Interaction of norepinephrine with Purkinje cell responses to cerebellar afferent inputs in aged rats

Age-related differences in the modulatory actions of NE on the evoked activity of cerebellar Purkinje cells were examined in young (3 month) and old (18-20 month) Fischer 344 rats. We have previously shown that NE is more potent in young than in old rats, in terms of its ability to inhibit spontaneous activity. In this investigation complex spike excitation, simple spike excitation, and inhibition of Purkinje cell discharge were elicited by stimulation of climbing fibers, mossy fibers, and cerebellar parallel fibers, and quantified by computing post-stimulus time histograms of the neuronal response, recorded extracellularly. Histograms were compared before, during and after local ejection of NE from multibarreled micropipettes. In young rats NE preferentially inhibits spontaneous discharge more than evoked excitations. The inhibitory response of the Purkinje cell to activation of basket and stellate cell afferents is potentiated by NE with respect to the inhibition of spontaneous discharge. In old rats the NE-induced potentiation of both excitatory and inhibitory responses was significantly diminished. The loss of noradrenergic enhancement of the relative responsiveness of Purkinje neurons to afferent inputs in senescent animals may relate to behavioral deficits seen in aging.     

Decreased sympathetic innervation of spleen in aged Fischer 344 rats

Splenic noradrenergic innervation in young adult and aged Fischer 344 rats was examined using fluorescence histochemistry for catecholamines and high performance liquid chromatography with electrochemical detection (LCEC) for the quantitation of norepinephrine (NE). In young adult rats, abundant noradrenergic plexuses followed the vasculature and trabeculae into splenic white pulp. In aged rats, noradrenergic innervation was reduced in density and in overall intensity of fluorescence, and splenic NE levels were significantly lower. The relationship between diminished noradrenergic innervation and diminished immune responsiveness in aging mammals, while not clear on a causal level, is presented as a hypothesis for further testing.     

Age-induced changes in single locus coeruleus brain transplants grown in oculo: an in vivo electrochemical study.

Brain stem tissue from fetal Sprague-Dawley rats containing the nucleus locus coeruleus (LC) was transplanted into the anterior chamber of the eye of young adult host rats and was studied at 4-6 months (young control) or 24-28 months after grafting (old). High-speed in vivo electrochemical measurements were used to characterize the potassium-evoked synaptic overflow of norepinephrine (NE) in both young and aged LC brain grafts. The amplitudes of potassium-evoked NE overflow were attenuated in the aged grafts as compared to the young LC grafts. In addition, the rise times of potassium-evoked responses were longer in the old LC grafts than in the young transplants. In contrast, the NE content of aged LC grafts, as determined by high-performance liquid chromatography coupled with electrochemical detection (HPLC-EC), was only slightly diminished and not significantly different from the NE levels seen in young LC grafts. However, light microscopical evaluation using tyrosine-hydroxylase immunocytochemistry revealed pyknotic cell bodies and fluorescent accumulations in aged locus coeruleus transplants which were indicative of degeneration in these grafts. The present data demonstrate a significant age-related decline in the presynaptic function of NE-containing neurons in intraocular locus coeruleus transplants of Sprague-Dawley rats.     

Roles of A- and D-type K channels in EPSP integration at a model dendrite

We examined whether a 2-week treatment with the spin-trapping agent MDL 101.002 (MDL) or a diet supplemented with vitamin E (Vit E) would alleviate age-related deficits in cerebellar noradrenergic function of male 18–20 month old F344 rats compared to age-matched controls. Cerebellar β-adrenergic receptor function was assessed using extracellular recordings of Purkinje cells during iontophoresis of GABA and isoproterenol (ISO). Noradrenergic receptor function of MDL and Vit E-treated rats was similar to young rats whereas for non-treated rats it was typical of that previously recorded in aged rats. Thus, treatment with MDL or Vit E reverses age-related deficits in cerebellar noradrenergic receptor function.     

Neuromodulation of lymphocyte reactivity in aged rats

Numerous reports indicate that both central nervous system (CNS) and immune system functions decline with age. We have previously shown that the CNS can modulate both mitogen-induced spleen cell proliferation and NK activity in young Fischer 344 rats. In the present study we have determined the effects of AHT lesions on the lymphocyte reactivity of aged Fischer 344 rats. These data show that lesions in the AHT of aged rats cannot modulate splenic mitogen responsiveness, however, NK activity is impaired. This differential effect may be due to multiple factors including enhanced splenic suppressor cell activity, the inability of the brain to send modulatory signals following lesioning, or the failure of the immune system to receive a neural signal and react to it.     

Neurochemical, endocrine and immunological responses to stress in young and old Fischer 344 male rats

Two experiments were performed. In the first, a 20 min conditioned emotional response (CER) paradigm was used to compare the neurochemical, endocrine and immunological responses to stress of 7- and 22-month-old Fischer 344 (F344) male rats. In the second, corticosterone levels 20 min following ether stress, and regional brain type I and II corticosterone receptor densities were examined using 7- and 17.5-month-old F344 male rats. Dopamine (DA) metabolism in old nonstressed rats was significantly reduced in the medial frontal cortex, neostriatum, nucleus accumbens and hypothalamus, but not in the amygdala. The CER procedure, nevertheless, increased medial frontal cortical, nucleus accumbens and amygdaloid DA turnover in both the young and old rats. The young and old nonstressed rats did not evidence differences in norepinephrine (NE) and serotonin (5-HT) concentrations. However, stress resulted in a decrease in medial frontal cortical 5-hydroxyindoleacetic acid (5-HIAA) and hypothalamic 5-HT levels in old but not in young animals. These observations suggest age-related differences in the response of central NE and 5-HT systems to stress. Ether and the CER procedure led to exaggerated corticosterone responses in the old rats (17.5 and 22 month, respectively). Hippocampal type I but not type II corticosterone receptors were decreased by 47% in the 17.5-month-old rats. Thus, age-related changes in hippocampal corticosterone receptor types do not occur in unison, and the exacerbated corticosterone response to stress precedes the reported down-regulation of hippocampal type II corticosterone receptors in aged rats. Age-related changes were not observed in the concentrations of corticosterone receptors in other brain regions, or in the prolactin response to stress. The old rats, however, evidenced a reduction in the availability of the renin substrate, angiotensinogen, and in stress-induced renin secretion. Immune function was impaired in the old nonstressed rats, and further compromised by exposure to the CER procedure. In comparison to the young control rats, the old nonstressed rats showed an increased percentage of splenic large granular lymphocytes, reduced splenic natural killer cytotoxicity, and impaired Con-A-stimulated splenic T lymphocyte proliferation. Reductions in T splenic cell proliferation and natural killer cytotoxicity were observed in the young rats subjected to the CER paradigm, but not to the same extent as in the old rats. These observations indicate that aging male F344 rats evidence major alterations in basal central monoamine, endocrine and immune functions, and an increased sensitivity of these systems to stress.     

Antioxidant protection of cerebellar β-adrenergic receptor function in aged F344 rats

We examined whether a 2-week treatment with the spin-trapping agent MDL 101.002 (MDL) or a diet supplemented with vitamin E (Vit E) would alleviate age-related deficits in cerebellar noradrenergic function of male 18–20 month old F344 rats compared to age-matched controls. Cerebellar β-adrenergic receptor function was assessed using extracellular recordings of Purkinje cells during iontophoresis of GABA and isoproterenol (ISO). Noradrenergic receptor function of MDL and Vit E-treated rats was similar to young rats whereas for non-treated rats it was typical of that previously recorded in aged rats. Thus, treatment with MDL or Vit E reverses age-related deficits in cerebellar noradrenergic receptor function.     

Age-related subsensitivity of cerebellar Purkinje neurons to locally applied beta 1-selective adrenergic agonist

Previous electrophysiological studies in aged rats have revealed a number of deficits in noradrenergic neurotransmission in the central nervous system. Such deficits include subsensitivity to the depressant effects of norepinephrine on cerebellar Purkinje neurons, which has been attributed specifically to altered beta adrenergic receptor-mediated processes. The objective of this study was to determine which beta adrenergic receptor subtype, beta₁ or beta₂, in responsible for this age-related subsensitivity. The effects of beta₁ and beta₂ agonists on spontaneous activity of Purkinje neurons was first examined in young rats and the selectivity of these agents was validated using selective beta₁ and beta₂ antagonists. The effects of the selective beta₁ and beta₂ agonists were then compared in young (3-month-old) and aged (18- and 26-month-old) Fischer 344 rats. These agents were applied to Purkinje neurons by pressure microejection from multibarreled micropipettes and the change in neuronal action potential discharge rate was recorded. Both dobutamine, a beta₁-selective agonist, and zinterol, a beta₂-selective agonist, induced dose-dependent inhibitions of Purkinje cell firing rate. Dobutamine-induced inhibitions were blocked by the selective beta₁ antagonist, ICI 89406 and not by the beta₂-selective antagonist, ICI 118551; conversely, zinterol-induced inhibitions were not blocked by ICI 89406 but were blocked by the presence of ICI 118551. Purkinje neurons of both groups of aged rats were significantly less sensitive to locally applied dobutamine than Purkinje cells of young rats. Subsensitivity to zinterol was not observed in either of the aged groups. These results suggest an age-related functional decline in beta₁ adrenergic receptor-mediated processes, with no concomitant change in beta₂ receptor-mediated processes. Since beta₁ adrenergic receptors modulate inhibitory afferent input to Purkinje neurons, a process that may be critical in maintaining intact motor function, this selective subsensitivity may be an important factor contributing to the motor dysfunctions often seen in the e elderly.     

Strain differences and laminar localization of structural neurochemical changes in aging rat

To obtain comparisons of age-related microchemical changes in cerebral cortex of two comonly employed rat strains (Fischer 344 and Sprague-Dawley), neurochemical assays of substances regarded as quantitative indices of structural entities in brain were performed. These included DNA as a marker for cells, lipid sialoganglioside as an index of neuronal membrane mass, and galactocerebroside as an index of myelin. Fischer 344 rats were studied at 3–4 months (young), 14–16 months (middle age) and 25–28 months (old). Sprague-Dawleys were examined at 3–6 months (young), 14–17 months (middle age) and 25–28 months (old). Significant differences in the time courses of changes occurred; Fischer rats increased their brain weight at each aging point, while Sprague-Dawley rats reached stable brain weights by 4 months of age. Neither strain had a significant change in cell packing density of somatosensory cortex as measured by DNA. However, total ganglioside sialic acid declined in both strains, occurring by middle age in the Fischer and not until senescence in the Sprague-Dawley cortex. Cerebroside galactose increased in the Fischer between young and middle age, and was not further elevated in the older group. The Sprague-Dawley had its major increase in this marker between the middle aged and senescent groups. Intralaminar assays of these same markers in young and old Fisher 344 rats again indicated that DNA did not change, and that sialoganglioside was lost from all layers of the cortex in equal amounts. However, the increase in galactocerebroside resulted entirely from increases in the lower lamina of somatosensory cortex (lamina IV and below), suggesting on-going myelination of afferent and efferent axons. The time course of lipid membrane alteration is strain-dependent and selective as to cortical laminar localization. The findings are discussed in reference to human aging change in the same neurochemical indices.     

Strain differences and laminar localization of structural neurochemical changes in aging rat

To obtain comparisons of age-related microchemical changes in cerebral cortex of two comonly employed rat strains (Fischer 344 and Sprague-Dawley), neurochemical assays of substances regarded as quantitative indices of structural entities in brain were performed. These included DNA as a marker for cells, lipid sialoganglioside as an index of neuronal membrane mass, and galactocerebroside as an index of myelin. Fischer 344 rats were studied at 3–4 months (young), 14–16 months (middle age) and 25–28 months (old). Sprague-Dawleys were examined at 3–6 months (young), 14–17 months (middle age) and 25–28 months (old). Significant differences in the time courses of changes occurred; Fischer rats increased their brain weight at each aging point, while Sprague-Dawley rats reached stable brain weights by 4 months of age. Neither strain had a significant change in cell packing density of somatosensory cortex as measured by DNA. However, total ganglioside sialic acid declined in both strains, occurring by middle age in the Fischer and not until senescence in the Sprague-Dawley cortex. Cerebroside galactose increased in the Fischer between young and middle age, and was not further elevated in the older group. The Sprague-Dawley had its major increase in this marker between the middle aged and senescent groups. Intralaminar assays of these same markers in young and old Fisher 344 rats again indicated that DNA did not change, and that sialoganglioside was lost from all layers of the cortex in equal amounts. However, the increase in galactocerebroside resulted entirely from increases in the lower lamina of somatosensory cortex (lamina IV and below), suggesting on-going myelination of afferent and efferent axons. The time course of lipid membrane alteration is strain-dependent and selective as to cortical laminar localization. The findings are discussed in reference to human aging change in the same neurochemical indices.     

Grafted granule and Purkinje cells can migrate into the mature cerebellum of normal adult rats

Summary Cerebellar primordia of different ages (embryonic days of 14 to 20) were transplanted into cerebella of normal syngeneic adult rats (Fischer 344). After 1–12 months (mostly 5–6 months), the host brains were examined by light and electron microscopy. In about 80% of our successful experiments, grafted Purkinje and/or granule cells migrated into the host molecular layer. Some granule cells migrated down along host Bergmann glia, reminiscent of their normal developmental migration route. Other granule cells migrated along astroglial processes that ensheathed capillary walls. Some grafted Purkinje cells were also located ectopically in the host molecular layer. They were identified as graft origins autoradiographically. This migration seemed to be encouraged under conditions where the subjacent host Purkinje cells had been lost. Where the grafted Purkinje cells were located on top of the host molecular layer, their primary dendrites faced vertically downwards into the host molecular layer. However, the position of the apical poles of migrated Purkinje cell bodies in the deeper aspect of the molecular layer varied considerably, suggesting that the orientation of the Purkinje cell dendrites is probably determined by the availability of afferent fibers. Thus, the present study has demonstrated that Purkinje and granule cells can migrate from embryonic cerebellar grafts into the molecular layer of the normal, adult host rat cerebellum.     

Effect of advanced age on p-aminohippurate-induced inhibition of renal tubular secretion in male Fischer 344 rats

The effect of aging on glomerular filtration, effective renal plasma flow and on the responsiveness of the renal tubular anion secretory system to inhibition by 4-aminobenzoylglycine (p-aminohippurate, PAH) was examined in young (5-month) and old (22-month) Fischer 344 male rats. Plasma clearance, protein binding and renal extraction of [131I]o-iodohippurate, [125I]iothalamate and HPLC-purified [99mTc]mercaptoacetyltriglycine (MAG3), were used as in vivo probes of renal function. The effect of advanced age, without concomitant PAH, on the disposition of these markers was initially determined in ketamine anesthetized, temperature-maintained male rats, ages 5, 14 and 22 months by means of constant infusion clearance studies. Aging per se decreased (P less than 0.05) the kidney-weight normalized or body weight-normalized GFR and effective renal plasma flow rates. GFR values averaged 1.67, 1.43 and 1.32 ml/min per g kidney for the 5-, 14- and 22-month-old rats, respectively. Kidney- or body weight-normalized clearances of MAG3 and o-iodohippurate showed similar (25-27%) decreases, whereas the absolute values (ml/min) for GFR, o-iodohippurate and MAG3 clearance rates were not altered by aging. The effective filtration fraction, extraction ratio and plasma protein binding were also unchanged by advanced age. Overall, the age-related decreases in renal function were minimal in Fischer-344 rats, compared to other species. Differences in data normalization, species and gender account, in part, for discrepancies observed when comparing results in different studies on the effects of advanced age on renal function. Subsequently, we examined the effect of aging on the renal responsiveness to inhibition of tubular anion secretion using constant rate PAH infusion studies, adjusted for age-related changes in renal function. Aging did not alter PAH-induced inhibition of iodohippurate secretion. Inhibition of MAG3 elimination was more pronounced in the old rats compared to the young controls.     

Multiple changes in noradrenergic mechanisms in the coeruleo-hippocampal pathway during aging. Structural and functional correlates in intraocular double grafts

Age-related changes of the coeruleo-hippocampal noradrenergic system were investigated using intraocular double transplants. Pieces of fetal hippocampus were grafted into the anterior chamber of the eye and placed into contact with previously inserted locus coeruleus grafts. Ages of both transplants and hosts were varied to enable studies of intrinsic versus extrinsic determinants of aging in an isolated neuronal circuit. Four different experimental groups, with the approximate age in months of grafts/hosts at the time of recording given in parentheses, were studied; young grafts in the eyes of young hosts (3/7), young grafts in the eyes of old hosts (3/23), mature transplants in adult host rats (8/12) and aged transplants in the eyes of aged rats (21/25). Extracellular recordings from the hippocampal part of the double grafts were performed. Superfusion with alpha-adrenergic antagonists and the alpha 2-agonist clonidine elicited significant increases in the discharge rate of the grafted hippocampal neurons in all groups except the aged transplants in the aged hosts (21/25), where a small excitation was elicited with clonidine and no effect at all was seen with alpha-adrenergic antagonists. The host age did not seem to be important since young transplants in the old hosts (3/23) showed a similar increase in discharge rate as transplants in the young and adult hosts. Tyrosine hydroxylase immunohistochemistry and high-performance liquid chromatography revealed that hippocampal transplants remaining in oculo for a minimum of 6-10 months became permanently hyperinnervated by noradrenergic fibers from the locus coeruleus grafts. The density of noradrenergic fibers was significantly lower in young transplants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes of the metabolic profile of rat cerebellar cortex: enzyme histochemical study

The influence of aging on the metabolic profile of cerebellar cortex was studied in young (3-month-old), adult (12-month-old) and aged (26-month-old) male Sprague-Dawley rats using enzyme histochemical techniques. The following enzymatic activities related to energy transduction were examined: lactate-(LDH) and succinate-(SDH) dehydrogenases; NADH2-tetrazolium reductase (NADHD) and alpha-glycerophosphate-dehydrogenase (GPDH). The intensity of enzymatic staining within the neuropil of molecular and granular layers as well as within the cytoplasm of Purkinje neurons of young, adult and aged animals was assessed microphotometrically. In the molecular layer LSH, SDH and NADHD levels were reduced in old rats; GPDH was decreased both in adult and old animals. In Purkinje neurons no age-related changes of the enzymatic activities under study were observed. In the granular layer LDH and GPDH showed an age-dependent loss; SDH and NADHD were unchanged. The possibility that age-related changes of the enzymatic activities under study may be due to impaired energy production mechanisms and/or represent the consequence of reduced energetic needs resulting from the documented age-dependent loss of synapses in the molecular or in the granular layers of cerebellar cortex is discussed.     

N-methyl-D-aspartate mediated responses decrease with age in Fischer 344 rat brain.

N-Methyl-D-aspartate (NMDA) receptor-mediated responses were studied in hippocampus, cortex, and striatum of Fischer 344 rats of various ages (3-5, 12-14, or 24-28 months old; young, middle-aged, and senescent or old, respectively) to determine whether aging alters the function of NMDA receptors. NMDA-induced inhibition of muscarinic-stimulated phosphoinositide hydrolysis in hippocampus, and NMDA-stimulated release of [3H]norepinephrine (NE) or [3H]dopamine (DA) were used as indices of NMDA receptor function. The muscarinic agonist carbachol (1 mM) stimulated PI hydrolysis in hippocampi from all three age groups with no significant differences between the groups. NMDA inhibited the carbachol-evoked PI response in a concentration-dependent manner (10-100 microM) in all age groups. However, the NMDA-induced (100 microM) inhibition of the carbachol-stimulated response was markedly reduced in an age-dependent manner with losses of 25% and 53% in middle-aged and senescent rats compared to young. Concentration-effect curves for NMDA-stimulated [3H]NE release were determined using hippocampal and cortical slices from rats of the three age groups. In the hippocampus the maximal response for NMDA was significantly decreased from 6.55 fractional [3H]NE release in young to 4.51 and 4.18 in middle-aged and old rats, respectively, with no age-related changes in the potency of NMDA or slope of the curves. In cortical slices the maximal response was significantly reduced in an age-dependent manner by 23% in the senescent rats compared to the young rats. NMDA-stimulated [3H]DA release from striatal slices was significantly lower in the senescent rats at concentrations of NMDA from 500-2000 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for nonrandom regression of dendrites of Purkinje neurons during aging

This study examined dendritic networks of Purkinje neurons for randomness of age-related changes in cerebella from Fischer 344 rats at three ages. Terminal dendritic segments were studied in relation to their distance from the neuronal soma (path length). The data indicated that the terminal dendritic segments furthest from the cell body were preferentially affected during aging and there was a redistribution of the remaining terminal segments in cells from rats of the intermediate age group. The data also suggested that dendritic regrowth occurred in proximal regions of networks from rats in the oldest group.     

Enzyme histochemistry of glutamate dehydrogenase in ageing rat cerebellar cortex

The influence of ageing on glutamate dehydrogenase activity was studied in the cerebellar cortex of 3-month-old (young), 12-month-old (adult) and 26-month-old (aged) male Sprague-Dawley rats by using an enzyme histochemical technique. In young rats the enzyme reactivity was observed in the neuropil of the molecular layer as well as in the perikarya of basket cells and of stellate cells; within the cytoplasm of Purkinje neurons and in synaptic glomeruli of the granular layer. Glutamate dehydrogenase activity was significantly increased in the cerebellar cortex of adult rats and decreased in old animals. The synaptic glomeruli of the granular layer were the structures of the cerebellar cortex more remarkably affected by age-related changes. The possibility that decreased glutamate catabolism occurring in the ageing cerebellar cortex may result in an excess of the amino acid and may contribute to the nerve cell loss occurring in the cerebellum of old rats is discussed.     

The poly(U) translational capacity of Fischer 344 rat liver does not deteriorate with age and is not affected by dietary regime

The translational capacity of ribosomes isolated from the livers of young (60-170 days, n = 13) and old (26.5 months, n = 11) Fischer 344 rats was assessed by poly(U)-directed [3H]phenylalanine incorporation. Run-off ribosomes and S100 supernatant were separately prepared from each individual liver. Ribosome concentrations were carefully measured. Individual differences were seen among young rats and among old rats; however, when considered as a group, the old rats, whether diet-restricted or fed ad libitum, translated poly(U) as well, or almost as well, as the young ones.     

Intracranial cerebellar grafts: Intermediate filament immunohistochemistry and electrophysiology

Summary Pieces of the developing cerebellar anlage were prepared from 13–15 day old rat embryos and transplanted to the cerebellar region of 5–7 and 13–14 day old rat pups. Approximately two months later, sections showed most grafts to consist of both cerebellar cortex, with a typical trilaminar organization, and white matter areas containing large neuronal perikarya.The astrocytic populations were studied using immunohistochemistry with antisera raised against the intermediate filaments, glial fibrillary acidic protein (GFA), and vimentin. The GFA-antiserum revealed a glial interface along most of the border between host brain and graft. Both antisera stained long, slender, although slightly distorted Bergmann fibers spanning the molecular layer. Using GF-Aantiserum, star-shaped fluorescent astrocytes were seen in the granular layer and in the white matter. Only in the white matter did the amount of GFA-like immunoreactivity suggest an astrocytic gliosis. With vimentin antiserum fluorescent astrocytes in the white matter were seen. There were no signs of increased amounts of vimentin-like immunoreactivity. Taken together, the amount and distribution of GFA-and vimentin-like immunoreactivity suggests a rather normal astrocytic development in the cerebellar grafts.Using an antiserum against the neurofilament (NF) triplet, delicate immunoreactive fibres were seen in both the molecular and the granular layer. No positive cell bodies could be visualized in the cortical areas. Although the Purkinje cells themselves were negative, fibre baskets around them were intensely stained. In the white matter a high density of NF-positive fibres and some positive perikarya were visualized. Thus the distribution of NF-like immunoreactivity in the grafts corresponded well to the normal NF distribution.The functional maturation of the cerebellar grafts was studied electrophysiologically. A spontaneous mean discharge rate of 19.3 + 1.7 Hz was recorded from the Purkinje cells. This compares with a discharge rate of 26.8 + 1.0 Hz for Purkinje neurons in situ. The difference was at least partly ascribable to the absence of climbing fibre bursts in the grafts. Local stimulation of the graft surface caused both decreased and increased Purkinje cell discharge. In conclusion, these experiments suggest that grafts of fetal cerebellar buds to the young cerebellum develop into cerebellar tissue having both morphological and electrophysiological characteristics quite similar to the normal cerebellum.