PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo . Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.

In mammalian cortex, feedforward excitatory connections recruit feedforward inhibition. This is often carried by parvalbumin (PV+) interneurons, which may densely connect to local pyramidal (Pyr) neurons. Whether this inhibition affects all local excitatory cells indiscriminately or is targeted to specific subnetworks is unknown. Here, we test how feedforward inhibition is recruited by using two-channel circuit mapping to excite cortical and thalamic inputs to PV+ interneurons and Pyr neurons to mouse primary vibrissal motor cortex (M1). Single Pyr and PV+ neurons receive input from both cortex and thalamus. Connected pairs of PV+ interneurons and excitatory Pyr neurons receive correlated cortical and thalamic inputs. While PV+ interneurons are more likely to form local connections to Pyr neurons, Pyr neurons are much more likely to form reciprocal connections with PV+ interneurons that inhibit them. This suggests that Pyr and PV ensembles may be organized based on their local and long-range connections, an organization that supports the idea of local subnetworks for signal transduction and processing. Excitatory inputs to M1 can thus target inhibitory networks in a specific pattern which permits recruitment of feedforward inhibition to specific subnetworks within the cortical column.

Intracortical inhibition in motor cortex (M1) regulates movement and motor learning. If cortical and thalamic inputs target different inhibitory cell types in different layers, then these afferents may play different roles in regulating M1 output. Using mice of both sexes, we quantified input to two main classes of M1 interneurons, parvalbumin+ (PV+) cells and somatostatin+ (SOM+) cells, using monosynaptic rabies tracing. We then compared anatomic and functional connectivity based on synaptic strength from sensory cortex and thalamus. Functionally, each input innervated M1 interneurons with a unique laminar profile. Different interneuron types were excited in a distinct, complementary manner, suggesting feedforward inhibition proceeds selectively via distinct circuits. Specifically, somatosensory cortex (S1) inputs primarily targeted PV+ neurons in upper layers (L2/3) but SOM+ neurons in middle layers (L5). Somatosensory thalamus [posterior nucleus (PO)] inputs targeted PV+ neurons in middle layers (L5). In contrast to sensory cortical areas, thalamic input to SOM+ neurons was equivalent to that of PV+ neurons. Thus, long-range excitatory inputs target inhibitory neurons in an area and a cell type-specific manner, which contrasts with input to neighboring pyramidal cells. In contrast to feedforward inhibition providing generic inhibitory tone in cortex, circuits are selectively organized to recruit inhibition matched to incoming excitatory circuits. SIGNIFICANCE STATEMENT M1 integrates sensory information and frontal cortical inputs to plan and control movements. Although inputs to excitatory cells are described, the synaptic circuits by which these inputs drive specific types of M1 interneurons are unknown. Anatomical results with rabies tracing and physiological quantification of synaptic strength shows that two main classes of inhibitory cells (PV+ and SOM+ interneurons) both receive substantial cortical and thalamic input, in contrast to interneurons in sensory areas (where thalamic input strongly prefers PV+ interneurons). Further, each input studied targets PV+ and SOM+ interneurons in a different fashion, suggesting that separate, specific circuits exist for recruitment of feedforward inhibition.

Somatic mutations have emerged as the likely cause of focal epilepsies associated with developmental malformations and epilepsy-associated glioneuronal tumors (GNT). Somatic BRAFV600E mutations in particular have been detected in the majority of low-grade neuroepithelial tumors (LNETS) and in neurons in focal cortical dysplasias adjacent to epilepsy-associated tumors. Furthermore, conditional expression of an activating BRAF mutation in neocortex causes seizures in mice. In this study we characterized the cellular electrophysiology of layer 2/3 neocortical pyramidal neurons induced to express BRAFV600E from neural progenitor stages. In utero electroporation of a piggyBac transposase plasmid system was used to introduce transgenes expressing BRAF wild type (BRAFwt), BRAFV600E, and/or enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein (mRFP) into radial glia progenitors in mouse embryonic cortex. Whole cell patch-clamp recordings of pyramidal neurons in slices prepared from both juvenile and adult mice showed that BRAFV600E resulted in neurons with a distinct hyperexcitable phenotype characterized by depolarized resting membrane potentials, increased input resistances, lowered action potential (AP) thresholds, and increased AP firing frequencies. Some of the BRAFV600E-expressing neurons normally destined for upper cortical layers by their birthdate were stalled in their migration and occupied lower cortical layers. BRAFV600E-expressing neurons also displayed increased hyperpolarization-induced inward currents (Ih) and decreased sustained potassium currents. Neurons adjacent to BRAFV600E transgene-expressing neurons, and neurons with TSC1 genetically deleted by CRISPR or those induced to carry PIK3CAE545K transgenes, did not show an excitability phenotype similar to that of BRAFV600E-expressing neurons. Together, these results indicate that BRAFV600E leads to a distinct hyperexcitable neuronal phenotype.NEW & NOTEWORTHY This study is the first to report the cell autonomous effects of BRAFV600E mutations on the intrinsic neuronal excitability. We show that BRAFV600E alters multiple electrophysiological parameters in neocortical neurons. Similar excitability changes did not occur in cells neighboring BRAFV600E-expressing neurons, after overexpression of wild-type BRAF transgenes, or after introduction of mutations affecting the mammalian target of rapamycin (mTOR) or the catalytic subunit of phosphoinositide 3-kinase (PIK3CA). We conclude that BRAFV600E causes a distinct, cell autonomous, highly excitable neuronal phenotype when introduced somatically into neocortical neuronal progenitors.

BACKGROUND: Frequent de-novo somatic mutations in major components (PI3KCA, AKT3, TSC1, TSC2, mTOR, BRAF) of molecular pathways crucial for cell differentiation, proliferation, growth and migration (mTOR, MAPK) has been previously implicated in malformations of cortical development (MCDs) and low-grade neuroepithelial tumors (LNETs) 1-7 . LNETs are the most frequent tumors found in patients undergoing resective surgery for refractory epilepsy treatment. BRAFV600E is found in up to 70% of LNETs. Previous studies suggest a causal relationship between those de-novo somatic mutations in mTOR, MAPK pathways and seizures occurrence, even without presence of malformation or a tumor 2, 3, 8-13. Recently Koh and colleagues 14 showed that BRAFV600E mutation may cause seizures through activation of RE1-silecing transcription factor (REST). Additionally, they showed a significant downregulation of synaptic transmission and plasticity pathways and decreased expression of multiple ion channels subunits including HCN1, KCNQ3, SCN2A and SCN3B. The downregulation of those genes including GABA receptors subunits and protein expression specific to interneurons subpopulations (SST, VIP) suggests that a dysregulated inhibitory circuits are responsible for seizures in GGs. The experimental manipulation - In-Utero electroporation of episomal activating Cre plasmids that they used to test their hypothesis in mice however activated mutant BRAFV637 only in excitatory neurons. And the downregulated genes in mice were confirmed by qRT-PCR in the whole tissue samples. The question of how electrophysiological properties of the affected and surrounding neurons are changed were not addressed. The changes in ion conductances and neuronal circuits responsible for seizures could be only inferred from gene expression profiles. Purpose of the current work was to investigate how overactive human BRAFV600E mutated protein incorporated into the mouse genome through piggyBase transposition increase neuronal excitability in ex-vivo mouse cortical slices and whether it induces histopathological features and gene expression profile alteration observed in low-grade neuroepithelial tumors (LNETs). METHODS: Using In-Utero Electroporation we have introduced human BRAFV600E protein into radial glia progenitors in mouse embryonic cortex on the background of piggyBac transposon system that allows incorporation of the DNA sequence of interest into the genome. Immunohistochemistry was used for examination of known markers in LNETs. RNA sequencing on Illumina NextSeq 500 was used to examine alterations in gene expression profiles. Whole-cell current- and voltage-clamp was used to examine changes in electrophysiological properties. Unsupervised Hierarchical Clustering Analysis was used to examine grouping of different conditions based on their gene expression profile and electrophysiological properties. Video electrocorticographic recordings were used to test whether BRAFV600E transgenic mice have spontaneous seizures. RESULTS: Under GLAST driving promoter BRAFV600E induced astrogenesis, caused morphological alterations in transgenic cells akin to balloon-like cells, and delayed neuronal migration. Under NESTIN driver promoter BRAFV600E increased neurogenesis, induced balloon-like cells and caused some cells to remain close to the lateral ventricle displaying large soma size compared to neurons in the upper cortical layers. Some of the balloon-like cells were immunopositive for astroglial marker glial fibrillary acidic protein (GFAP), and for both upper and lower cortical layers markers (Cux1 and Ctip2). Gene ontology analysis for BRAFV600E gene expression profile showed that there is a tissue-wide increased inflammatory immune response, complement pathway activation, microglia recruitment and astrocytes activation, which supported increased immunoreactivity to microglial marker iba1, and to GFAP respectively. In current clamp BRAFV600E neurons have increased excitability properties including more depolarized resting membrane potential, increased input resistance, low capacitance, low rheobase, low action potential (AP) voltage threshold, and increased AP firing frequency. Additionally, BRAFV600E neurons have increased SAG and rebound excitation, indicative of increased hyperpolarization activated depolarizing conductance (IH), which is confirmed in voltage-clamp. The sustained potassium current sensitive to tetraethylammonium was decreased in BRAFV600E neurons.. In 4 out of 59 cells, we have also observed a post-action potential depolarizing waves, frequencies of which increased in potassium current recording when Ca2+ was substituted to Co2+ in the extracellular solution (5/24). We show that using 20 electrophysiological properties BRAFV600E neurons segregate separately from other conditions. Comparison of electrophysiological properties of those neurons with neurons bearing somatic mutations in mechanistic target of rapamycin (MTOR) pathway regulatory components, overactivation of which is been shown in malformations of cortical development (MCDs), showed that expression of PIK3CAE545K under GLAST+ promoter and TSC1 knockdown (KD) with CRISPR-Cas9 have different effects on neuronal excitability.

Hebbian-type learning rules, which underlie learning and refinement of neuronal connectivity, postulate input specificity of synaptic changes. However, theoretical analyses have long appreciated that additional mechanisms, not restricted to activated synapses, are needed to counteract positive feedback imposed by Hebbian-type rules on synaptic weight changes and to achieve stable operation of learning systems. The biological basis of such mechanisms has remained elusive. Here we show that, in layer 2/3 pyramidal neurons from slices of visual cortex of rats, synaptic changes induced at individual synapses by spike timing-dependent plasticity do not strictly follow the input specificity rule. Spike timing-dependent plasticity is accompanied by changes in unpaired synapses: heterosynaptic plasticity. The direction of heterosynaptic changes is weight-dependent, with balanced potentiation and depression, so that the total synaptic input to a cell remains preserved despite potentiation or depression of individual synapses. Importantly, this form of heterosynaptic plasticity is induced at unpaired synapses by the same pattern of postsynaptic activity that induces homosynaptic changes at paired synapses. In computer simulations, we show that experimentally observed heterosynaptic plasticity can indeed serve the theoretically predicted role of robustly preventing runaway dynamics of synaptic weights and activity. Moreover, it endows model neurons and networks with essential computational features: enhancement of synaptic competition, facilitation of the development of specific intrinsic connectivity, and the ability for relearning. We conclude that heterosynaptic plasticity is an inherent property of plastic synapses, crucial for normal operation of learning systems. SIGNIFICANCE STATEMENT We show that spike timing-dependent plasticity in L2/L3 pyramids from rat visual cortex is accompanied by plastic changes in unpaired synapses. These heterosynaptic changes are weight-dependent and balanced: individual synapses expressed significant LTP or LTD, but the average over all synapses did not change. Thus, the rule of input specificity breaks down at individual synapses but holds for responses averaged over many inputs. In model neurons and networks, this experimentally characterized form of heterosynaptic plasticity prevents runaway dynamics of synaptic weights and activity, enhances synaptic competition, facilitates development of specific intrinsic connectivity, and enables relearning. This new form of heterosynaptic plasticity represents the cellular basis of a theoretically postulated mechanism, which is additional to Hebbian-type rules, and is necessary for stable operation of learning systems.

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ‐aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABAA receptors (GABAARs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CBSH3− or CBSH3+ in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2SH3− or CB2SH3+ in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CBSH3− or CBSH3+ DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291–1311, 2017. © 2016 Wiley Periodicals, Inc.

Understanding single-neuron computations and encoding performed by spike-generation mechanisms of cortical neurons is one of the central challenges for cell electrophysiology and computational neuroscience. An established paradigm to study spike encoding in controlled conditions in vitro uses intracellular injection of a mixture of signals with fluctuating currents that mimic in vivo-like background activity. However this technique has two serious limitations: it uses current injection, while synaptic activation leads to changes of conductance, and current injection is technically most feasible in the soma, while the vast majority of synaptic inputs are located on the dendrites. Recent progress in optogenetics provides an opportunity to circumvent these limitations. Transgenic expression of light-activated ionic channels, such as Channelrhodopsin2 (ChR2), allows induction of controlled conductance changes even in thin distant dendrites. Here we show that photostimulation provides a useful extension of the tools to study neuronal encoding, but it has its own limitations. Optically induced fluctuating currents have a low cutoff (~70Hz), thus limiting the dynamic range of frequency response of cortical neurons. This leads to severe underestimation of the ability of neurons to phase-lock their firing to high frequency components of the input. This limitation could be worked around by using short (2 ms) light stimuli which produce membrane potential responses resembling EPSPs by their fast onset and prolonged decay kinetics. We show that combining application of short light stimuli to different parts of dendritic tree for mimicking distant EPSCs with somatic injection of fluctuating current that mimics fluctuations of membrane potential in vivo, allowed us to study fast encoding of artificial EPSPs photoinduced at different distances from the soma. We conclude that dendritic photostimulation of ChR2 with short light pulses provides a powerful tool to investigate population encoding of simulated synaptic potentials generated in dendrites at different distances from the soma.

There is increasing concern over the widespread use of perfluorinated chemicals, which accumulate in various tissues and penetrate the mammalian fetus. A chick model was established for the rapid evaluation of teratogenicity of these chemicals, an important issue because developmental defects often occur at lower exposures than those required for adult systemic toxicity. Chicken eggs were injected with varying doses of perfluorooctanoic acid prior to incubation. Observed were defects in hatching, increased incidence of splayed legs, and interference with the appropriate development of yellow plumage. All these defects are potentially related to essential molecular/biochemical and functional development of the chick. Because of the relationship between structural defects and vulnerability of the developing brain, our model points to the need to evaluate neurobehavioral teratogenicity, which may involve even lower doses.