Neuronal nuclear antigen (NeuN): a new tool in the diagnosis of central neurocytoma

The use of neuronal nuclear antigen (NeuN) as a reliable neuronal marker in the differential diagnosis of clear cell neoplasms of the central nervous system was determined in a biopsy series of 23 cases. Immunohistochemical analyses were carried out by antisera against neuronal nuclear antigen, synaptophysin, neuron-specific enolase, microtubule-associated protein 2, and glial fibrillary acidic protein. All eight central neurocytomas were characteristically immunolabeled by NeuN. NeuN immunoreactivity was uniformly strong and basically located in the nuclei of neurocytes. Despite this uniform staining pattern of central neurocytomas, 12 cases of oligodendrogliomas and three cases of ependymoma were negative for NeuN. As the diagnostic criteria for central neurocytoma include immunohistochemical and/or ultrastructural evidence for neuronal differentiation, NeuN as a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues may greatly facilitate the differential diagnosis of central neurocytomas.     

Caudal end of the rat spinal dorsal horn

We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons.     

大鼠视网膜神经细胞体外培养

目的 建立视网膜神经层分散细胞的培养方法，以进一步对视网膜神经元细胞(尤其是视网膜神经节细胞)和神经胶质细胞进行体外实验研究。方法 取出生后1～3d的SD乳鼠的视网膜，用胰酶消化法制备分散细胞悬液后，接种于置有包被poly-D-lysine玻片的24孔培养板中培养，倒置相差显微镜观察细胞生长规律。培养第7d，用抗神经元特异性核(NeuN)抗体和抗神经丝(Neurofilament-200，NF-200)抗体分别标记视网膜神经元细胞和神经节细胞(RGCs)，并计算每10个高倍镜下的细胞数以及RGCs所占百分数。结果 体外培养的视网膜神经元细胞经历了贴壁、重聚、迁移和相互接触的过程，有突起样生长，其中RGCs占神经元的55％左右。结论 视网膜神经层分散细胞体外培养成功，并有较好的RGCs生长率，为进一步进行视网膜疾病的研究创造了条件。     

The total number of white matter interstitial neurons in the human brain

In the adult human brain, the interstitial neurons (WMIN) of the subcortical white matter are the surviving remnants of the fetal subplate zone. It has been suggested that they perform certain important functions and may be involved in the pathogenesis of several neurological and psychiatric disorders. However, many important features of this class of human cortical neurons remain insufficiently explored. In this study, we analyzed the total number, and regional and topological distribution of WMIN in the adult human subcortical white matter, using a combined immunocytochemical (NeuN) and stereological approaches. We found that the average number of WMIN in 1 mm³ of the subcortical white matter is 1.230 ± 549, which translates to the average total number of 593 811 183.6 ± 264 849 443.35 of WMIN in the entire subcortical telencephalic white matter. While there were no significant differences in their regional distribution, the lowest number of WMIN has been consistently observed in the limbic cortex, and the highest number in the frontal cortex. With respect to their topological distribution, the WMIN were consistently more numerous within gyral crowns, less numerous along gyral walls and least numerous at the bottom of cortical sulci (where they occupy a narrow and compact zone below the cortical‐white matter border). The topological location of WMIN is also significantly correlated with their morphology: pyramidal and multipolar forms are the most numerous within gyral crowns, whereas bipolar forms predominate at the bottom of cortical sulci. Our results indicate that WMIN represent substantial neuronal population in the adult human cerebral cortex (e.g. more numerous than thalamic or basal ganglia neurons) and thus deserve more detailed morphological and functional investigations in the future.     

Age-related changes in rostral basal forebrain cholinergic and GABAergic projection neurons: relationship with spatial impairment

Both cholinergic and GABAergic projections from the rostral basal forebrain contribute to hippocampal function and mnemonic abilities. While dysfunction of cholinergic neurons has been heavily implicated in age-related memory decline, significantly less is known regarding how age-related changes in codistributed GABAergic projection neurons contribute to a decline in hippocampal-dependent spatial learning. In the current study, confocal stereology was used to quantify cholinergic (choline acetyltransferase [ChAT] immunopositive) neurons, GABAergic projection (glutamic decarboxylase 67 [GAD67] immunopositive) neurons, and total (neuronal nuclei [NeuN] immunopositive) neurons in the rostral basal forebrain of young and aged rats that were first characterized on a spatial learning task. ChAT immunopositive neurons were significantly but modestly reduced in aged rats. Although ChAT immunopositive neuron number was strongly correlated with spatial learning abilities among young rats, the reduction of ChAT immunopositive neurons was not associated with impaired spatial learning in aged rats. In contrast, the number of GAD67 immunopositive neurons was robustly and selectively elevated in aged rats that exhibited impaired spatial learning. Interestingly, the total number of rostral basal forebrain neurons was comparable in young and aged rats, regardless of their cognitive status. These data demonstrate differential effects of age on phenotypically distinct rostral basal forebrain projection neurons, and implicate dysregulated cholinergic and GABAergic septohippocampal circuitry in age-related mnemonic decline.     

Astrogliosis is temporally correlated with enhanced neurogenesis in adult rat hippocampus following a glucoprivic insult

2-Deoxy-d-glucose (2-DG) administration causes transient depletion of glucose derivates and ATP. Hence, it can be used in a model system to study the effects of a mild glycoprivic brain insult mimicking transient hypoglycemia, which often occurs when insulin or oral hypoglycemic agents are administered for diabetes control. In the present study, the effect of a single 2-DG application (500 mg/kg, a clinically applicable dose) on glial reactivity and neurogenesis in adult rat hippocampus was examined, as well as a possible temporal correlation between these two phenomena. Post-insult (PI) glial reactivity time course was assessed by immunoreaction against glial-fibrillary acidic protein (GFAP) during the following 5 consecutive days. A clear increase of GFAP immunoreactivity in hilus was observed from 48 to 96 h PI. Moreover, enhanced labeling of long radial processes in the granule cell layer adjacent to hilus was evidenced. On the other hand, a transient increase of progenitor cell proliferation was detected in the subgranular zone, prominently at 48 h PI, coinciding with the temporal peak of glial activation. This increase resulted in an augment of neuroblasts double labeled with 5-bromo-deoxyuridine (BrdU) and with double cortin (DCX) at day 7 PI. Around half of these cells survived 28 days showing matured neuronal phenotype double labeled by BrdU and a neuronal specific nuclear protein marker (NeuN). These findings suggest that a transient neuroglycoprivic state exerts a short-term effect on glial activation that possibly triggers a long-term effect on neurogenesis in hippocampus.     

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.     

The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds specifically to microglia in a rat model of traumatic brain injury: implications for PET imaging

Traumatic brain injury (TBI) is a significant cause of mortality, morbidity, and disability. Microglial activation is commonly observed in response to neuronal injury which, when prolonged, is thought to be detrimental to neuronal survival. Activated microglia can be labeled using PK11195, a ligand that binds the peripheral benzodiazepine receptor (PBR), receptors which are increased in activated microglia and sparse in the resting brain. We compared the binding properties of two PBR ligands PK11195 and DAA1106 in rats using the controlled cortical impact (CCI) model of experimental TBI. While both ligands showed relative increases with specific binding in the cortex ipsilateral to injury compared to the contralateral side, [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195. Combined immunohistochemistry and autoradiography in brain tissues near the injury site showed that [(3)H]DAA1106 binding co-registered with activated microglia more than astrocytes. Further, increased [(3)H]DAA1106-specific binding positively correlated with the degree of microglial activation, and to a lesser degree with reactive astrocytosis. Finally, in vivo administration of each ligand in rats with TBI showed greater retention of [(11)C]DAA1106 compared to [(11)C](R)-PK11195 at the site of the contusion as assessed by ex vivo autoradiography. These results in a rat model of TBI indicate that [(11)C]DAA1106 binds with higher affinity to microglia when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a better ligand than [(11)C](R)-PK11195 for in vivo PET imaging of activated microglia in TBI.     

Persistent impairment of hippocampal neurogenesis in young adult rats following early postnatal alcohol exposure

Background: Prenatal alcohol exposure can cause damage to the developing fetus with outcomes including growth deficiency, facial dysmorphology, brain damage, and cognitive and behavioral deficits. Smaller brains in children with FASD have been linked both with reduced cell proliferation in the developing CNS and with apoptotic cell loss of postmitotic neurons. Prenatal alcohol exposure in rodents during the period of brain development comparable to that of the first and second trimesters of human pregnancy persistently alters adult neurogenesis. Long‐term effects of alcohol exposure during the third trimester equivalent, which occurs postnatally in the rat, on adult neurogenesis have not been previously reported. The goal of this study was to examine the effect of postnatal binge‐like alcohol exposure on cell proliferation and neurogenesis in hippocampal dentate gyrus during adolescence and young adulthood. Methods: Male Long‐Evans rat pups were assigned to 3 groups: alcohol‐exposed (AE), sham‐intubated (SI) or suckle control (SC). AE pups received ethanol in a milk formula in a binge manner (2 feedings, 2 hours apart, total dose 5.25 g/kg/day) on postnatal days (PD) 4–9. BrdU was injected every other day on PD30–50. Animals were perfused either on PD50 to examine cytogenesis and neurogenesis in hippocampal dentate gyrus at the end of BrdU injections or on PD80 to evaluate new cell survival. Dorsal hippocampal sections were immunostained for BrdU, a marker for proliferating cells, Ki67, endogenous marker of proliferation, and NeuN, a marker for mature neurons. Results: Binge‐like alcohol exposure on PD4–9 significantly reduced the number of mature neurons in adult hippocampal dentate gyrus (DG) both on PD50 and PD80, without altering cumulative cytogenesis on PD50. In addition, the number of new neurons, that were generated between PD30 and 50, was further reduced after 30 days of survival in all 3 groups (SC, SI, and AE). Conclusions: These observations suggest that early postnatal binge alcohol exposure results in long‐term deficits of adult hippocampal neurogenesis, providing a potential basis for the deficits of hippocampus‐dependent behaviors reported for this model.     

Targeting whole body metabolism and mitochondrial bioenergetics in the drug development for Alzheimer's disease

Aging is by far the most prominent risk factor for Alzheimer's disease (AD), and both aging and AD are associated with apparent metabolic alterations. As developing effective therapeutic interventions to treat AD is clearly in urgent need, the impact of modulating whole-body and intracellular metabolism in preclinical models and in human patients, on disease pathogenesis, have been explored. There is also an increasing awareness of differential risk and potential targeting strategies related to biological sex, microbiome, and circadian regulation. As a major part of intracellular metabolism, mitochondrial bioenergetics, mitochondrial quality-control mechanisms, and mitochondria-linked inflammatory responses have been considered for AD therapeutic interventions. This review summarizes and highlights these efforts.