Age-dependent effects of deafferentation of the rat superior cervical ganglion on expression of P65 (synaptotagmin) during postnatal development.

Previous studies have shown that deafferentation of the rat superior cervical ganglion (SCG) alters the levels of p65 (synaptotagmin), a synaptic vesicle integral membrane protein, within the ganglion. Neonatal deafferentation blocks normal postnatal increases in p65, while deafferentation in adult animals produces a transient increase in p65 expression. The present study examines the time course of the shift from the neonatal to adult pattern of response to deafferentation. Neonatal and 7 day old rats showed the neonatal response to deafferentation. Ganglia from rats aged 14 days or older at deafferentation exhibited the transient increase in p65 at 7 days after surgery. The shift from the neonatal to adult response occurs during the second postnatal week. The change in response to deafferentation may be associated with refinement of synaptic function in a manner yet to be determined.     

Deafferentation-induced increases in a synaptic vesicle protein in the adult rat superior cervical ganglion are associated with new protein synthesis

Previous studies have shown that deafferentation of the adult rat superior cervical ganglion results in a transient increase in levels of a 65 kDa synaptic vesicle membrane protein (SV). The present study indicates that the observed increase in SV after deafferentation is the result of new protein synthesis. Treatment with cycloheximide, a protein synthesis inhibitor, for 8 h at selected times after surgery produces decreases in SV which are greater than that observed after treatment of unoperated animals. The results suggest that an increased rate of synthesis of this protein is induced by deafferentation. Transsynaptic factors may play important roles in regulation of protein synthesis in sympathetic ganglia.     

In vivo expression of ganglionic long-term potentiation in superior cervical ganglia from hypertensive aged rats

Sustained increase in central sympathetic outflow to ganglia may provide the repeated high frequency presynaptic activity required for induction of long-term potentiation in sympathetic ganglia (gLTP), which is known to be involved in the manifestation of a neurogenic form of hypertension, namely stress-hypertension. Aging is often viewed as a progressive decline in physiological competence with a corresponding impaired ability to adapt to stressful stimuli. Old animals have exaggerated sympathetic activity as well as increased morbidity and mortality during prolonged exposure to stressful stimuli. Using the superior cervical ganglion (SCG) as a model for sympathetic ganglia, electrophysiological and biochemical evidence show that mildly hypertensive aged rats (22-month old) have expressed gLTP in vivo. This is suggested by a number of lines of evidence. Firstly, a shift in input/output (I/O) curve of ganglia from aged rats to the left side of I/O curve of ganglia from 6-month old (adult) rats indicating expression of gLTP. Secondly, failure of in vitro high frequency stimulation to induce gLTP in ganglia isolated from aged rats, which indicates occlusion due to saturation, which, in turn, suggests in vivo expression of gLTP in these ganglia. Thirdly, in vitro inhibition of basal ganglionic transmission by blockers of gLTP (5-HT(3) antagonists) is observed in ganglia isolated from aged rats, but not in those from adult rats. Finally, immunoblot analysis revealed that protein levels of signaling molecules such as calcium-calmodulin kinase II (CaMKII; phosphorylated and total), which normally increase during expression of LTP, are elevated in ganglia isolated from aged rats compared to those from adult ones. Protein levels of calcineurin, which dephosphorylates P-CaMKII, were reduced in ganglia isolated from aged rats, probably as a support mechanism to allow prolonged phosphorylation of CaMKII. Our findings suggest in vivo expression of gLTP in sympathetic ganglia of aged animals, which may contribute to the moderate hypertension often seen in aged subjects.     

The Effects of Forelimb Deafferentation on the Post-Natal Development of Synaptic Plasticity in the Hippocampus

The effects of partial deafferentation of the forelimb on the development of long-term potentiation in the hippocampus of rats aged 13–18 days were studied. Long-term potentiation in hippocampus field CA1 was of greater amplitude and duration in control rats at 16–18 days of post-natal ontogenesis than in adult animals. Partial deafferentation by section of the median nerve in the forelimb on the 13th day of life led to the disappearance of this excess at 16–18 days. The peak in synaptic plasticity occurred later in operated animals – on day 17 – and was much less marked than in controls. The decreases in the amplitude and duration of long-term potentiation in hippocampal field CA1 in operated animals provides evidence for a decrease in the sensitivity and/or number of NMDA receptors. This suggests that partial deafferentation of one limb may lead not to a decrease but to an increase in spike and synaptic activity in the hippocampus, which in normal conditions may affect the maturation of the plastic properties of synaptic transmission associated with the expression and positions of NMDA receptors. The level of long-term potentiation in sham-operated rats was significantly greater than in controls of the same age. This significant increase in NMDA-dependent long-term potentiation may be explained by a decrease in the level of activation due to anesthesia. It is suggested that the decrease in the spike activity of cells receiving signals from the median nerve may be compensated for by activation of other specific and non-specific inputs.     

The release of sympathetic neurotransmitters is impaired in aged rats after an inflammatory stimulus: a possible link between cytokine production and sympathetic transmission

Aging results in a general decline in the response to external insults, including acute inflammatory challenges. In young animals, the inflammatory response requires activation of the sympathetic system, including neurotransmitters such as ATP, and catecholamines (epinephrine and norepinephrine). To test whether aging affects activation of this axis, and whether this in turn might affect cytokine release, we administered lipopolysaccharide (LPS) i.p. to adult, middle-aged and aged Fisher 344 rats (6-, 15- and 23-month old, respectively) and evaluated the early (0-12 h) serum levels of Neuropeptide-Y (NP-Y), ATP and vanillyl mandelic acid (VMA, as an indirect measurement of catecholamine levels). In addition, we evaluated the association between these factors and serum levels of the cytokines tumor necrosis factor-alpha (TNFα) and interleukin-10 (IL-10). Induction of both ATP and NP-Y was markedly reduced in the serum of aged animals, when compared to their younger counterparts, while induction of VMA was not affected by age. In spite of these changes, serum levels of TNFα and IL-10 were strongly hyper induced and delayed in aged rats. The results suggest that during aging there is a dysregulation in sympathetic neurotransmitter regulatory mechanisms, and this might play a role in the impairment of the inflammatory response.     

Delayed angiogenesis in aging rats and therapeutic effect of adenoviral gene transfer of VEGF

We have studied an age-related impairment in angiogenesis and evaluated the effect of overexpressing VEGF in this situation. Polyvinyl alcohol sponges were implanted subcutaneously into aged (24-month), adult (12-month), and young (2-month) rats. Blood vessel ingrowth and proliferative activity in the sponges were assessed by histology with immunostaining for von Willebrand's factor and proliferating cell nuclear antigen (PCNA), respectively. The percentage of total sponge area filled with ingrowing fibrovascular tissue was minimal in aged rats, intermediate in adult rats and highest in young rats. A similar pattern was observed for the total blood vessel numbers in the sponges from old to young animals. The percentage of total sponge endothelial cells (ECs) showing proliferative activity (PCNA positive) was lowest in the aged animals, intermediate in the adult rats and highest in the young rats. To further explore the mechanism of impaired angiogenesis in aged animals, we investigated and found a reduced level of endogenous VEGF protein expression in 12-month-old rats compared to that in 2-month-old rats. VEGF121 gene transfer significantly enhanced blood vessel and fibrovascular tissue ingrowth in adult/aged rats. Adenoviral-VEGF gene transfer also significantly stimulated EC proliferation in aged and adult rats. However, identical treatment failed to further stimulate the already more robust angiogenesis in young animals. The different angiogenic response in adult vs. young rats was not due to differences in gene transfer efficiency, since similar levels of human VEGF121 protein was detected in adult and young rats. Our results indicate that the decreased angiogenic response with aging is associated with reduced EC proliferation and reduced endogenous VEGF production. Adenoviral-VEGF121 gene transfer is effective in augmenting angiogenesis, particularly in older animals.     

Age-related structural and neurochemical changes of the human superior cervical ganglion.

The aim of this study was to investigate age-related morphological and neurochemical changes in the human superior cervical ganglion (SCG). Thirty-seven superior sympathetic human cervical ganglia of young, adult, and aged subjects were examined using morphometric analysis, biotin-streptavidin immunohistochemistry for detecting neurofilament, myelin protein, protein gene product 9.5, nerve growth factor receptor p75 in sympathetic neurons and nerve fibers. Morphometric parameters of neurons (area, long and short axis, shape factor of the neuron body, nucleus, cytoplasm, and lipofuscin) were investigated in every sixth serial section of the ganglion. Seven hundred neurons with clearly visible nuclei were measured in each studied group. The present study showed that human SCG of older subjects had larger areas of neuron body, cytoplasm and nucleus, a lower shape factor, an increased amount of lipofuscin, and a greater number of large-size neurons, as compared to SCG obtained from young subjects. Neuronal cytoskeletal alterations manifested themselves through a decreased number of neurofilament-positive neurons were detected in old human SCG. The amount of myelinated fibers decreased with age, although the amount of myelinated fibers in the young and the adult subjects varied from few to a moderate number. PGP 9.5 immunoreactivity varied in different age groups. A marked reduction of nerve growth factor receptor p75 in old human sympathetic neurons was detected. In conclusion, the findings of this study confirm age-related morphological changes in the human SCG. Structural neuronal changes may influence the deterioration of neuronal functional capacity, neuronal plasticity, and regenerative characteristics.     

Effects of 2-deoxyglucose on plasma catecholamines in adult and aged rats

To examine the effects of aging on the responsiveness of the sympathetic-adrenal medullary system, I have measured plasma levels of norepinephrine (NE) and epinephrine (EPI) in adult (6 months old) and aged (24 months old) Fischer 344 male rats. Two days prior to testing, rats were surgically prepared with chronic tail artery catheters to permit remote sampling of blood in conscious, unrestrained animals. Following collection of basal blood samples, each rat received a single injection of 2-deoxyglucose (2-DG, 250 or 500 mg/kg, IP) and additional blood samples were collected 1, 2 and 4 hours later. 2-DG, a glucose analogue, stimulates a centrally mediated activation of the adrenal medulla and to a lesser extent the postganglionic sympathetic neurons. For purposes of analysis, data were excluded from animals which died within 4 hours after injection. Basal plasma levels of both catecholamines were similar in adult and aged rats. Administration of 2-DG was attended by significant and sustained increases in plasma NE and EPI in rats of both ages. A greater proportion of aged rats died following administration of 2-DG compared to adult rats. At the higher dose of 2-DG, plasma levels of NE were significantly higher in 6 month old rats at 1 and 2 hours post-injection. In contrast, plasma levels of EPI were significantly higher in 24 month old rats at 1 and 2 hours after administration of 250 mg/kg 2-DG and at 1 hour after administration of 500 mg/kg 2-DG. These findings indicated that the adrenal medullary response of aged rats to an intense and prolonged stressor is greater than that of adult rats. The attenuated plasma NE response of aged rats to 2-DG may be due to a lessor release of NE from the adrenal medulla and/or a diminished activation of postganglionic sympathetic nerves.     

Effects of 2-deoxyglucose on plasma catecholamines in adult and aged rats

To examine the effects of aging on the responsiveness of the sympathetic-adrenal medullary system, I have measured plasma levels of norepinephrine (NE) and epinephrine (EPI) in adult (6 months old) and aged (24 months old) Fischer 344 male rats. Two days prior to testing, rats were surgically prepared with chronic tail artery catheters to permit remote sampling of blood in conscious, unrestrained animals. Following collection of basal blood samples, each rat received a single injection of 2-deoxyglucose (2-DG, 250 or 500 mg/kg, IP) and additional blood samples were collected 1, 2 and 4 hours later. 2-DG, a glucose analogue, stimulates a centrally mediated activation of the adrenal medulla and to a lesser extent the postganglionic sympathetic neurons. For purposes of analysis, data were excluded from animals which died within 4 hours after injection. Basal plasma levels of both catecholamines were similar in adult and aged rats. Administration of 2-DG was attended by significant and sustained increases in plasma NE and EPI in rats of both ages. A greater proportion of aged rats died following administration of 2-DG compared to adult rats. At the higher dose of 2-DG, plasma levels of NE were significantly higher in 6 month old rats at 1 and 2 hours post-injection. In contrast, plasma levels of EPI were significantly higher in 24 month old rats at 1 and 2 hours after administration of 250 mg/kg 2-DG and at 1 hour after administration of 500 mg/kg 2-DG. These findings indicated that the adrenal medullary response of aged rats to an intense and prolonged stressor is greater than that of adult rats. The attenuated plasma NE response of aged rats to 2-DG may be due to a lessor release of NE from the adrenal medulla and/or a diminished activation of postganglionic sympathetic nerves.     

Repeated inspiratory occlusions acutely impair myocardial function in rats

Repeated episodes of hypoxia and sympathetic activation during obstructive sleep apnoea are implicated in the initiation and progression of cardiovascular diseases, but the acute effects are unknown. We hypothesized that repeated inspiratory occlusions cause acute myocardial dysfunction and injury. In 22 spontaneously breathing pentobarbital-anaesthetized rats, inspiration was occluded for 30 s every 2 min for 3 h. After ∼1.5 h, mean arterial pressure started to fall; heart rate between occlusions was stable throughout, consistent with only transient increases in sympathetic activity during each occlusion. Three hours of occlusions resulted in ventricular diastolic dysfunction (reduced peak rate of change of ventricular pressure and slower relaxation). Post-occlusions, the left ventricular contractile response to dobutamine was blunted. After 1 h of recovery, left ventricular pressure generation had returned to values no different from those in sham animals in 5 of 9 of the animals. Cardiac myofibrils from rats subjected to occlusions had depressed calcium-activated myosin ATPase activity, indicating myofilament contractile dysfunction that was not due to breakdown of contractile proteins. Haematoxylin and eosin-stained cross-sections revealed multifocal areas of necrosis within the septum and both ventricles. Repeated inspiratory occlusions, analogous to moderately severe obstructive sleep apnoea, acutely cause global cardiac dysfunction with multifocal myocardial infarcts.     

The effect of age and injury on the expression of inducible nitric oxide synthase in facial motor neurons in F344 rats

Nitric oxide has been implicated in both normal neuronal aging as well as nerve repair events because of its known roles in synaptic plasticity, synaptogenesis and neuroplathologic processes. In this study, we have determined the effect of aging, by comparing brainstem facial motor neurons (FMNs) as well as blood vessels, from adult F344 rats to those in old animals. Inducible nitric oxide synthase (iNOS) expression was determined both by immunohistochemistry using an antibody to iNOS on tissue sections and slot blots. In adult rats, iNOS expression was detectable only in FMNs and not in blood vessels. In old rats, there were robust levels of iNOS protein in blood vessels, while iNOS protein was not detectable in FMNs from old rats. There was also a 12-fold increase in iNOS expression in isolated blood vessels from old rats compared to vessels from adult animals. To determine the effect of injury on iNOS expression, the facial nerve was transected and immunocytochemistry performed as above. After nerve transection in adult rats, iNOS was demonstrable only in blood vessels after 1 day, but by 7 days iNOS protein immunoreactivity was robust in FMNs. In old animals, iNOS protein expression was observed only in FMNs at 1 day, but by 7 days after injury, protein immunoreactivity was localized to the blood vessels. These data suggest that aging and injury differentially affect the expression of iNOS and that the up-regulation of iNOS may be important for the availability of nitric oxide in the aged or injured nervous system.     

Age-related loss of synaptic terminals in the rat medial nucleus of the trapezoid body

The effect of aging on axosomatic synaptic terminals in the rat medial nucleus of the trapezoid body was studied using quantitative electron microscopy. In young adult rats (3 months of age), the mean percentage of the surface area of principal cells covered by synaptic terminals is 61.7% (S.E.M. = 4.1) while in aged animals (27-33 months of age) the per cent coverage is 43.7% (S.E.M. = 3.3). Likewise, between 3 and 27-33 months of age, the average number of synaptic terminals present along a 100 micron length of principal cell surface decreases significantly (P less than 0.001) from 28.3 (S.E.M. = 1.3) to 18.9 (S.E.M. = 1.3). Only terminals derived from calyces of Held are lost in the aged animals, displaying a 37% reduction between 3 and 27-33 months of age. The length of apposition by synaptic terminals in the medial nucleus of the trapezoid body does not change significantly with aging. We conclude that because of a significant loss of calycine synaptic endings, the structure of calyces of Held becomes less complex with advancing age in rats. This would presumably result in an age-related partial deafferentation of principal cells, causing significant alterations in the processing of auditory information in the medial nucleus of the trapezoid body.     

Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex

Age-related changes within the auditory brainstem typically include alterations in inhibitory neurotransmission and coding mediated by GABA and glycinergic circuits. As part of an effort to evaluate the impact of aging on neurotransmission in the higher auditory centers, the present study examined age-related changes in the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in rat primary auditory cortex (AI), which contains a vast network of intrinsic and extrinsic GABAergic circuits throughout its layers. Message levels of the two GAD isoforms found in brain, GAD(65) and GAD(67), and GAD(67) protein levels were compared in young adult, middle-aged and aged rats using in situ hybridization and quantitative immunocytochemistry, respectively. For comparison, age-related GAD changes were also assessed in the parietal cortex and hippocampus. Significant age-related decreases in GAD(65&67) messages were observed in AI layers II-VI of aged rats relative to their young adult cohorts. The largest changes were identified in layer II (GAD(65): -26.6% and GAD(67): -40.1%). GAD(67) protein expression decreased significantly in parallel with mRNA decreases in all layers of AI. Adjacent regions of parietal cortex showed no significant GAD(67) protein changes among the age groups, except in layer IV. As previously described, GAD(67) message and protein levels in selected hippocampal regions were significantly reduced in aged rats. Age-related GAD reductions likely reflect decreases in both metabolic and pre-synaptic GABA levels suggesting a plastic down-regulation of normal adult inhibitory GABA neurotransmission. Consistent with the present findings, functional studies in primate visual cortex and preliminary studies in AI find coding changes suggestive of altered inhibitory processing in aged animals. An age-related loss of normal adult GABA neurotransmission in AI would likely alter temporal coding properties and could contribute to the loss in speech understanding observed in the elderly.     

Neuronal and inducible nitric oxide synthase expressions and activities in the hippocampi and cortices of young adult,aged cognitively unimpaired,and impaired Long-Evans rats

Nitric oxide (NO) is a neurosignaling molecule that appears to play a significant role in learning and memory. This molecule has also been implicated in neurotoxicity due to its oxidative properties. Previous experiments from our laboratories have demonstrated elevated hippocampal and cortical neuronal nitric oxide synthase (NOS) mRNA levels in aged cognitively unimpaired and impaired Long-Evans rats, which could represent either increased neuronal NOS activity thereby leading to NO-mediated neurotoxicity, or a compensatory response by aged neurones to maintain physiological nitric oxide output. The current study measured the protein expression and activity levels of neuronal and inducible NOS in young adult (6 months) and aged (24-26 months) Long-Evans rats by means of western blotting and NOS activity assay. Aged animals were assigned as either cognitively unimpaired or aged with moderate cognitive impairments based on their performances in the Morris water maze behavioural task. Our results showed that hippocampal and cortical neuronal NOS expressions were significantly decreased in aged animals. These aged animals also exhibited increased hippocampal and cortical inducible NOS expressions. Between the two aged animal groups, cognitively impaired rats showed significantly lower hippocampal and cortical neuronal but higher hippocampal inducible NOS expressions. Young adult rats exhibited significantly higher hippocampal and cortical NOS activities than the aged animals. Aged animals with cognitive deficits showed significantly lower hippocampal NOS activity than cognitively unimpaired aged rats.Our data indicate that aging is associated with a decline in neuronal but elevated inducible NOS functioning in brain areas involved in learning and memory. These phenomena could contribute to the cognitive deficits observed in a sub-population of aged animals.     

Responsiveness of sympathetic and sensory iridial nerves to NGF treatment in young and aged rats

Altered neuronal responses to trophic factors may play a role in neuronal maintenance in adulthood and may also be involved in neuronal atrophy in old age. We have investigated this issue in the rat iris, studying responsiveness of sympathetic and sensory iridial nerves to a range of NGF concentrations in mature and aged rats. We show here that growth responses of sensory nerves to NGF, as measured by quantitative immunohistochemistry and image analysis, were unchanged in old rats. In contrast, there was a small but significant reduction in responsiveness of aged sympathetic neurons. The shapes of the dose-response curves for sensory and sympathetic neurons were different, with a larger response over a narrower range of concentrations in sensory neurons. Lower levels of p75 immunoreactivity were observed in iridial nerves from old compared to young rats. NGF treatment had no effect on receptor staining in young rats but restored 'young' levels of p75 staining in old rats. Our results do not support the hypothesis of a primary role for NGF in maintenance or atrophy of nerves in ageing.     

Persistence of sympathetic ingrowth fibers in aged rat hippocampus

Sympathetic fibers appear in the rat hippocampus following interruption of the septal-hippocampal pathway. The current study examined the relative ability of young (3 month) and old (24 month) rats to produce sympathetic ingrowth fibers. Sympathetic ingrowth was present in the hippocampus of young adult rats 2 months after fimbria/fornix transection. The aged rats either lacked, or had very limited, hippocampal ingrowth two months after fimbria/fornix transections. In contrast, aged rats which had fimbria/fornix transections as young adults had extensive hippocampal ingrowth fibers. The topographical distribution of ingrowth fibers in the hippocampal formation was similar in young 3-month-old rats and rats which were lesioned young and sacrificed when aged. Counts of sympathetic fibers found similar numbers of hippocampal ingrowth fibers at both of the post-operative ages. Thus, although aged animals were acutely impaired in their ability to initiate the ingrowth response, the sympathetic fibers which were established at a young age persisted into senescence.     

Effects of long-term administration and withdrawal of clonidine on activity of sympathetic efferent nerve unit in spontaneously hypertensive rats

Daily administration of clonidine for 5 weeks (approx. 300 micrograms/kg/day, p.o.) produced significant decreases in blood pressure and renal sympathetic efferent activity in spontaneously hypertensive rats (SHR). After cessation of clonidine administration, blood pressure and sympathetic nerve activity recovered rapidly from the depressed levels to the untreated control levels. These findings could indicate that clonidine-induced hypotension and its rapid recovery to control after withdrawal of clonidine are caused by corresponding changes in sympathetic efferent nerve activity.     

Alterations in beta adrenergic and muscarinic receptors in aged rat heart. Effects of chronic administration of propranolol and atropine.

The cardiac responses to sympathetic and vagal stimulations are attenuated with ageing. To understand these findings, the densities of beta adrenergic (beta R) and muscarinic (MR) receptors in the left ventricles have been quantitated in parallel in male Wistar rats (4- and 24-month-old) using [125I]iodocyanopindolol and [3H]quinuclidinyl benzilate as specific radioligands. The homologous regulation of these receptor densities was also explored after a 7-day continuous infusion of propranolol or atropine. As compared to young rats, the beta R and MR densities in aged animals were decreased (from 31 +/- 2 to 23 +/- 2 fmol/mg protein, P less than 0.05 for beta R; from 104 +/- 7 to 54 +/- 3 fmol/mg protein, P less than 0.001 for MR) but the diminution in MR was more pronounced (-48%) than that in beta R (-26%), resulting in a drop in the beta R/MR ratio. Continuous infusion of propranolol or atropine up-regulated the beta R and MR densities (respectively +50%, P less than 0.01 and +33%, P less than 0.05) in aged but not in young adult rats. We therefore conclude: (i) that the diminution of the cardiac response to the sympathetic and vagal stimulations during ageing may be partly explained by a decrease in the corresponding receptor density; (ii) these changes are reversible and the density of these two groups of receptors can return to adult control values by chronic administration of the appropriate antagonist.