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.     

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.     

BDNF is necessary for maintenance of noradrenergic innervations in the aged rat brain

In the axon terminals of the locus coeruleus (LC) neurons, a high level of axonal branching was occurred in the middle-aged brain, and the increased branching was maintained in the aged brain. In the present study, we hypothesized that neurotrophic support is necessary for the morphological age-related changes seen in the noradrenergic innervations from the LC to frontal cortex. Through immunohistochemical and quantitative image analyses, we examined the age-dependent effects of brain-derived neurotrophic factor (BDNF) on the noradrenergic axon terminals in the frontal cortex of F344 rats. We continuously infused BDNF into the frontal cortex of young (6-months-old), middle-aged (13-months-old), or aged (25-months-old) rats. Exogenous BDNF infusion caused a marked increase in the density of noradrenergic axons in the aged brain, but no trophic action of BDNF was observed in the young and middle-aged brain. Neutralization of endogenous BDNF with a specific function-blocking antibody to BDNF led to a reduction in noradrenergic axons in the frontal cortex of 19-month-old rats. The present results suggest that BDNF is not involved in the augmentation of noradrenergic innervations in the aging brain, but it is necessary for the maintenance of noradrenergic innervations in the aged brain.     

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.     

Age-dependent changes in dopaminergic projections from the substantia nigra pars compacta to the neostriatum

Age-dependent changes in dopaminergic (DA) innervation of the neostriatum (Str) were studied in male F344/N rats. Projections from the substantia nigra pars compacta (SNc) to the neostriatum were quantified using electrophysiological methods at age points from 6 to 24 months. The percentage of DA neurons activated antidromically by electrical stimulation (P-index) of Str increased between 18 and 24 months. Additionally, the percentage of DA neurons showing multiple antidromic latencies from striatal stimulation (M-index), which suggests axonal branching of individual DA neurons, increased significantly between 6 and 12 months and 6 and 24 months. These results suggest that DA neurons exhibit increased axonal branching in the aged brain.     

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-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-dependent changes in axonal branching of single locus coeruleus neurons projecting to two different terminal fields

Age-dependent changes in the axonal branching patterns of single locus coeruleus neurons, which innervate both the frontal cortex and hippocampus dentate gyrus, have been studied in male F344 rats. We used an electrophysiological approach involving antidromic activation to differentiate single from multi-threshold locus coeruleus neurons in each terminal field with age (7-27 mo of age). Most of these neurons have a single threshold in the young rats, whereas in the older brains, the neurons have multi-threshold responses. This implies an increased amount of axonal branching in the older brains. The time course of the increase differs in the two terminal fields, suggesting that the degree of plasticity or age-dependent increase in branching can differ across terminal fields.     

Duration-dependent effects of repeated restraint stress on cortical projections of locus coeruleus neurons

Using electrophysiological techniques, changes in noradrenergic fiber innervation in the cerebral cortex following repeated stress (restraint in a small cage for either 1 or 6 h daily) were examined by quantifying the density of cortical terminal axons of locus coeruleus (LC) neurons in the rat. After termination of the stress treatment, the single-unit activity of LC neurons was recorded extracellularly under urethane anesthesia, and antidromic activation from 7 cortical points covering nearly the entire cerebral cortex was examined. The percentage of LC neurons activated anti-dromically from the medial frontal cortex was higher in the animals stressed for 1 h daily for 2 weeks. In contrast, the percentage of LC neurons activated antidromically from the cerebral cortex decreased in the animals who received 6 h stress for 2 weeks. These results suggest that stress can cause dual effects, either sprouting or retraction of cortical LC axons depending upon the duration of stress treatment.     

Endogenous glutamate release from frontal cortex of adult and aged rats

Glutamate (GLU) is a major excitatory neurotransmitter in the frontal cortex. Alterations in GLU neurotransmission are present in a number of neurodegenerative diseases, however, little is known about the normal aging process of GLU utilizing neurons. GLU release, uptake and content were examined in the frontal cortex of adult (6 months old) and aged (24 months old) male, Fisher 344 rats. These markers were used to assess the functional integrity of intrinsic and extrinsic GLU utilizing pathways innervating the frontal cortex. Basal- and potassium- (56 mM) evoked GLU release from brain slices of aged rats were not significantly different from that of adults. Kainic acid (1.0 mM) failed to significantly augment basal or potassium-stimulated GLU release in the frontal cortex of either aged or adult rats. Uptake of [³H] GLU into brain slices was also unaltered as a function of age. In contrast, GLU content was decreased 17% in the frontal cortex of aged rats when compared to the adults. These results suggest that the functional integrity of GLU utilizing nerve terminals in the frontal cortex is maintained in 24-month-old Fisher 344 rats. The decrease in GLU content may reflect a generalized neuronal loss or a defect in neuronal and/or glial GLU metabolism in the metabolic compartment.     

Changes in the electrical activity of locus coeruleus neurons in pregnant rats

Under urethane anesthesia, unit activity of locus coeruleus (LC) neurons was recorded extracellularly in pregnant and non-pregnant rats. The spontaneous firing of LC neurons was found to be reduced in pregnant rats. In addition, biochemical studies indicated that noradrenaline contents in the cerebral cortex, one of the major projection sites of LC neurons, were significantly higher in pregnant than non-pregnant rats. These results suggest that the electrical activity of noradrenergic neurons in the LC is reduced during gestation.     

The distribution of dopamine-beta-hydroxylase, neuropeptide Y and galanin in locus coeruleus neurons

The locus coeruleus (LC) is composed of noradrenaline-producing neurons that project widely throughout the neuraxis. Subpopulations of LC neuron perikarya have been shown to contain neuropeptide Y (NPY) and galanin (GAL). In the major terminal fields of LC projections, the cerebral cortex, dorsal thalamus and cerebellar cortex, there are differing plexuses of dopamine-beta-hydroxylase (DBH), NPY and GAL immunoreactive axons. DBH immunoreactive plexuses are found in all areas which conform in appearance to previous demonstrations of noradrenaline localization by fluorescence histochemistry. In contrast, there are few NPY immunoreactive axons in thalamus and cerebellum, and the cortical plexus, while similar to the DBH immunoreactive plexus, is not affected by 6-hydroxydopamine treatment. Similarly, there are few GAL immunoreactive axons in either cerebral cortex, dorsal thalamus or cerebellar cortex. Transection of ascending LC axons results in accumulation of DBH but not NPY or GAL immunoreactivity proximal to the lesion. These observations indicate that NPY and GAL are distributed differently in LC neurons from noradrenaline and DBH.     

Quantitative Changes in the Fibroarchitectonics of the Human Cortex from Birth to the Age of 12 Years

Brains from 74 children aged from birth to 12 years were used to study fibroarchitectonic characteristics in topographically and functional diverse cortical zones (the temporal-parietal-occipital subregion, occipital, precentral, postcentral, and frontal areas) of the cerebral cortex; children aged up to 12 months were studied on the basis of one-month age intervals. Studies were performed by computer analysis of optical images on frontal sections stained with Nissl cresyl violet and silver nitrate impregnation by the modified Peters method. Data on the rate of increase in the thickness of radial bundles of fibers, the distances between bundles, and the age dynamics of the ratios of the specific volumes of neurons and fibers in fields 3, 4p, 6op, 17, 19, 37ac, 44, and 32/10 were obtained. These measurements showed that age-related transformation of fibroarchitectonics in fields of the sensorimotor, somatosensory, occipital, temporal-parietal-occipital, and frontal areas occurred at different times and with different intensities; the most significant changes were seen in the first 2-3 years of life, with changes continuing at a less intense level to age 9-12 years.     

神经元型一氧化氮合酶在快速衰老小鼠额叶皮质中的增龄性改变

为了观察快速衰老小鼠(senescence accelerated mouse,SAM)衰老过程中大脑额叶皮质中nNOS的分布和表达变化,探讨NO/nNOS在中枢神经系统衰老中的作用。采用雄性快速衰老亚系8小鼠(senescence accelerated mouse/prone8,SAMP8)及抗快速衰老亚系1小鼠(senescence accelerated mouse/resistance1,SAMR1)为研究对象,其中SAMP8为实验组,SAMR1为对照组,每组动物再分为青年组(2月龄)和老年组(10月龄)两组。用免疫组织化学方法观察SAM额叶皮质中的nNOS神经元的形态和分布,并计数nNOS阳性神经元在额叶皮质中的数量;用RT-PCR法检测额叶皮质中nNOS mRNA表达水平。结果显示:SAMP8老年组与青年组相比,额叶皮质中nNOS阳性神经元的数量显著增加(15.8±6.3vs8.0±4.9,P<0.05);SAMP8与SAMR1比较,青年组额叶皮质nNOS阳性神经元的数量差异无统计学意义,老年组额叶皮质nNOS阳性神经元的数量显著增加(15.8±6.3vs7.5±5.3,P<0.05)。SAMP8老年组额叶皮质nNOS mRNA水平明显高于SAMP8青年组(1.00±0.17vs0.67±0.13,P<0.01)和老年组SAMR1(1.00±0.17vs0.67±0.11,P<0.01)。以上结果提示:额叶皮质中nNOS神经元的数量增加可能产生过量NO,NO可能参与了SAMP8快速衰老的过程。本研究的结果为通过调节额叶皮质NO产量来延缓衰老及衰老相关功能障碍提供了依据。     

Ovariectomy up-regulates neuronal neurofilament light chain mRNA expression with regional and temporal specificity

Estrogens can influence the survival, plasticity and function of many adult neurons. Many of these effects, such as neurite outgrowth and increased dendritic spine density, are mediated by changes in neuronal cytoskeletal architecture. Since neurofilament proteins play a key role in the maintenance and remodeling of the neuronal cytoskeleton, we postulated that changes in neurofilament light chain mRNA may parallel some of the alterations in neuronal architecture which follow bilateral ovariectomy. We measured neurofilament light chain mRNA levels using a ribonuclease protection assay at two time-points after ovariectomy in mature female rats. One week after ovariectomy, neurofilament light chain mRNA levels (corrected for glucose-6-phosphate dehydrogenase mRNA) did not differ from sham-operated animals in the five brain regions examined (hypothalamus, striatum, hippocampus, frontal cortex and occipital cortex). Four months after ovariectomy, neurofilament light chain mRNA levels were similarly unchanged in the hypothalamus and striatum. In contrast, statistically significant increases in neurofilament light chain mRNA expression were observed in the three regions receiving basal forebrain projections (hippocampus, frontal cortex and occipital cortex). In situ hybridization demonstrated increases in neurofilament light chain mRNA expression involving subpopulations of smaller medial septal neurons. There also appeared to be an increased number of larger septal neurons following long-term ovariectomy.We propose that atrophic changes involving basal forebrain projection fibers are followed by compensatory axonal growth by other 'intact' basal forebrain neurons. Increased neurofilament light chain mRNA expression and somatic hypertrophy in medial septal neurons may both be reflective of the need to sustain an axonal network which is larger and more complex. In contrast, increased neurofilament light chain mRNA expression observed in basal forebrain targets following long-term ovariectomy may be reflective of compensatory changes taking place in local neurons.     

Age-correlated changes in dopaminergic nigrostriatal perikarya of the C57BL/6NNia mouse

Alterations in neurotransmitter systems of the basal ganglia have been postulated to contribute to the disruption of motor function and balance associated with aging. This study examined nigrostriatal (A9) and mesolimbic (A10) dopamine neurons for qualitative age-correlated changes using fluorescence histochemistry for catecholamines and immunocytochemical techniques for the catecholamine-synthesizing enzyme, tyrosine hydroxylase. Results from this study suggest that age-correlated morphological changes in A9 but not all A10 neurons in the midbrain are present in mature adult (10-month) C57BL/6NNia mice and show a progressive increase in severity until at least 30 months of age. These changes are characterized by a progressive accumulation of lipofuscin in dopamine-containing perikarya, a markedly reduced dopamine content per cell as determined visually by histofluorescence, and an increase in the number of large, fluorescent axonal dilations in dopamine-containing fibers of the mesolimbic and nigrostriatal systems. These data suggest that heterogeneous morphological aging patterns exist within dopamine-containing neurons of the midbrain and that based upon their terminal projection sites, various regions of the striatum and cortex may be differentially affected in the aged brain. In addition, these findings support the belief that age-related changes in neural structure are not generalized to an entire brain nucleus or cell type but are selective for individual cells within an affected area.     

Anatomical organization of the eye fields in the human and non-human primate frontal cortex

There are several eye fields in the primate frontal cortex. The number and location of these oculomotor control zones remain controversial, especially in the human brain. In the monkey, the frontal eye field (FEF) is located in the rostral bank of the arcuate sulcus at approximately the level of the posterior end of the sulcus principalis, the supplementary eye field (SEF) is located on the dorsomedial frontal cortex, and the cingulate eye field (CEF) in the dorsal bank of the cingulate sulcus. In the human frontal cortex, the location of the FEF varies depending on the method used, electrical stimulation or functional neuroimaging, to establish it. Some investigators have argued that the SEF is located on the medial wall of the frontal lobe but its presumed location remains controversial. The location of the CEF in the human brain is not known. The present article reviews electrophysiological and functional neuroimaging evidence regarding the location of these frontal oculomotor areas in the macaque monkey and human brains and, in light of new findings in the human brain, attempts to reconcile the differences observed in the location of these eye fields using the different techniques. Together, these data suggest the existence of at least four eye fields in the frontal cortex, i.e. the FEF, the SEF, the CEF, and a premotor eye field, and suggest that their anatomical relationships are preserved from monkey to human brain.     

Localization of cellular changes within multimodal sensory regions in aged monkey brain: Possible implications for age-related cognitive loss

Young (4 to 7 years) and aged (18 to 28 years) rhesus monkeys were sacrificed and various neuromorphometric analyses performed to determine age differences in gross topography, cell population and patterns of cellular degeneration. Two brain regions implicated for their role in age-related cognitive disturbances, the hippocampus and the gyri bordering the principal sulcus in the frontal cortex were selected for these comparisons. Reliable morphometric differences between age groups were observed in both neural areas. One significant difference observed in the hippocampus was a reduced mean depth of the pyramidal layer of the CA-1 zone in the aged monkeys. Also, the mean number of neurons per transverse section in the CA-1 zone of the pyramidal layer was significantly less in the aged monkeys, and in certain instances cell gaps were observed in this region. In the lateral principal gyrus of the frontal cortex, the number of neurons in full-depth “cores” was lower in the old monkeys, glial count was higher and the ratio of neurons to neuroglia, therefore, lower in the older monkeys. Further, the mean area of the principal gyri, measured planimetrically from the apex of the medial gyrus to that of the lateral gyrus was significantly smaller in the aged monkeys. These findings indicate that significant age-dependent cellular differences occur in brain areas thought to be functionally involved in the particular cognitive behaviors most severely impaired in aged monkeys. These differences in brain morphology may, therefore, help provide some leads into the types of neurological changes contributing to the severe cognitive disorders suffered by the elderly.     

Non-dopaminergic catecholaminergic neurons of mesencephalic and medullary nuclei contain different levels of dopamine

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within nondopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltansferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the nomdrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.     

The mode of projections of single locus coeruleus neurons to the cerebral cortex in rats

Axonal distributions of single locus coeruleus neurons within the cerebral cortex were examined with antidromic stimulation technique combined with cortical lesions (frontal lobotomy and lobectomy). In urethan-anesthetized rats, stimulating electrodes were implanted in 10 points extending over nearly the entire cerebral cortex, and antidromic responses of single locus coeruleus neurons to stimulation of these stimulus sites were analysed. Fifty percent of locus coeruleus neurons examined were activated antidromically from at least one cortical point in the cerebral cortex. The pattern and extent of axonal distributions of single locus coeruleus neurons in the cortex appeared to vary from cell to cell. From the results obtained in rats with the cortical lesions, it is concluded that in addition to locus coeruleus neurons with intracortical axons running from rostral to caudal, there are the neurons projecting to the occipital cortex without innervating the frontal cortex and those projecting simultaneously to the frontal and occipital cortex with two axonal branches. There was no topographic order between the recording sites within the locus coeruleus and the projection sites in the cortex.