Dendritic Pruning Research Articles

The spliceosome-associated protein Mfap1 binds to VCP in Drosophila.

Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP) regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da) neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to vcp mutations, Mfap1 overexpression causes c4da neuron dendrite pruning defects and mislocalization of TDP-43 in these cells, but genetic analyses show that Mfap1 is not a crucial VCP target during dendrite pruning. Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity.

Distinct effects on the dendritic arbor occur by microbead versus bath administration of brain-derived neurotrophic factor

Proper communication among neurons depends on an appropriately formed dendritic arbor, and thus, aberrant changes to the arbor are implicated in many pathologies, ranging from cognitive disorders to neurodegenerative diseases. Due to the importance of dendritic shape to neuronal network function, the morphology of dendrites is tightly controlled and is influenced by both intrinsic and extrinsic factors. In this work, we examine how brain-derived neurotrophic factor(BDNF), one of the most well-studied extrinsic regulators of dendritic branching, affects the arbor when it is applied locally via microbeads to cultures of hippocampal neurons. We found that local application of BDNF increases both proximal and distal branching in a time-dependent manner and that local BDNF application attenuates pruning of dendrites that occurs with neuronal maturation. Additionally, we examined whether cytosolic PSD-95 interactor(cypin), an intrinsic regulator of dendritic branching, plays a role in these changes and found strong evidence for the involvement of cypin in BDNF-promoted increases in dendrites after 24 but not 48h of application. This current study extends our previous work in which we found that bath application of BDNF for 72h, but not shorter times, increases proximal dendrite branching and that this increase occurs through transcriptional regulation of cypin. Moreover, this current work illustrates how dendritic branching is regulated differently by the same growth factor depending on its spatial localization, suggesting a novel pathway for modulation of dendritic branching locally.

Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons

Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons

Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons

Pruning, whereby neurons eliminate their excess neurites, is central for the maturation of the nervous system. In Drosophila, sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. Here, we show that the small GTPase Arf1, an important regulator of the secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP- and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. We further identified Sec71 as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not for apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, we show that the Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell-adhesion molecule Neuroglian (Nrg).

PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila.

Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. Here, we show that the kinase PAR-1 is required for pruning and dendritic microtubule breakdown. Our data show that neurons lacking PAR-1 fail to break down dendritic microtubules, and PAR-1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR-1 is a known Tau kinase, and genetic interactions suggest that PAR-1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila Finally, PAR-1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. Our results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.

Extrinsic Repair of Injured Dendrites as a Paradigm for Regeneration by Fusion in Caenorhabditis elegans.

Injury triggers regeneration of axons and dendrites. Research has identified factors required for axonal regeneration outside the CNS, but little is known about regeneration triggered by dendrotomy. Here, we study neuronal plasticity triggered by dendrotomy and determine the fate of complex PVD arbors following laser surgery of dendrites. We find that severed primary dendrites grow toward each other and reconnect via branch fusion. Simultaneously, terminal branches lose self-avoidance and grow toward each other, meeting and fusing at the tips via an AFF-1-mediated process. Ectopic branch growth is identified as a step in the regeneration process required for bypassing the lesion site. Failure of reconnection to the severed dendrites results in degeneration of the distal end of the neuron. We discover pruning of excess branches via EFF-1 that acts to recover the original wild-type arborization pattern in a late stage of the process. In contrast, AFF-1 activity during dendritic auto-fusion is derived from the lateral seam cells and not autonomously from the PVD neuron. We propose a model in which AFF-1-vesicles derived from the epidermal seam cells fuse neuronal dendrites. Thus, EFF-1 and AFF-1 fusion proteins emerge as new players in neuronal arborization and maintenance of arbor connectivity following injury in Caenorhabditis elegans Our results demonstrate that there is a genetically determined multi-step pathway to repair broken dendrites in which EFF-1 and AFF-1 act on different steps of the pathway. EFF-1 is essential for dendritic pruning after injury and extrinsic AFF-1 mediates dendrite fusion to bypass injuries.

Electrical brain stimulation induces dendritic stripping but improves survival of silent neurons after optic nerve damage

Repetitive transorbital alternating current stimulation (rtACS) improves vision in patients with chronic visual impairments and an acute treatment increased survival of retinal neurons after optic nerve crush (ONC) in rodent models of visual system injury. However, despite this protection no functional recovery could be detected in rats, which was interpreted as evidence of “silent survivor” cells. We now analysed the mechanisms underlying this “silent survival” effect. Using in vivo microscopy of the retina we investigated the survival and morphology of fluorescent neurons before and after ONC in animals receiving rtACS or sham treatment. One week after the crush, more neurons survived in the rtACS-treated group compared to sham-treated controls. In vivo imaging further revealed that in the initial post-ONC period, rtACS induced dendritic pruning in surviving neurons. In contrast, dendrites in untreated retinae degenerated slowly after the axonal trauma and neurons died. The complete loss of visual evoked potentials supports the hypothesis that cell signalling is abolished in the surviving neurons. Despite this evidence of “silencing”, intracellular free calcium imaging showed that the cells were still viable. We propose that early after trauma, complete dendritic stripping following rtACS protects neurons from excitotoxic cell death by silencing them.

Postnatal Dendritic Growth and Spinogenesis of Layer-V Pyramidal Cells Differ between Visual, Inferotemporal, and Prefrontal Cortex of the Macaque Monkey.

Pyramidal cells in the primate cerebral cortex, particularly those in layer III, exhibit regional variation in both the time course and magnitude of postnatal growth and pruning of dendrites and spines. Less is known about the development of pyramidal cell dendrites and spines in other cortical layers. Here we studied dendritic morphology of layer-V pyramidal cells in primary visual cortex (V1, sensory), cytoarchitectonic area TE in the inferotemporal cortex (sensory association), and granular prefrontal cortex (Walker's area 12, executive) of macaque monkeys at the ages of 2 days, 3 weeks, 3.5 months, and 4.5 years. We found that changes in the basal dendritic field area of pyramidal cells were different across the three areas. In V1, field size became smaller over time (largest at 2 days, half that size at 4.5 years), in TE it did not change, and in area 12 it became larger over time (smallest at 2 days, 1.5 times greater at 4.5 years). In V1 and TE, the total number of branch points in the basal dendritic trees was similar between 2 days and 4.5 years, while in area 12 the number was greater in the adult monkeys than in the younger ones. Spine density peaked at 3 weeks and declined in all areas by adulthood, with V1 exhibiting a faster decline than area TE or area 12. Estimates of the total number of spines in the dendritic trees revealed that following the onset of visual experience, pyramidal cells in V1 lose more spines than they grow, whereas those in TE and area 12 grow more spines than they lose during the same period. These data provide further evidence that the process of synaptic refinement in cortical pyramidal cells differs not only according to time, but also location within the cortex. Furthermore, given the previous finding that layer-III pyramidal cells in all these areas exhibit the highest density and total number of spines at 3.5 months, the current results indicate that pyramidal cells in layers III and V develop spines at different rates.

Maternal care associates with differences in morphological complexity in the medial preoptic area

The medial preoptic area (MPOA) is implicated in the expression of maternal behavior including the frequency of pup licking/grooming (LG) in the rat. Cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) is a transcription factor that regulates the expression of many genes. We found that lactating rats that are more maternal towards their pups showing increased licking/grooming (i.e. high-LG mothers) had increased levels of phosphorylated CREB (pCREB) in the MPOA following a nursing bout and they displayed a reduced population of greater dendritic complexity index (DCI) neurons compared to less maternal rats showing decreased licking/grooming (i.e. low-LG mothers). CREB overexpression in MPOA neuronal cultures associated with a decrease in dendritic complexity and an increase in the expression of Rem2 and brain-derived neurotrophic factor (BDNF), genes implicated in dendritic pruning. While there were no differences in Rem2 expression in virgin high and low-LG female rats, Rem2 was significantly increased in the MPOA of high-LG compared to low-LG lactating rats. CREB activity in the MPOA associates with maternal behavior and reduced dendritic complexity possibly by increasing Rem2 expression.

Rabconnectin-3\u03b1 is required for the morphological maturation of GnRH neurons and kisspeptin responsiveness

A few hundred hypothalamic neurons form a complex network that controls reproduction in mammals by secreting gonadotropin-releasing hormone (GnRH). Timely postnatal changes in GnRH secretion are essential for pubertal onset. During the juvenile period, GnRH neurons undergo morphological remodeling, concomitantly achieving an increased responsiveness to kisspeptin, the main secretagogue of GnRH. However, the link between GnRH neuron activity and their morphology remains unknown. Here, we show that brain expression levels of Dmxl2, which encodes the vesicular protein rabconnectin-3α, determine the capacity of GnRH neurons to be activated by kisspeptin and estradiol. We also demonstrate that Dmxl2 expression levels control the pruning of GnRH dendrites, highlighting an unexpected role for a vesicular protein in the maturation of GnRH neuronal network. This effect is mediated by rabconnectin-3α in neurons or glial cells afferent to GnRH neurons. The widespread expression of Dmxl2 in several brain areas raises the intriguing hypothesis that rabconnectin-3α could be involved in the maturation of other neuronal populations.

A compact divisor based on SN P systems along with dendritic behavior

The Spiking Neural P (SN P) system is defined as a type of parallel computing mechanism bio-inspired by the behavior of the soma. Several authors have been employing these systems in order to create efficient arithmetic divisor circuits exploiting at maximum their intrinsic parallel processing. However, the current neural divisors expend a large amount of neurons with complex spiking rules to synchronize the input information to be processed by the soma. This work proposes a compact neural divisor that uses eight neurons and two type spiking rules per neuron. In addition, the proposed circuit includes the dendrite’s behavior as feedback connections, dendritic delays, reduction in the dendrite length and dendritic pruning into the conventional SN P systems in order to simplify the synchronization of the neural processing carried out by the soma. The results show that the proposed neural divisor can be implemented in embedded neuromorphic circuits. This, potentially allows its use in portable applications such as vision processing systems for mobile robots and cryptographic systems for mobile communication devices.

Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons.

Pruning, whereby neurons eliminate their excess neurites, is central for the maturation of the nervous system. In Drosophila, sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. Here, we show that the small GTPase Arf1, an important regulator of the secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP- and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. We further identified Sec71 as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not for apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, we show that the Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell-adhesion molecule Neuroglian (Nrg).

Transplanted embryonic neurons integrate into adult neocortical circuits.

The ability of the adult mammalian brain to compensate for neuronal loss caused by injury or disease is very limited. Transplantation aims to replace lost neurons, but the extent to which new neurons can integrate into existing circuits is unknown. Here, using chronic in vivo two-photon imaging, we show that embryonic neurons transplanted into the visual cortex of adult mice mature into bona fide pyramidal cells with selective pruning of basal dendrites, achieving adult-like densities of dendritic spines and axonal boutons within 4-8 weeks. Monosynaptic tracing experiments reveal that grafted neurons receive area-specific, afferent inputs matching those of pyramidal neurons in the normal visual cortex, including topographically organized geniculo-cortical connections. Furthermore, stimulus-selective responses refine over the course of many weeks and finally become indistinguishable from those of host neurons. Thus, grafted neurons can integrate with great specificity into neocortical circuits that normally never incorporate new neurons in the adult brain.

Segmenting and validating brain tissue definitions in the presence of varying tissue contrast

We propose a method for segmenting brain tissue as either gray matter or white matter in the presence of varying tissue contrast, which can derive from either differential changes in tissue water content or increasing myelin content of white matter. Our method models the spatial distribution of intensities as a Markov Random Field (MRF) and estimates the parameters for the MRF model using a maximum likelihood approach. Although previously described methods have used similar models to segment brain tissue, accurate model of the conditional probabilities of tissue intensities and adaptive estimates of tissue properties to local intensities generates tissue definitions that are accurate and robust to variations in tissue contrast with age and across illnesses. Robustness to variations in tissue contrast is important to understand normal brain development and to identify the brain bases of neurological and psychiatric illnesses. We used simulated brains of varying tissue contrast to compare both visually and quantitatively the performance of our method with the performance of prior methods. We assessed validity of the cortical definitions by associating cortical thickness with various demographic features, clinical measures, and medication use in our three large cohorts of participants who were either healthy or who had Bipolar Disorder (BD), Autism Spectrum Disorder (ASD), or familial risk for Major Depressive Disorder (MDD).We assessed validity of the tissue definitions using synthetic brains and data for three large cohort of individuals with various neuropsychiatric disorders. Visual inspection and quantitative analyses showed that our method accurately and robustly defined the cortical mantle in brain images with varying contrast. Furthermore, associating the thickness with various demographic and clinical measures generated findings that were novel and supported by histological analyses or were supported by previous MRI studies, thereby validating the cortical definitions generated by the proposed method: (1) Although cortical thickness decreased with age in adolescents, in adults cortical thickness did not correlate significantly with age. Our synthetic data showed that the previously reported thinning of cortex in adults is likely due to decease in tissue contrast, thereby suggesting that the method generated cortical definitions in adults that were invariant to tissue contrast. In adolescents, cortical thinning with age was preserved likely due to widespread dendritic and synaptic pruning, even though the effects of decreasing tissue contrast were minimized. (3) The method generated novel finding of both localized increases and decreases in thickness of males compared to females after controlling for the differing brain sizes, which are supported by the histological analyses of brain tissue in males and females. (4) The proposed method, unlike prior methods, defined thicker cortex in BD individuals using lithium. The novel finding is supported by the studies that showed lithium treatment increased dendritic arborization and neurogenesis, thereby leading to thickening of cortex. (5) In both BD and ASD participants, associations of more severe symptoms with thinner cortex showed that correcting for the effects of tissue contrast preserved the biological consequences of illnesses. Therefore, consistency of the findings across the three large cohorts of participants, in images acquired on either 1.5T or 3T MRI scanners, and with findings from prior histological analyses provides strong evidence that the proposed method generated valid and accurate definitions of the cortex while controlling for the effects of tissue contrast.

Adult-born dentate granule cells show a critical period of dendritic reorganization and are distinct from developmentally born cells.

Adult-born dentate granule cells (abGCs) exhibit a critical developmental phase during function integration. The time window of this phase is debated and whether abGCs become indistinguishable from developmentally born mature granule cells (mGCs) is uncertain. We analyzed complete dendritic reconstructions from abGCs and mGCs using viral labeling. AbGCs from 21-77days post intrahippocampal injection (dpi) exhibited comparable dendritic arbors, suggesting that structural maturation precedes functional integration. In contrast, significant structural differences were found compared to mGCs: AbGCs had more curved dendrites, more short terminal segments, a different branching pattern, and more proximal terminal branches. Morphological modeling attributed these differences to developmental dendritic pruning and postnatal growth of the dentate gyrus. We further correlated GC morphologies with the responsiveness to unilateral medial perforant path stimulation using the immediate-early gene Arc as a marker of synaptic activation. Only abGCs at 28 and 35dpi but neither old abGCs nor mGCs responded to stimulation with a remodeling of their dendritic arbor. Summarized, abGCs stay distinct from mGCs and their dendritic arbor can be shaped by afferent activity during a narrow critical time window.

Social play in juvenile hamsters alters dendritic morphology in the medial prefrontal cortex and attenuates effects of social stress in adulthood.

Social play is a fundamental aspect of behavioral development in many species. Social play deprivation in rats alters dendritic morphology in the ventromedial prefrontal cortex (vmPFC) and we have shown that this brain region regulates responses to social defeat stress in Syrian hamsters. In this study, we tested whether play deprivation during the juvenile period disrupts dendritic morphology in the prefrontal cortex and potentiates the effects of social defeat stress. At weaning, male hamsters were either group-housed with peers or pair-housed with their mother, with whom they do not play. In adulthood, animals received acute social defeat stress or no-defeat control treatment. The hamsters were then tested for a conditioned defeat response in a social interaction test with a novel intruder, and were also tested for social avoidance of a familiar opponent. Brains were collected for Golgi-Cox staining and analysis of dendritic morphology in the infralimbic (IL), prelimbic (PL), and orbitofrontal cortex (OFC). Play-deprived animals showed an increased conditioned defeat response and elevated avoidance of a familiar opponent compared with play-exposed animals. Furthermore, play-deprived animals showed increased total length and branch points in apical dendrites of pyramidal neurons in the IL and PL cortices, but not in the OFC. These findings suggest that social play deprivation in juvenile hamsters disrupts neuronal development in the vmPFC and increases vulnerability to the effects of social stress in adulthood. Overall, these results suggest that social play is necessary for the natural dendritic pruning process during adolescence and promotes coping with stress in adulthood. (PsycINFO Database Record

Red Light Treatment in an Axotomy Model of Neurodegeneration.

Red light has been shown to provide neuroprotective effects. Axotomizing the optic nerve initiates retinal ganglion cell (RGC) degeneration, and an early marker of this is dendritic pruning. We hypothesized that 670nm light can delay axotomy-induced dendritic pruning in the retinal explant. To test this hypothesis, we monitored the effects of 670nm light (radiant exposure of 31.7Jcm(-2) ), on RGC dendritic pruning in retinal explants from C57BL/6J mice, at 40min, 8h and 16h post axotomy. For sham-treated retinae, area under the Sholl curve, peak of the Sholl curve and dendritic length at 8h post axotomy showed statistically significant reductions by 42.3% (P=0.008), 29.8% (P=0.007) and 38.4% (P=0.038), respectively, which were further reduced after 16h by 40.56% (P<0.008), 33.9% (P<0.007), 45.43% (P<0.006), respectively. Dendritic field area was also significantly reduced after 16h, by 44.23% (P<0.019). Such statistically significant reductions were not seen in light-treated RGCs at 8 or 16h post axotomy. The results demonstrate the ability of 670nm light to partially prevent exvivo dendropathy in the mouse retina, suggesting that it is worthexploring as a treatment option for dendropathy-associatedneurodegenerative diseases, including glaucoma and Alzheimer's disease.

Research advances on animal genetics in China in 2015.

Chinese scientists have made significant achievements in the field of animal genetics in 2015. Incomplete statistics show that among all the publications of 2015 involving nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), zebrafish (Danio rerio), African clawed frog (Xenopus) or mice (Mus musculus), about 1/5 publications are from China. Many innovative studies were published in high-impact international academic journals by Chinese scientists, including the identification of a putative magnetic receptor MagR, the genetic basis for the regulation of wing polyphenism in the insect brown planthopper (Nilaparvata lugens), DNA N6-methyladenine (6mA) modification in the Drosophila genome, a novel molecular mechanism regarding the dendritic spine pruning and maturation in the mammals, the mechanism for the CREB coactivator CRTC2 in the regulation of hepatic lipid metabolism, the control of systemic inflammation by neurotransmitter dopamine, the role of Gasdermin protein family in triggering pyroptosis, a parvalbumin-positive excitatory visual pathway to trigger fear responses in mice, etc. Chinese scientists have also made important contributions in genome editing via TALEN or CRISPR/Cas system. According to incomplete statistics, more than 1/5 of the publications related to genome editing in 2015 are from China, where a variety of animals with different approaches were targeted, ranging from the worm to primates. Particularly, CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes was successfully achieved for the first time. China has been one of the leading countries in genome sequencing in recent years, and Chinese scientists reported the sequence and annotation of the genomes of several important animal species in 2015, including goose (Anser cygnoides), Schlegel's Japanese Gecko (Gekko japonicus), grass carp (Ctenopharyngodon idellus), large yellow croaker (Larimichthys crocea) and pig (Sus scrofa). They further analyzed the genome-wide genetic basis of the species-specific physiological and pathological characteristics as well as their adaptation to environmental conditions. In this review, we make a first attempt to summarize the research advances on animal genetics in China in 2015, with an emphasis on the achievements led by Chinese scientists and carried out in Chinese institutions. We will briefly discuss the significance of their research and contributions of Chinese scientists in animal genetics.

Sex differences in impulsivity and brain morphometry in methamphetamine users.

Methamphetamine (METH) is an addictive stimulant, and METH users have abnormal brain structures and function. The aims of this study were to investigate the relationships between impulsivity, brain structures, and possible sex-specific differences between METH users and non-drug using Controls. Structural MRI and the Barratt Impulsiveness Scale (BIS) questionnaire were completed in 124 subjects: 62 METH (ages 41.2±1.4years, 34 males) and 62 Controls (ages 43.3±2.3years, 36 males). Independent and interactive effects of METH use status and sex were evaluated. Relationships between METH usage characteristics, brain morphometry, and impulsivity scores were examined. METH users had higher impulsivity scores, on both the Cognitive and Behavioral Factors from the BIS (p<0.0001-0.0001). Compared with same-sex Controls, male METH users had larger, while female METH users had smaller, right superior frontal cortex (interaction-p=0.0005). The male METH users with larger frontal volumes and female METH users with smaller or thinner frontal cortices had greater Cognitive impulsivity (interaction-p≤0.05). Only female METH users showed relatively larger nucleus accumbens (interaction-p=0.03). Greater impulsivity and thinner frontal cortices in METH users are validated. Larger superior frontal cortex in male METH users with greater cognitive impulsivity suggest decreased dendritic pruning during adolescence might have contributed to their impulsive and drug use behaviors. In the female METH users, smaller frontal cortices and the associated greater impulsivity suggest greater neurotoxicity to these brain regions, while their relatively larger nucleus accumbens suggest an estrogen-mediated neuroprotective glial response. Men and women may be affected differently by METH use.

Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture.

PurposeTo develop an ex vivo organotypic retinal explant culture system suitable for multiple time-point imaging of retinal ganglion cell (RGC) dendritic arbors over a period of 1 week, and capable of detecting dendrite neuroprotection conferred by experimental treatments.MethodsThy1-YFP mouse retinas were explanted and maintained in organotypic culture. Retinal ganglion cell dendritic arbors were imaged repeatedly using confocal laser scanning microscopy. Maximal projection z-stacks were traced by two masked investigators and dendritic fields were analyzed for characteristics including branch number, size, and complexity. One group of explants was treated with brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) added to the culture media. Changes in individual dendritic fields over time were detected using pair-wise comparison testing.ResultsRetinal ganglion cells in mouse retinal explant culture began to degenerate after 3 days with 52.4% surviving at 7 days. Dendritic field parameters showed minimal change over 8 hours in culture. Intra- and interobserver measurements of dendrite characteristics were strongly correlated (Spearman rank correlations consistently > 0.80). Statistically significant (P < 0.001) dendritic tree degeneration was detected following 7 days in culture including: 40% to 50% decreases in number of branch segments, number of junctions, number of terminal branches, and total branch length. Scholl analyses similarly demonstrated a significant decrease in dendritic field complexity. Treatment of explants with BDNF+CNTF significantly attenuated dendritic field degeneration.ConclusionsRetinal explant culture of Thy1-YFP tissue provides a useful model for time-lapse imaging of RGC dendritic field degeneration over a course of several days, and is capable of detecting neuroprotective amelioration of dendritic pruning within individual RGCs.