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1. Restrained dendritic growth of adult-born granule cells innervated by transplanted fetal GABAergic interneurons in mice with temporal lobe epilepsy.

   https://doi.org/10.1523/ENEURO.0110-18.2019  

   Gupta, J ; Bromwich, M ; Radell, J ; Arshad, MN ; Gonzalez, S ; Luikart, BW ; Aaron, GB ; Naegele, JR ( March 2019 , ENeuro)

   null (Ed.)

   The dentate gyrus (DG) is a region of the adult rodent brain that undergoes continuous neurogenesis. Seizures and loss or dysfunction of GABAergic synapses onto adult-born dentate granule cells (GCs) alter their dendritic growth and migration, resulting in dysmorphic and hyperexcitable GCs. Additionally, transplants of fetal GABAergic interneurons in the DG of mice with temporal lobe epilepsy (TLE) result in seizure suppression, but it is unknown whether increasing interneurons with these transplants restores GABAergic innervation to adult-born GCs. Here we address this question by retroviral birth-dating GCs at different times up to 12 weeks after pilocarpine-induced TLE in adult mice. ChR2-EYFP-expressing MGE-derived GABAergic interneurons from E13.5 mouse embryos were transplanted into the DG of the TLE mice and GCs with transplant-derived inhibitory post-synaptic currents were identified by patch-clamp electrophysiology and optogenetic interrogation. Putative synaptic sites between GCs and GABAergic transplants were also confirmed by intracellular biocytin staining, immunohistochemistry, and confocal imaging. 3D reconstructions of dendritic arbors and quantitative morphometric analyses were carried out in >150 adult-born GCs. GABAergic inputs from transplanted interneurons correlated with markedly shorter GC dendrites, compared to GCs that were not innervated by the transplants. Moreover, these effects were confined to distal dendritic branches and a short time window of 6-8 weeks. The effects were independent of seizures as they were also observed in naïve mice with MGE transplants. These findings are consistent with the hypothesis that increased inhibitory currents over a smaller dendritic arbor in adult-born GCs may reduce their excitability and lead to seizure suppression. 

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2. Induction of Temporal Lobe Epilepsy in Mice with Pilocarpine

   https://doi.org/10.21769/BioProtoc.3533  

   Arshad, M ; Naegele, J. ( January 2020 , Bioprotocol)

   null (Ed.)

   In the pilocarpine model of temporal lobe epilepsy (TLE) in rodents, systemic injections of pilocarpine induce continuous, prolonged limbic seizures, a condition termed “Status Epilepticus” (SE). With appropriate doses, many inbred strains of mice show behavioral seizures within an hour after pilocarpine is injected. With the behavioral scoring system based on a modification of the original Racine scale, one can monitor the seizures behaviorally, as they develop into more prolonged seizures and SE. SE is typically associated with damage to subsets of hippocampal neurons and other structural changes in the hippocampus and generally subsides on its own. However, more precise control of the duration of SE is commonly achieved by injecting a benzodiazepine into the mouse 1 to 3 h after the onset of SE to suppress the seizures. Several days following pilocarpine-induced SE, electrographic and behavioral seizures begin to occur spontaneously. The goal of this protocol is to reliably generate mice that develop spontaneous recurrent seizures (SRS) and show the typical neuropathological changes in the brain characteristic of severe human mesial temporal lobe epilepsy (mTLE), without high mortality. To reduce mortality, multiple subthreshold injections of pilocarpine are administered, which increases the percentage of mice developing SE without concomitant mortality. Precise control of the duration of SE (1 or 3 h) is achieved by suppressing SE with the benzodiazepine Midazolam (Versed). We have found that this protocol is an efficient means for generating mice that subsequently develop characteristics of human mTLE including high-frequency interictal spike and wave activity and SRS. In addition, we and others have shown that this protocol produces mice that show excitotoxic cell death of subsets of hippocampal GABAergic interneurons, particularly in the dentate gyrus and compensatory sprouting of excitatory projections from dentate granule cells (mossy fiber sprouting). Aspects of this protocol have been described in several of our previous publications. 

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3. Adult‐born granule cell mossy fibers preferentially target parvalbumin‐positive interneurons surrounded by perineuronal nets

   https://doi.org/10.1002/hipo.23296  

   Briones, Brandy A. ; Pisano, Thomas J. ; Pitcher, Miah N. ; Haye, Amanda E. ; Diethorn, Emma J. ; Engel, Esteban A. ; Cameron, Heather A. ; Gould, Elizabeth ( January 2021 , Hippocampus)

   Adult‐born granule cells (abGCs) integrate into the hippocampus and form connections with dentate gyrus parvalbumin‐positive (PV+) interneurons, a circuit important for modulating plasticity. Many of these interneurons are surrounded by perineuronal nets (PNNs), extracellular matrix structures known to participate in plasticity. We compared abGC projections to PV+ interneurons with negative‐to‐low intensity PNNs to those with high intensity PNNs using retroviral and 3R‐Tau labeling in adult mice, and found that abGC mossy fibers and boutons are more frequently located near PV+ interneurons with high intensity PNNs. These results suggest that axons of new neurons preferentially stabilize near target cells with intense PNNs. Next, we asked whether the number of abGCs influences PNN formation around PV+ interneurons, and found that near complete ablation of abGCs produced a decrease in the intensity and number of PV+ neurons with PNNs, suggesting that new neuron innervation may enhance PNN formation. Experience‐driven changes in adult neurogenesis did not produce consistent effects, perhaps due to widespread effects on plasticity. Our study identifies abGC projections to PV+ interneurons with PNNs, with more presumed abGC mossy fiber boutons found near the cell body of PV+ interneurons with strong PNNs.

    

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4. Elevated zinc transporter ZnT3 in the dentate gyrus of mast cell‐deficient mice

   https://doi.org/10.1111/ejn.14575  

   Wiqas, Amen ; LeSauter, Joseph ; Taub, Alana ; Austin, Rachel Narehood ; Silver, Rae ( September 2019 , European Journal of Neuroscience)

   Zinc is important in neurogenesis, but excessive levels can cause apoptosis and other pathologies leading to cognitive impairments. Mast cells are present in many brain regions including the hippocampus, an area rich in vesicular zinc. Mast cells contain zinc‐rich granules and a well‐developed mechanism for uptake of zinc ions; both features point to the potential for a role in zinc homeostasis. Prior work using the Timm stain supported this hypothesis, as increased labile zinc was detected in the hippocampus of mast cell‐deficient mice compared to wild‐type mice while no differences in total zinc were found between the two genotypes in the whole brain or other tissues. The current report further examines differences in zinc homeostasis between wild‐type and mast cell‐deficient mice by exploring the zinc transporter ZnT3, which transports labile zinc into synaptic vesicles. The first study used immunocytochemistry to localize ZnT3 within the mossy fibre layer of the hippocampus to determine whether there was differential expression of ZnT3 in wild‐type versus mast cell‐deficient mice. The second study used inductively coupled plasma mass spectrometry (ICP‐MS) to determine total zinc content in the whole dentate gyrus of the two genotypes. The immunocytochemical results indicate that there are higher levels of ZnT3 localized to the mossy fibre layer of the dentate gyrus of mast cell‐deficient mice than in wild‐type mice. TheICP‐MSdata reveal no differences in total zinc in dentate gyrus as a whole. The results are consistent with the hypothesis that mast cells participate in zinc homeostasis at the level of synaptic vesicles.

    

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5. Comparative performance of double‐digest RAD sequencing across divergent arachnid lineages

   https://doi.org/10.1111/1755-0998.12575  

   Burns, Mercedes ; Starrett, James ; Derkarabetian, Shahan ; Richart, Casey H. ; Cabrero, Allan ; Hedin, Marshal ( August 2016 , Molecular Ecology Resources)

   Next‐generation sequencing technologies now allow researchers of non‐model systems to perform genome‐based studies without the requirement of a (often unavailable) closely related genomic reference. We evaluated the role of restriction endonuclease (RE) selection in double‐digest restriction‐site‐associatedDNAsequencing (ddRADseq) by generating reduced representation genome‐wide data using four differentREcombinations. Our expectation was thatREselections targeting longer, more complex restriction sites would recover fewer loci thanREwith shorter, less complex sites. We sequenced a diverse sample of non‐model arachnids, including five congeneric pairs of harvestmen (Opiliones) and four pairs of spiders (Araneae). Sample pairs consisted of either conspecifics or closely related congeneric taxa, and in total 26 sample pair analyses were tested. Sequence demultiplexing, read clustering and variant calling were performed in thepyRADprogram. The 6‐base pair cutterEcoRIcombined with methylated site‐specific 4‐base pair cutterMspIproduced, on average, the greatest numbers of intra‐individual loci and shared loci per sample pair. As expected, the number of shared loci recovered for a sample pair covaried with the degree of genetic divergence, estimated with cytochrome oxidase I sequences, although this relationship was non‐linear. Our comparative results will prove useful in guiding protocol selection for ddRADseq experiments on many arachnid taxa where reference genomes, even from closely related species, are unavailable.

    

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