Involvement of neurotrophic factors in aging of noradrenergic innervations in hippocampus and frontal cortex

In the present study, we investigated the age-dependent changes in the axon terminals of the locus coeruleus (LC) neurons in the frontal cortex and hippocampus, in which a high degree of axonal branching in the middle-aged brain was suggested to occur in our previous electrophysiological study. We used 6-, 13- and 25-month-old male F344/N rats, and performed Western blot analysis of the norepinephrine transporter (NET), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). NET expression level increased in the 13-month-old hippocampus, but was not altered by aging in the frontal cortex. BDNF expression level increased in the hippocampus, but did not change with age in the frontal cortex. On the other hand, GDNF expression level was increased with age in the frontal cortex, but was not in the hippocampus. These results suggest that the LC noradrenergic innervations may be locally regulated by different neurotrophic factors that exert their trophic actions at different target sites.     

Effects of BDNF infusion on the axon terminals of locus coeruleus neurons of aging rats

Using in vivo electrophysiological techniques and continuous local infusion methods, we examined the effects of brain-derived neurotrophic factor (BDNF) and its specific antibody (anti-BDNF) on the noradrenergic axon terminals of the locus coeruleus (LC) neurons in the frontal cortex of aging rats. Recently, we observed that LC neurons with multiple-threshold antidromic responses (multi-threshold LC neurons) increased critically between 15 and 17 months of age. To examine whether the BDNF is involved in this change occurred in the aging brain, we continuously infused BDNF into the frontal cortex for 14 days. Exogenous BDNF produced a marked increase in the multi-threshold LC neurons in the 13-month-old brain, accompanied with a decrease in threshold current. However, no morphological change in the noradrenergic axons was observed in the BDNF-infused cortex. In contrast, infusion of anti-BDNF led to a dose-dependent reduction of the multi-threshold LC neurons in the 19-month-old brain, accompanied with an increase in threshold current. These findings suggest that BDNF may contribute to functional changes in the presynaptic axon terminals of LC neurons in the aging brain.     

Changes in cortical noradrenergic axon terminals of locus coeruleus neurons in aged F344 rats.

The noradrenergic innervations and noradrenaline contents of the frontal cortex in two age groups (9 and 25 months) of male F344 rats have been quantified by electrophysiological and biochemical methods. In the electrophysiological study, the percentage of locus coeruleus (LC) neurons activated antidromically from the frontal cortex decreased with age. In contrast, the percentage of LC neurons showing multiple antidromic latencies, which suggests axonal branching of individual LC neurons, increased markedly between 9 and 25 months in the frontal cortex. In the biochemical study, we found no significant difference in noradrenaline levels in the cortical terminal fields of LC neurons during aging. These results suggest that LC neurons give rise to axonal branches to retain noradrenaline levels in their target fields in the aged brain. Our findings show that LC neurons preserve a strong capability for remodeling their axon terminals even in the aged brain.     

Age-dependent changes in noradrenergic innervations of the frontal cortex in F344 rats

Noradrenergic innervations of the frontal cortex with advancing age (9, 13 and 25 months) in male F344 rats were quantified by immunohistochemistry for dopamine-beta-hydroxylase (DBH), which is a marker enzyme for noradrenergic axons. The density of DBH-positive axons, varicosities (swellings along an axon from which noradrenaline is released), and the number of varicosities per unit length of axon were measured in the frontal cortex. We found that the density of axons and varicosities significantly decreased at an earlier stage of aging (9-13 months), but not at a later stage (13-25 months). On the other hand, the number of varicosities per unit length of axon did not change with age. The result shows that the density of varicosities, which represent the synapses of noradrenergic neurons, decrease in the frontal cortex in the early aging process.     

Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus neurons

Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus (LC) noradrenergic neurons were studied in the frontal cortex using an extracellular single unit recording technique in vivo. Clonidine, a selective alpha(2) adrenergic agonist, and nisoxetine, a selective noradrenaline uptake inhibitor, were infused locally into the frontal cortex to examine the effects of these drugs on release and uptake activities of the axon terminals of LC neurons. Although the infusion of clonidine produced a marked suppression of release, the effect did not change with age. Infusion of nisoxetine caused an inhibition of uptake, but the effect was attenuated in aged rats. These results suggest that the release activity mediated by the presynaptic autoreceptor did not change with age, but the uptake activity mediated by the NA transporter declined with age in the axon terminals of LC neurons.     

Age-related deficits as working memory load increases: relationships with growth factors

Young and aged female rats were tested on a water radial-arm maze designed to measure performance as working memory load increased, followed by brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin 3 (NT3) protein assessments in hippocampus and frontal cortex. Aged rats showed deficiencies in both working and reference memory. There were also profound age-related working memory load effects. Aged rats made more errors as working memory load increased and showed learning only during early trials when memory load was low, while young rats exhibited learning over all trials. Neurotrophin assessment showed that frontal cortex NGF and BDNF levels were positively, and hippocampal NT3 negatively, correlated with number of errors made during specific trials in aged animals. Comparison to untested rats showed that testing increased NT3, but not BDNF or NGF, protein levels in both age groups. Findings suggest that young rats learn to handle a higher working memory load as testing progresses, while aged rats do not, and that frontal cortex and hippocampal neurotrophin levels may relate to working memory proficiency in aged female rats.     

人参皂甙对老龄大鼠Meynert核及大脑皮质脑源性神经营养因子含量的影响

目的观察Meyaert核（NBM）与大脑皮质神经元内脑源性神经营养因子（BDNF）的老龄性改变,探讨人参皂甙对BDNF蛋白含量表达的影响.方法雌性Wistar大鼠随机分为青年组、老龄组及老龄给药组.老龄给药组大鼠自18个月开始饲以人参皂甙至27月龄.对各组NBM及大脑皮层神经元进行免疫组织化学法染色、图像分析及统计学处理.结果老龄组大鼠NBM及大脑皮质内BDNF含量均显著低于青年组（P＜0.01）.给药组大脑皮质内BDNF含量较老龄组显著增加（P＜0.01）,但NBM内BDNF含量变化无统计学意义.结论老龄大鼠NBM及大脑皮质内BDNF含量显著降低,给予人参皂甙可显著提高大脑皮质神经元BDNF的表达.     

The cholinergic innervation develops early and rapidly in the rat cerebral cortex: a quantitative immunocytochemical study.

A recently developed method for determining the length of cholinergic axons and number of cholinergic axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase was used to estimate the areal and laminar densities of the cholinergic innervation in rat frontal (motor), parietal (somatosensory) and occipital (visual) cortex at different postnatal ages. This cortical innervation showed an early beginning, a few immunostained fibers being already present in the cortical subplate at birth. In the first two postnatal weeks, it developed rapidly along three parameters: a progressive increase in the number of varicosities per unit length of axon, and a lengthening and branching of the axons. Between postnatal days 4 and 16, the number of varicosities increased steadily from two to four per 10 microm of cholinergic axon. The mean densities of cholinergic axons increased from 1.4 to 9.6, 1.7 to 9.3 and 0.7 to 7.2 m/mm(3), and the corresponding densities of varicosities from 0.4 to 3.9, 0.4 to 3.5, and 0.2 to 2.6x10(6)/mm(3) in the frontal, parietal and occipital areas, respectively. The rate of growth was maximal during these first two weeks, after which the laminar pattern characteristic of each area appeared to be established. Adult values were almost reached by postnatal day 16 in the parietal cortex, but maturation proceeded further in the frontal and particularly in the occipital cortex.These quantitative data on the ingrowth and maturation of the cholinergic innervation in postnatal rat cerebral cortex substantiate a role for acetylcholine in the development of this brain region and emphasize the striking growth capacity of individual cholinergic neurons.     

大鼠视皮层中脑源性神经营养因子及γ-氨基丁酸表达的年龄相关性变化

目的 比较不同年龄组大鼠初级视皮层中脑源性神经营养因子（BDNF）及抑制性神经递质γ-氨基丁酸（GABA）表达的年龄相关性变化，为探讨老年性视觉功能衰退的细胞分子机制提供线索。方法 Nissl染色显示视皮层分层并用于神经元计数；免疫组织化学法标记大鼠视皮层中BDNF及GABA免疫阳性神经元。光镜下观察并用Image-Pro Express 6.0分析软件进行细胞密度统计和免疫反应吸光度值测量。结果 青年、中年及老年组(每组n=6)大鼠初级视皮层各层的神经元平均密度差异不显著；但与青年组大鼠相比，中年组及老年组大鼠初级视皮层各层中BDNF及GABA免疫阳性神经元密度显著下降，BDNF及GABA免疫反应强度明显减弱；与中年组大鼠相比，老年组大鼠视皮层各层中BDNF及GABA免疫阳性神经元密度及其反应强度亦明显降低。在衰老过程中，大鼠初级视皮层各层内GABA表达的减少与BDNF表达的降低具有高度的相关性。结论 大鼠初级视皮层中BDNF和GABA的表达随着衰老出现进行性下降，衰老过程中BDNF分泌的减少可能引起脑内抑制性神经递质GABA表达下调，这可能是介导老年性视觉功能衰退的重要途径之一。     

BDNF-induced LTP in dentate gyrus is impaired with age: analysis of changes in cell signaling events

Brain-derived neurotrophic factor (BDNF) has emerged as a major regulator of synaptic plasticity in the adult brain and acute BDNF infusion has been shown to trigger long-term potentiation (BDNF-LTP) in adult rats. Here we compared the effects of acute BDNF infusion in young adult and aged anesthetized rats. In young rats, BDNF-LTP was accompanied by increased activation of the BDNF receptor TrkB, and extracellular signal-regulated kinase (ERK), as well as enhanced evoked release of glutamate in synaptosomes prepared from DG. In aged rats, both BDNF-LTP and the associated signaling were significantly impaired, while analysis of untreated hippocampal tissue from aged rats showed that activation of TrkB and ERK were decreased. In addition to effects in the DG, BDNF-LTP was accompanied by robust phosphorylation of the calcium/cAMP-responsive element binding protein (CREB) in tissue prepared from entorhinal cortex of both young and aged rats. These results suggest a cascade of presynaptic changes contributing to the expression of BDNF-induced LTP and show that BDNF-induced transduction mechanisms are attenuated with age.     

Stress-induced changes in brain-derived neurotrophic factor expression are attenuated in aged Fischer 344/N rats

Aging and stress can sometimes result in a decline in brain function. We addressed the question whether changes in the expression of neurotrophic factors, which are necessary for the survival and maintenance of neurons, might occur during aging and stress. Therefore, we used in situ hybridization to investigate the effects of aging and stress on neurotrophic factor expression in young (3–4 month) and old (24 month) male Fischer 344/N rats. The ability of acute immobilization stress (2 h) to modulate BDNF mRNA levels in old rats was significantly reduced both in the hippocampus (a smaller decrease in BDNF) and the PVN (a smaller increase in BDNF) compared to young rats. In contrast, the induction of nerve growth factor and neurotrophin 3 (NT-3) by stress was not influenced by age. The diminished BDNF responses to stress in aged rats may be relevant to difficulties in adaptation to stress encountered during old age.     

Aging and cholinergic deafferentation alter GluR1 expression in rat frontal cortex

Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of aged rats compared to young adults. Cholinergic afferents from the NBM modulate glutamatergic transmission in neocortex, and glutamate is known to be involved in dendritic plasticity. To begin to identify possible mechanisms underlying age-related differences in plasticity after NBM lesion, we assessed the effect of cholinergic deafferentation on expression of the AMPA receptor subunit GluR1 in frontal cortex of young adult and aging rats. Young adult, middle-aged, and aged rats received sham or 192 IgG-saporin lesions of the NBM, and an unbiased stereological technique was used to estimate the total number of intensely GluR1-immunopositive neurons in layer II-III of frontal cortex. While the number of GluR1-positive neurons was increased in both middle-aged and aged rats, lesions markedly increased the number of intensely GluR1-immunopositive neurons in frontal cortex of young adult rats only. This age-related difference in lesion-induced expression of AMPA receptor subunit protein could underlie the age-related differences in dendritic plasticity after NBM lesions.     

Age-dependent interactive changes in serotonergic and noradrenergic cortical axon terminals in F344 rats

In the frontal cortex of aging rats, we found an increase in sprouting of the noradrenergic (NA) axons originated from the locus coeruleus (LC). The serotonergic (5-HT) axons originating from the dorsal raphe (DR) share the same cortical area and their age-dependent changes and interactions with NA axons were still unclear. To compare quantitatively the extent of axonal sprouting of DR and LC neurons in the frontal cortex, we extracellularly recorded from both DR and LC neurons in the same animals and antidromically stimulated 32 cortical sites (a pair of stimulating electrodes was moved at 100-mum intervals from 500 to 2000 microm in depth). In addition, to examine the effects of degeneration of 5-HT axons on NA axons, and vice versa, we used specific neurotoxins for 5-HT (PCA) or NA (DSP-4) axons. We also used noradrenaline uptake inhibitor (maprotiline) to verify the effects of NA on degeneration of 5-HT axons. Results suggested that 5-HT axons sprouted between 15 and 17 months of age and noradrenaline accelerated the age-dependent change of 5-HT axons.     

Curcumin reverses corticosterone-induced depressive-like behavior and decrease in brain BDNF levels in rats

A rat model of depression has been recently developed using exogenous corticosterone (CORT) administration. This study aimed to examine the antidepressant-like effect and the possible mechanisms of curcumin in a CORT-induced depression model in rats. The results showed that 3-week CORT injections caused depression-like behavior in rats, as indicated by the significant decrease in sucrose consumption and increase in immobility time in the forced swim test. Repeated CORT injections also significantly decreased brain-derived neurotrophic factor (BDNF) protein levels in the hippocampus and frontal cortex of the rats. Treatment of the rats with curcumin significantly suppressed the depression-like behavior and the decrease in brain BDNF levels induced by the repeated CORT injections. The results suggest that curcumin produces an antidepressant-like effect in CORT-treated rats, which is possibly mediated by increasing BDNF expression in the hippocampus and frontal cortex.     

Sympathetic neuroaxonal dystrophy in the aged rat pineal gland

Dysfunction of circadian melatonin production by the pineal gland in aged humans and rats is thought to reflect the functional loss of its sympathetic innervation. Our ultrastructural neuropathologic studies of the sympathetic innervation of the pineal gland of aged (24 months old) Fischer-344 and Sprague-Dawley rats showed loss of nerve terminals as well as the development of neuroaxonal dystrophy (NAD), an ultrastructurally distinctive distal axonopathy, far in excess of that in young control rats. Immunolocalization of tyrosine hydroxylase confirmed the age-related loss of normal noradrenergic innervation and development of NAD. NAD was more frequent in aged female rats compared to males and was particularly severe in aged female Sprague-Dawley rats compared to Fischer-344 rats. Pineal NGF content was significantly increased or unchanged in female and male aged Fischer-344 rats, respectively, compared to young controls. The rat pineal is a sensitive experimental model for the quantitative ultrastructural examination of age-related neuropathological changes in nerve terminals of postganglionic noradrenergic sympathetic axons, changes which may reflect similar changes in the diffusely distributed sympathetic innervation of other targeted endorgans.     

The MAP kinase phosphatase MKP-1 regulates BDNF-induced axon branching

The refinement of neural circuits during development depends on a dynamic process of branching of axons and dendrites that leads to synapse formation and connectivity. The neurotrophin brain-derived neurotrophic factor (BDNF) is essential for the outgrowth and activity-dependent remodeling of axonal arbors in vivo. However, the mechanisms that translate extracellular signals into the formation of axonal branches are incompletely understood. We found that MAP kinase phosphatase-1 (MKP-1) controls axon branching. MKP-1 expression induced by BDNF signaling caused spatiotemporal deactivation of c-jun N-terminal kinase (JNK), which reduced the phosphorylation of JNK substrates that destabilize microtubules. Indeed, neurons from mkp-1 null mice could not produce axon branches in response to BDNF. Our results identify a signaling mechanism that regulates axonal branching and provide a framework for studying the molecular mechanisms of innervation and axonal remodeling under normal and pathological conditions.     

Age-dependent effect of cholinergic lesion on dendritic morphology in rat frontal cortex

Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: 3 months after surgery, excitotoxic lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of aged rats relative to young adults. To determine whether the differential effect of the lesion was due specifically to loss of cholinergic input from the NBM, we assessed dendritic morphology in frontal cortex after specific cholinergic depletion in young adult, middle-aged, and aged male rats. Rats received unilateral sham or 192-IgG-saporin lesions of the NBM. Two weeks after surgery, brains were stained using a Golgi-Cox procedure. Dendritic morphology was quantified in pyramidal neurons in layers II-III of frontal cortex. Although lesions altered apical dendrites at all ages, these effects were most pronounced in aged rats. In addition, lesions produced marked atrophy of basilar dendrites in middle-aged and aged rats only. Thus, the differential dendritic atrophy resulting from NBM lesions in aged rats occurs within 2 weeks after lesion, and results specifically from loss of cholinergic innervation.     

Expression of mRNA of neurotrophic factors and their receptors are significantly altered after subchronic ketamine treatment

The neurotrophic factors play an important role in the maintenance of neurone viability and neuronal communication which are considered to be altered in schizophrenia. Subchronic application of ketamine (Ket) was found to be a useful model in schizophrenia research. To further validate this model the mRNA levels of neurotrophic factors NGF, NT-3, and BDNF and their receptors TrkA, TrkB, and TrkC, respectively, were measured in different brain areas in Ket-pretreated rats subchronically dosed with the atypical antipsychotic drug risperidone (Ris). With the exception of NGF in the frontal cortex, Ket pretreatment did change NGF, NT-3, and BDNF mRNA levels in the frontal cortex, the hippocampus, the striatum, the thalamus/hypothalamus region, and in the cerebellum. These changes correspond with changes at their tyrosine kinase receptors. Ris treatment normalised altered NT-3 levels in the hippocampus and balanced BDNF levels in the same structure. It was concluded that the Ket model might reflect distinct alterations in neurotrophic factor activity as found in schizophrenic patients and, moreover, that Ris treatment rebalances disturbed neurotrophic factor activity.     

Evidence for up-regulation of brain-derived neurotrophic factor mRNA by soy phytoestrogens in the frontal cortex of retired breeder female rats

In this study, we examined the effects of oral micronized estradiol and soy phytoestrogens on the mRNA levels of brain-derived neurotrophic factor (BDNF) in the frontal cortex of ovariectomized retired breeder female rats. Fifteen ovariectomized rats were randomized into three groups and fed either a soy-free control diet (OVX), a control diet with added soybean estrogens (SBE), or a control diet with added estradiol (E2) for 8 weeks. Frontal cortex samples were collected for RNA isolation. Northern and Phosphorlmager analyses were used to determine the relative levels of BDNF mRNA. Both estradiol and soy phytoestrogens significantly increased the mRNA levels of BDNF compared to ovariectomized controls, suggesting that soy phytoestrogens may act as estrogen agonists in regulating BDNF mRNA in the frontal cortex of retired breeder female rats.