Granule Cell Neurons Research Articles

Structural plasticity of pyramidal cell neurons measured after FLASH and conventional dose-rate irradiation.

Evidence shows that ultra-high dose-rate FLASH-radiotherapy (FLASH-RT) protects against normal tissue complications and functional decrements in the irradiated brain. Past work has shown that radiation-induced cognitive impairment, neuroinflammation and reduced structural complexity of granule cell neurons were not observed to the same extent after FLASH-RT (> MGy/s) compared to conventional dose-rate (CONV, 0.1 Gy/s) delivery. To explore the sensitivity of different neuronal populations to cranial irradiation and dose-rate modulation, hippocampal CA1 and medial prefrontal cortex (PFC) pyramidal neurons were analyzed by electron and confocal microscopy. Neuron ultrastructural analyses by electron microscopy after 10 Gy FLASH- or CONV-RT exposures indicated that irradiation had little impact on dendritic complexity and synapse density in the CA1, but did increase length and head diameter of smaller non-perforated synapses. Similarly, irradiation caused no change in PFC prelimbic/infralimbic axospinous synapse density, but reductions in non-perforated synapse diameters. While irradiation resulted in thinner myelin sheaths compared to controls, none of these metrics were dose-rate sensitive. Analysis of fluorescently labeled CA1 neurons revealed no radiation-induced or dose-rate-dependent changes in overall dendritic complexity or spine density, in contrast to our past analysis of granule cell neurons. Super-resolution confocal microscopy following a clinical dosing paradigm (3×10Gy) showed significant reductions in excitatory vesicular glutamate transporter 1 and inhibitory vesicular GABA transporter puncta density within the CA1 that were largely dose-rate independent. Collectively, these data reveal that, compared to granule cell neurons, CA1 and mPFC neurons are more radioresistant irrespective of radiation dose-rate.

Extracellular vesicles from GABAergic but not glutamatergic neurons protect against neurological dysfunction following cranial irradiation

Cranial irradiation used to control brain malignancies invariably leads to progressive and debilitating declines in cognition. Clinical efforts implementing hippocampal avoidance and NMDAR antagonism, have sought to minimize dose to radiosensitive neurogenic regions while normalizing excitatory/inhibitory (E/I) tone. Results of these trials have yielded only marginal benefits to cognition, prompting current studies to evaluate the potential of systemic extracellular vesicle (EV) therapy to restore neurocognitive functionality in the irradiated brain. Here we tested the hypothesis that EVs derived from inhibitory but not excitatory neuronal cultures would prove beneficial to cognition and associated pathology. Rats subjected to a clinically relevant, fractionated cranial irradiation paradigm were given multiple injections of either GABAergic- or glutamatergic-derived EV and subjected to behavioral testing. Rats treated with GABAergic but not glutamatergic EVs showed significant improvements on hippocampal- and cortical-dependent behavioral tasks. While each treatment enhanced levels of the neurotrophic factors BDNF and GDNF, only GABAergic EVs preserved granule cell neuron dendritic spine density. Additional studies conducted with GABAergic EVs, confirmed significant benefits on amygdala-dependent behavior and modest changes in synaptic plasticity as measured by long-term potentiation. These data point to a potentially more efficacious approach for resolving radiation-induced neurological deficits, possibly through a mechanism able to restore homeostatic E/I balance.

Evaluation of histological and ultrastructural changes provoked by prenatal tramadol on postnatal cortical cerebellar neuronal development in rats: possible implication of Ki67, GFAP and MicroRNA-7/P53 signalling trajectories

Tramadol is a novel centrally acting analgesic. Despite, its implementation during pregnancy may impair neuronal survival and synaptic development in neonatal cerebella. The current investigation assessed the histological and ultrastructural alterations in postnatal cortical cerebellar neuronal development induced by prenatal tramadol. 30 offsprings were divided to control group I: fifteen pups born to mothers given saline from D10 till D21 of gestation. Tramadol-treated group II: fifteen pups born to mothers received tramadol HCL (50 mg/kg/day) from D10 till D21 of gestation. Pups were categorized into three subgroups (a, b, and c) and offered for sacrifice on the seventh, fourteenth and twenty-first post-natal days. Light microscopic examination revealed the overcrowding and signs of red degeneration affecting purkinje cell layer. Neurodegenerative signs of both purkinje and granule cell neurons were also confirmed by TEM in form of chromatin condensation, dilated Golgi channels, disrupted endoplasmic reticulum, marked infolding of the nuclear envelope and decrease in granule cell precursors. In addition, the astrocytic processes and terminal nerve axons appeared with different degrees of demyelination and decreased number of oligodendrocytes and degenerated mitochondria. Furthermore, group II exhibited an increase in P53 immune expression. The area percentage of apoptotic cells detected by TUNEL assay was significantly increased. Besides to the significant decrease of Ki67 immunoreactivity in the stem neuronal cell progenitors. Quantitative PCR results showed a significant decline in micro RNA7 gene expression in tramadol treated groups resulting in affection of multiple target genes in P53 signaling pathways, improper cortical size and defect in neuronal development.

Canonical Wnt activator Chir99021 prevents epileptogenesis in the intrahippocampal kainate mouse model of temporal lobe epilepsy

The Wnt signaling pathway mediates the development of dentate granule cell neurons in the hippocampus. These neurons are central to the development of temporal lobe epilepsy and undergo structural and physiological remodeling during epileptogenesis, which results in the formation of epileptic circuits. The pathways responsible for granule cell remodeling during epileptogenesis have yet to be well defined, and represent therapeutic targets for the prevention of epilepsy. The current study explores Wnt signaling during epileptogenesis and for the first time describes the effect of Wnt activation using Wnt activator Chir99021 as a novel anti-epileptogenic therapeutic approach.Focal mesial temporal lobe epilepsy was induced by intrahippocampal kainate (IHK) injection in wild-type and POMC-eGFP transgenic mice. Wnt activator Chir99021 was administered daily, beginning 3 h after seizure induction, and continued up to 21-days. Immature granule cell morphology was quantified in the ipsilateral epileptogenic zone and the contralateral peri-ictal zone 14 days after IHK, targeting the end of the latent period. Bilateral hippocampal electrocorticographic recordings were performed for 28-days, 7-days beyond treatment cessation. Hippocampal behavioral tests were performed after completion of Chir99021 treatment.Consistent with previous studies, IHK resulted in the development of epilepsy after a 14 day latent period in this well-described mouse model. Activation of the canonical Wnt pathway with Chir99021 significantly reduced bilateral hippocampal seizure number and duration. Critically, this effect was retained after treatment cessation, suggesting a durable antiepileptogenic change in epileptic circuitry. Morphological analyses demonstrated that Wnt activation prevented pathological remodeling of the primary dendrite in both the epileptogenic zone and peri-ictal zone, changes in which may serve as a biomarker of epileptogenesis and anti-epileptogenic treatment response in pre-clinical studies. These findings were associated with improved object location memory with Chir99021 treatment after IHK.This study provides novel evidence that canonical Wnt activation prevents epileptogenesis in the IHK mouse model of mesial temporal lobe epilepsy, preventing pathological remodeling of dentate granule cells. Wnt signaling may therefore play a key role in mesial temporal lobe epileptogenesis, and Wnt modulation may represent a novel therapeutic strategy in the prevention of epilepsy.

RIPK3 promotes brain region-specific interferon signaling and restriction of tick-borne flavivirus infection.

Innate immune signaling in the central nervous system (CNS) exhibits many remarkable specializations that vary across cell types and CNS regions. In the setting of neuroinvasive flavivirus infection, neurons employ the immunologic kinase receptor-interacting kinase 3 (RIPK3) to promote an antiviral transcriptional program, independently of the traditional function of this enzyme in promoting necroptotic cell death. However, while recent work has established roles for neuronal RIPK3 signaling in controlling mosquito-borne flavivirus infections, including West Nile virus and Zika virus, functions for RIPK3 signaling in the CNS during tick-borne flavivirus infection have not yet been explored. Here, we use a model of Langat virus (LGTV) encephalitis to show that RIPK3 signaling is specifically required in neurons of the cerebellum to control LGTV replication and restrict disease pathogenesis. This effect did not require the necroptotic executioner molecule mixed lineage kinase domain like protein (MLKL), a finding similar to previous observations in models of mosquito-borne flavivirus infection. However, control of LGTV infection required a unique, region-specific dependence on RIPK3 to promote expression of key antiviral interferon-stimulated genes (ISG) in the cerebellum. This RIPK3-mediated potentiation of ISG expression was associated with robust cell-intrinsic restriction of LGTV replication in cerebellar granule cell neurons. These findings further illuminate the complex roles of RIPK3 signaling in the coordination of neuroimmune responses to viral infection, as well as provide new insight into the mechanisms of region-specific innate immune signaling in the CNS.

Distinct Actions of the Thyroid Hormone Transporters Mct8 and Oatp1c1 in Murine Adult Hippocampal Neurogenesis.

Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in Allan-Herndon-Dudley Syndrome, a severe form of psychomotor retardation, while inactivating mutations in another TH transporter, organic anion transporting polypeptide 1c1 (OATP1C1), are linked to juvenile neurodegeneration. These diseases point to essential roles for TH transporters in CNS function. We recently defined the presence of Mct8 in adult hippocampal progenitors and mature granule cell neurons and unraveled cell-autonomous and indirect requirements for Mct8 in adult hippocampal neurogenesis. Here, we investigated whether Oatp1c1 is involved in the hippocampal neurogenic process in concert with Mct8. We detected Oatp1c1 gene expression activity and transcripts in subsets of progenitors, neurons and niche cells in the dentate gyrus. Absence of Oatp1c1 resulted in increased neuroblast and reduced immature neuron numbers in 6-month-old Oatp1c1ko and Mct8/Oatp1c1 double knockout (M/Odko) mice. Reduced EdU-label retention in Mct8ko and M/Odko mice confirmed the impact of Mct8 on neuron formation. In contrast, no significant effect of Oatp1c1 loss on granule cell neuron production and anxiety-like behavior in the open field arena were seen. Together, our results reinforce that distinct actions of each TH transporter are required at multiple stages to ensure proper adult hippocampal neurogenesis.

Isoxazole-9 reduces enhanced fear responses and retrieval in ethanol-dependent male rats.

Plasticity in the dentate gyrus (DG) is strongly influenced by ethanol, and ethanol experience alters long-term memory consolidation dependent on the DG. However, it is unclear if DG plasticity plays a role in dysregulation of long-term memory consolidation during abstinence from chronic ethanol experience. Outbred male Wistar rats experienced 7weeks of chronic intermittent ethanol vapor exposure (CIE). Seventy-two hours after CIE cessation, CIE and age-matched ethanol-naïve Air controls experienced auditory trace fear conditioning (TFC). Rats were tested for cue-mediated retrieval in the fear context either twenty-four hours (24hr), ten days (10days), or twenty-one days (21days) later. CIE rats showed enhanced freezing behavior during TFC acquisition compared to Air rats. Air rats showed significant fear retrieval, and this behavior did not differ at the three time points. In CIE rats, fear retrieval increased over time during abstinence, indicating an incubation in fear responses. Enhanced retrieval at 21days was associated with reduced structural and functional plasticity of ventral granule cell neurons (GCNs) and reduced expression of synaptic proteins important for neuronal plasticity. Systemic treatment with the drug Isoxazole-9 (Isx-9; small molecule that stimulates DG plasticity) during the last week and a half of CIE blocked altered acquisition and retrieval of fear memories in CIE rats during abstinence. Concurrently, Isx-9 modulated the structural and functional plasticity of ventral GCNs and the expression of synaptic proteins in the ventral DG. These findings identify that abstinence-induced disruption of fear memory consolidation occurs via altered plasticity within the ventral DG, and that Isx-9 prevented these effects.

Cell Type-specific Knockout with Gli1-mediated Cre Recombination in the Developing Cerebellum.

The inducible Cre-loxP system provides a useful tool for inducing the selective deletion of genes that are essential for proper development and enables the study of gene functions in properly developed animals. Here, we show that inducible Cre-loxP driven by the Gli1-promoter can induce cell-type-specific deletion of target genes in cerebellar cortical neurons. We used reporter mice containing the YFP (yellow fluorescence protein) gene at the Gt(ROSA)26Sor locus with a loxP-flanked transcriptional stop sequence, in which successful Cre-mediated excision of the stop sequence is indicated by YFP expression in Cre-expressing cells. Administration of tamoxifen during early postnatal days (P4~7) induces Cre-dependent excision of stop sequences and allows YFP expression in proliferating neuronal progenitor cells in the external granule layer and Bergmann glia in the Purkinje cell layer. A substantial number of YFP-positive progenitor cells in the external granule layer migrated to the internal granule cell layer and became granule cell neurons. By comparison, injection of tamoxifen during late postnatal days (P19~22) induces YFP expression only in Bergmann glia, and most granule cell neurons were devoid of YFP expression. The results indicate that the Gli1 promoter is temporarily active in progenitor cells in the external granule layer during the early postnatal period but constitutively active in Bergmann glia. We propose that the Gli1-mediated CreER system can be applied for the conditional deletion of genes of interest from cerebellar granule cell neurons and/or Bergmann glia.

Intrinsic Neuronal Activity during Migration Controls the Recruitment of Specific Interneuron Subtypes in the Postnatal Mouse Olfactory Bulb.

Neuronal activity has been identified as a key regulator of neuronal network development, but the impact of activity on migration and terminal positioning of interneuron subtypes is poorly understood. The absence of early subpopulation markers and the presence of intermingled migratory and postmigratory neurons make the developing cerebral cortex a difficult model to answer these questions. Postnatal neurogenesis in the subventricular zone (SVZ) offers a more accessible and compartmentalized model. Neural stem cells regionalized along the border of the lateral ventricle produce two main subtypes of neural progenitors, granule cells and periglomerular neurons that migrate tangentially in the rostral migratory stream (RMS) before migrating radially in the olfactory bulb (OB) layers. Here, we used targeted postnatal electroporation to compare the migration of these two populations in male and female mice. We do not observe any obvious differences regarding the mode of tangential or radial migration between these two subtypes. However, we find a striking increase of intrinsic calcium activity in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. By decreasing neuronal excitability in GC-Ps, we find that neuronal activity has little effect on migration but is required for normal positioning and survival of GC-Ps in the OB layers. Strikingly, decreasing activity of periglomerular neuron precursors (PGN-Ps) did not impact their positioning or survival. Altogether these findings suggest that neuronal excitability plays a subtype specific role during the late stage of migration of postnatally born OB interneurons.SIGNIFICANCE STATEMENT While neuronal activity is a critical factor regulating different aspects of neurogenesis, it has been challenging to study its role during the migration of different neuronal subpopulations. Here, we use postnatal targeted electroporation to label and manipulate the two main olfactory bulb (OB) interneuron subpopulations during their migration: granule cell and periglomerular neuron precursors (PGN-Ps). We find a very striking increase of calcium activity only in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. Interestingly, blocking activity in GC-Ps affected mainly their positioning and survival while PGN-Ps were not affected. These results suggest that neuronal activity is required specifically for the recruitment of GC-Ps in the OB layers.

Nuclear Reorganization in Hippocampal Granule Cell Neurons from a Mouse Model of Down Syndrome: Changes in Chromatin Configuration, Nucleoli and Cajal Bodies.

Down syndrome (DS) or trisomy of chromosome 21 (Hsa21) is characterized by impaired hippocampal-dependent learning and memory. These alterations are due to defective neurogenesis and to neuromorphological and functional anomalies of numerous neuronal populations, including hippocampal granular cells (GCs). It has been proposed that the additional gene dose in trisomic cells induces modifications in nuclear compartments and on the chromatin landscape, which could contribute to some DS phenotypes. The Ts65Dn (TS) mouse model of DS carries a triplication of 92 genes orthologous to those found in Hsa21, and shares many phenotypes with DS individuals, including cognitive and neuromorphological alterations. Considering its essential role in hippocampal memory formation, we investigated whether the triplication of this set of Hsa21 orthologous genes in TS mice modifies the nuclear architecture of their GCs. Our results show that the TS mouse presents alterations in the nuclear architecture of its GCs, affecting nuclear compartments involved in transcription and pre-rRNA and pre-mRNA processing. In particular, the GCs of the TS mouse show alterations in the nucleolar fusion pattern and the molecular assembly of Cajal bodies (CBs). Furthermore, hippocampal GCs of TS mice present an epigenetic dysregulation of chromatin that results in an increased heterochromatinization and reduced global transcriptional activity. These nuclear alterations could play an important role in the neuromorphological and/or functional alterations of the hippocampal GCs implicated in the cognitive dysfunction characteristic of TS mice.

Chronic ethanol exposure differentially alters neuronal function in the medial prefrontal cortex and dentate gyrus

Alterations in the function of prefrontal cortex (PFC) and hippocampus have been implicated in underlying the relapse to alcohol seeking behaviors in humans and animal models of moderate to severe alcohol use disorders (AUD). Here we used chronic intermittent ethanol vapor exposure (CIE), 21d protracted abstinence following CIE (21d AB), and re-exposure to one vapor session during protracted abstinence (re-exposure) to evaluate the effects of chronic ethanol exposure on basal synaptic function, neuronal excitability and expression of key synaptic proteins that play a role in neuronal excitability in the medial PFC (mPFC) and dentate gyrus (DG). CIE consistently enhanced excitability of layer 2/3 pyramidal neurons in the mPFC and granule cell neurons in the DG. In the DG, this effect persisted during 21d AB. Re-exposure did not enhance excitability, suggesting resistance to vapor-induced effects. Analysis of action potential kinetics revealed that altered afterhyperpolarization, rise time and decay time constants are associated with the altered excitability during CIE, 21d AB and re-exposure. Molecular adaptations that may underlie increases in neuronal excitability under these different conditions were identified. Quantitative polymerase chain reaction of large-conductance potassium (BK) channel subunit mRNA in PFC and DG tissue homogenates did not show altered expression patterns of BK subunits. Western blotting demonstrates enhanced phosphorylation of Ca2⁺/calmodulin-dependent protein kinase II (CaMKII), and reduced phosphorylation of glutamate receptor GluN2A/2B subunits. These results suggest a novel relationship between activity of CaMKII and GluN receptors in the mPFC and DG, and neuronal excitability in these brain regions in the context of moderate to severe AUD.

Mechanical injury and blood are drivers of spatial memory deficits after rapid intraventricular hemorrhage

Aneurysmal intraventricular hemorrhage (IVH) survivors may recover with significant deficits in learning and memory. The goal of this study was to investigate the mechanism of memory decline after intraventricular aneurysm rupture. We developed an aneurysmal IVH rat model by injecting autologous, arterial blood over the period of two minutes into the right lateral ventricle. We also evaluated the effects of a volume-matched artificial cerebrospinal fluid (CSF) control, thrombin and the mode of delivery (pulsed hand injection versus continuous pump infusion). We performed magnetic resonance brain imaging after 1 and 5 weeks to evaluate for hydrocephalus and histological analysis of the dentate gyrus after 6 weeks. Only animals which underwent a whole blood pulsed hand injection had a spatial memory acquisition and retention deficit 5 weeks later. These animals had larger ventricles at 1 and 5 weeks than animals which underwent a continuous pump infusion of whole blood. We did not find a decline in dentate gyrus granule cell neurons or an impairment in dentate gyrus neurogenesis or differentiation 6 weeks after IVH. Rapid injections of blood or volume resulted in microglial activation in the dentate gyrus. In conclusion, our results point to mechanical injury as the predominant mechanism of memory decline after intraventricular aneurysmal rupture. However, volume-matched pulsed injections of artificial CSF did not create a spatial memory deficit at 5 weeks. Therefore, whole blood itself must play a role in the mechanism. Further research is required to evaluate whether the viscosity of blood causes additional mechanical disruption and hydrocephalus through a primary injury mechanism or whether the toxicity of blood causes a secondary injury mechanism that leads to the observed spatial memory deficit after 5 weeks.

The amino acid transporter Slc7a5 regulates the mTOR pathway and is required for granule cell development.

Pathogenic mutations in the solute carrier family 7 member 5 (SLC7A5) gene, which encodes an amino acid transporter cause microcephaly and seizures, yet the mechanisms responsible for these phenotypes are unclear. Models have demonstrated that Slc7a5 deletion is embryonic lethal and that these embryos lack a fully formed telencephalon. This phenotype is similar to that of mammalian target of rapamycin (mTOR) protein kinase deletion or mTOR inhibition. Notably, in many cells, Slc7a5 import of amino acids is required to maintain mTOR activity. Slc7a5 is present within neurogenic regions during embryogenesis, is found in cultured neurons and can modulate neuronal electrophysiological properties. However, Slc7a5 is also highly expressed within endothelial cells of the blood–brain barrier where removal in conditional mice leads to severe behavioral defects and non-cell autonomous changes in neurons. Therefore, the extent that neural Slc7a5 is required for development is unclear. Here, subventricular zone neural stem cells that generate olfactory bulb granule cell neurons were electroporated with SLC7A5 or Slc7a5 short hairpin RNA encoding plasmids. Although early phases of neural development were unaltered, Slc7a5 knockdown effected late phases of GC dendrite maturation and survival. Slc7a5 knockdown also decreased mTOR pathway activity. Ras homolog enriched in brain, an mTOR activator, rescued the effect of Slc7a5 knockdown on mTOR pathway activity and dendrite arbors. The data presented here demonstrate that Slc7a5 is required for GC mTOR pathway activity, maturation and survival, which may help explain why Slc7a5 mutations prevent normal brain development and function.

Chronic DREADD Isn't As Bad As It Sounds.

Long-Term Chemogenetic Suppression of Spontaneous Seizures in a Mouse Model for Temporal Lobe Epilepsy Desloovere J, Boon P, Larsen LE, et al. Epilepsia. 2019;60(11):2314-2324. doi:10.1111/epi.16368.Objective:More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for TLE.Methods:Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO; 1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg), and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs), and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days, and the effect on seizures was monitored.Results:Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. Clozapine-N-oxide and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals.Significance:This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.

Hippocampal Neurogenesis Requires Cell-Autonomous Thyroid Hormone Signaling.

SummaryAdult hippocampal neurogenesis is strongly dependent on thyroid hormone (TH). Whether TH signaling regulates this process in a cell-autonomous or non-autonomous manner remains unknown. To answer this question, we used global and conditional knockouts of the TH transporter monocarboxylate transporter 8 (MCT8), having first used FACS and immunohistochemistry to demonstrate that MCT8 is the only TH transporter expressed on neuroblasts and adult slice cultures to confirm a necessary role for MCT8 in neurogenesis. Both mice with a global deletion or an adult neural stem cell-specific deletion of MCT8 showed decreased expression of the cell-cycle inhibitor P27KIP1, reduced differentiation of neuroblasts, and impaired generation of new granule cell neurons, with global knockout mice also showing enhanced neuroblast proliferation. Together, our results reveal a cell-autonomous role for TH signaling in adult hippocampal neurogenesis alongside non-cell-autonomous effects on cell proliferation earlier in the lineage.

Long-term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy.

More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. IntrahippocampalIHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10mg/kg) and clozapine (0.03 and 0.1mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.

Reduced Hippocampal Neurogenesis in Mice Deficient in Apoptosis Repressor with Caspase Recruitment Domain (ARC)

In the adult hippocampal dentate gyrus (DG), the majority of newly generated cells are eliminated by apoptotic mechanisms. The apoptosis repressor with caspase recruitment domain (ARC), encoded by the Nol3 gene, is a potent and multifunctional death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. The aim of the present study was to parse the role of ARC in the development of new granule cell neurons. Nol3 gene expression as revealed by in situ hybridization is present in the entire dentate granule cell layer. Moreover, a comparison of Nol3 expression between FACS-sorted Sox2-positive neural stem cells and Doublecortin (DCX)-positive immature neurons demonstrates upregulation of Nol3 during neurogenesis. Using ARC-deficient mice, we show that proliferation and survival of BrdU birth-dated cells are strongly reduced in the absence of ARC while neuronal-glial fate choice is not affected. Both the number of DCX-positive cells and the number of calretinin (CR)-positive immature postmitotic neurons are reduced in the hippocampus of ARC−/− mice. ARC knockout is not associated with increased numbers of microglia or with microglia activation. However, hippocampal brain-derived neurotrophic factor (BDNF) protein content is significantly increased in ARC−/− mice, possibly representing a compensatory response. Collectively, our results suggest that ARC plays a critical cell-autonomous role in preventing cell death during adult granule cell neogenesis.

Sex Differences in Context-Driven Reinstatement of Methamphetamine Seeking is Associated with Distinct Neuroadaptations in the Dentate Gyrus.

The present study examined differences in operant responses in adult male and female rats during distinct phases of addiction. Males and females demonstrated escalation in methamphetamine (0.05 mg/kg, i.v.) intake with females showing enhanced latency to escalate, and bingeing. Following protracted abstinence, females show reduced responses during extinction, and have greater latency to extinguish compared with males, indicating reduced craving. Females demonstrated lower context-driven reinstatement compared to males, indicating that females have less motivational significance to the context associated with methamphetamine. Whole-cell patch-clamp recordings on dentate gyrus (DG) granule cell neurons (GCNs) were performed in acute brain slices from controls and methamphetamine experienced male and female rats, and neuronal excitability was evaluated from GCNs. Reinstatement of methamphetamine seeking reduced spiking in males, and increased spiking in females compared to controls, demonstrating distinct neuroadaptations in intrinsic excitability of GCNs in males and females. Reduced excitability of GCNs in males was associated with enhanced levels of neural progenitor cells, expression of plasticity-related proteins including CaMKII, and choline acetyltransferase in the DG. Enhanced excitability in females was associated with an increased GluN2A/2B ratio, indicating changes in postsynaptic GluN subunit composition in the DG. Altered intrinsic excitability of GCNs was associated with reduced mossy fiber terminals in the hilus and pyramidal projections, demonstrating compromised neuroplasticity in the DG in both sexes. The alterations in excitability, plasticity-related proteins, and mossy fiber density were correlated with enhanced activation of microglial cells in the hilus, indicating neuroimmune responses in both sexes. Together, the present results indicate sexually dimorphic adaptive biochemical changes in excitatory neurotransmitter systems in the DG and highlight the importance of including sex as a biological variable in exploring neuroplasticity and neuroimmune changes that predict enhanced relapse to methamphetamine-seeking behaviors.

Chronic Neurological Disease Due to Methylmercury Poisoning.

Organic mercury, especially methylmercury, poisoning causes chronic neurological disease predominantly affecting the brain. There have been documented exposures from eating fish from contaminated waters in Japan and in northwestern Ontario and in Iraq from eating bread made from seed wheat treated with methylmercuric fungicide. The neurological disease is called Minamata disease in Japan. Visual field constriction due to involvement of the calcarine cortex, sensory disturbance due to involvement of the somatosensory cortex, and cerebellar ataxia due to involvement of granule cell neurons of the cerebellum are common and characteristic features due to methylmercury poisoning. Other neurological features include dysarthria, postural and action tremor, cognitive impairment, and hearing loss and dysequilibrium. In contrast, peripheral nerve disease is more characteristic of inorganic mercury intoxication. Similarly, psychosis is more typical of exposure to inorganic mercury, which has been documented in the felt hat industry ("mad hatter"). Laboratory tests (e.g., on blood and hair and toenail samples) are of limited value in the assessment of chronic neurological disease due to mercury poisoning because they may not reflect remote neuronal injury due to mercury. Methylmercury also causes injury to fetal brains during development. There is no effective treatment.

The role of hippocampal adult neurogenesis in methamphetamine addiction.

One of the consequences of chronic methamphetamine (Meth) abuse and Meth addiction is impaired hippocampal function which plays a critical role in enhanced propensity for relapse. This impairment is predicted by alterations in hippocampal neurogenesis, structural- and functional-plasticity of granule cell neurons (GCNs), and expression of plasticity-related proteins in the dentate gyrus. This review will elaborate on the effects of Meth in animal models during different stages of addiction-like behavior on proliferation, differentiation, maturation, and survival of newly born neural progenitor cells. We will then discuss evidence for the contribution of adult neurogenesis in context-driven Meth-seeking behavior in animal models. These findings from interdisciplinary studies suggest that a subset of newly born GCNs contribute to context-driven Meth-seeking in Meth addicted animals.