Glioblastomas are invasive brain tumors with high therapeutic resistance. Neuron-to-glioma synapses have been shown to promote glioblastoma progression. However, a characterization of tumor-connected neurons has been hampered by a lack of technologies. Here, we adapted retrograde tracing using rabies viruses to investigate and manipulate neuron-tumor networks. Glioblastoma rapidly integrated into neural circuits across the brain, engaging in widespread functional communication, with cholinergic neurons driving glioblastoma invasion. We uncovered patient-specific and tumor-cell-state-dependent differences in synaptogenic gene expression associated with neuron-tumor connectivity and subsequent invasiveness. Importantly, radiotherapy enhanced neuron-tumor connectivity by increased neuronal activity. In turn, simultaneous neuronal activity inhibition and radiotherapy showed increased therapeutic effects, indicative of a role for neuron-to-glioma synapses in contributing to therapeutic resistance. Lastly, rabies-mediated genetic ablation of tumor-connected neurons halted glioblastoma progression, offering a viral strategy to tackle glioblastoma. Together, this study provides a framework to comprehensively characterize neuron-tumor networks and target glioblastoma.

The brain constantly adapts to environmental changes by modifying the expression of genes that enable synaptic plasticity, learning and memory. The expression of several of these genes requires nuclear calcium (Ca2+) signals, which in turn requires that Ca2+ signals generated by neuronal activity at the synapses or the soma propagate to the nucleus. Since cytoplasmic Ca2+ diffusion is highly restricted, Ca2+ signal propagation to the nucleus requires the participation of other cellular mechanisms. The inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) channels, both of which reside in the endoplasmic reticulum (ER) membrane, play key roles in cellular Ca2+ signal generation. Yet, their roles in the generation of nuclear and mitochondrial Ca2+ signals induced by neuronal activity require further investigation. Here, the impact of IP3R1 or RyR2 knockdown on gabazine-induced nuclear and mitochondrial Ca2+ signals in neurons was evaluated. To this aim, recombinant adeno-associated viruses (rAAVs) were used to introduce small hairpin RNAs (shRNAs) to knockdown type-1 (IP3R1) and type-2 (RyR2) channel expression in cultured rat hippocampal neurons. Additionally, synaptic contact numbers were assessed through immunocytochemistry. Knockdown of IP3R1 or RyR2 channels significantly reduced their protein contents and the generation of gabazine-induced nuclear and mitochondrial Ca2+ signals, without altering synaptic contact numbers. Our results highlight the contribution of IP3R1 and RyR2 channels to the generation of nuclear and mitochondrial Ca2+ signal induced by neuronal activity, reinforcing the role that these Ca2+ release channels play in hippocampal synaptic plasticity and memory formation.

BACKGROUND: Long-term fear memory storage involves gradual reorganization of supporting brain regions over time, a process termed systems consolidation. Memories initially rely on the hippocampus but gradually shift dependence to the neocortex. Although hippocampal activity drives this transfer, the molecular basis of systems consolidation is largely unknown. DNA methylation changes accompany persistent fear memory formation in the hippocampus and cortex, but its causal role in memory storage and systems consolidation remains unclear. METHODS: We investigated the role of hippocampal DNA methylation in fear memory persistence through multiple approaches. Using recombinant adeno-associated virus (rAAV)-mediated gene transfer, we overexpressed or knocked down a DNA methyltransferase (DNMT3A2) in the dorsal hippocampus of mice and assessed its impact on fear memory duration. Engram tagging and manipulation tools were applied to study cortical fear engram stabilization. Finally, RNA sequencing analysis was used to identify transcriptional changes driven by DNMT3A2 overexpression. RESULTS: Overexpression of hippocampal DNMT3A2 induced a persistent fear memory, while its knockdown impaired remote memory recall. RNA sequencing revealed that DNMT3A2 overexpression modified the expression of synaptic transmission regulatory genes. Furthermore, genetic engram tagging and manipulation revealed that hippocampal DNA methylation promoted the transfer of the fear memory trace from the hippocampus to the cortex and the stabilization of cortical fear memory traces. CONCLUSIONS: Our findings demonstrate that hippocampal DNA methylation regulates the long-term storage of persistent fear memories by facilitating the transfer of memory traces from the hippocampus to the cortex and cortical stabilization. These results highlight DNA methylation as a key molecular mechanism underlying systems consolidation and long-term fear memory storage.

The long-term stabilization of memory traces or engram involves the rapid formation of cortical engrams during encoding that mature functionally over time guided by the activity of the hippocampus. The molecular mechanisms that regulate this process remain largely unknown. Here, we found that hippocampal DNA methylation converts short-lasting into long-lasting memories by promoting systems consolidation and the stabilization of cortical engrams.### Competing Interest StatementThe authors have declared no competing interest.

Abstract Glioblastomas are primary brain tumors known for their whole-brain invasion and high therapeutic resistance. In previous studies, neuron-to-glioma synapses have been shown to promote glioblastoma progression. A deep characterization of this communication and of tumor-connected neurons has not been possible due to technological limitations. We adapted a rabies virus-based retrograde tracing approach to investigate and manipulate neuron-tumor networks. Interestingly, we found that glioblastoma cells rapidly integrated into neural circuits across the brain, engaging with distal neurons of diverse subtypes in very early stages of tumor development. Beyond glutamatergic neurons, we identified cholinergic neurons as drivers of glioblastoma cell invasion. We revealed patient- and tumor cell state-dependent differences in the expression of synaptogenic genes which also correlated with neuron-tumor connectivity and tumor cell invasivity. Notably, radiotherapy led to increased neuron-tumor connectivity mediated via neuronal hyperexcitability. The anti-epileptic drug perampanel alongside radiotherapy mitigated this effect and enhanced treatment efficacy by simultaneous neuronal activity inhibition. Utilizing our rabies-based approach, we genetically ablated tumor-connected neurons leading to a significantly decreased tumor progression offering a potential novel therapeutic avenue. Taken together, we developed a framework for dedicated analysis of multicellular neuron-tumor networks and propose our methodology as a versatile and adaptable approach to tackle glioblastoma.

Long-term memories are believed to be encoded by unique transcriptional signatures in the brain. The expression of immediate early genes (IEG) promotes structural and molecular changes required for memory consolidation. Recent evidence has shown that the brain is equipped with mechanisms that not only promote, but actively constrict memory formation. However, it remains unknown whether IEG expression may play a role in memory suppression. Here we uncovered a novel function of the IEG neuronal PAS domain protein 4 (Npas4), as an inducible memory suppressor gene of highly salient aversive experiences. Using a contextual fear conditioning paradigm, we found that low stimulus salience leads to monophasic Npas4 expression, while highly salient learning induces a biphasic expression of Npas4 in the hippocampus. The later phase requires N-methyl-D-aspartate (NMDA) receptor activity and is independent of dopaminergic neurotransmission. Our in vivo pharmacological and genetic manipulation experiments suggested that the later phase of Npas4 expression restricts the consolidation of a fear memory and promote behavioral flexibility, by facilitating fear extinction and the contextual specificity of fear responses. Moreover, immunofluorescence and electrophysiological analysis revealed a concomitant increase in synaptic input from cholecystokinin (CCK)-expressing interneurons. Our results demonstrate how salient experiences evoke unique temporal patterns of IEG expression that fine-tune memory consolidation. Moreover, our study provides evidence for inducible gene expression associated with memory suppression as a possible mechanism to balance the consolidation of highly salient memories, and thereby to evade the formation of maladaptive behavior.

Abstract Glioblastomas, primary brain tumors known for their extensive brain colonization and high therapeutic resistance, pose significant treatment challenges. Recent research has revealed that glioblastoma cells can form functional multicellular networks both with themselves, surrounding astrocytes and neurons. However, the investigation of the glioblastoma neuronal connectome has been limited by a lack of suitable methodologies. In this study, we utilized an adapted rabies-based retrograde tracing approach to characterize tumor-connected neurons and examine the neuronal connectome of the tumor in the context of therapy resistance. Our initial findings demonstrated that radiotherapy induces hyperexcitability in tumor-connected neurons, as shown by patch-clamp electrophysiology. This hyperexcitability was associated with an increase in neuron-tumor connectivity post-irradiation. To mitigate this increased neuronal input to therapy-resistant glioblastoma cells, we combined radiotherapy with the anti-epileptic drug perampanel. This combination therapy, which inhibits overall neuronal activity while delivering tumor cell-toxic photon irradiation, proved significantly more effective in reducing the number of therapy-resistant tumor cells. In summary, we employed a novel methodology to investigate the role of the neuronal tumor connectome in therapeutic resistance, identifying tumor-connected neurons as a potential target for improving glioblastoma treatment.

The LINC00473 (Lnc473) gene has previously been shown to be associated with cancer and psychiatric disorders. Its expression is elevated in several types of tumors and decreased in the brains of patients diagnosed with schizophrenia or major depression. In neurons, Lnc473 transcription is strongly responsive to synaptic activity, suggesting a role in adaptive, plasticity-related mechanisms. However, the function of Lnc473 is largely unknown. Here, using a recombinant adeno-associated viral vector, we introduced a primate-specific hu-man Lnc473 RNA into mouse primary neurons. We show that this resulted in a transcriptomic shift comprising down -regulation of epilepsy-associated genes and a rise in cAMP response element-binding protein (CREB) activity, which was driven by augmented CREB-regulated transcription coactivator 1 nuclear localization. Moreover, we demonstrate that ectopic Lnc473 expression increased neuronal excitability as well as network excitability. These findings suggest that primates may possess a lineage-specific activity-dependent modulator of CREB-regulated neuronal excitability.

Differences in the learning associated transcriptional profiles between mouse strains with distinct learning abilities could provide insight into the molecular basis of learning and memory. The inbred mouse strain DBA/2 shows deficits in hippocampus-dependent memory, yet the transcriptional responses to learning and the underlying mechanisms of the impairments are unknown. Comparing DBA/2J mice with the reference standard C57BL/6N mouse strain we verify an enhanced susceptibility to kainic acid induced seizures, confirm impairments in hippocampus-dependent spatial memory tasks and uncover additional behavioral abnormalities including deficits in hippocampus-independent learning. Surprisingly, we found no broad dysfunction of the DBA/2J strain in immediate early gene (IEG) activation but instead report brain region-specific and gene-specific alterations. The learning-associated IEGs Arc, c-Fos, and Nr4a1 showed no DBA/2J deficits in basal or synaptic activity induced gene expression in hippocampal or cortical primary neuronal cultures or in the CA1, CA3, or retrosplenial cortex following spatial object recognition (SOR) training in vivo. However, the parietal cortex showed reduced and the dentate gyrus showed enhanced SOR-evoked induction of most IEGs. All DBA/2J hippocampal regions exhibited elevated basal expression of inhibin β A (Inhba) and a learning-associated superinduction of the transcription factor neuronal Per-Arnt-Sim domain protein 4 (Npas4) known to regulate the synaptic excitation-inhibition balance. In line with this, CA1 pyramidal neurons of DBA/2J mice showed fewer inhibitory and more excitatory miniature postsynaptic currents but no alteration in most other electrophysiological properties or gross dendritic morphology. The dysregulation of Npas4 and Inhba expression and synaptic connectivity may underlie the cognitive deficits and increased susceptibility to seizures of DBA/2J mice.

The morphology of dendritic arbors determines the location, strength and interaction of synaptic inputs. It is therefore important to understand the factors regulating dendritic arborization both during development and in situations of physiological or pathological plasticity. We have recently shown that VEGF-D (Vascular Endothelial Growth Factor D) is required to maintain length and complexity of basal dendrites in mouse hippocampal pyramidal cells. Lack of VEGF-D resulted in long-term memory deficits, suggesting a link between dendritic morphology and cognitive function. Here, we compared the effect of VEGF-D expression on basal versus apical dendrites of CA1 pyramidal cells, as well as its importance for synaptic processing of network oscillations. We report opposing, layer-specific effects of VEGF-D knockdown which resulted in shrinkage of basal and increased complexity of apical dendrites. Synaptic potentials and layer-specific voltage gradients during network oscillations remained, however, unaltered. These findings reveal a high spatial selectivity of VEGF-D effects at the sub-cellular level, and strong homeostatic mechanisms which keep spatially segregated synaptic inputs in a balance.

Excitotoxicity induced by NMDA receptors (NMDARs) is thought to be intimately linked to high intracellular calcium load. Unexpectedly, NMDAR-mediated toxicity can be eliminated without affecting NMDAR-induced calcium signals. Instead, excitotoxicity requires physical coupling of NMDARs to TRPM4. This interaction is mediated by intracellular domains located in the near-membrane portions of the receptors. Structure-based computational drug screening using the interaction interface of TRPM4 in complex with NMDARs identified small molecules that spare NMDAR-induced calcium signaling but disrupt the NMDAR/TRPM4 complex. These interaction interface inhibitors strongly reduce NMDA-triggered toxicity and mitochondrial dysfunction, abolish cyclic adenosine monophosphate-responsive element-binding protein (CREB) shutoff, boost gene induction, and reduce neuronal loss in mouse models of stroke and retinal degeneration. Recombinant or small-molecule NMDAR/TRPM4 interface inhibitors may mitigate currently untreatable human neurodegenerative diseases.

Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N -methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstream of toxic NMDA receptor signaling and can protect mitochondria from glutamate toxicity, we developed a cell viability screening assay in primary mouse cortical neurons. In a proof-of-principle screen we tested 146 natural products and 424 FDA-approved drugs for their ability to protect neurons against NMDA-induced cell death. We confirmed several known neuroprotective drugs that include Dutasteride, Enalapril, Finasteride, Haloperidol, and Oxybutynin, and we identified neuroprotective properties of Elvitegravir. Using live imaging of tetramethylrhodamine ethyl ester-labelled primary cortical neurons, we found that Elvitegravir, Dutasteride, and Oxybutynin attenuated the NMDA-induced breakdown of the mitochondrial membrane potential. Patch clamp electrophysiological recordings in NMDA receptor-expressing HEK293 cell lines and primary mouse hippocampal neurons revealed that Elvitegravir does not act at the NMDA receptor and does not affect the function of glutamatergic synapses. In summary, we have developed a cost-effective and easy-to-implement screening assay in primary neurons and identified Elvitegravir as a neuro- and mitoprotective drug that acts downstream of the NMDA receptor.

Key points We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi‐selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK‐801 on both synaptic and brief, local agonist application‐evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ∼77 nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK‐801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29–39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death. AbstractSynaptic NMDA receptors (NMDARs) play a central role in pro‐survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES‐NMDARs) activate pro‐death pathways and have been implicated in many neurodegenerative diseases. ES‐NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity‐dependent NMDAR antagonist MK‐801 to block synaptic NMDARs. This approach is limited by the concomitant MK‐801 blockade of ES‐NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35–42 postnatal days, we estimated ambient glutamate to be 72–83 nM resulting in a block of more than 82% of ES‐NMDARs during a 5 min MK‐801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK‐801 to preferentially block ES‐NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK‐801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES‐NMDARs were estimated to comprise 29–39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES‐NMDAR signalling.

Coordinated collective electrochemical signals in multicellular assemblies, such as ion fluxes, membrane potentials, electrical gradients, and steady electric fields, play an important role in cell and tissue spatial organization during many physiological processes like wound healing, inflammatory responses, and hormone release. This mass of electric actions cumulates in an en masse activity within cell collectives which cannot be deduced from considerations at the individual cell level. However, continuously sampling en masse collective electrochemical actions of the global electrochemical activity of large-scale electrically coupled cellular assemblies with intracellular resolution over long time periods has been impeded by a lack of appropriate recording techniques. Here we present a bioelectrical interface consisting of low impedance vertical gold nanoelectrode interfaces able to penetrate the cellular membrane in the course of cellular adhesion, thereby allowing en masse recordings of intracellular electrochemical potentials that transverse electrically coupled NRK fibroblast, C2C12 myotube assemblies, and SH-SY5Y neuronal networks of more than 200,000 cells. We found that the intracellular electrical access of the nanoelectrodes correlates with substrate adhesion dynamics and that penetration, stabilization, and sealing of the electrode-cell interface involves recruitment of surrounding focal adhesion complexes and the anchoring of actin bundles, which form a caulking at the electrode base. Intracellular recordings were stable for several days, and monitoring of both basal activity as well as pharmacologically altered electric signals with high signal-to-noise ratios and excellent electrode coupling was performed.

Despite the reduced life expectancy and staggering financial burden of medical treatment associated with tobacco smoking, the molecular, cellular, and ensemble adaptations associated with chronic nicotine consumption remain poorly understood. Complex circuitry interconnecting dopaminergic and cholinergic regions of the midbrain and mesopontine tegmentum are critical for nicotine associated reward. Yet our knowledge of the nicotine activation of these regions is incomplete, in part due to their cell type diversity. We performed double immunohistochemistry for the immediate early gene and surrogate activity sensor, c-Fos, and markers for either cholinergic, dopaminergic or GABAergic cell types in mice treated with nicotine. Both acute (0.5mg/kg) and chronic (0.5mg/kg/day for 7 days) nicotine strongly activated GABAergic neurons of the interpeduncular nucleus and medial terminal nucleus of the accessory optic tract (MT). Acute but not chronic nicotine also activated small percentages of dopaminergic and other neurons in the ventral tegmental area (VTA) as well as noncholinergic neurons in the pedunculotegmental and laterodorsal tegmental nuclei (PTg/LDTg). Twenty four hours of nicotine withdrawal after chronic nicotine treatment suppressed c-Fos activation in the MT. In comparison to nicotine, a single dose of cocaine caused a similar activation in the PTg/LDTg but not the VTA where GABAergic cells were strongly activated but dopaminergic neurons were not affected. These results indicate the existence of drug of abuse specific ensembles. The loss of ensemble activation in the VTA and PTg/LDTg after chronic nicotine represents a molecular and cellular tolerance which may have implications for the mechanisms underlying nicotine dependence.

Nuclear calcium is an important signaling end point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here, we show that nuclear calcium acting via a target gene, VEGFD, is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds the structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Therefore, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help to stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies.SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline.

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.41 +/- 5.1 action potentials during a 6.7 +/- 1.9 s depolarization of 19.5 +/- 2.0 mV causing a 9.3 +/- 0.9 s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5-8 consecutive burst-induced nuclear calcium transients at 0.05-0.15 Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased. (C) 2017 Elsevier Ltd. All rights reserved.

Long-term adaptive responses in the brain, such as learning and memory, require synaptic activity-regulated gene expression, which has been thoroughly investigated in rodents. Using human iPSC-derived neuronal networks, we show that the human and the mouse synaptic activity-induced transcriptional programs share many genes and both require Ca2+-regulated synapse-to-nucleus signaling. Species-specific differences include the noncoding RNA genes BRE-AS1 and LINC00473 and the protein-coding gene ZNF331, which are absent in the mouse genome, as well as several human genes whose orthologs are either not induced by activity or are induced with different kinetics in mice. These results indicate that lineage-specific gain of genes and DNA regulatory elements affects the synaptic activity-regulated gene program, providing a mechanism driving the evolution of human cognitive abilities.