Age-related changes in the synapses of the rat's neostriatum.

By means of transmission electron microscopy, the age-related changes in axospinous (ASS) and axodendritic (ADS) synapses in the dorsal part of the rostral neostriatum in two groups of Wistar rats: young (3-month-old), and senescent (25-month-old) were examined. The changes in different parameters, characterizing the ASS and ADS: synaptic density (SD), number of synaptic vesicles (SV), number of synaptic contact zone (SCZ), and number of dendritic spines, bearing synapses (DS) were investigated morphometrically. The SD of the ASS decreased significantly during aging, but the SD of the ADS did not changed significantly. The mean area of the synaptic boutons increased significantly during aging in two types of synapses. The mean number of vesicles per synaptic bouton increased, but the number of vesicles per microm2 of synaptic bouton, and per microm3 of the neuropil decreased. The mean SCZ length increased in both types of synapses. The total SCZ length per 1000 microm2 of the neuropil, and the total area of the SCZ per 1000 microm3 of the neuropil decreased in ASS, but the same parameters of the ADS did not changed significantly. The mean number of synaptic DS per 1000 microm2 of the neropil decreased during aging, but the mean area of the synaptic DS increased. The present results support the hypothesis that the synaptic contacts change significantly during aging, and the ASS are more vulnerable during aging than the ADS.

Age-related changes in the NADPH-diaphorase-positive dendrites in the dorsolateral neuronal column of the periaqueductal gray in rat.

The effects of age on the dendritic tree of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in the dorsolateral neuronal column of the periaqueductal gray (DLPAG) in the rat were examined, using NADPH-d histochemistry and computer-assisted analysis. In 26-month-old rats some of the neurons exhibited considerably branched dendrites, while others show amputation stumps, or local swellings in the initial dendritic portion. Quantitative analysis demonstrated that the number of dendritic segments in DLPAG increased with advancing age. The mean segment length and the mean length of the terminal segment significantly increased in 12-month-old rats compared to 3-month-old rats and decreased in 26-month-old rats in comparison to 12-month-old, as the reduction was significant for the terminal segments only. The total dendritic length of NADPH-d-stained neurons increased with aging. The increase was more pronounced in 12-month-old rats compared to 3-month-old rats and the difference was significant in 26-month-old rats. The number of the nodal vertices, Va and Vb, significantly increased between 12- and 26-month of age. The dendritic reorganization was considered a sign of compensatory mechanism counteracting the degenerative changes.

Age-related changes in the NADPH-diaphorase-positive neuronal perikarya of the dorsolateral column of the periaqueductal gray in the rat.

The distribution of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) positive neuronal perikarya in the rostral, middle and caudal parts of the dorsolateral periaqueductal gray (DLPAG) in 3-, 12- and 26-month-old rats was compared by means of histochemistry and computer assisted image analysis. The total number, the maximum diameter, the cross-sectional area and the optical density (OD) of the NADPH-d positive neurons were analyzed. The results demonstrate that there are no significant differences in any of the investigated parameters between the left and the right parts of the same age and of the same level. The total cell number in the DLPAG of 26-month-old rats was significantly decreased in comparison with 3- and 12-month-old rats. The cross-sectional area increased between 3 and 12 months of age, and decreased in 26-month-old rats in comparison with 12-month-old rats. The OD increased significantly between 3 and 12 months of age, and decreased significantly between 12 and 26 months of age only at caudal level. The observed mild changes could lead to alterations in the brain physiology of aging.

Contribution of nitric oxide and substance P to nonadrenergic, noncholinergic transmission in the guinea pig ileum.

1. The possible contribution of the nonadrenergic noncholinergic (NANC) transmitters nitric oxide (NO) and substance P (SP) to the contractility of guinea pig isolated ileum was studied by the responses of the longitudinal muscle to electrical field stimulation (0.8 msec, 40 V, 1-20 Hz, 20 sec) of the intrinsic nerves and by the presence and distribution of NADPH-diaphorase- and SP-positive nerve structures in the myenteric plexus. 2. The electrically elicited, tetrodotoxin (0.3 microM)-sensitive responses, in the presence of phentolamine (5 microM), propranol (5 microM), and atropine (3 microM) consisted of relaxation, followed by twitch and tonic contraction on which phasic contractions were superimposed. 3. NG-nitro-L-arginine (L-NNA; 0.1 mM or 0.5 mM), an inhibitor of NO synthesis abolished the relaxation. L-arginine (0.1 mM), a substrate for NO synthesis, but not D-arginine, restored it. L-NNA concentration dependently increased the twitch and tonic contractions. Sodium nitroprusside (1 microM or 10 (M), an exogenous donor of NO, was without effect on the electrically evoked responses. 4. AP 13.2 ACOH (AP; 0.1 microM or 10 microM), a blocker of SP receptors, frequency dependently inhibited or even prevented the twitch and tonic contractions. AP concentration-dependently increased the relaxation or reversed the responses to electrical stimulation into a deep relaxation. 5. The concentration-response curve for SP (1 nM-0.1 microM) was shifted to the right by AP, the EC50 values being 5.2 +/- 0.4 nM and 88.0 +/- 3.0 nM, respectively. The effects of SP were not altered by L-NNA (0.1 mM). 6. These findings, supported by morphological data about distribution of NADPH-diaphorase-positive nerve cell bodies and processes and SP-positive varicose fibers, suggest the contribution of NO and SP to NANC transmission. It appears that NO inhibits prejunctionally the SP transmission whereas SP counteracts the NO effect at the postjunctional level.

Histological and immunocytochemical data on the differentiation of intestinal endocrine cells in human fetus.

The differentiation of the argyrophil, argentaffin, 5-hydroxytryptamine (5-HT)-, somatostatin (SOM)-, cholecystokinin (CCK)-, substance P (SP)-, methionine-enkephalin (Met-Enk)- and vasoactive intestinal polypeptide (VIP)-immunoreactive entero-endocrine cells (EECs) was examined in human fetuses. A great increase in the frequency of EECs in the duodenum and the rectum was observed between the 7th and 12th gestation week. The differentiation of the EECs advanced distally in the small intestine and proximally in the large intestine. In 24-25-week-old fetuses the frequency of the EECs was also increased in the ileum and the colon. A different time-course of the appearance and differentiation of the types of EECs was observed. Met-Enk- and VIP-immunoreactive endocrine cells were not detected at any age. A regional difference in the frequency and morphology of the endocrine cell types was observed in the eldest fetuses.

Aging and the dendritic morphology of the rat laterodorsal and pedunculopontine tegmental nuclei.

The morphological appearance and quantitative parameters characterizing the dendrites of NADPH-diaphorase-stained neurons in the laterodorsal (LDT) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats were studied. All dendritic segments were classified according to the number of terminal and link segments they drain and the vertex analysis was used to quantify the dendritic tree and to determine its configuration. Morphological aberrations of the dendrites as local swelling, nodulation, thinning, shrinkage, folding and even the appearance of stumps were observed with advancing age. The quantitative analysis demonstrated a significant reduction (one-way ANOVA) of the total dendritic length, mean terminal path length, maximal segment length, total segment number and number of terminal segments at the rostral two thirds of the LDT and in the PPN. The mean vertex path length and the mean segment length significantly decreased only at the rostral level of the LDT. Plotting of the segment length against equivalent orders showed a decrease in all generations of the dendritic segments. The vertex ratios remained constant and indicated that the configuration of the dendritic tree remained unchanged during aging. The alterations in the dendrites mainly developed after 12 months of age. The age-related changes in the morphology and quantitative parameters of the dendrites in the rostral two thirds of the LDT and PPN were rather similar, which could be explained by the common anatomical, neurochemical and electrophysiological features. Thus, the present results suggest a mild, but continuous regression of the dendritic tree of the rat LDT and PPN in normal aging.

Changes in NADPH-diaphorase neurons of the rat laterodorsal and pedunculopontine tegmental nuclei in aging.

The aim of the present study was to compare the morphological pattern and the quantitative parameters of nitric oxide (NO)-containing neurons in the laterodorsal (LTD) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats. NADPH-diaphorase (NADPH-d) histochemical reaction, as a marker of the cholinergic neurons in the two mesopontine nuclei, and computer-assisted image analysis were used. The relationships between the neurons stained for NADPH-d and choline acetyltransferase (ChAT) were examined using a double-labelling procedure. The results demonstrated only occasional ChAT positive somata that did not exhibit NADPH-d staining. The volume of the LTD and PPN and the number of NADPH-d neurons remained unaltered with advancing age. However, ANOVA demonstrated a significant effect of age and level on the cross-sectional areas, maximum diameters and staining intensity of NADPH-d somata in the LTD and PPN. The three parameters were reduced in 26-month-old rats compared to 3-month-old rats. The changes in the morphological appearance of NADPH-d somata and processes as well as the quantitative analysis pointed to age-related neuronal atrophy. It was accompanied by hypertrophy of some neighbouring neurons, suggesting a compensatory mechanism which would counteract the degenerative changes. The age-dependent alterations in the LTD and PPN were rather similar.

Vasopressin- and oxytocin-immunoreactive nerve cells in the aging rat hypothalamus.

Immunohistochemistry and morphometry were used to study the age-related changes in the vasopressin (AVP) and oxytocin (OXT) nerve cells in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic (SCN) nuclei of 3-, 11- and 28-month-old rats. The results showed a statistically significant reduction in the mean number of AVP cells in the PVN, SON and SCN, and of OXT cells in the PVN with advancing age. Different age-related changes in the mean size of the immunoreactive cells were found in the three nuclei: a significant and transitory increase in the AVP and OXT cell sizes in the PVN, a tendency towards increasing the AVP and OXT cell sizes in the SON, and a significant and gradual decrease in the AVP cell size in the SCN. The combination of the morphometric data and staining patterns of the AVP and OXT perikarya and fibers in the PVN and SON pointed to an increased transport of AVP and OXT in 11-month-old rats as well as to a decreased production of these peptides in the PVN of 28-month-old rats. Taken together the staining pattern and the morphometric results showed a progressive loss of AVP cells in the SCN in aging.

Morphological evidence for effects of the aging on the serotoninergic neurons in the rat brainstem nuclei.

The effects of age on the 5-hydroxytryptamine-immunoreactive (5-HT-IR) neurons in raphe and extrarapheal nuclei of rats were examined using morphometry. Quantitative parameters of the neuronal density and cross-sectional areas of the cell bodies as well as of the length and numbers of the processes of the 5-HT-IR neurons were analyzed in 3-, 12- and 26-month-old-rats. The results showed that aging affected the 5-HT-IR neurons in the rat brainstem nuclei. The age-related changes in the immunostaining pattern and quantitative parameters were more or less pronounced and earlier or later developed depending on the nucleus studied. It is most probably connected with specific functions and projections of each raphe nucleus.

Age-related changes in basic fibroblast growth factor-immunoreactive cells of rat substantia nigra.

Immunohistochemistry and computer-assisted image analysis were used to examine the age-related changes in bFGF-immunoreactivity in rat substantia nigra (SN). Distribution pattern, number, size and staining intensity of bFGF-immunoreactive (bFGF-ir) cells in pars compacta and pars reticulata of 3-, 12- and 26-month-old rats were compared. The overall distribution of bFGF-immunoreactivity was similar in the three age groups, but changes in the morphological appearance of bFGF-ir somata and processes occurred in aging. The results demonstrated a significantly reduced number of bFGF-ir cells in pars compacta (by 56.87%) and pars reticulata (by 30.4%) in 26-month-old rats compared to 3-month-old rats. The reduction of the cell number did not occur smoothly and equally in the two parts of SN. The quantitative analysis clearly indicated a significant decrease in the size of bFGF-ir neurons in pars compacta (by 18.1%) and pars reticulata (by 14.15%) of 26-month-old rats compared to 3-month-old rats. Compared to 3-month-old rats, a 19.77% and 17.83% increase in the staining intensity was observed in the remaining bFGF-ir neurons of pars compacta and pars reticulata, respectively in 26-month-old rats. Since there was no correlation between the decreased size and increased staining, it is most probable that the intensification of the staining intensity of bFGF-ir neurons was a compensatory response to the cell death.

Age-related morphological and morphometrical changes in parvalbumin- and calbindin-immunoreactive neurons in the rat hippocampal formation.

Parvalbumin (PV)- and calbindin (CaBP)-immunostaining in the hippocampal formation of 3-, 11- and 28-month-old Wistar rats was studied using monoclonal antibodies. A quantitative analysis of the densities, cross-sectional areas, length and number of processes of PV-immunoreactive neurons in the hippocampal dentata and CA1 areas of the three age groups was employed. Marked age-related changes in the morphological appearance and in the quantitative parameters characterizing the PV-immunoreactive neurons in both hippocampal regions were observed. The intensity of CaBP-immunostaining of the hippocampal principle cells and interneurons remained the same but the immunoreactive fibers were structurally altered in aging.

Age-related changes in cholinergic and noradrenergic transmission in the rat cerebellum. A histochemical and immunocytochemical study.

The histochemical and immunocytochemical distribution of some cholinergic and noradrenergic markers was compared in the cerebellum of young adult (3-month old) and aged (24-month old) Wistar rats. A decrease in the density and staining of acetyl cholinesterase (AChE) positive fibers, puncta and Golgi cells was found in both the cerebellar cortex and nuclei of aged rats. The age-related changes in choline acetyltransferase immunoreactivity were less pronounced than the changes in AChE activity. A reduction in the density of catecholamine fluorescent fibers and puncta was observed in the cerebellar cortex during aging. In aged rats the increase in monoamine oxidase (MAO)-A activity was more pronounced than the increase in MAO-B activity.

Age-related changes in serotonin-immunoreactive neurons in the rat nucleus raphe dorsalis and nucleus centralis superior: a light microscope study.

Comparison of serotonin-immunoreactive (SER-IR) neurons in the nucleus raphe dorsalis (NRD) and the nucleus centralis superior (NCS) of 3-month-old and 28-month-old rats was made using qualitative and quantitative immunohistochemical analysis. Significant age-related changes in size and density of the SER-IR somata as well as in the length and number of their processes were demonstrated. A different vulnerability of the SER-IR neurons in both raphe nuclei to aging was observed that may be related to their different structural and functional features.

Age-related changes in serotonin-immunoreactivity in the telencephalon and diencephalon of rats.

Comparison of the appearance and density of the serotoninergic fibers and terminals in some tel- and diencephalon areas of young adult (3 months) and aged (28 months) rats was made immunohistochemically using an antibody to serotonin. In young adult rats a characteristic arrangement of the serotonin-immunoreactive (SER-IR) fibers and terminals in the tel- and diencephalon was observed. In aged rats the distribution pattern was the same but changes in the appearance and density of the SER-IR fibers were demonstrated. Many swollen and folded fibers forming cluster-like structures as well as reduced fiber networks were seen in the neocortex, hippocampal formation and striatum of aged rats. Aberrant fibers and decreased fiber density were also observed in the bed nucleus of the stria terminalis, lateral septum, nucleus accumbens and thalamic nuclei. Reduced fine granular immunostaining and scattered swollen varicosities were found in some amygdaloid and hypothalamic nuclei of aged rats. These results provide further morphological evidence for changes in the serotoninergic innervation during aging.

Changes in GABA-immunoreactivity and GABA-transaminase activity in rat amygdaloid complex in aging.

The distribution of gamma-aminobutyric acid (GABA), gamma-aminobutyric acid-transaminase (GABA-T) and glial fibrillary acid protein (GFAP) in the amygdaloid complex of young adult (3-month old) and aged (26-month old) Wistar rats was compared by using histo- and immunohistochemical techniques. The study revealed a heterogeneous distribution of GABA-immunoreactive and GABA-T positive nerve cells, fibers and terminals in various amygdaloid nuclei of young adult rats. A significant reduction in the density of GABA-immunoreactive neurons was demonstrated in the amygdala of aged rats. Reduced density and staining of GABA- and GABA-T-positive fibers and terminals were also seen in aged rat amygdala. Heavily GABA- and GABA-T-stained aberrant fibers and coarse puncta were clustered around blood vessels as well as under the pial and ependymal surfaces. GFAP-immunostaining demonstrated numerous hypertrophic astrocytes in these areas.

Qualitative and quantitative glial changes in the hippocampus of aged rats.

Ultrastructure of the glial cells in the CA 1 hippocampal field and some quantitative parameters characterizing the astrocytes in stratum lacunosum-moleculare (str. L-M) of the CA 1 hippocampal field of young-mature (3-month old) and aged (24-month old) rats were examined. The qualitative observations showed structural changes in astrocytes, oligodendrocytes and microglial cells in all layers of the CA1 hippocampal field in aged rats. The quantitative analysis demonstrated a considerable increase in total and individual volume fractions of astrocyte processes, in number of astrocyte profiles per square area of neuropil and in percentage area of neuropil occupied by astrocyte process profiles. These changes as well as the increased number of astrocyte nuclei suggest the appearance of hypertrophy and hyperplasie of astrocytes in str. L-M of the CA 1 hippocampal field of rats in aging.

Immuno- and histochemical data on changed GABA transmission in aged rat cerebellum.

The distribution of gamma-aminobutyric acid-immunoreactivity (GABA-I) and activity of GABA-transaminase (GABA-T) was compared in the cerebellum of young adult (3-month old) and aged (24-month old) Wistar rats. A decrease in the density and staining of GABA-immunoreactive and GABA-T positive nerve cells, fibers and puncta were observed in both cerebellar cortex and nuclei of aged rats as compared to those in young adult rats. In contrast, an increased number of Bergmann glia cell and astrocyte processes and bodies in aged rats were demonstrated in GABA immunostaining and histochemical GABA-T reaction. The changes in GABA-I and GABA-T activity provide evidence for altered GABA transmission during normal aging of rat cerebellum.

Changes in the synapses in the parietal cortex of senescent rats.

Quantitative parameters characterizing the density of the synapses, the pre- and postsynaptic elements, and the synaptic contact zones (SCZ) in the parietal cortex of young adult (3-month-old) and aged rats (24 months) were investigated using an automated computer system. A big loss of axospinous, but not of axodendritic synapses, was observed in the aged rats. The parameters for the synaptic vesicles and SCZ were changed also only in the axospinous synapses. The changes in the quantitative parameters are accompanied by changes in the structure of synapses. The structural changes may be considered as being degenerative and regenerative.

Age-related abnormalities of oxidoreductase activity and mitochondria in rat hippocampal formation.

The activity of oxidoreductases in the hippocampal formation of young adult (3-month old) and aged (24-month old) Wistar rats was compared by histochemical methods. A decreased activity of NADH-diaphorase and dehydrogenases involved in the Krebs cycle and glycolysis as well as an increased activity of glucose-6-phosphate dehydrogenase were demonstrated in the hippocampal nerve cells of aged rats. The activity of oxidoreductases in the glial cells of aged rats was increased. Degenerative changes in scattered mitochondria were seen ultrastructurally. No significant differences in the relative volume fraction of mitochondria in the presynaptic axon terminals and dendrites were found between young adult and aged rats.

Histo- and immunohistochemical changes in acetylcholinesterase and choline acetyltransferase activities in the amygdaloid complex in aged rats.

Histo- and immunohistochemical distribution of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the amygdaloid nuclei of young adult (3 month old) and aged (26 month old) Wistar rats was compared. AChE staining and ChAT immunoreactivity showed the same regional variations in the amygdaloid nuclei of young adult rats. The density and staining of AChE- and ChAT-positive fibres, terminals, and nerve cells were reduced in aged rat amygdala. Moreover, heavily stained aberrant fibres and coarse terminals were located around the nerve cells, blood vessels, and occasionally in patches. In aged rats, atrophic AChE positive and ChAT immunoreactive nerve cells exhibited serpentine-like, thicker, and less extensively branched dendrites than those in young adult rats. These changes are similar to the age-related changes in the cholinergic enzymes in other brain regions which are targets to the basal forebrain.