumulation of lipopigments in both young and aged neurons. Sialectomy resulted in (a) reduced catecholamine fluorescence, (b) reduced tyrosine hydroxylase immunoreactivity, (c) reduced number of catecholamine neurons, (d) increased autofluorescent lipopigment. Ageing resulted in (a) reduced number of neurons, (b) increased ratio of large to small neurons, (c) increased autofluorescent lipopigment. Alterations after sialectomy were more detrimental in the aged ganglia than in the young adult ganglia. The discontinuation of the retrograde supply of nerve growth factor may contribute to these alterations.

The localization of PKC-beta was studied in rat sympathetic neurons using a polyclonal antibody specific for the beta 1- and beta 2-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC-beta-LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC-beta-LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC-beta-immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC-beta-LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC-beta in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC-beta in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.

Adrenal medullary tissue was autotransplanted to the superior cervical ganglion (SCG) of aged (26 months old) and young adult (3 months old) rats. Four and 20 weeks after operation, the viability of the transplants was evaluated using the formaldehyde-induced fluorescence (FIF) technique and tyrosine hydroxylase (TH) immunocytochemistry. Four weeks postgrafting, the transplant consisted of a densely-packed group of intensively fluorescent chromaffin cells in both age groups. The cells showed strong TH immunoreactivity and some of them were elongated, but only a few displayed short processes. At 20 weeks, most of the cells were spindle shaped and sent out fluorescent processes and a few of them were transformed toward ganglion-like cells. The results suggest that both young and old adrenal chromaffin cells are able to survive, produce neuronal processes and transform toward a neuronal phenotype in the rat superior cervical ganglion.

The accumulation of lipopigments during aging in several peripheral organs and in the nervous system is considered to be related to the peroxidation of unsaturated fatty acids. In this study the effect of lifelong (until to 18 months) dietary antioxidants selenium and vitamin-E on pigment accumulation in some peripheral tissues was estimated using fluorescence and electron microscopy. In the vitamin E deficiency group, there was increased pigment accumulation in all peripheral tissues studied except the hypogastric ganglion, where no change was observed. The vitamin E supplementation degreased the pigment accumulation in older animals in some of the tissues studied. At the electron microscopical level the accumulated pigment in the adrenal cortex showed a lipofuscin-like structure. Lifelong selenium supplementation or deficiency did not significantly alter pigment accumulation in any of the tissues studied. It is possible that in many organs dietary selenium may not play a critical role in lipofuscin formation.

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.

The expression of c-fos protein in cultured human glial cells derived from the brain and spinal cord was investigated immunocytochemically. Primary cultures of fetal glial cells were maintained in culture for three weeks and deprived of animal sera for 22 h. The glial cell nature of the cells was ascertained by GFAP-immunoreactivity. Incubations with phorbol dibutyrate, 8-Br-cAMP and sodium nitroprusside representing signal transduction pathways of PKC, PKA and cyclic GMP kinase, respectively, were carried out for 60 and 120 min. The control serum-deprived cultures did not display c-fos protein immunoreactivity (c-fos-IR), whereas phorbol dibutyrate incubation for 120 min induced strong c-fos-IR in the nuclei of both brain and spinal cord derived glial cells. Semiquantitative intensity measurements revealed a slight c-fos-IR induction after 8-Br-cAMP as well, but not after sodium nitroprusside. The observations suggest that c-fos protein is involved in PKC and PKA signal transduction in cultured human glial cells.

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 distribution of neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the rat superior cervical and hypogastric ganglia. NPY-LI was localized in the majority of the sympathetic neurons, a few small granule-containing (SGC) cells and nerve terminals. Most of the NPY-immunoreactive sympathetic neurons were also tyrosine hydroxylase (TH)-immunoreactive but in hypogastric ganglia few neurons with NPY-LI were devoid of TH-immunoreactivity. Electron microscopically NPY-LI was found in the Golgi complexes of sympathetic neurons, in large cytoplasmic granules (100–150 nm in diameter) of the SGC cells and in large dense-cored vesicles (80–100 nm in diameter) of the nerve terminals. NPY-LI coexists mainly with noradrenaline in sympathetic neurons, and may have regulatory functions in sympathetic ganglia and in target organs.

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.

The age-related changes in lipopigment autofluorescence were studied by micro-spectrofluorometry in three different types of human neurons: the sympathetic neurons of the stellate and superior mesenteric ganglion and pyramidal neurons of the frontal cortex. The age-related increase in lipopigment autofluorescence was more rapid in stellate ganglion but similar linear increases were found also in superior mesenteric ganglion and frontal cortex. There was an age-related shift in the autofluorescence from yellow to orange in the ganglia. This may be due to the accumulation of neuromelanin in noradrenergic neurons. Lipopigments were identified in sympathetic neurons at the age of 4 months and all neurons carried pigment granules after the age of 64 years. It is concluded that lipopigment autofluorescence is a useful marker for cellular ageing in both the peripheral and the central nervous system.

The effect of immobilization stress on the expression of c-fos protein in the adrenal cortex of adult rats was investigated immunocytochemically. After immobilization stress lasting for longer than 30 min, an enhanced c-fos-like immunoreactivity was observed in the cortical cells of the zona fasciculata and zona reticulata. Compared to unstressed controls, an about 5-fold increase in the density of the immunoreactive cells in a unit of the cortical area was seen following a 1-h immobilization. The enhanced immunoreactivity lasted for at least 3 h after 1-h immobilization and it began to diminish 5 h after the stress. Furthermore, administration of dexamethasone 2 h prior to 1-h immobilization attenuated the stress-enhanced immunostaining for the c-fos-like protein. These results suggest that an acute stress may cause a dramatic and long-persisting induction of c-fos-like protein in the cortical cells of rat adrenals. The characteristic zonal distribution of the c-fos induction in rat adrenals as well as the effect of dexamethasone suggest involvement of the pituitary adrenocorticotropic hormone (ACTH) in the induction.

Neurotensin-like immunoreactivity was localized in nerve fibers and terminals of hamster adrenal medulla at light and electron microscopy using the peroxidase-antiperoxidase method. Numerous varicose neurotensin-immunoreactive nerves and terminals were found among nonlabeled cell groups situated peripherally in the adrenal medulla. Combined formaldehyde-glutaraldehyde (Faglu) fluorescence and immunohistochemistry of the same vibratome section showed that only norepinephrine cells were innervated by neurotensin-immunoreactive nerves. All norepinephrine cells seemed to be innervated by neurotensin-immunoreactive nerves. Neurotensin-immunoreactive nerves disappeared after extrinsic denervation of the adrenal gland. By electron microscopy numerous neurotensin-immunoreactive terminals were seen to make synaptic contacts with norepinephrine cells and with autonomic ganglion cells present in small numbers among norepinephrine cells. In the terminals neurotensin-like immunoreactivity was localized mainly in large dense-cored vesicles, but some precipitates were also associated with small vesicles, diffusely scattered in the axoplasm. The present findings suggest that in the hamster adrenal medulla part of the nerve terminals arising from splanchnic nerves contain neurotensin-like peptide. The functional significance of these nerves in the hamster adrenal medulla remains to be elucidated.

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

Cervical sympathetic ganglia from nine human fetuses in the second trimester were studied by light-, fluorescence- and electronmicroscopy. Within ganglia granule-containing cells, corresponding to the small intensely fluorescent (SIF) cells, were sparsely distributed along capillaries. Satellite cells were interspersed among neuronal cell types. All stages of the developing sympathetic ganglion cells were present. Primitive sympathetic ganglion cells (PSC) had dark nuclei and scanty cytoplasm in which the rough endoplasmic reticulum (RER) was not organized. These cells often contacted each other vs the more mature pattern of satellite cell support. Neuroblasts had more voluminous cytoplasm, RER was more apparent than in the PSCs and dense-cored vesicles and mitochondria were more frequently seen. Their diameter was between 5 and 20 μm. The most mature neuronal cell type, the young sympathetic ganglion cell, was often covered by satellite cells, but synaptic contacts often appeared on the somata. Maturity of the cells was underscored by an abundant RER and ribosomes organized as Nissl bodies. Indeed all the organelles typical of an adult ganglion cell were present and fully developed, although in smaller amounts. However, the diameter (20–30 μm) of the cells was smaller than in adult ganglia. All ganglia examined possessed mature axodendritic and axosomatic synapses. Large dense-cored vesicles were found in some neuroblasts and in young sympathetic ganglion cells. However, neither cytoplasmic nor synaptic small dense-cored vesicles were seen. These morphological results correlate with pharmacological and immunohistochemical data on human fetal tissue which show that both cholinergic and adrenergic receptors, catecholamine-synthesizing enzymes and enkephalin are developed by the end of the first trimester.

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.

The effect of daily physical training for 24 months on the sympathetic neurons, adrenal gland, extra-adrenal catecholamine storing cells and on the heart was investigated in rats. The tissue catecholamine fluorescence intensity was determined by microfluorimetric quantitation of catecholamines. The maximal and final body weights were significantly lower in trained animals. The trained rats showed prominent increase of heart weight relative to body weight, while the adrenals did not enlarge. The adrenergic nerve fiber density of the heart and the fluorescence intensity of the terminal axons were significantly increased. There were no changes in the fluorescence intensity of the perikarya of the sympathetic neurons and the amount of extra-adrenal catecholamine storing cells after physical exercise. The volume of the superior cervical ganglion was doubled and the neuronal perikarya were enlarged in trained animals. The prolonged physical training throughout the life span of the rat gave new information about the reactions of the sympathetic nervous system to physical exercise.

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.

Choline acetyltransferase (ChAT) activity in the cerebral cortex and the different solubility fractions of acetylcholinesterase (AChE) in the cerebral cortex and cerebellum were investigated in rats of different ages. ChAT activity was not decreased markedly in the cerebral cortex of 24- to 25-month-old rats compared to 3- to 4-month-old rats. The activity of detergent-soluble (DS) AChE in the cerebral cortex and cerebellum was lower in the older rats (24–25 months) rats than in younger (3–4 months) ones. The activity of DS-AChE in the cerebral cortex and cerebellum did not differ between 10- to 11-month-old and 24- to 25-month-old rats. The activity of low salt soluble (LSS) AChE in the cerebral cortex and cerebellum did not differ between older and younger rats.

There is recent evidence that the amine storing cells of mammalian adrenal medulla also contain bioactive peptides. In the present study we examined human adrenal glands with the immunoperoxidase-bridge method using specific antisera raised against [Met 5 ]- and [Leu 5 ]-enkephalin, vasoactive intestinal polypeptide hormone (VIP), and substance P. Approximately one-third of the cells in the adrenal medulla demonstrated enkephalin-like immunoreactivity. The intensity of the immunostain varied among individual cells but did not appear to correlate with amine content, as determined by the formaldehyde-induced fluorescence of catecholamines. An abundant network of varicose fibre-like structures and dots, representing preterminal and terminal nerves, demonstrated vasoactive intestinal polypeptide-like immunoreactivity and were found in close proximity to medullary gland cells. Substance P-like immunoreactivity was observed in a few fibres in the medulla and cortex. However, we could not detect cells containing vasoactive intestinal polypeptide- or substance P-like immunoreactivity in adrenal glands. p ]The present findings suggest that human adrenal medullary cells contain both [Met 5 ]- and [Leu 5 ]-enkephalin and are richly innervated by peptidergic nerves containing vasoactive intestinal polypeptide. These peptides may modulate the release and effects of catecholamines in the adrenal medulla. The nerves with substance P-like immunoreactivity may represent a separate peptidergic neuronal system.