Cortex Invivo Research Articles

In Vitro Reconstitution of Dynein Force Exertion in a Bulk Viscous Medium

The forces generated by microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. To date, most studies have focused on MT force exertion by motors anchored to a static surface, such as the cell cortex invivo or glass surfaces invitro [2-4]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization, and orientation [5-14] but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo sizes and medium viscosities shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study reinforces the notion that endomembrane transport can exert significant forces on MTs.

Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex invivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. Invivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 invivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Interleukin-4 and Interleukin-13 Exacerbate Neurotoxicity of Prothrombin Kringle-2 in Cortex In Vivo via Oxidative Stress.

The present study investigated the effects of activated microglia-derived interleukin-4 (IL-4) and IL-13 on neurodegeneration in prothrombin kringle-2 (pKr-2)-treated rat cortex. pKr-2 was unilaterally injected into the Sprague–Dawley rat cerebral cortex and IL-4 and IL-13 neutralizing antibody was used to block the function of IL-4 and IL-13. Immunohistochemical analysis showed a significant loss of NeuN+ and Nissl+ cells and an increase of OX-42+ cells in the cortex at seven days post pKr-2. The levels of IL-4 and IL-13 expression were upregulated in the activated microglia as early as 12 hours post pKr-2 and sustained up to seven days post pKr-2. Neutralization by IL-4 or IL-13 antibodies (NA) significantly increased neuronal survival in pKr-2-treated rat cortex in vivo by suppressing microglial activation and the production of reactive oxygen species, as analyzed by immunohisotochemistry and hydroethidine histochemistry. These results suggest that IL-4 and IL-13 that were endogenously expressed from reactive microglia may play a critical role on neuronal death by regulating oxidative stress during the neurodegenerative diseases, such as Alzheimer’s disease and dementia.

PET imaging of dopamine release in the frontal cortex of manganese-exposed non-human primates.

Humans and non-human primates exposed to excess levels of manganese (Mn) exhibit deficits in working memory and attention. Frontal cortex and fronto-striatal networks are implicated in working memory and these circuits rely on dopamine for optimal performance. Here, we aimed to determine if chronic Mn exposure alters invivo dopamine release (DAR) in the frontal cortex of non-human primates. We used [11 C]-FLB457 positron emission tomography with amphetamine challenge to measure DAR in Cynomolgus macaques. Animals received [11 C]-FLB457 positron emission tomography scans with and without amphetamine challenge prior to Mn exposure (baseline), at different time points during the Mn exposure period, and after 10months of Mn exposure cessation. Four of six Mn-exposed animals expressed significant impairment of frontal cortex invivo DAR relative to baseline. One Mn animal had no change in DAR and another Mn animal expressed increased DAR relative to baseline. In the reversal studies, one Mn-exposed animal exhibited complete recovery of DAR while the second animal had partial recovery. In both animals, frontal cortex Mn concentrations normalized after 10months of exposure cessation based on T1-weighted magnetic resonance imaging. D1-dopamine receptor (D1R) autoradiography in frontal cortex tissue indicates that Mn animals that experienced cessation of Mn exposure expressed D1R levels that were approximately 50% lower than Mn animals that did not experience cessation of Mn exposure or control animals. The present study provides evidence of Mn-induced alterations in frontal cortex DAR and D1R that may be associated with working memory and attention deficits observed in Mn-exposed subjects.

Patchwork-Type Spontaneous Activity in Neonatal Barrel Cortex Layer 4 Transmitted via Thalamocortical Projections

Establishment of precise neuronal connectivity in the neocortex relies on activity-dependent circuit reorganization during postnatal development; however, the nature of cortical activity during this period remains largely unknown. Using two-photon calcium imaging of the barrel cortex invivo during the first postnatal week, we reveal that layer 4 (L4) neurons within the same barrel fire synchronously in the absence of peripheral stimulation, creating a "patchwork" pattern of spontaneous activity corresponding to the barrel map. By generating transgenic mice expressing GCaMP6s in thalamocortical axons, we show that thalamocortical axons also demonstrate the spontaneous patchwork activity pattern. Patchwork activity is diminished by peripheral anesthesia but is mostly independent of self-generated whisker movements. The patchwork activity pattern largely disappeared during postnatal week 2, as even L4 neurons within the same barrel tended to fire asynchronously. This spontaneous L4 activity pattern has features suitable for thalamocortical (TC) circuit refinement in the neonatal barrel cortex.

Micro-coil-induced Inhomogeneous Electric Field Produces sound-driven-like Neural Responses in Microcircuits of the Mouse Auditory Cortex In Vivo

Magnetic stimulation is widely used in neuroscience research and clinical treatment. Despite recent progress in understanding the neural modulation mechanism of conventional magnetic stimulation methods, the physiological mechanism at the cortical microcircuit level is not well understood due to the poor stimulation focality and large electric artifact in the recording. To overcome these issues, we used a sub-millimeter-sized coil (micro-coil) to stimulate the mouse auditory cortex in vivo. To determine the mechanism, we conducted the first direct electrophysiological recording of micro-coil-driven neural responses at multiple sites on the horizontal surface and laminar areas of the auditory cortex. The laminar responses of local field potentials (LFPs) to the magnetic stimulation reached layer 6, and the spatiotemporal profiles were very similar to those of the acoustic stimulation, suggesting the activation of the same cortical microcircuit. The horizontal LFP responses to the magnetic stimulation were evoked within a millimeter-wide area around the stimulation coil. The activated cortical area was dependent on the coil orientation, providing useful information on the effective position of the coil relative to the brain surface for modulating cortical circuitry activity. In addition, numerical calculation of the induced electric field in the brain revealed that the inhomogeneity of the horizontal electric field to the surface is critical for micro-coil-induced cortical activation. The results suggest that our micro-coil technique has the potential to be used as a chronic, less-invasive and highly focal neuro-stimulator, and is useful for investigating microcircuit responses to magnetic stimulation for clinical treatment.

Acetazolamide potentiates the afferent drive to prefrontal cortex invivo.

The knowledge on real‐time neurophysiological effects of acetazolamide is still far behind the wide clinical use of this drug. Acetazolamide – a carbonic anhydrase inhibitor – has been shown to affect the neuromuscular transmission, implying a pH‐mediated influence on the central synaptic transmission. To start filling such a gap, we chose a central substrate: hippocampal‐prefrontal cortical projections; and a synaptic phenomenon: paired‐pulse facilitation (a form of synaptic plasticity) to probe this drug's effects on interareal brain communication in chronically implanted rats. We observed that systemic acetazolamide potentiates the hippocampal‐prefrontal paired‐pulse facilitation. In addition to this field electrophysiology data, we found that acetazolamide exerts a net inhibitory effect on prefrontal cortical single‐unit firing. We propose that systemic acetazolamide reduces the basal neuronal activity of the prefrontal cortex, whereas increasing the afferent drive it receives from the hippocampus. In addition to being relevant to the clinical and side effects of acetazolamide, these results suggest that exogenous pH regulation can have diverse impacts on afferent signaling across the neocortex.

Mitochondrial Dynamics in Visual Cortex Are Limited In Vivo and Not Affected by Axonal Structural Plasticity

Mitochondria buffer intracellular Ca2+ and provide energy [1]. Because synaptic structures with high Ca2+ buffering [2-4] or energy demand [5] are often localized far away from the soma, mitochondria are actively transported to these sites [6-11]. Also, the removal and degradation of mitochondria are tightly regulated [9, 12, 13], because dysfunctional mitochondria are a source of reactive oxygen species, which can damage the cell [14]. Deficits in mitochondrial trafficking have been proposed to contribute to the pathogenesis of Parkinson's disease, schizophrenia, amyotrophic lateral sclerosis, optic atrophy, and Alzheimer's disease [13, 15-19]. In neuronal cultures, about a third of mitochondria are motile, whereas the majority remains stationary for several days [8, 20]. Activity-dependent mechanisms cause mitochondria to stop at synaptic sites [7, 8, 20, 21], which affects synapse function and maintenance. Reducing mitochondrial content in dendrites decreases spine density [22, 23], whereas increasing mitochondrial content or activity increases it [7]. These bidirectional interactions between synaptic activity and mitochondrial trafficking suggest that mitochondria may regulate synaptic plasticity. Here we investigated the dynamics of mitochondria in relation to axonal boutons of neocortical pyramidal neurons for the first time invivo. We find that underthese circumstances practically all mitochondria are stationary, both during development and in adulthood. In adult visual cortex, mitochondria are preferentially localized at putative boutons, where they remain for several days. Retinal-lesion-induced cortical plasticity increases turnover of putative boutons but leaves mitochondrial turnover unaffected. We conclude that in visual cortex invivo, mitochondria are less dynamic than invitro, and that structural plasticity does not affect mitochondrial dynamics.

Sensory Response of Transplanted Astrocytes in Adult Mammalian Cortex In Vivo.

Glial precursor transplantation provides a potential therapy for brain disorders. Before its clinical application, experimental evidence needs to indicate that engrafted glial cells are functionally incorporated into the existing circuits and become essential partners of neurons for executing fundamental brain functions. While previous experiments supporting for their functional integration have been obtained under in vitro conditions using slice preparations, in vivo evidence for such integration is still lacking. Here, we utilized in vivo two-photon Ca2+ imaging along with immunohistochemistry, fluorescent indicator labeling-based axon tracing and correlated light/electron microscopy to analyze the profiles and the functional status of glial precursor cell-derived astrocytes in adult mouse neocortex. We show that after being transplanted into somatosensory cortex, precursor-derived astrocytes are able to survive for more than a year and respond with Ca2+ signals to sensory stimulation. These sensory-evoked responses are mediated by functionally-expressed nicotinic receptors and newly-established synaptic contacts with the host cholinergic afferents. Our results provide in vivo evidence for a functional integration of transplanted astrocytes into adult mammalian neocortex, representing a proof-of-principle for sensory cortex remodeling through addition of essential neural elements. Moreover, we provide strong support for the use of glial precursor transplantation to understand glia-related neural development in vivo.

Simultaneous Multi-plane Imaging of Neural Circuits

Recording the activity of large populations of neurons is an important step toward understanding theemergent function of neural circuits. Here we present a simple holographic method to simultaneously perform two-photon calcium imaging of neuronal populations across multiple areas and layers of mouse cortex invivo. We use prior knowledge of neuronal locations, activity sparsity, and a constrained nonnegative matrix factorization algorithm to extract signals from neurons imaged simultaneously and located in different focal planes or fields of view. Our laser multiplexing approach is simple and fast, and could be used as a general method to image the activity of neural circuits in three dimensions across multiple areas in the brain.

Subnetwork-Specific Homeostatic Plasticity in Mouse Visual Cortex In Vivo

SummaryHomeostatic regulation has been shown to restore cortical activity in vivo following sensory deprivation, but it is unclear whether this recovery is uniform across all cells or specific to a subset of the network. To address this issue, we used chronic calcium imaging in behaving adult mice to examine the activity of individual excitatory and inhibitory neurons in the same region of the layer 2/3 monocular visual cortex following enucleation. We found that only a fraction of excitatory neurons homeostatically recover activity after deprivation and inhibitory neurons show no recovery. Prior to deprivation, excitatory cells that did recover were more likely to have significantly correlated activity with other recovering excitatory neurons, thus forming a subnetwork of recovering neurons. These network level changes are accompanied by a reduction in synaptic inhibition onto all excitatory neurons, suggesting that both synaptic mechanisms and subnetwork activity are important for homeostatic recovery of activity after deprivation.

Promoter Decommissioning by the NuRD Chromatin Remodeling Complex Triggers Synaptic Connectivity in the Mammalian Brain

Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by invivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex invivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum invivo. Finally, in a targeted invivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain.

GABAAReceptor-Mediated Bidirectional Control of Synaptic Activity, Intracellular Ca2+, Cerebral Blood Flow, and Oxygen Consumption in Mouse Somatosensory Cortex In Vivo

Neural activity regulates local increases in cerebral blood flow (ΔCBF) and the cortical metabolic rate of oxygen (ΔCMRO2) that constitutes the basis of BOLD functional neuroimaging signals. Glutamate signaling plays a key role in brain vascular and metabolic control; however, the modulatory effect of GABA is incompletely understood. Here we performed in vivo studies in mice to investigate how THIP (which tonically activates extrasynaptic GABAARs) and Zolpidem (a positive allosteric modulator of synaptic GABAARs) impact stimulation-induced ΔCBF, ΔCMRO2, local field potentials (LFPs), and fluorescent cytosolic Ca(2+) transients in neurons and astrocytes. Low concentrations of THIP increased ΔCBF and ΔCMRO2 at low stimulation frequencies. These responses were coupled to increased synaptic activity as indicated by LFP responses, and to Ca(2+) activities in neurons and astrocytes. Intermediate and high concentrations of THIP suppressed ΔCBF and ΔCMRO2 at high stimulation frequencies. Zolpidem had similar but less-pronounced effects, with similar dependence on drug concentration and stimulation frequency. Our present findings suggest that slight increases in both synaptic and extrasynaptic GABAAR activity might selectively gate and amplify transient low-frequency somatosensory inputs, filter out high-frequency inputs, and enhance vascular and metabolic responses that are likely to be reflected in BOLD functional neuroimaging signals.

Laminar and Columnar Structure of Sensory-Evoked Multineuronal Spike Sequences in Adult Rat Barrel Cortex In Vivo

One of the most relevant questions regarding the function of the nervous system is how sensory information is represented in populations of cortical neurons. Despite its importance, the manner in which sensory-evoked activity propagates across neocortical layers and columns has yet not been fully characterized. In this study, we took advantage of the distinct organization of the rodent barrel cortex and recorded with multielectrode arrays simultaneously from up to 74 neurons localized in several functionally identified layers and columns of anesthetized adult Wistar rats in vivo. The flow of activity within neuronal populations was characterized by temporally precise spike sequences, which were repeatedly evoked by single-whisker stimulation. The majority of the spike sequences representing instantaneous responses were led by a subgroup of putative inhibitory neurons in the principal column at thalamo-recipient layers, thus revealing the presence of feedforward inhibition. However, later spike sequences were mainly led by infragranular excitatory neurons in neighboring columns. Although the starting point of the sequences was anatomically confined, their ending point was rather scattered, suggesting that the population responses are structurally dispersed. Our data show for the first time the simultaneous intra- and intercolumnar processing of information at high temporal resolution.

Synaptic Scaling and Homeostatic Plasticity in the Mouse Visual Cortex In Vivo

Homeostatic plasticity is important to maintain a set level of activity in neuronal circuits and has been most extensively studied in cell cultures following activity blockade. It is still unclear, however, whether activity changes associated with mechanisms of homeostatic plasticity occur in vivo, for example after changes in sensory input. Here, we show that activity levels in the visual cortex are significantly decreased after sensory deprivation by retinal lesions, followed by a gradual increase in activity levels in the 48 hr after deprivation. These activity changes are associated with synaptic scaling, manifested in vitro by an increase in mEPSC amplitude and in vivo by an increase in spine size. Together, these data show that homeostatic activity changes occur in vivo in parallel with synaptic scaling.

The XLID Protein PQBP1 and the GTPase Dynamin 2 Define a Signaling Link that Orchestrates Ciliary Morphogenesis in Postmitotic Neurons

Intellectual disability (ID) is a prevalent developmental disorder of cognition that remains incurable. Here, we report that knockdown of the X-linked ID(XLID) protein polyglutamine-binding protein 1 (PQBP1) in neurons profoundly impairs the morphogenesis of the primary cilium, including in the mouse cerebral cortex invivo. PQBP1 is localized at the base of the neuronal cilium, and targeting its WW effector domain to the cilium stimulates ciliary morphogenesis. We also find that PQBP1 interacts with Dynamin 2 and thereby inhibits its GTPase activity. Accordingly, Dynamin 2 knockdown in neurons stimulates ciliogenesis and suppresses the PQBP1 knockdown-induced ciliary phenotype. Strikingly, a mutation of the PQBP1 WW domain that causes XLID disrupts its ability to interact with and inhibit Dynamin 2 and to induce neuronal ciliogenesis. These findings define PQBP1 and Dynamin 2 as components of a signaling pathway that orchestrates neuronal ciliary morphogenesis in the brain.

The X-Linked Intellectual Disability Protein PHF6 Associates with the PAF1 Complex and Regulates Neuronal Migration in the Mammalian Brain

Intellectual disability is a prevalent disorder that remains incurable. Mutations of the X-linked proteinPHF6 cause the intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS). However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. We report that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex invivo, leading to the formation of white matter heterotopias displaying neuronal hyperexcitability. We find that PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype invivo. We also identify Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5), a potential schizophrenia susceptibility gene, as a critical downstream target of PHF6 in the control of neuronal migration. These findings define PHF6, PAF1, and NGC/CSPG5 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of developmental disorders of cognition.

Trnp1 Regulates Expansion and Folding of the Mammalian Cerebral Cortex by Control of Radial Glial Fate

Evolution of the mammalian brain encompassed a remarkable increase in size of the cerebral cortex, which includes tangential and radial expansion. However, the mechanisms underlying these key features are still largely unknown. Here, we identified the DNA-associated protein Trnp1 as a regulator of cerebral cortex expansion in both of these dimensions. Gain- and loss-of-function experiments in the mouse cerebral cortex invivo demonstrate that high Trnp1 levels promote neural stem cell self-renewal and tangential expansion. In contrast, lower levels promote radial expansion, with a potent increase of the number of intermediate progenitors and basal radial glial cells leading to folding of the otherwise smooth murine cerebral cortex. Remarkably, TRNP1 expression levels exhibit regional differences in the cerebral cortex of human fetuses, anticipating radial or tangential expansion. Thus, the dynamic regulation of Trnp1 is critical to control tangential and radial expansion of the cerebral cortex in mammals.

Long-Term Potentiation in the Neonatal Rat Barrel Cortex In Vivo

Long-term potentiation (LTP) is important for the activity-dependent formation of early cortical circuits. In the neonatal rodent barrel cortex, LTP has been studied only in vitro. We combined voltage-sensitive dye imaging with extracellular multielectrode recordings to study whisker stimulation-induced LTP in the whisker-to-barrel cortex pathway of the neonatal rat barrel cortex in vivo. Single whisker stimulation at 2 Hz for 10 min induced an age-dependent expression of LTP in postnatal day (P) 0 to P14 rats, with the strongest expression of LTP at P3-P5. The magnitude of LTP was largest in the activated barrel-related column, smaller in the surrounding septal region, and no LTP could be observed in the neighboring barrel. Current source density analyses revealed an LTP-associated increase of synaptic current sinks in layer IV/lower layer II/III at P3-P5 and in the cortical plate/upper layer V at P0-P1. Our study demonstrates for the first time an age-dependent and spatially confined LTP in the barrel cortex of the newborn rat in vivo.

Two-Photon Imaging of Microglia in the Mouse Cortex In Vivo

Microglia are the primary immune effector cells of the brain parenchyma. They are distributed throughout the brain at various densities. Two-photon fluorescence microscopy, together with expression of fluorescent proteins in microglia, has enabled study of these fascinating cells in vivo. Imaging studies have shown, for example, that microglia continually survey their cellular environment and immediately respond to injury. However, we still know very little about their roles in various parts of the developing and adult brain or their diverse effector functions in aging and different disease states. Experimental procedures have been developed for minimally invasive short- and long-term two-photon imaging of microglial cells in cortical regions of the intact mouse brain. This protocol describes two-photon imaging of microglia in the mouse cortex in vivo, using mice which have had a head plate implanted and have been prepared with either a thinned skull or optical window. Technical pitfalls, limitations, and alternative approaches are also discussed.