C9ORF72 knockdown triggers FTD-like symptoms and cell pathology in mice.

The GGGGCC intronic repeat expansion within C9ORF72 is the most common genetic cause of ALS and FTD. This mutation results in toxic gain of function through accumulation of expanded RNA foci and aggregation of abnormally translated dipeptide repeat proteins, as well as loss of function due to impaired transcription of C9ORF72. A number of in vivo and in vitro models of gain and loss of function effects have suggested that both mechanisms synergize to cause the disease. However, the contribution of the loss of function mechanism remains poorly understood. We have generated C9ORF72 knockdown mice to mimic C9-FTD/ALS patients haploinsufficiency and investigate the role of this loss of function in the pathogenesis. We found that decreasing C9ORF72 leads to anomalies of the autophagy/lysosomal pathway, cytoplasmic accumulation of TDP-43 and decreased synaptic density in the cortex. Knockdown mice also developed FTD-like behavioral deficits and mild motor phenotypes at a later stage. These findings show that C9ORF72 partial loss of function contributes to the damaging events leading to C9-FTD/ALS.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue.

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

Lipid markers and related transcripts during excitotoxic neurodegeneration in kainate-treated mice.

Lipid homeostasis is dysregulated in some neurodegenerative diseases and after brain injuries due to excess glutamate or lack of oxygen. However the kinetics and cell specificity of dysregulation in different groups of lipids during excitotoxic neuronal death are not clear. Here we examined the changes during excitotoxic neuronal death induced by injecting kainic acid (KA) into the CA1 region of mouse hippocampus. We compared neuronal loss and glial cell proliferation with changes in lipid-related transcripts and markers for different lipid groups, over 12 days after KA-treatment. As neurons showed initial signs of damage, transcripts and proteins linked to fatty acid oxidation were up-regulated. Cholesterol biosynthesis induced by transcripts controlled by the transcription factor Srebp2 seems to be responsible for a transient increase in neuronal free cholesterol at 1 to 2 days. In microglia, but not in neurons, Perilipin-2 associated lipid droplets were induced and properties of Nile red emissions suggest lipid contents change over time. After microglial expression of phagocytotic markers at 2 days, some neutral lipid deposits co-localized with lysosome markers of microglia and were detected within putative phagocytotic cups. These data delineate distinct lipid signals in neurons and glial cells during excitotoxic processes from initial neuronal damage to engagement of the lysosome-phagosome system.

New role of P2X7 receptor in an Alzheimer's disease mouse model.

Extracellular aggregates of amyloid ß (Aß) peptides, which are characteristic of Alzheimer's disease (AD), act as an essential trigger for glial cell activation and the release of ATP, leading to the stimulation of purinergic receptors, especially the P2X7 receptor (P2X7R). However, the involvement of P2X7R in the development of AD is still ill-defined regarding the dual properties of this receptor. Particularly, P2X7R activates the NLRP3 inflammasome leading to the release of the pro-inflammatory cytokine, IL-1ß; however, P2X7R also induces cleavage of the amyloid precursor protein generating Aß peptides or the neuroprotective fragment sAPPα. We thus explored in detail the functions of P2X7R in AD transgenic mice. Here, we show that P2X7R deficiency reduced Aß lesions, rescued cognitive deficits and improved synaptic plasticity in AD mice. However, the lack of P2X7R did not significantly affect the release of IL-1ß or the levels of non-amyloidogenic fragment, sAPPα, in AD mice. Instead, our results show that P2X7R plays a critical role in Aß peptide-mediated release of chemokines, particularly CCL3, which is associated with pathogenic CD8+ T cell recruitment. In conclusion, our study highlights a novel detrimental function of P2X7R in chemokine release and supports the notion that P2X7R may be a promising therapeutic target for AD.

Microglial phenotypes in the human epileptic temporal lobe.

Microglia, the immune cells of the brain, are highly plastic and possess multiple functional phenotypes. Differences in phenotype in different regions and different states of epileptic human brain have been little studied. Here we use transcriptomics, anatomy, imaging of living cells and ELISA measurements of cytokine release to examine microglia from patients with temporal lobe epilepsies. Two distinct microglial phenotypes were explored. First we asked how microglial phenotype differs between regions of high and low neuronal loss in the same brain. Second, we asked how microglial phenotype is changed by a recent seizure. In sclerotic areas with few neurons, microglia have an amoeboid rather than ramified shape, express activation markers and respond faster to purinergic stimuli. The repairing interleukin, IL-10, regulates the basal phenotype of microglia in the CA1 and CA3 regions with neuronal loss and gliosis. To understand changes in phenotype induced by a seizure, we estimated the delay from the last seizure until tissue collection from changes in reads for immediate early gene transcripts. Pseudotime ordering of these data was validated by comparison with results from kainate-treated mice. It revealed a local and transient phenotype in which microglia secrete the human interleukin CXCL8, IL-1B and other cytokines. This secretory response is mediated in part via the NRLP3 inflammasome.

Imaging pathological activities of human brain tissue in organotypic culture.

BACKGROUND: Insights into human brain diseases may emerge from tissue obtained after operations on patients. However techniques requiring transduction of transgenes carried by viral vectors cannot be applied to acute human tissue. NEW METHOD: We show that organotypic culture techniques can be used to maintain tissue from patients with three different neurological syndromes for several weeks in vitro. Optimized viral vector techniques and promoters for transgene expression are described. RESULTS: Region-specific differences in neuronal form, firing pattern and organization as well as pathological activities were maintained over 40-50 days in culture. Both adeno-associated virus and lentivirus based vectors were persistently expressed from ∼10 days after application, providing 30-40 days to exploit genetically expressed constructs. Different promoters, including hSyn, e/hSyn, CMV and CaMKII, provided cell-type specific transgene expression. The Ca probe GCaMP let us explore epileptogenic synchrony and a FRET-based probe was used to follow activity of the kinase mTORC1. COMPARISON WITH EXISTING METHODS: The use of a defined culture medium, with low concentrations of amino acids and no growth factors, permitted organotypic culture of tissue from humans aged 3-62 years. Epileptic activity was maintained and excitability changed relatively little until ∼6 weeks in culture. CONCLUSIONS: Characteristic morphology and region-specific neuronal activities are maintained in organotypic culture of tissue from patients diagnosed with mesial temporal lobe epilepsy, cortical dysplasia and cortical glioblastoma. Viral vector techniques permit expression of probes for long-term measurements of multi-cellular activity and intra-cellular signaling.

Induction of Anti-Hebbian LTP in CA1 Stratum Oriens Interneurons: Interactions between Group I Metabotropic Glutamate Receptors and M1 Muscarinic Receptors.

An anti-Hebbian form of LTP is observed at excitatory synapses made with some hippocampal interneurons. LTP induction is facilitated when postsynaptic interneurons are hyperpolarized, presumably because Ca(2+) entry through Ca(2+)-permeable glutamate receptors is enhanced. The contribution of modulatory transmitters to anti-Hebbian LTP induction remains to be established. Activation of group I metabotropic receptors (mGluRs) is required for anti-Hebbian LTP induction in interneurons with cell bodies in the CA1 stratum oriens. This region receives a strong cholinergic innervation from the septum, and muscarinic acetylcholine receptors (mAChRs) share some signaling pathways and cooperate with mGluRs in the control of neuronal excitability.We therefore examined possible interactions between group I mGluRs and mAChRs in anti-Hebbian LTP at synapses which excite oriens interneurons in rat brain slices. We found that blockade of either group I mGluRs or M1 mAChRs prevented the induction of anti-Hebbian LTP by pairing presynaptic activity with postsynaptic hyperpolarization. Blocking either receptor also suppressed long-term effects of activation of the other G-protein coupled receptor on interneuron membrane potential. However, no crossed blockade was detected for mGluR or mAchR effects on interneuron after-burst potentials or on the frequency of miniature EPSPs. Paired recordings between pyramidal neurons and oriens interneurons were obtained to determine whether LTP could be induced without concurrent stimulation of cholinergic axons. Exogenous activation of mAChRs led to LTP, with changes in EPSP amplitude distributions consistent with a presynaptic locus of expression. LTP, however, required noninvasive presynaptic and postsynaptic recordings. SIGNIFICANCE STATEMENT: In the hippocampus, a form of NMDA receptor-independent long-term potentiation (LTP) occurs at excitatory synapses made on some inhibitory neurons. This is preferentially induced when postsynaptic interneurons are hyperpolarized, depends on Ca(2+) entry through Ca(2+)-permeable AMPA receptors, and has been labeled anti-Hebbian LTP. Here we show that this form of LTP also depends on activation of both group I mGluR and M1 mAChRs. We demonstrate that these G-protein coupled receptors (GPCRs) interact, because the blockade of one receptor suppresses long-term effects of activation of the other GPCR on both LTP and interneuron membrane potential. This LTP was also detected in paired recordings, although only when both presynaptic and postsynaptic recordings did not perturb the intracellular medium. Changes in EPSP amplitude distributions in dual recordings were consistent with a presynaptic locus of expression.

Increasing the effectiveness of intracerebral injections in adult and neonatal mice: a neurosurgical point of view.

Intracerebral injections of tracers or viral constructs in rodents are now commonly used in the neurosciences and must be executed perfectly. The purpose of this article is to update existing protocols for intracerebral injections in adult and neonatal mice. Our procedure for stereotaxic injections in adult mice allows the investigator to improve the effectiveness and safety, and save time. Furthermore, for the first time, we describe a two-handed procedure for intracerebral injections in neonatal mice that can be performed by a single operator in a very short time. Our technique using the stereotaxic arm allows a higher precision than freehand techniques previously described. Stereotaxic injections in adult mice can be performed in 20 min and have >90% efficacy in targeting the injection site. Injections in neonatal mice can be performed in 5 min. Efficacy depends on the difficulty of precisely localizing the injection sites, due to the small size of the animal. We describe an innovative, effortless, and reproducible surgical protocol for intracerebral injections in adult and neonatal mice.

An organotypic brain slice preparation from adult patients with temporal lobe epilepsy.

BACKGROUND: A long-term in vitro preparation of diseased brain tissue would facilitate work on human pathologies. Organotypic tissue cultures retain an appropriate neuronal form, spatial arrangement, connectivity and electrical activity over several weeks. However, they are typically prepared with tissue from immature animals. In work using tissue from adult animals or humans, survival times longer than a few days have not been reported and it is not clear that pathological neuronal activities are retained. NEW METHOD: We modified tissue preparation procedures and used a defined culture medium to make organotypic cultures of temporal lobe tissue obtained after operations on adult patients with pharmaco-resistant mesial temporal lobe epilepsies. RESULTS: Organototypic culture preparation and maintenance techniques were judged on criteria of morphology and the generation of epileptiform activities. Short-duration (30-100 ms) interictal-like population activities were initiated spontaneously in either the subiculum, dentate gyrus or the CA2/CA3 region, but not the cortex, for up to 3-4 weeks in culture. Ictal-like discharges, of duration greater than 10s, were induced by convulsants. Epileptiform activities were modulated by both glutamatergic and GABAergic receptor antagonists. COMPARISON WITH EXISTING METHODS: Our methods now permit the maintenance in organotypic culture of epileptic adult human tissue, generating appropriate epileptiform activity over 3-4 weeks. CONCLUSIONS: We have shown that characteristic morphology and pathological activities are maintained in organotypic cultures of adult human tissue. These cultures should permit studies on the effects of prolonged drug treatments and long-term procedures such as viral transduction.

Recurrent synapses and circuits in the CA3 region of the hippocampus: an associative network.

In the CA3 region of the hippocampus, pyramidal cells excite other pyramidal cells and interneurons. The axons of CA3 pyramidal cells spread throughout most of the region to form an associative network. These connections were first drawn by Cajal and Lorente de No. Their physiological properties were explored to understand epileptiform discharges generated in the region. Synapses between pairs of pyramidal cells involve one or few release sites and are weaker than connections made by mossy fibers on CA3 pyramidal cells. Synapses with interneurons are rather effective, as needed to control unchecked excitation. We examine contributions of recurrent synapses to epileptiform synchrony, to the genesis of sharp waves in the CA3 region and to population oscillations at theta and gamma frequencies. Recurrent connections in CA3, as other associative cortices, have a lower connectivity spread over a larger area than in primary sensory cortices. This sparse, but wide-ranging connectivity serves the functions of an associative network, including acquisition of neuronal representations as activity in groups of CA3 cells and completion involving the recall from partial cues of these ensemble firing patterns.

The paradox of the paroxysm: can seizure precipitants help explain human ictogenesis?

An epileptic brain is permanently in a diseased state, but seizures occur rarely and without warning. Here we examine this paradox, common to paroxysmal diseases. We review the problem in the context of the prototypic acquired epilepsies of the medial temporal lobe. We ask how an epileptic temporal lobe differs from a healthy one and examine biological mechanisms that may explain the transition to seizure. Attempts to predict seizure timing from analyses of brain electrical activity suggest that the neurological processes involved may be initiated significantly before a seizure. Furthermore, whereas seizures are said to occur without warning, some patients say they know when a seizure is imminent. Several factors, including sleep deprivation, oscillations in hormonal levels, or withdrawal from drugs, increase the probability of a seizure. We ask whether these seizure precipitants might act through common neuronal mechanisms. Several precipitating factors seem to involve relief from a neurosteroid modulation of gamma-amino butyric acid receptor type A (GABAA) receptors. We propose tests of this hypothesis.

Group I mGluR agonist-evoked long-term potentiation in hippocampal oriens interneurons.

Several subtypes of interneurons in the feedback circuit in stratum oriens of the hippocampus exhibit NMDA receptor-independent long-term potentiation (LTP) at glutamatergic synapses made by local pyramidal neurons. LTP has been reported with both "Hebbian" and "anti-Hebbian" induction protocols, where high-frequency presynaptic stimulation is paired with either postsynaptic depolarization or hyperpolarization. Do these phenomena represent distinct forms of plasticity, dependent on group I metabotropic receptors (mGluRs) and rectifying Ca2+ -permeable AMPA receptors, respectively? Blockade of either mGluR1 or mGluR5 prevented anti-Hebbian LTP induction in stratum oriens interneurons in rat hippocampal slices. Exogenous activation of group I mGluRs by the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was unable to induce LTP on its own, and instead depressed excitatory transmission. However, when paired with postsynaptic hyperpolarization, DHPG or the group I metabotropic receptor (mGluR5)-selective agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) elicited a delayed long-lasting potentiation, which was accompanied by a decrease in paired-pulse facilitation. Anti-Hebbian LTP occluded the effect of DHPG paired with hyperpolarization, implying that the induction cascades triggered by both conjunctions of stimuli converge on common expression mechanisms.

Short- and long-term depression at glutamatergic synapses on hippocampal interneurons by group I mGluR activation.

Group I metabotropic glutamate receptors (mGluRs) are expressed by many interneurons of the hippocampus. Although they have been implicated in short- and long-term synaptic plasticity of glutamatergic transmission, their roles in modulating transmission to interneurons are incompletely understood. The selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) acutely depressed transmission at synapses in the feed-forward inhibitory pathway made by Schaffer collaterals on interneurons in the rat hippocampal CA1 sub-field. DHPG elicited a qualitatively similar depression at synapses made by pyramidal neuron axon collaterals on interneurons in the feedback circuit in stratum oriens. Selective blockers revealed a link from mGluR1 to reversible, and mGluR5 to long-lasting, depression. The acute DHPG-induced depression was consistently accompanied by an elevation in paired-pulse ratio, implying a presynaptic decrease in release probability. However, it was also attenuated by blocking G-protein and Ca(2+) signalling within the postsynaptic neuron, arguing for a retrograde signalling cascade. The DHPG-evoked depression was unaffected by antagonists of CB1 and GABA(B) receptors but was occluded when presynaptic P/Q-type Ca(2+) channels were blocked. Finally, high-frequency stimulation delivered to an independent conditioning pathway evoked a heterosynaptic reversible depression, which was sensitive to group I mGluR antagonists. Group I mGluRs thus powerfully modulate synaptic excitation of hippocampal interneurons and mediate inter-synaptic cross-talk. This article is part of a Special Issue entitled 'Synaptic Plasticity & Interneurons'.

Gene expression analysis of the emergence of epileptiform activity after focal injection of kainic acid into mouse hippocampus.

We report gene profiling data on genomic processes underlying the progression towards recurrent seizures after injection of kainic acid (KA) into the mouse hippocampus. Focal injection enabled us to separate the effects of proepileptic stimuli initiated by KA injection. Both the injected and contralateral hippocampus participated in the status epilepticus. However, neuronal death induced by KA treatment was restricted to the injected hippocampus, although there was some contralateral axonal degeneration. We profiled gene expression changes in dorsal and ventral regions of both the injected and contralateral hippocampus. Changes were detected in the expression of 1526 transcripts in samples from three time-points: (i) during the KA-induced status epilepticus, (ii) at 2 weeks, before recurrent seizures emerged, and (iii) at 6 months after seizures emerged. Grouping genes with similar spatio-temporal changes revealed an early transcriptional response, strong immune, cell death and growth responses at 2 weeks and an activation of immune and extracellular matrix genes persisting at 6 months. Immunostaining for proteins coded by genes identified from array studies provided evidence for gliogenesis and suggested that the proteoglycan biglycan is synthesized by astrocytes and contributes to a glial scar. Gene changes at 6 months after KA injection were largely restricted to tissue from the injection site. This suggests that either recurrent seizures might depend on maintained processes including immune responses and changes in extracellular matrix proteins near the injection site or alternatively might result from processes, such as growth, distant from the injection site and terminated while seizures are maintained.

Epileptiform activities in slices of hippocampus from mice after intra-hippocampal injection of kainic acid.

Intra-hippocampal kainate injection induces the emergence of recurrent seizures after a delay of 3-4 weeks. We examined the cellular and synaptic basis of this activity in vitro using extracellular and intracellular records from longitudinal hippocampal slices. These slices permitted recordings from the dentate gyrus, the CA3 and CA1 regions and the subiculum of both the injected and the contralateral non-injected hippocampus. A sclerotic zone was evident in dorsal regions of slices from the injected hippocampus, while ventral regions and tissue from the contralateral hippocampus were not sclerotic. Interictal field potentials of duration 50-200 ms were generated spontaneously in both ipsilateral and contralateral hippocampal slices, but not in the sclerotic region, at 3-12 months after injection. They were initiated in the CA1 and CA3 regions and the subiculum. They were blocked by antagonists at glutamatergic receptors and were transformed into prolonged epileptiform events by GABAergic receptor antagonists. The membrane potential and the reversal potential of GABAergic synaptic events were more depolarized in CA1 pyramidal cells from kainate-treated animals than in control animals. Ictal-like events of duration 8-80 s were induced by tetanic stimulation (50 Hz, 0.2-1 s) preferentially in dorsal contralateral and ventral ipsilateral slices. Similar events were initiated by focal application of a combination of high K(+) and GABA. These data show that both interictal and ictal-like activities can be induced in slices of both ipsilateral and contralateral hippocampus from kainate-treated animals and suggest that changes in cellular excitability and inhibitory synaptic signalling may contribute to their generation.

Effects of focal injection of kainic acid into the mouse hippocampus in vitro and ex vivo.

Intra-hippocampal kainate injection induces an epileptiform activity termed status epilepticus. We examined the emergence of this activity with extracellular and intracellular records of responses (1) to focal kainate (KA) application in slices of mouse hippocampus and (2) of slices from mice injected with KA. The effects varied with distance from the injection site of KA. At distances less than approximately 800 microm, KA injection induced a strong increase in extracellular firing which ceased after 2-4 min. Pyramidal cells in this zone fired and depolarized to a potential at which action potentials were no longer evoked. No further activity was detected near the injection site for 3-5 h. In longitudinal slices of the CA3 region, firing induced by KA injection spread at a velocity close to 1 x 10(-)(4) mm ms(-)(1). The velocity increased to approximately 1 x 10(-)(1) mm ms(-)(1) when synaptic inhibition was blocked, suggesting that inhibitory processes normally restrict the spread of firing. At distances of 1.5-2.5 mm, KA injection induced a short-term increase in firing which was maintained, and often increased and rhythmic at gamma frequencies at 2-5 h after injection. We also examined slices prepared from animals injected with KA, at a delay of 2-5 h corresponding to the expression of status epilepticus. Near the injection site, Gallyas silver staining revealed cellular degeneration, and no activity was recorded. Interictal-like activity was generated by ipsilateral slices distant from KA injection. Contralateral slices also generated an interictal-like activity, but no cell death was detected. Hippocampal oscillations generated at distant sites may be associated with status epilepticus.

Mesial temporal lobe epilepsy: a pathological replay of developmental mechanisms?

Focal epilepsies of the mesial temporal lobe are often associated with a hippocampal sclerosis. Changes in Cl homeostasis and GABAergic signalling in downstream regions, may contribute to the human pathology. A review of changes in cellular and synaptic function and connectivity after de-afferentation suggests this epileptic syndrome may involve a pathological replay of developmental mechanisms.