Grey matter heterotopia subtypes show specific morpho-electric signatures and network dynamics.

Grey matter heterotopia (GMH) are neurodevelopmental disorders associated with abnormal cortical function and epilepsy. Subcortical band heterotopia (SBH) and periventricular nodular heterotopia (PVNH) are two well-recognized GMH subtypes in which neurons are misplaced, either forming nodules lining the ventricles in PVNH, or forming bands in the white matter in SBH. Although both PVNH and SBH are commonly associated with epilepsy, it is unclear whether these two GMH subtypes differ in terms of pathological consequences or, on the contrary, share common altered mechanisms. Here, we studied two robust preclinical models of SBH and PVNH, and performed a systematic comparative assessment of the physiological and morphological diversity of heterotopia neurons, as well as the dynamics of epileptiform activity and input connectivity. We uncovered a complex set of altered properties, including both common and distinct physiological and morphological features across heterotopia subtypes, and associated with specific dynamics of epileptiform activity. Taken together, these results suggest that pro-epileptic circuits in GMH are, at least in part, composed of neurons with distinct, subtype-specific, physiological and morphological properties depending on the heterotopia subtype. Our work supports the notion that GMH represent a complex set of disorders, associating both shared and diverging pathological consequences, and contributing to forming epileptogenic networks with specific properties. A deeper understanding of these properties may help to refine current GMH classification schemes by identifying morpho-electric signatures of GMH subtypes, to potentially inform new treatment strategies.

GluK2 Is a Target for Gene Therapy in Drug-Resistant Temporal Lobe Epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE. METHODS: We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE. RESULTS: Here, we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated interictal-like epileptiform discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9-anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs. INTERPRETATION: Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients. ANN NEUROL 2023;94:745-761.

Kainate receptors modulate the microstructure of synchrony during dentate gyrus epileptiform activity.

Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. In TLE, recurrent mossy fiber (rMF) sprouting from dentate gyrus granule cells (DGCs) forms an aberrant epileptogenic network between dentate granule cells (DGCs) that operates via ectopically expressed kainate receptors (KARs). It was previously shown that KARs expressed at the rMF-DGC synapses play a prominent role in epileptiform network events in TLE. However, it is not well understood how KARs influence neuronal network dynamics and contribute to the generation of epileptiform network activity in the dentate gyrus. To address this question, we monitored the activity of DGCs using single-cell resolution calcium imaging performed in a reliable in vitro model of TLE. Under our experimental conditions, the most prominent DGC activity patterns were interictal-like epileptiform network events, which were correlated with high levels of neuronal synchronization. The pharmacological blockade of KARs reduced the frequency as well as the number of neurons involved in these events, without altering their spatiotemporal dynamics. Analysis of the microstructure of synchrony showed that blockade of KARs diminished the fraction of neurons forming the main functional cluster. Therefore, we propose that KARs act as modulators in the epileptic network by facilitating the recruitment of neurons into coactive cell assemblies, thereby contributing to the occurrence of epileptiform network events.

Substantial outcome improvement using a refined pilocarpine mouse model of temporal lobe epilepsy.

Systemic pilocarpine treatment is one of the most reliable means of inducing temporal lobe epilepsy (TLE). However, the traditional pilocarpine injection protocol using mice was associated with a high death rate, possibly because of cardiorespiratory collapse following status epilepticus (SE). To prevent this, we developed a modified procedure of pilocarpine SE induction, which included a single injection of a moderate dose of caffeine during the induction phase. That new protocol was based on the use of young male mice as well as on a refined Racine's scale. Using that protocol, we report a substantially increased survival rate, thus enabling the generation of a large cohort of mice that exhibited cardinal histological (e.g., mossy fiber sprouting) and electrophysiological (e.g., chronic interictal events and ictal seizures) characteristics associated with TLE. In conclusion, our refined caffeine- and pilocarpine-based protocol substantially improves the outcome of the reliable pilocarpine mouse model of TLE.

Calreticulin levels determine onset of early muscle denervation by fast motoneurons of ALS model mice.

Although the precise signaling mechanisms underlying the vulnerability of some sub-populations of motoneurons in ALS remain unclear, critical factors such as metallo-proteinase 9 expression, neuronal activity and endoplasmic reticulum stress have been shown to be involved. In the context of SOD1(G93A) ALS mouse model, we previously showed that a two-fold decrease in calreticulin (CRT) is occurring in the vulnerable fast motoneurons. Here, we asked to which extent the decrease in CRT levels was causative to muscle denervation and/or motoneuron degeneration. Toward this goal, a hemizygous deletion of the crt gene in SOD1(G93A) mice was generated since the complete ablation of crt is embryonic lethal. We observed that SOD1(G93A);crt(+/-) mice display increased and earlier muscle weakness and muscle denervation compared to SOD1(G93A) mice. While CRT reduction in motoneurons leads to a strong upregulation of two factors important in motoneuron dysfunction, ER stress and mTOR activation, it does not aggravate motoneuron death. Our results underline a prevalent role for CRT levels in the early phase of muscle denervation and support CRT regulation as a potential therapeutic approach.

Paradoxical neuronal hyperexcitability in a mouse model of mitochondrial pyruvate import deficiency.

Neuronal excitation imposes a high demand of ATP in neurons. Most of the ATP derives primarily from pyruvate-mediated oxidative phosphorylation, a process that relies on import of pyruvate into mitochondria occuring exclusively via the mitochondrial pyruvate carrier (MPC). To investigate whether deficient oxidative phosphorylation impacts neuron excitability, we generated a mouse strain carrying a conditional deletion of MPC1, an essential subunit of the MPC, specifically in adult glutamatergic neurons. We found that, despite decreased levels of oxidative phosphorylation and decreased mitochondrial membrane potential in these excitatory neurons, mice were normal at rest. Surprisingly, in response to mild inhibition of GABA mediated synaptic activity, they rapidly developed severe seizures and died, whereas under similar conditions the behavior of control mice remained unchanged. We report that neurons with a deficient MPC were intrinsically hyperexcitable as a consequence of impaired calcium homeostasis, which reduced M-type potassium channel activity. Provision of ketone bodies restored energy status, calcium homeostasis and M-channel activity and attenuated seizures in animals fed a ketogenic diet. Our results provide an explanation for the seizures that frequently accompany a large number of neuropathologies, including cerebral ischemia and diverse mitochondriopathies, in which neurons experience an energy deficit.

Dentate Granule Cells Recruited in the Home Environment Display Distinctive Properties.

The dentate granule cells (DGCs) play a crucial role in learning and memory. Many studies have described the role and physiological properties of these sparsely active neurons using different behavioral contexts. However, the morpho-functional features of DGCs recruited in mice maintained in their home cage (without training), considered as a baseline condition, have not yet been established. Using fosGFP transgenic mice, we observed ex vivo that DGCs recruited in animals maintained in the home cage condition are mature neurons that display a longer dendritic tree and lower excitability compared with non-activated cells. The higher GABAA receptor-mediated shunting inhibition contributes to the lower excitability of DGCs activated in the home environment by shifting the input resistance towards lower values. Remarkably, that shunting inhibition is neither observed in non-activated DGCs nor in DGCs activated during training in virtual reality. In short, our results suggest that strong shunting inhibition and reduced excitability could constitute a distinctive neural signature of mature DGCs recruited in the context of the home environment.

Spontaneous glutamatergic activity induces a BDNF-dependent potentiation of GABAergic synapses in the newborn rat hippocampus.

Spontaneous ongoing synaptic activity is thought to play an instructive role in the maturation of the neuronal circuits. However the type of synaptic activity involved and how this activity is translated into structural and functional changes is not fully understood. Here we show that ongoing glutamatergic synaptic activity triggers a long-lasting potentiation of gamma-aminobutyric acid (GABA) mediated synaptic activity (LLP(GABA-A)) in the developing rat hippocampus. LLP(GABA-A) induction requires (i) the activation of AMPA receptors and L-type voltage-dependent calcium channels, (ii) the release of endogenous brain-derived neurotrophic factor (BDNF), and (iii) the activation of postsynaptic tropomyosin-related kinase receptors B (TrkB). We found that spontaneous glutamatergic activity is required to maintain a high level of native BDNF in the newborn rat hippocampus and that application of exogenous BDNF induced LLP(GABA-A) in the absence of glutamatergic activity. These results suggest that ongoing glutamatergic synaptic activity plays a pivotal role in the functional maturation of hippocampal GABAergic synapses by means of a cascade involving BDNF release and downstream signalling through postsynaptic TrkB receptor activation.

Restoring wild-type-like CA1 network dynamics and behavior during adulthood in a mouse model of schizophrenia.

Schizophrenia is a severely debilitating neurodevelopmental disorder. Establishing a causal link between circuit dysfunction and particular behavioral traits that are relevant to schizophrenia is crucial to shed new light on the mechanisms underlying the pathology. We studied an animal model of the human 22q11 deletion syndrome, the mutation that represents the highest genetic risk of developing schizophrenia. We observed a desynchronization of hippocampal neuronal assemblies that resulted from parvalbumin interneuron hypoexcitability. Rescuing parvalbumin interneuron excitability with pharmacological or chemogenetic approaches was sufficient to restore wild-type-like CA1 network dynamics and hippocampal-dependent behavior during adulthood. In conclusion, our data provide insights into the network dysfunction underlying schizophrenia and highlight the use of reverse engineering to restore physiological and behavioral phenotypes in an animal model of neurodevelopmental disorder.

Pioneer glutamatergic cells develop into a morpho-functionally distinct population in the juvenile CA3 hippocampus.

The developing CA3 hippocampus is comprised by highly connected hub neurons that are particularly effective in achieving network synchronization. Functional hub neurons were shown to be exclusively GABAergic, suggesting that the contribution of glutamatergic neurons to physiological synchronization processes at early postnatal stages is minimal. However, without fast GABAergic transmission, a different situation may prevail. In the adult CA3, blocking fast GABAergic transmission induces the generation of network bursts that can be triggered by the stimulation of single pyramidal neurons. Here we revisit the network function of CA3 glutamatergic neurons from a developmental viewpoint, without fast GABAergic transmission. We uncover a sub-population of early-generated glutamatergic neurons that impacts network dynamics when stimulated in the juvenile hippocampus. Additionally, this population displays characteristic morpho-physiological features in the juvenile and adult hippocampus. Therefore, the apparently homogeneous glutamatergic cell population likely displays a morpho-functional diversity rooted in temporal embryonic origins.

Onset of Pup Locomotion Coincides with Loss of NR2C/D-Mediated Cortico-Striatal EPSCs and Dampening of Striatal Network Immature Activity.

Adult motor coordination requires strong coincident cortical excitatory input to hyperpolarized medium spiny neurons (MSNs), the dominant neuronal population of the striatum. However, cortical and subcortical neurons generate during development large ongoing patterns required for activity-dependent construction of networks. This raises the question of whether immature MSNs have adult features from early stages or whether they generate immature patterns that are timely silenced to enable locomotion. Using a wide range of techniques including dynamic two-photon imaging, whole cell or single-channel patch clamp recording in slices from Nkx2.1-GFP mice, we now report a silencing of MSNs that timely coincides with locomotion. At embryonic stage (as early as E16) and during early postnatal days, genetically identified MSNs have a depolarized resting membrane potential, a high input resistance and lack both inward rectifying (IK(IR)) and early slowly inactivating (I(D)) potassium currents. They generate intrinsic voltage-gated clustered calcium activity without synaptic components. From postnatal days 5-7, the striatal network transiently generates synapse-driven giant depolarizing potentials when activation of cortical inputs evokes long lasting EPSCs in MSNs. Both are mediated by NR2C/D-receptors. These immature features are abruptly replaced by adult ones before P10: MSNs express IK(IR) and I(D) and generate short lasting, time-locked cortico-striatal AMPA/NMDA EPSCs with no NR2C/D component. This shift parallels the onset of quadruped motion by the pup. Therefore, MSNs generate immature patterns that are timely shut off to enable the coordination of motor programs.