Changes In Neuronal Morphology Research Articles

Effect of sub-optimal doses of fluoxetine plus estradiol on antidepressant-like behavior and hippocampal neurogenesis in ovariectomized rats

Estrogens and antidepressants synergize to reduce depressive symptoms and stimulate neurogenesis and neuroplastic events. The aim of this study was to explore whether the antidepressant-like effect induced by the combination of low doses of estradiol (E2) and fluoxetine (FLX) involves changes in cell proliferation, early survival, morphology and dendrite complexity of hippocampal new-immature neurons. The antidepressant-like effects of E2 and/or FLX were evaluated by the forced swimming test (FST), cell proliferation was determined with the endogenous marker Ki67, survival of newborn cells was established with bromo-deoxiuridine (BrdU) and immature neurons were ascertained by doublecortin (DCX) labeling while their dendrite complexity was evaluated with Sholl analysis. Ovariectomized Wistar rats were randomly assigned to one of the following groups: Vehicle (saline/14 days+Oil/-8h before FST); E2 (saline/14 days + E2 2.5 or 10 μg/rat; -8 h before FST); FLX (1.25 or 10 mg/kg for 14 days + oil -8h before FST), and FLX plus E2 (FLX 1.25 mg/kg for 14 days + E2 2.5 μg/rat -8 h before FST). The combination of sub-threshold doses of FLX plus E2 produced antidepressant-like actions similar to those induced by FLX or E2 given independently at optimal doses. Only FLX at an optimal dose and the combination of FLX plus E2 increased cell proliferation, the number of DCX-labeled immature neurons and the complexity of their dendritic tree, suggesting that these events may be responsible for their antidepressant-like effect.

Alaskan Botanicals Influence Neuronal Aging

Aging is a risk factor for many life‐threatening disorders, including neurodegenerative diseases. Many genetic mutations and environmental factors that increase lifespan may also postpone these age‐related diseases. The overall goal of this project is to examine the effect of Alaskan botanicals on the aging nervous system, using the nematode model system, Caenorhabditis elegans. We tested the impact of crude extracts from three edible, medicinally beneficial Interior Alaskan botanicals (Vaccinium uliginosum [bog blueberry], Empetrum nigrum [crowberry], Vaccinium vitis‐idaea [lowbush cranberry], Inonotus obliquus [chaga]) on whole animal lifespan and markers of neuronal aging. Specifically, we evaluated age‐ and botanical‐related changes in neuronal morphology and function of mechanosensory and GABAergic motor neurons using fluorescently labeled C. elegans strains. We find that these botanicals are able to extend wildtype C. elegans lifespan by up to 35%. Preliminary results suggest that mechanosensory neuronal morphology and function are also affected, but not GABA‐ergic neurons. We explored the mechanism of action of these botanicals through determining the extracts' phenolic, flavonoid, and anthocyanin composition and in vivo DAF‐16, SKN‐1, and HSF‐1 transcription factor activation following treatments. Through this botanical screen, we will scientifically validate and underscore the value of ecological resources endemic to the Arctic, elucidate the mechanism of changing neuronal morphology and functionality with age, and, perhaps, establish precedent for novel therapies for neurodegenerative diseases.

Effects of agmatine on permeability of blood-brain barrier and expression of aquaporin 4 and matrix metalloproteinase 9

Objective To investigate the protection of agmatine on blood brain barrier (BBB) permeability and the effect on the expression of aquaporin 4 (AQP4) and matrix metalloproteinase 9 (MMP9) in ischemic reperfusion injury rats. Methods Sixty healthy male Sprague-Dawley rats were randomly divided into normal control group (NC), model group and agmatine group, and there were 20 rats in each group. Normal control group was treated with intraperitoneal injection of saline in 2 hours after incision and suture of skin. Model group was treated with intraperitoneal injection of saline in 2 hours after the establishment of middle cerebral artery occulation (MCAO) model. Agmatine group was treated with intraperitoneal injection of agmatine (AGM) in 2 hours after the establishment of MCAO model. The damage of blood brain barrier was detected by measuring the permeability of blood brain barrier.The infarct size of brain was observed with TTC staining.The morphological changes of neurons were observed with electron microscope.The expressions of AQP4 and MMP9 were detected using immunohistochemical method. Results The permeability of BBB of agmatine treatment group (0.31±0.10) decreased significantly compared with the model group (0.46±0.09)(P<0.05), but was still significantly higher than the normal control group (0.24±0.12) (P<0.05). There was no infarction area in normal control group and the infarction areas of agmatine group decreased obviously compared with the model group. Compared with model group, the number of neurons with morphological changes reduced significantly and the degree of pathological changes of neurons was obviously improved in agmatine group. Compared with normal control group, the expression of AQP4 and MMP9 in ischemic penumbra of left cerebral hemisphere parietal cortex in the rats of model group and agmatine group increased significantly(P<0.05). And the expression of AQP4 and MMP9 in model group was significantly higher than that of agmatine group(P<0.05). Conclusion Agmatine has a protective effect on BBB with ischemia-reperfusion injury due to its down-regulation the expression of AQP-4 and MMP-9. Key words: Aquaporin 4; Matrix metalloproteinase 9; Agmatine; Cerebral ischemic reperfusion

Chemicals eluting from disposable plastic syringes and syringe filters alter neurite growth, axogenesis and the microtubule cytoskeleton in cultured hippocampal neurons.

Cultures of dissociated hippocampal neurons are often used to study neuronal cell biology. We report that the development of these neurons is strongly affected by chemicals leaching from commonly used disposable medical-grade syringes and syringe filters. Contamination of culture medium by bioactive substance(s) from syringes and filters occurred with multiple manufacturing lots and filter types under normal use conditions and resulted in changes to neurite growth, axon formation and the neuronal microtubule cytoskeleton. The effects on neuronal morphology were concentration dependent and significant effects were detected even after substantial dilution of the contaminated medium. Gas chromatography-mass spectrometry analyses revealed many chemicals eluting from the syringes and filters. Three of these chemicals (stearic acid, palmitic acid and 1,2-ethanediol monoacetate) were tested but showed no effects on neurite growth. Similar changes in neuronal morphology were seen with high concentrations of bisphenol A and dibutyl phthalate, two hormonally active plasticisers. Although no such compounds were detected by gas chromatography–mass spectrometry, unknown plasticisers in leachates may affect neurites. This is the first study to show that leachates from laboratory consumables can alter the growth of cultured hippocampal neurons. We highlight important considerations to ensure leachate contamination does not compromise cell biology experiments.

Membrane-Initiated Non-Genomic Signaling by Estrogens in the Hypothalamus: Cross-Talk with Glucocorticoids with Implications for Behavior

The estrogen receptor and glucocorticoid receptor are members of the nuclear receptor superfamily that can signal using both non-genomic and genomic transcriptional modes. Though genomic modes of signaling have been well characterized and several behaviors attributed to this signaling mechanism, the physiological significance of non-genomic modes of signaling has not been well understood. This has partly been due to the controversy regarding the identity of the membrane ER (mER) or membrane GR (mGR) that may mediate rapid, non-genomic signaling and the downstream signaling cascades that may result as a consequence of steroid ligands binding the mER or the mGR. Both estrogens and glucocorticoids exert a number of actions on the hypothalamus, including feedback. This review focuses on the various candidates for the mER or mGR in the hypothalamus and the contribution of non-genomic signaling to classical hypothalamically driven behaviors and changes in neuronal morphology. It also attempts to categorize some of the possible functions of non-genomic signaling at both the cellular level and at the organismal level that are relevant for behavior, including some behaviors that are regulated by both estrogens and glucocorticoids in a potentially synergistic manner. Lastly, it attempts to show that steroid signaling via non-genomic modes may provide the organism with rapid behavioral responses to stimuli.

Ethanol deregulates Mecp2/MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

Methyl CpG Binding Protein 2 (MeCP2) is an important epigenetic factor in the brain. MeCP2 expression is affected by different environmental insults including alcohol exposure. Accumulating evidence supports the role of aberrant MeCP2 expression in ethanol exposure-induced neurological symptoms. However, the underlying molecular mechanisms of ethanol-induced MeCP2 deregulation remain elusive. To study the effect of ethanol on Mecp2/MeCP2 expression during neurodifferentiation, we established an in vitro model of ethanol exposure, using differentiating embryonic brain-derived neural stem cells (NSC). Previously, we demonstrated the impact of DNA methylation at the Mecp2 regulatory elements (REs) on Mecp2/MeCP2 expression in vitro and in vivo. Here, we studied whether altered DNA methylation at these REs is associated with the Mecp2/MeCP2 misexpression induced by ethanol. Binge-like and continuous ethanol exposure upregulated Mecp2/MeCP2, while ethanol withdrawal downregulated its expression. DNA methylation analysis by methylated DNA immunoprecipitation indicated that increased 5-hydroxymethylcytosine (5hmC) and decreased 5-methylcytosine (5mC) enrichment at specific REs were associated with upregulated Mecp2/MeCP2 following continuous ethanol exposure. The reduced Mecp2/MeCP2 expression upon ethanol withdrawal was associated with reduced 5hmC and increased 5mC enrichment at these REs. Moreover, ethanol altered global DNA methylation (5mC and 5hmC). Under the tested conditions, ethanol had minimal effects on NSC cell fate commitment, but caused changes in neuronal morphology and glial cell size. Taken together, our data represent an epigenetic mechanism for ethanol-mediated misexpression of Mecp2/MeCP2 in differentiating embryonic brain cells. We also show the potential role of DNA methylation and MeCP2 in alcohol-related neurological disorders, specifically Fetal Alcohol Spectrum Disorders.

The development of lasting impairments: A mild pediatric brain injury alters gene expression, dendritic morphology, and synaptic connectivity in the prefrontal cortex of rats

Apart from therapeutic discovery, the study of mild traumatic brain injury (mTBI) has been focused on two challenges: why do a majority of individuals recover with little concern, while a considerable proportion suffer with persistent and often debilitating symptomology; and, how do mild injuries significantly increase risk for an early-onset neurodegeneration? Owing to a lack of observable damage following mTBI, this study was designed to determine if there were changes in neuronal morphology, synaptic connectivity, and epigenetic patterning that could contribute to the manifestation of persistent neurological dysfunction. Prefrontal cortex tissue from male and female rats was used for Golgi-Cox analysis along with the profiling of changes in gene expression (BDNF, DNMT1, FGF2, IGF1, Nogo-A, OXYR, and TERT) and telomere length (TL), following a single mTBI or sham injury in the juvenile period. Golgi-Cox analysis of dendritic branch order, dendritic length, and spine density demonstrate that an early mTBI increases complexity of pyramidal neurons in the mPFC. Furthermore, there are also substantial changes in the expression levels of the seven genes of interest and TL following a single mild injury in this brain region. The results from the neuroanatomical measures and changes in gene expression indicate that the mTBI disrupts normal pruning processes that are typically underway at this point in development. In addition, there are significant interactions between the social environment and epigenetic processes that work in concert to perpetuate neurological dysfunction.

Enhanced neurite outgrowth and branching precede increased amyloid-β-induced neuronal apoptosis in a novel Alzheimer's disease model.

Though it is widely accepted that amyloid-β (Aβ) is a key factor in Alzheimer's disease (AD) pathology, its underlying mechanism remains unclear. In order to study the association between Aβ and neural circuitry dysfunction, we developed a primary culture preparation derived from the nervous system of transgenic Drosophila melanogaster larvae expressing human Aβ1-42 (Aβ42). Cultured neurons undergo a consistent developmental process, culminating in an elaborate neuronal network with distinct functional and morphological characteristics. Throughout this development, a time-dependent increase in intracellular expression levels of Aβ42 was detected, followed by extracellular staining at a later time point. When compared to controls, Aβ42 cultures exhibited enhanced levels of apoptosis, resulting in reduced cell viability. Moreover, as primary culture preparations enable high resolution monitoring of neuronal phenotypes, we were able to detect subtle morphological changes in neurons expressing Aβ42, namely an enhancement in neurite outgrowth and arborization, which preceded the effect of neurodegeneration. Our results establish D. melanogaster primary neuronal cultures as a rapid, accessible and cost-effective platform for AD molecular studies and drug screening, and suggest a possible role for Aβ42 in the organization of neuronal processes.

Transgenerational sex-specific impact of preconception stress on the development of dendritic spines and dendritic length in the medial prefrontal cortex.

Perinatal adverse experience programs social and emotional behavioral traits and is a major risk factor for the development of behavioral and psychiatric disorders. Little information is available on how adversity to the mother prior to her first pregnancy (preconception stress, PCS) may affect brain structural development, which may underlie behavioral dysfunction in the offspring. Moreover, little is known about possible sex-dependent consequences of PCS in the offspring. This study examined spine number/densityand dendritic length/complexity of layer II/III pyramidal neurons in the anterior cingulate (ACd), prelimbic/infralimbic (PL/IL) and orbitofrontal cortex (OFC) of male and female rats born to mothers exposed to unpredictable variable stress at different time points prior to reproduction. Our main findings are that in line with our hypothesis adversity to the mother before her pregnancy results in highly complex changes in neuronal morphology in the medial prefrontal, but not in the orbitofrontal cortical regions of her future offspring that persist into adulthood. Moreover, our study revealed that (1) in the PCS2 group (offspring of dams mated two weeks after stress) spine numbers and dendritic length and complexity were increased in response to PCS in the ACd and PL/IL, (2) these regional effects depended on the temporal proximity of adversity and conception, (3) in the ACd of the PCS2 group only males and the left hemispheres were affected. We speculate that these transgenerational brain structural changes are mediated by stress-induced epigenetic (re)programming of future gene activity in the oocyte.

Stress-induced alterations in large-scale functional networks of the rodent brain

Stress-related psychopathology is associated with altered functioning of large-scale brain networks. Animal research into chronic stress, one of the most prominent environmental risk factors for development of psychopathology, has revealed molecular and cellular mechanisms potentially contributing to human mental disease. However, so far, these studies have not addressed the system-level changes in extended brain networks, thought to critically contribute to mental disorders. We here tested the effects of chronic stress exposure (10days immobilization) on the structural integrity and functional connectivity patterns in the brain, using high-resolution structural MRI, diffusion kurtosis imaging, and resting-state functional MRI, while confirming the expected changes in neuronal dendritic morphology using Golgi-staining. Stress effectiveness was confirmed by a significantly lower body weight and increased adrenal weight. In line with previous research, stressed animals displayed neuronal dendritic hypertrophy in the amygdala and hypotrophy in the hippocampal and medial prefrontal cortex. Using independent component analysis of resting-state fMRI data, we identified ten functional connectivity networks in the rodent brain. Chronic stress appeared to increase connectivity within the somatosensory, visual, and default mode networks. Moreover, chronic stress exposure was associated with an increased volume and diffusivity of the lateral ventricles, whereas no other volumetric changes were observed. This study shows that chronic stress exposure in rodents induces alterations in functional network connectivity strength which partly resemble those observed in stress-related psychopathology. Moreover, these functional consequences of stress seem to be more prominent than the effects on gross volumetric change, indicating their significance for future research.

An in vitro study of the neuroprotective effect of propofol on hypoxic hippocampal slice

Primary objective: To determine whether propofol has a neuroprotective effect on hypoxic brain injury.Research design: A hippocampal slice, in artificial cerebrospinal fluid (ASCF) with glucose and oxygen deprivation (OGD), was used as an in vitro model for brain hypoxia.Methods and procedures: The orthodromic population spike (OPS) and hypoxic injury potentia1 (HIP) were recorded in the CA1 region when Schaffer collateral was stimulated in the CA3 region of the hippocampal slices during hypoxia. The concentrations of amino acid neurotransmitters in perfusion solution of hippocampal slices were directly measured using high performance liquid chromatography (HPLC). Morphological changes of neurons, astrocytes and mitochondria in CA1 region were observed using histology and electron microscopy. Neuronal apoptosis was evaluated with TUNEL assay.Main outcome and results: Propofol treatment delayed the elimination of OPS and improved the recovery of OPS; decreased frequency of HIP, postponed the onset of HIP and increased the duration of HIP. Propofol treatment also decreased the release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but elevated the release of γ-aminobutyric acid (GABA). Morphological studies showed that propofol treatment attenuated oedema of pyramid neurons in the CA1 region and reduced apoptosis.Conclusions: Propofol has a neuro-protective effect on hippocampal neuron injury induced by hypoxia.

072 — (SIM0179) Enriched environment attenuates some neuronal and glial morphological changes induced by undernourishment and seizures during early life

072 — (SIM0179) Enriched environment attenuates some neuronal and glial morphological changes induced by undernourishment and seizures during early life

Unilateral injection of Aβ25-35 in the hippocampus reduces the number of dendritic spines in hyperglycemic rats.

Alzheimer's disease (AD) is a neurodegenerative process exacerbated by several risk factors including impaired glucose metabolism in the brain that could cause molecular and neurochemical alterations in cognitive regions such as the hippocampus (Hp). Consequently, this process could cause neuronal morphological changes; however, the mechanism remains elusive. We induced chronic hyperglycemia after streptozotocin (STZ) administration. Then, we examined spatial learning and memory using the Morris water maze test and evaluated neuronal morphological changes using the Golgi-Cox stain procedure in hyperglycemic rats that received a Aβ25-35 unilateral injection into the Hp. Our results demonstrate that STZ combined with Aβ25-35 induced significant deficits in the spatial memory. In addition, we observed a significant reduction in the number of dendritic spines of pyramidal neurons in the dorsal Hp of rats with STZ plus Aβ25-35 . In conclusion, the reduced spine density of pyramidal neurons in the CA1 dorsal Hp could produce the spatial memory deficit observed in these animals. These results suggest that hyperglycemia can trigger Aβ-induced neurodegeneration and thus the appearance of AD symptoms would be accelerated. Synapse 68:585-594, 2014. © 2014 Wiley Periodicals, Inc.

Deletion of the NMDA receptor GluN2A subunit significantly decreases dendritic growth in maturing dentate granule neurons.

It is known that NMDA receptors can modulate adult hippocampal neurogenesis, but the contribution of specific regulatory GluN2 subunits has been difficult to determine. Here we demonstrate that mice lacking GluN2A (formerly NR2A) do not show altered cell proliferation or neuronal differentiation, but present significant changes in neuronal morphology in dentate granule cells. Specifically, GluN2A deletion significantly decreased total dendritic length and dendritic complexity in DG neurons located in the inner granular zone. Furthermore, the absence of GluN2A also resulted in a localized increase in spine density in the middle molecular layer, a region innervated by the medial perforant path. Interestingly, alterations in dendritic morphology and spine density were never seen in dentate granule cells located in the outer granular zone, a region that has been hypothesized to contain older, more mature, neurons. These results indicate that although the GluN2A subunit is not critical for the cell proliferation and differentiation stages of the neurogenic process, it does appear to play a role in establishing synaptic and dendritic morphology in maturing dentate granule cells localized in the inner granular zone.

Effects of simvastatin on heat hock protein 70 after spinal cord ischemia reperfusion injury

Objective To investigate the protective effect of simvastatin on spinal cord ischemia/reperfusion injury through the observation of the changes of heat shock protein 70 (Hsp70).Methods 24healthy adult Tibet mini-pigs of either sex,weighted 20-30 kg,were randomly divided into three groups (n =8),including sham operation group (group C),ischemia/reperfusion group (group I/R) and simvastatin experimental group (group Sim).In group I/R and Sim,the models of spinal cord ischemia/reperfusion were established by the occlusion of thoracic aorta and subclavian artery (70 min).The accessory hemiazygos vein was isolated and the distal end was ligated.In group I/R,1000 ml lactated ringer solution were pumped into the vein and the rate was 860 ml/h during the ischemia time.In group Sim,1000 ml simvastatin solution were pumped into the vein and the dose was 0.25 mg/kg during the ischemia time.Feed the swines of the experimental group simvastatin tablets 80 mg/d,orally for 30 days.After the operation,the motor function scores were recorded at 6,24,48 h (see Taylor motor function scoring system).All eight swines of each group were killed at 30 days after reperfusion,taking the L2-L5 and sacral spinal cord as quickly as possible.The hematoxylin and eosin (HE) staining method and the NISSLE staining method were adopted to observe the morphological changes of neurons and the immunohistochemical method was adopted to detect the changes of Hsp70.Results In group Sim,the 24,48 h motor function scores (3.2 ± 0.3,3.6 ± 0.2 respectively) after operation were significantly higher than that in group I/R (2.2 ±0.4,2.8 ± 0.4 respectively) (P ＜ 0.05).Compared with group I/R,the morphology of neurons in group Sim had a slight damage and higher expression changes of Hsp70 can be found in group Sim (P ＜ 0.05).Conclusion Simvastatin has a significant protective effect on the spinal cord ischemia reperfusion injury and the potential mechanism may be related to the up-regulation of Hsp70. Key words: Simvastatin; Spinal cord; Ischemia/reperfusion injury; Heat shock protein 70; Protection

Suppressor of cytokine signaling 1 regulates the neuronal differentiation of C17.2 neuronal stem cells

Objective To investigate the role of suppressor of cytokine signaling 1 (SOCS1) in regulating the differentiation of C17.2 neuronal stem cells (NSCs).Methods C17.2 NSCs were transfected by Lentiviral vector which included objective gene-SOCSl.The experiment was individed into LV-SOCS1 group,LV group and PBS group.The morphological change of C17.2NSCs after nransfection was observed by light microscopy.The expression of Nestin,microtubule-associated proteins 2 (MAP2),β-tubulinⅢ,myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in C17.2 NSCs was detected by immunocytochemistry,Western blotting and reverse transcription-polymerase chain reaction (RT-PCR).The protein expression of β-tubulinⅢ in C17.2 NSCs was examined by immunocytochemistry and Western blotting.Results An decreased expression of Nestin and an increased expression of MAP2 in LV-SOCS1 group was found as compared with LV group and PBS group after transfection (P ＜ 0.01).Some C17.2 NSCs in LV-SOCS1 group underwent prominent neuronal morphological changes after transfection and almost all the cells with morphological changes were β-tubulin Ⅲ-positive,and the other two groups had less cells with morphological changes and β-tubulin Ⅲ-positive (LV-SOCS1 group:10.05％,LV group:0.71％,PBS group:0.65％) (P＜0.01).The β-tubulin lⅢ expression in LV-SOCS1 group was significantly higher than in other two groups (P ＜0.01).Conclusion SOCS1 may play a role in promoting the neuronal differentiation of C17.2 NSCs. Key words: Suppressor of cytokine signaling 1 ; Neuronal stem cells; Neuron; Differentiation

Relationship between monocularly deprivation and amblyopia rats and visual system development

ObjectiveTo explore the changes of lateral geniculate body and visual cortex in monocular strabismus and form deprived amblyopic rat, and visual development plastic stage and visual plasticity in adult rats. MethodsA total of 60 SD rats ages 13 d were randomly divided into A, B, C three groups with 20 in each group, group A was set as the normal control group without any processing, group B was strabismus amblyopic group, using the unilateral extraocular rectus resection to establish the strabismus amblyopia model, group C was monocular form deprivation amblyopia group using unilateral eyelid edge resection + lid suture. At visual developmental early phase (P25), meta phase (P35), late phase (P45) and adult phase (P120), the lateral geniculate body and visual cortex area 17 of five rats in each group were exacted for C-fos Immunocytochemistry. Neuron morphological changes in lateral geniculate body and visual cortex was observed, the positive neurons differences of C-fos expression induced by light stimulation was measured in each group, and the condition of radiation development of P120 amblyopic adult rats was observed. ResultsIn groups B and C, C-fos positive cells were significantly lower than the control group at P25 (P<0.05), there was no statistical difference of C-fos protein positive cells between group B and group A (P>0.05), C-fos protein positive cells level of group B was significantly lower than that of group A (P<0.05). The binoculus C-fos protein positive cells level of groups B and C were significantly higher than that of control group at P35, P45 and P120 with statistically significant differences (P<0.05). ConclusionsThe increasing of C-fos expression in geniculate body and visual cortex neurons of adult amblyopia suggests the visual cortex neurons exist a certain degree of visual plasticity.

Modified neocortical and cerebellar protein expression and morphology in adult rats following prenatal inhibition of the kynurenine pathway

Inhibition of the kynurenine pathway of tryptophan metabolism during gestation can lead to changes in synaptic transmission, neuronal morphology and plasticity in the rat hippocampus. This suggests a role for the kynurenine pathway in early brain development, probably caused by kynurenine modulation of N-methyl-d-aspartate (NMDA) glutamate receptors which are activated by the tryptophan metabolite quinolinic acid and blocked by kynurenic acid. We have now examined samples of neocortex and cerebellum of adult animals to assess the effects of a prenatally administered kynurenine-3-monoxygenase inhibitor (Ro61-8048) on protein and mRNA expression, dendritic structure and immuno-histochemistry. No changes were seen in mRNA expression using quantitative real-time polymerase chain reaction. Changes were detected in the expression of several proteins including the GluN2A subunit, unco-ordinated-5H3 (unc5H3), doublecortin, cyclo-oxygenase, sonic hedgehog and Disrupted in schizophrenia-1 (DISC1), although no differences in immunoreactive cell numbers were observed. In the midbrain, dependence receptor expression was also changed. The numbers and lengths of individual dendritic regions were not changed but there were significant increases in the overall complexity values of apical and basal dendritic trees. The data support the hypothesis that constitutive kynurenine metabolism plays a critical role in early, embryonic brain development, although fewer effects are produced in the neocortex and cerebellum than in the hippocampus and the nature of the changes seen are qualitatively different. The significant changes in DISC1 and unc5H3 may be relevant to cerebellar dysfunction and schizophrenia respectively, in which these proteins have been previously implicated.

Existence of glia mitigated ketamine-induced neurotoxicity in neuron–glia mixed cultures of neonatal rat cortex and the glia-mediated protective effect of 2-PMPA

The present study compared ketamine-induced neurotoxicity in the neuron–glia mixed cultures and neuronal cultures and further explored the neuroprotective effect of the NAAG peptidase inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA). Firstly, Rosenfeld's staining and immunofluorescence staining of microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) were used to address the difference of morphology in the mixed cultures and neuronal cultures. Our results showed that neurons and astrocytes grew in good conditions. The ratio of neurons and astrocytes in the mixed cultures was around 1:1, and the purity of neurons in the neuronal cultures is 91.3%. Furthermore, ketamine was used to test the hypothesis that the presence of a higher proportion of glia in the mixed cultures would be protective against ketamine-induced neurotoxicity in the mixed cultures compared with neuronal cultures. The results showed that ketamine-induced morphological changes, cell viability decrease and lactate dehydrogenase (LDH) levels increase were significantly mitigated in neuron–glia mixed cultures compared with neuronal cultures. Furthermore, 2-PMPA was included to further explore efficient protective drug for ketamine-induced neurotoxicity. Our results showed that 2-PMPA reduced ketamine-induced decrease of cell viability and increase of LDH levels in the mixed cultures but not in the neuronal cultures. Further morphological changes of neurons and astrocytes also indicated that 2-PMPA could improve ketamine damaged neurons in the mixed cultures instead of neuronal cultures. These results indicate that glia protect neurons from ketamine-induced neurotoxicity. These data further suggest that glia mediate the neuroprotective effect of 2-PMPA and 2-PMPA has the potential to treat ketamine-induced neurotoxicity in vivo. Delineating the mechanisms underlying the communication between neurons and glia and the neuroprotective effects of 2-PMPA in the mixed cultures to ketamine-induced neurotoxicity require further investigation.

Activation of microglia bolsters synapse formation.

Activation of microglia bolsters synapse formation.