Glial cell differentiation in neuron-free and neuron-rich regions

The proliferative cells of the developing hippocampal fiber tract fimbria have only the potential for gliogenesis; thus the developing fimbria provides an ideal model for the study of the development and differentiation of its constituent glial cells. In the first stage of development, the fimbrial primordium can be distinguished morphologically, and during the second stage, the fimbria becomes a well-defined fiber tract. In the third stage, a divergent immunocytochemical staining pattern clearly demarcates the neuron-free fimbria from the hippocampus, where a mixed neuro- and gliogenesis occurs. The distinct expression of S-100 protein in radial glial cells is restricted to the fimbria. During the final stage of development, the ventricular lining of the fimbria will mature into an ependyma. It is suggested that the S-100-positive radial glial cells of the fimbria, which probably retain their proliferative capacity, represent a homogeneous population of precursor cells that will give rise to the glial cells of the adult fimbria. The appearance of S-100 in the fimbrial radial glial cells seems to occur coincidentally with the establishment of hippocampal commissural connections. The S-100-positive radial glial cells of the fimbria may guide and segregate populations of growing axons by providing physical and chemical cues. Thus, S-100 protein per se seems to be intimately involved in modulation and regulation of axonal growth and patterning.     

Glial cell line-derived neurotrophic factor in the substantia nigra from control and parkinsonian brains

Glial cell line-derived neurotrophic factor (GDNF) was measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme-linked immunosorbent assay. In both groups, the levels of GDNF in the various brain regions were lower (pg/mg protein) than those of brain-derived growth factor (ng/mg order), and were significantly higher in the nigro-striatal dopaminergic regions (substantia nigra, caudate nucleus, putamen) than in the cerebellum and frontal cortex (P<0.05). However, the content of GDNF in the dopaminergic regions showed no significant difference between parkinsonian and control patients.     

Synapses in the granule cell layer of the rat dentate gyrus: serial-sectioning study

Synaptic contacts on the granule cell somata as well as on their axon initial segments in the dentate gyrus of one juvenile 5-week-old rat and one adult 12-week-old rat were analyzed in an electron microscopic serial-sectioning study. In the dentate gyrus of the juvenile rat, somata of 17 granule cells were nearly completely reconstructed from a series of 183 serial sections, and the axon initial segments of 15 of these granule cells were traced in various lengths. On the other hand, in the dentate gyrus of the adult rat, somata of 31 granule cells were almost completely reconstructed from a series of 238 serial sections, and the axon initial segments of 23 of these granule cells were traced in various lengths. Both symmetrical and asymmetrical synapses were observed on the somata, whereas almost all synapses on the axon initial segments were of symmetrical type and asymmetrical synapses on the axon initial segments were rather exceptional. Although we confirmed two conclusions from previous random-section studies to some extent, that is, the superficial-to-deep gradient of synaptic densities on granule cell somata and the presence of a substantial number of asymmetrical synapses on granule cell somata (about 23% of total somatic synapses), the present serial-sectioning study clearly revealed that granule cells vary greatly with regard to the number of synapses on their somata (15–186 in a 5-week-old rat and 9–144 in a 12-week-old rat) and axon initial segments. The granule cells also differed in the proportion of somatic asymmetrical synapses to total synapses they received (0–44% in a 5-week-old rat and 0–60% in a 12-week-old rat). The results of the present study indicated that, when a relatively small number of granule cells are analyzed, one should take the heterogeneity of synaptic contacts on granule cells in number and type into consideration.     

Region and lamina-specific distribution of extracellular matrix proteoglycans,hyaluronan and tenascin-R in the mouse hippocampal formation

The extracellular matrix is known to show region-specific characteristics in the adult brain. Our comparative cytochemical study is focused on the laminar organisation of major extracellular matrix constituents in the murine hippocampal formation, including the regions CA1, CA2 and CA3 of the hippocampus proper, the dentate gyrus, the subiculum and the presubiculum. Components related to chondroitin sulphate proteoglycans were detected by N-acetylgalactosamine-binding Wisteria floribunda agglutinin, colloidal iron staining, and antibodies to different proteoglycan domains, including the Cat-301 and Cat-315 epitopes of aggrecan, as well as neurocan, brevican and phosphacan. The distribution patterns of these components were correlated with the patterns revealed for hyaluronan and the brain-specific extracellular matrix glycoprotein, tenascin-R, known to be ligands of extracellular matrix proteoglycans. Lectin binding clearly labelled perineuronal nets of the extracellular matrix around interneurons, which were preferentially located within or near the principal cell layers in all regions. In the hippocampus proper, the CA2 subfield showed an intense labelling of the neuropil around pyramidal cell bodies and the neuropil zones in the strata oriens and radiatum. These patterns were also seen after immunoreaction for chondroitin proteoglycan domains, brevican and phosphacan, as well as after detection of hyaluronan and tenascin-R. Characteristic laminar and intralaminar patterns were additionally expressed in the neuropil in all regions. In the dentate gyrus, the staining intensity for brevican, phosphacan and tenascin-R was predominant in the middle molecular layer, and for Cat-315 in the inner molecular layer, whereas immunoreactivity for neurocan increased within the outer molecular layer towards the hippocampal fissure. Our findings indicate that proteoglycans, hyaluronan and tenascin-R show differential patterns of co-expression in the individual regions and laminae of the hippocampal formation. The inhomogeneous composition of these major components suggests that the extracellular matrix is specifically adapted to the functional domains of intrahippocampal connections and afferent fibre systems.     

Molarless condition suppresses proliferation but not differentiation rates into neurons in the rat dentate gyrus

The dentate gyrus (DG) of the hippocampal complex is one of the few areas of the rodent brain where neurogenesis continues throughout adulthood. We investigated the effects of the molarless condition on cell proliferation, rate of differentiation into neurons in the subgranular zone of the DG, and plasma corticosterone levels. The molarless condition decreased cell proliferation in the DG and increased plasma corticosterone levels. Approximately 80% of newly generated cells differentiated into neurons and the remaining 20% of the cells differentiated into astrocytes. These ratios were not significantly different between control and molarless rats. In conclusion, the rates of neurogenesis and gliogenesis in the DG are suppressed by the molarless condition, and this suppression might be associated with the increased corticosteroid levels in molarless subjects.     

A study of glial cell proliferation in the molecular layer of the dentate gyrus of the rat following interruption of the ventral hippocampal commissure

Summary The proliferation of glial cells in the molecular layer of the dentate gyrus in response to lesions of the ventral hippocampal commissure, has been studied autoradiographically following intraventricular injections of ³H-thymidine. Within 24h of commissurotomy there is an appreciable increase in the number of labeled cells throughout the molecular layer which reaches its peak at approximately 36h. This generalized glial hyperplasia persists for at least 5–6 weeks and there does not appear to be a secondary re-distribution of the newly-generated glial cells as has been reported after entorhinal lesions (Gall et al., 1979). In semi-thin plastic sections most of the proliferating cells more closely resemble the medium-shade oligodendrocytes of Ling et al. (1973) than typical microglia; the reactive astrocytes do not appear to participate in the glial proliferation.This work was supported in part by grants NS-10943 from the NINCDS and DA-00259 from ADAMAH, and was carried out while C.A. was on leave of absence from the Department of Morphology, Autonoma University School of Medicine, Madrid, and was in receipt of a Fogarty International Fellowship (1F05 TWO 2580)     

出生前人脑齿状回颗粒细胞的梯度发育：钙结合素免疫细胞化学研究

为了探索人类齿状回颗粒细胞的出生前发育规律，用免疫细胞化学方法研究了１３～３８周人胎齿状回钙结合素（ＣＢ）阳性颗粒细胞的分布和发育。结果发现，在发育过程中，齿状回外肢的颗粒细胞最早表达ＣＢ，而内肢的颗粒细胞最晚表达ＣＢ；ＣＢ阳性颗粒细胞的数量及染色深度按先外肢后内肢的顺序逐渐增加。然而，ＣＢ阳性颗粒细胞的这种梯度分布在３８周时已不明显。本实验结果提示，出生前人脑齿状回各部位颗粒细胞并不是均匀地发育，而是呈现梯度发育     

大鼠创伤性脑损伤后海马齿状回中脑脂结合蛋白的表达变化

Objective To investigate the change of brain lipid binding protein(BLBP) expression in hippocampus dentate gyrus(DG) at different time points after traumatic brain injury (TBI). Methods Seventy-two rats were divided into the injured group, sham group and control group randomly, and then were subjected to a lateral fluid percussion injury (FPI). Western blotting was used to detect BLBP expression. Brains were sectioned for immunofluorescence staining of BLBP and Vimentin at the time points of 1, 3, 7, 14 days after TBI.Results The results of Western blotting showed that the BLBP expression was lower than that of control group at 1 day post injury（EM>P/EM><0.01） and reached the peak compared with the other groups at 7 days after injury（EM>P/EM><0.01）, then descended at 14 days compared with control group after injury（EM>P/EM><0.01）. The changes of BLBP and Vimentin double-label positive cells were consistent with the results of Western blotting. The BLBP and Vimentin double-label positive cells were found mainly at the subgranular zone of ipsilateral injured hippocampus DG, and most of them were radial glia like cells; BLBP and Vimentin double-labelled positive cells were found at the hilus of DG at 7days after injury, and most of them looked like reactive astrocytes. Conclusion The expression of BLBP in DG after TBI decreased firstly, then increased and reached peak at 7 days after injury, decreased dramatically again at last. The     

Glial cell line-derived neurotrophic factor does not enter normal mouse brain

Glial cell line-derived neurotrophic factor (GDNF) is produced both in the central nervous system (CNS) and the periphery. Effective in ameliorating neurodegeneration in several animal models of CNS disease, its promise as a therapeutic agent would be greatly enhanced if it readily crossed the blood–brain barrier (BBB) in unmodified form. Here, we used the sensitive techniques of multiple-time regression analysis and ex-vivo perfusion in blood-free buffer to examine the entry of ¹²⁵I-GDNF into mouse brain. The integrity of GDNF in blood and brain was examined by high performance liquid chromatography and the physicochemical properties determining permeability were measured by octanol/buffer partition coefficient and hydrogen bonding. The efflux of ¹²⁵I-GDNF was determined to test for the presence of a bidirectional transport system. The results show that ¹²⁵I-GDNF differs from other peptides and polypeptides in that it does not enter brain any faster than ^99mTc-albumin, an effect that cannot be explained by degradation, rapid efflux, protein binding, or inadequate lipophilicity. Thus, GDNF shows a different type of interaction with the BBB. In normal mice, the BBB functions as a substantial physical barrier; in pathological or traumatic situations when the barrier is partially disrupted, the lack of restriction by a saturable transport system could make GDNF a suitable candidate for peripheral delivery in promoting neuroregeneration.     

Stereological cell counts of GABAergic neurons in rat dentate hilus following transient cerebral ischemia

We have previously demonstrated a 60-80% ischemic loss of somatostatinergic neurons in the dorsal dentate hilus of the rat. However, several studies have failed to demonstrate ischemic loss of GABAergic neurons in hilus, although one study reports that 96% of the somatostatinergic neurons in the dorsal hilus colocalize GABA. In order to understand this paradox, we have now estimated, using unbiased stereology, the total number of neurons immunohistochemically stained against glutamic acid decarboxylase-65 (GAD65) and GAD67 in the dorsal dentate hilus. Rats were divided into groups subjected to either sham operation (n=8) or 8 min of transient global ischemia during systemic hypotension (n=8) and allowed to survive for 7-9 days. Results from cell counts (mean +/- SD) in sham rats demonstrated that the dorsal hilus contains 9,189+/-3,957 GAD65 neurons and 6,991+/-2,784 GAD67 neurons. After ischemia, corresponding cell counts demonstrated 10,216+/-4,866 GAD65 neurons and 10,119+/-5,906 GAD67 neurons, and these results were not significantly different (P>0.05) from results in sham rats. Power analysis of the t-test informs that losses less than 80% are not significant and reflects the excessive variance in our material. For comparison, we estimated a total of 21% ischemic neuron death in the dorsal hilus on cresyl violet-stained sections from other corresponding sham (n=7) and ischemic rats (n=7). This explains why ischemic loss of hilar GABAergic neurons can only be detected by counts of the vulnerable subpopulation colocalizing somatostatin. Our investigation has demonstrated a surprisingly high variation between rats in a number of GAD-immunopositive neurons located in the dorsal dentate hilus, which is related to variations between the individual rats and neurons in their endogenous GAD expression.     

Enhanced acoustic startle responding in rats with radiation-induced hippocampal granule cell hypoplasia

Summary Irradiation of the neonatal rat hippocampus reduces the proliferation of granule cells in the dentate gyrus and results in locomotor hyperactivity, behavioral perseveration and deficits on some learned tasks. In order to address the role of changes in stimulus salience and behavioral inhibition in animals with this type of brain damage, irradiated and normal rats were compared in their startle reactions to an acoustic stimulus. A portion of the brain of 10 rats was exposed to a fractionated total dose of 13 Gy during the first 16 days post partum. This procedure produced selective hypoplasia (91% reduction) of the granule cells in the hippocampal dentate gyrus. Other rats (N = 10) were sham irradiated. Sudden tones were presented to each adult rat at a rate of 1 every 30 s (spaced trials) during an initial 10-min session and 1 every 15 s (massed trials) during a subsequent session. Irradiated rats startled with a consistently higher amplitude than controls and were more likely to exhibit startle responses. These animals with hippocampal damage also failed to habituate to the startle stimulus and, under certain circumstances, showed potentiated startle responses after many tone presentations.     

Potentiation of excitatory synaptic transmission in the normal and in the reinnervated dentate gyrus of the rat

Summary Following destruction of the ipsilateral temporo-ammonic tract, which originates in the entorhinal cortex, and terminates on the granule cells of the dentate gyrus, fibers from the surviving contralateral entorhinal area proliferate forming extensive new connections with the denervated dentate granule cells. Utilizing extracellular recording techniques, we have compared the characteristics of synaptic transmission in the lesion induced afferents with the characteristics of the normal ipsilateral afferents by analyzing the responses of dentate granule cells to paired pulse activation of temporo-dentate circuitry.In the dentate gyrus of the normal rat, an extracellularly recorded EPSP evoked by stimulation of the ipsilateral entorhinal cortex is enhanced by as much as 100% by a conditioning pulse to the same afferent system. This is called paired pulse potentiation. In the reinnervated dentate gyrus, the extracellular EPSP evoked by a test stimulus delivered to the contralateral entorhinal cortex is also potentiated by a conditioning pulse. The paired pulse potentiation in the reinnervated dentate gyrus has a time course which is comparable to that of the normal ipsilateral afferent system, but the magnitude of the potentiation is somewhat less, averaging approximately 140% of control.A second manifestation of paired pulse potentiation in the normal ipsilateral temporo-dentate circuit is that more granule cells discharge in response to the second of a pair of stimuli. Potentiation of granule cell discharge, as measured by the increase in the size of the population spike in the test response, may be as much as 500% of control at the optimal interstimulus interval. In the operated animals, however, paired pulse stimulation of the lesion induced crossed temporo-dentate circuit results in little, if any, enhancement of granule cell discharge in response to the second stimulus, despite the fact that theextracellulrly recorded EPSP is potentiated.These results are discussed in relation to the similarity between the normal and the lesion induced afferents to the dentate cells, with consideration for the normal functioning of the circuit from the entorhinal cortex to the dentate gyrus.Some of the material in this paper was included in a dissertation submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at the University of California at Irvine, Irvine, California 92717 (1974).     

Age-related effects on hippocampal precursor cell subpopulations and neurogenesis

Hippocampal neurogenesis continuously declines in the aging brain but only little is known about age-related alterations in the subgranular zone (SGZ) of the dentate gyrus which accommodates different subpopulations of precursor cells. Here, we examined the age-related effects on total number and proliferation rate of distinct precursor cell populations in the dentate gyrus of 3 and 16 months old transgenic pNestin-GFP mice. Following a single injection of bromodeoxyuridine (BrdU) we observed a significant reduction of all proliferating precursor subtypes in aged mice compared to young controls. Stereological analysis further revealed that this decreased proliferation was not only caused by a general reduction in total number of precursor subtypes but also by a subtype-specific alteration of the proliferation rate. Whereas radial glia-like and early neuronal precursor cells demonstrate decreased proliferation rates, no difference was found for doublecortin-positive precursors. Additional long-term experiments further revealed that these age-related alterations in the proliferative zone were accompanied by a strongly decreased neurogenesis while hippocampal function was not impaired.     

A novel monoclonal antibody recognizes lysosome-like structures and reflects regional and age-related differences in the rat dentate gyrus

The granule cells (GCs) of dentate gyrus exhibit regionally specific morphology, and continue to be born and to develop well into adult life. We used a novel monoclonal antibody, MAb2G7, elicited by immunization of a mouse with a microsome fraction of the hippocampus, to evaluate regional and age-related differences in GCs immunohistochemically. Weak cytoplasmic reactions were observed in many neurons, but intense MAb2G7-positive dots were observed only in GCs. Using electron microscopy, we observed that these dots were localized in the internal droplets of secondary lysosome-like structures in GCs. The MAb2G7-positive granules were quantitatively analyzed in young adult and middle-aged rats. Larger numbers of reactive granules were observed in the infrapyramidal blade (IPB) than in the suprapyramidal blade (SPB) and the numbers of positive granules were proportionally reduced in the two areas in middle-aged rats. The changes in the MAb2G7 immunoreactivity may reflect different activation or neurogeneration of GCs in the IPB versus the SPB, and in middle-aged versus young adult rats.     

Sustained increase in adult neurogenesis in the rat hippocampal dentate gyrus after transient brain ischemia

It is known that the number of newly generated neurons is increased in the young and adult rodent subventricular zone (SVZ) and dentate gyrus (DG) after transient brain ischemia. However, it remains unclear whether increase in neurogenesis in the adult DG induced by ischemic stroke is transient or sustained. We here reported that from 2 weeks to 6 months after transient middle cerebral artery occlusion (MCAO), there were more doublecortin positive (DCX+) cells in the ipsilateral compared to the sham-control and contralateral DG of the adult rat. After the S-phase marker 5-bromo-2'-deoxyuridine (BrdU) was injected 2 days after MCAO to label newly generated cells, a large number of BrdU-labeled neuroblasts differentiated into mature granular neurons. These BrdU-labeled neurons survived for at least 6 months. When BrdU was injected 6 weeks after injury, there were still more newly generated neuroblasts differentiated into mature neurons in the ipsilateral DG. Altogether, our data indicate that transient brain ischemia initiates a prolonged increase in neurogenesis and promotes the normal development of the newly generated neurons in the adult DG.     

Decreased levels of disrupted-in-schizophrenia 1 (DISC1) are associated with expansion of the dentate granule cell layer in normal and kindled rats

Disrupted-in-schizophrenia 1 (DISC1) is a candidate gene involved in the pathogenesis of schizophrenia. DISC1 expression is particularly abundant in the adult dentate gyrus, in which decreased levels lead to aberrant growth, impaired migration, and accelerated integration of adult generated neurons. Because seizures can also result in similar changes, we tested the hypothesis that DISC1 expression may be altered in an animal model of epilepsy. We found that extended amygdala kindling (i.e., 99-electrical stimulations) significantly decreased DISC1 labeling in the dentate granule cell layer and subgranular zone. Extended kindling also led to an increase in the number of ectopic granule cells in the hilus. In addition, although the width of the granule cell layer was not generally affected by kindling, decreased levels of DISC1 in the subgranular zone and granule cell layer were associated with an expansion of the upper blade and crest of the dentate gyrus in both normal and kindled rats. These novel findings suggest that seizure activity affects DISC1 signaling in the dentate gyrus and that DISC1 expression may regulate the cytoarchitectural organization of the granule cell layer.     

Growth of a new fiber projection in the brain of adult rats: Re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions

Summary Ablation of the entorhinal cortex of the rat removes the major synaptic input to the granule cells of the ipsilateral dentate gyrus. Following unilateral entorhinal lesions in adult rats, we have examined the efferent projections of the remaining contralateral entorhinal cortex to determine if these might sprout to re-innervate the deafferented dentate gyrus. Autoradiographical tracing of the fiber projections of the remaining contralateral entorhinal cortex 60 days following lesions indicates that new fibers sprout and grow for several hundred microns into the denervated regions, to terminate on portions of the granule cell dendrites which would normally receive ipsilateral entorhinal afferents.These re-innervating fibers form electrophysiologically functional synaptic connections with the denervated dentate granule cells. In the normal animal, unilateral stimulation of the entorhinal cortex does not result in short latency activation of the contralateral dentate gyrus whereas following ipsilateral entorhinal lesions, re-innervation by contralateral entorhinal afferents is reflected electrophysiologically by the appearance of a new short latency evoked potential to contralateral entorhinal stimulation. By field potential analysis, we demonstrate that this new short latency evoked potential is a reflection of mono-synaptic activation of the denervated dentate granule cells by the re-innervating contralateral entorhinal fibers.In addition, the time course of contralateral entorhinal re-innervation is determined electrophysiologically. The new short latency response to contralateral entorhinal stimulation appears as early as 9 days post-lesion, matures functionally between 9 and 15 days, and after 15 days, remains apparently undiminished for as long as 200 days. This implies that the new synapses formed in response to a deafferenting lesion are formed rapidly and remain permanently capable of activating the dentate granule cells which had been deprived of ipsilateral entorhinal input.The material in this paper was included in a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of California at Irvine, Irvine, California 92664.     

胶质细胞源性神经营养因子对猴骨髓间充质干细胞分化为神经元样细胞的影响

背景：鉴于骨髓间充质干细胞体外分化为神经样细胞的最终研究目的,是将诱导后的细胞移植入体内参与损伤神经系统的修复过程,因此,保证移植细胞的活性显得十分重要。 目的：探讨胶质细胞源性神经营养因子对隐丹参酮体外诱导猴骨髓间充质干细胞分化为神经元样细胞的保护作用。 方法：以隐丹参酮为诱导剂诱导第8代猴骨髓间充质干细胞分化为神经元样细胞,应用流式细胞仪检测不同时段诱导细胞的凋亡百分比(每间隔0.5 h为1组,共12组)。选择细胞凋亡百分比较高的一个时段,观察添加不同质量浓度胶质细胞源性神经营养因子(0-100 μg/L,共11组)对诱导细胞凋亡的影响。 结果与结论：诱导后细胞凋亡百分比逐渐升高,约4 h时达到峰值,维持约1 h后下降(P < 0.05)。 随着胶质细胞源性神经营养因子质量浓度由0 μg/L提高到30 μg/L,细胞凋亡百分比逐渐下降(P < 0.05),当胶质细胞源性神经营养因子质量浓度超过30 μg/L后,细胞凋亡水平受胶质细胞源性神经营养因子质量浓度影响不再显著。结果可见胶质细胞源性神经营养因子在隐丹参酮体外诱导猴骨髓间充质干细胞分化为神经元样细胞过程中具有保护作用。     

Comparison of electron microscopy and silver staining for the detection of the first entorhinal synapses to develop in the dentate gyrus

Summary The development of the projection from the entorhinal cortex to the dentate gyrus (perforant path) has been studied with the electron microscope. The projection was lesioned in baby rats 5–13 days old and the dentate gyrus examined after 6–72 hr. Degenerating synapses first appeared in small numbers in the dentate neuropile at 7 days and in greater numbers in progressively older animals. There was a sixteen-fold increase in the number of synapses undergoing degeneration between 7 and 13 days. This investigation provides a calibration for the reduced silver method which has been used to trace developing axons (Singh, 1977). By this method the first signs of Wallerian degeneration, after cutting axons in the perforant path, were seen in the dentate neuropile at 9 days.