Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis.

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.

Distinct Mechanisms of Over-Representation of Landmarks and Rewards in the Hippocampus.

In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.

Long-term hippocampal interneuronopathy drives sex-dimorphic spatial memory impairment induced by prenatal THC exposure.

Prenatal exposure to Δ9-tetrahydrocannabinol (THC), the most prominent active constituent of cannabis, alters neurodevelopmental plasticity with a long-term functional impact on adult offspring. Specifically, THC affects the development of pyramidal neurons and GABAergic interneurons via cannabinoid CB1 receptors (CB1R). However, the particular contribution of these two neuronal lineages to the behavioral alterations and functional deficits induced by THC is still unclear. Here, by using conditional CB1R knockout mice, we investigated the neurodevelopmental consequences of prenatal THC exposure in adulthood, as well as their potential sex differences. Adult mice that had been exposed to THC during embryonic development showed altered hippocampal oscillations, brain hyperexcitability, and spatial memory impairment. Remarkably, we found a clear sexual dimorphism in these effects, with males being selectively affected. At the neuronal level, we found a striking interneuronopathy of CCK-containing interneurons in the hippocampus, which was restricted to male progeny. This THC-induced CCK-interneuron reduction was not evident in mice lacking CB1R selectively in GABAergic interneurons, thus pointing to a cell-autonomous THC action. In vivo electrophysiological recordings of hippocampal LFPs revealed alterations in hippocampal oscillations confined to the stratum pyramidale of CA1 in male offspring. In addition, sharp-wave ripples, a major high-frequency oscillation crucial for learning and memory consolidation, were also altered, pointing to aberrant circuitries caused by persistent reduction of CCK+ basket cells. Taken together, these findings provide a mechanistic explanation for the long-term interneuronopathy responsible for the sex-dimorphic cognitive impairment induced by prenatal THC.

KCC2 Regulates Neuronal Excitability and Hippocampal Activity via Interaction with Task-3 Channels.

KCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention.

Proximodistal Organization of the CA2 Hippocampal Area.

The proximodistal axis is considered a major organizational principle of the hippocampus. At the interface between the hippocampus and other brain structures, CA2 apparently breaks this rule. The region is involved in social, temporal, and contextual memory function, but mechanisms remain elusive. Here, we reveal cell-type heterogeneity and a characteristic expression gradient of the transcription factor Sox5 within CA2 in the rat. Using intracellular and extracellular recordings followed by neurochemical identification of single cells, we find marked proximodistal trends of synaptic activity, subthreshold membrane potentials, and phase-locked firing coupled to theta and gamma oscillations. Phase-shifting membrane potentials and opposite proximodistal correlations with theta sinks and sources at different layers support influences from different current generators. CA2 oscillatory activity and place coding of rats running in a linear maze reflect proximodistal state-dependent trends. We suggest that the structure and function of CA2 are distributed along the proximodistal hippocampal axis.

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.

Altered Oscillatory Dynamics of CA1 Parvalbumin Basket Cells during Theta-Gamma Rhythmopathies of Temporal Lobe Epilepsy.

Recent reports in human demonstrate a role of theta-gamma coupling in memory for spatial episodes and a lack of coupling in people experiencing temporal lobe epilepsy, but the mechanisms are unknown. Using multisite silicon probe recordings of epileptic rats engaged in episodic-like object recognition tasks, we sought to evaluate the role of theta-gamma coupling in the absence of epileptiform activities. Our data reveal a specific association between theta-gamma (30-60 Hz) coupling at the proximal stratum radiatum of CA1 and spatial memory deficits. We targeted the microcircuit mechanisms with a novel approach to identify putative interneuronal types in tetrode recordings (parvalbumin basket cells in particular) and validated classification criteria in the epileptic context with neurochemical identification of intracellularly recorded cells. In epileptic rats, putative parvalbumin basket cells fired poorly modulated at the falling theta phase, consistent with weaker inputs from Schaffer collaterals and attenuated gamma oscillations, as evaluated by theta-phase decomposition of current-source density signals. We propose that theta-gamma interneuronal rhythmopathies of the temporal lobe are intimately related to episodic memory dysfunction in this condition.

Determinants of different deep and superficial CA1 pyramidal cell dynamics during sharp-wave ripples.

Sharp-wave ripples represent a prominent synchronous activity pattern in the mammalian hippocampus during sleep and immobility. GABAergic interneuronal types are silenced or fire during these events, but the mechanism of pyramidal cell (PC) participation remains elusive. We found opposite membrane polarization of deep (closer to stratum oriens) and superficial (closer to stratum radiatum) rat CA1 PCs during sharp-wave ripples. Using sharp and multi-site recordings in combination with neurochemical profiling, we observed a predominant inhibitory drive of deep calbindin (CB)-immunonegative PCs that contrasts with a prominent depolarization of superficial CB-immunopositive PCs. Biased contribution of perisomatic GABAergic inputs, together with suppression of CA2 PCs, may explain the selection of CA1 PCs during sharp-wave ripples. A deep-superficial gradient interacted with behavioral and spatial effects to determine cell participation during sleep and awake sharp-wave ripples in freely moving rats. Thus, the firing dynamics of hippocampal PCs are exquisitely controlled at subcellular and microcircuit levels in a cell type-selective manner.

Dampened hippocampal oscillations and enhanced spindle activity in an asymptomatic model of developmental cortical malformations.

Developmental cortical malformations comprise a large spectrum of histopathological brain abnormalities and syndromes. Their genetic, developmental and clinical complexity suggests they should be better understood in terms of the complementary action of independently timed perturbations (i.e., the multiple-hit hypothesis). However, understanding the underlying biological processes remains puzzling. Here we induced developmental cortical malformations in offspring, after intraventricular injection of methylazoxymethanol (MAM) in utero in mice. We combined extensive histological and electrophysiological studies to characterize the model. We found that MAM injections at E14 and E15 induced a range of cortical and hippocampal malformations resembling histological alterations of specific genetic mutations and transplacental mitotoxic agent injections. However, in contrast to most of these models, intraventricularly MAM-injected mice remained asymptomatic and showed no clear epilepsy-related phenotype as tested in long-term chronic recordings and with pharmacological manipulations. Instead, they exhibited a non-specific reduction of hippocampal-related brain oscillations (mostly in CA1); including theta, gamma and HFOs; and enhanced thalamocortical spindle activity during non-REM sleep. These data suggest that developmental cortical malformations do not necessarily correlate with epileptiform activity. We propose that the intraventricular in utero MAM approach exhibiting a range of rhythmopathies is a suitable model for multiple-hit studies of associated neurological disorders.

Systemic injection of kainic acid differently affects LTP magnitude depending on its epileptogenic efficiency.

Seizures have profound impact on synaptic function and plasticity. While kainic acid is a popular method to induce seizures and to potentially affect synaptic plasticity, it can also produce physiological-like oscillations and trigger some forms of long-term potentiation (LTP). Here, we examine whether induction of LTP is altered in hippocampal slices prepared from rats with different sensitivity to develop status epilepticus (SE) by systemic injection of kainic acid. Rats were treated with multiple low doses of kainic acid (5 mg/kg; i.p.) to develop SE in a majority of animals (72-85% rats). A group of rats were resistant to develop SE (15-28%) after several accumulated doses. Animals were subsequently tested using chronic recordings and object recognition tasks before brain slices were prepared for histological studies and to examine basic features of hippocampal synaptic function and plasticity, including input/output curves, paired-pulse facilitation and theta-burst induced LTP. Consistent with previous reports in kindling and pilocapine models, LTP was reduced in rats that developed SE after kainic acid injection. These animals exhibited signs of hippocampal sclerosis and developed spontaneous seizures. In contrast, resistant rats did not become epileptic and had no signs of cell loss and mossy fiber sprouting. In slices from resistant rats, theta-burst stimulation induced LTP of higher magnitude when compared with control and epileptic rats. Variations on LTP magnitude correlate with animals' performance in a hippocampal-dependent spatial memory task. Our results suggest dissociable long-term effects of treatment with kainic acid on synaptic function and plasticity depending on its epileptogenic efficiency.

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells.

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1­S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.