Restoring activity in the thalamic reticular nucleus improves sleep architecture and reduces Aß accumulation in mice.

Sleep disruptions promote increases of amyloid ß (Aß) and tau in the brain and increase Alzheimer's disease (AD) risk, but the precise mechanisms that give rise to sleep disturbances have yet to be defined. The thalamic reticular nucleus (TRN) is essential for sleep maintenance and for the regulation of slow-wave sleep (SWS). We examined the TRN in transgenic mice that express mutant human amyloid precursor protein (APP) and found reduced neuronal activity, increased sleep fragmentation, and decreased SWS time as compared to nontransgenic littermates. Selective activation of the TRN using excitatory DREADDs restored sleep maintenance, increased time in SWS, and reduced amyloid plaque load in both hippocampus and cortex. Our findings suggest that the TRN may play a major role in symptoms associated with AD. Enhancing TRN activity might be a promising therapeutic strategy for AD.

Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies.

Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a "fully-humanized" set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/.

SOX9-induced Generation of Functional Astrocytes Supporting Neuronal Maturation in an All-human System.

Astrocytes, the main supportive cell type of the brain, show functional impairments upon ageing and in a broad spectrum of neurological disorders. Limited access to human astroglia for pre-clinical studies has been a major bottleneck delaying our understanding of their role in brain health and disease. We demonstrate here that functionally mature human astrocytes can be generated by SOX9 overexpression for 6 days in pluripotent stem cell (PSC)-derived neural progenitor cells. Inducible (i)SOX9-astrocytes display functional properties comparable to primary human astrocytes comprising glutamate uptake, induced calcium responses and cytokine/growth factor secretion. Importantly, electrophysiological properties of iNGN2-neurons co-cultured with iSOX9-astrocytes are indistinguishable from gold-standard murine primary cultures. The high yield, fast timing and the possibility to cryopreserve iSOX9-astrocytes without losing functional properties makes them suitable for scaled-up production for high-throughput analyses. Our findings represent a step forward to an all-human iPSC-derived neural model for drug development in neuroscience and towards the reduction of animal use in biomedical research.

TRPM4 Conductances in Thalamic Reticular Nucleus Neurons Generate Persistent Firing during Slow Oscillations.

During sleep, neurons in the thalamic reticular nucleus (TRN) participate in distinct types of oscillatory activity. While the reciprocal synaptic circuits between TRN and sensory relay nuclei are known to underlie the generation of sleep spindles, the mechanisms regulating slow (<1 Hz) forms of thalamic oscillations are not well understood. Under in vitro conditions, TRN neurons can generate slow oscillations in a cell-intrinsic manner, with postsynaptic Group 1 metabotropic glutamate receptor activation triggering long-lasting plateau potentials thought to be mediated by both T-type Ca2+ currents and Ca2+-activated nonselective cation currents (ICAN). However, the identity of ICAN and the possible contribution of thalamic circuits to slow rhythmic activity remain unclear. Using thalamic slices derived from adult mice of either sex, we recorded slow forms of rhythmic activity in TRN neurons, which were driven by fast glutamatergic thalamoreticular inputs but did not require postsynaptic Group 1 metabotropic glutamate receptor activation. For a significant fraction of TRN neurons, synaptic inputs or brief depolarizing current steps led to long-lasting plateau potentials and persistent firing (PF), and in turn, resulted in sustained synaptic inhibition in postsynaptic relay neurons of the ventrobasal thalamus (VB). Pharmacological approachesindicated that plateau potentials were triggered by Ca2+ influx through T-type Ca2+ channels and mediated by Ca2+- and voltage-dependent transient receptor potential melastatin 4 (TRPM4) channels. Together, our results suggest that thalamic circuits can generate slow oscillatory activity, mediated by an interplay of TRN-VB synaptic circuits that generate rhythmicity and TRN cell-intrinsic mechanisms that control PF and oscillation frequency.SIGNIFICANCE STATEMENT Slow forms of thalamocortical rhythmic activity are thought to be essential for memory consolidation during sleep and the efficient removal of potentially toxic metabolites. In vivo, thalamic slow oscillations are regulated by strong bidirectional synaptic pathways linking neocortex and thalamus. Therefore, in vitro studies in the isolated thalamus offer important insights about the ability of individual neurons and local circuits to generate different forms of rhythmic activity. We found that circuits formed by GABAergic neurons in the thalamic reticular nucleus and glutamatergic relay neurons in the ventrobasal thalamus generated slow oscillatory activity, which was accompanied by persistent firing in thalamic reticular nucleus neurons. Our results identify both cell-intrinsic and synaptic mechanisms that mediate slow forms of rhythmic activity in thalamic circuits.

Synaptic Release of Acetylcholine Rapidly Suppresses Cortical Activity by Recruiting Muscarinic Receptors in Layer 4.

Cholinergic afferents from the basal forebrain (BF) can influence cortical activity on rapid time scales, enabling sensory information processing and exploratory behavior. However, our understanding of how synaptically released acetylcholine (ACh) influences cellular targets in distinct cortical layers remains incomplete. Previous studies have shown that rapid changes in cortical dynamics induced by phasic BF activity can be mediated by the activation of nicotinic ACh receptors (nAChRs) expressed in distinct types of GABAergic interneurons. In contrast, muscarinic ACh receptors (mAChRs) are assumed to be involved in slower and more diffuse ACh signaling following sustained increases in afferent activity. Here, we examined the mechanisms underlying fast cholinergic control of cortical circuit dynamics by pairing optical stimulation of cholinergic afferents with evoked activity in somatosensory cortical slices of mice of either sex. ACh release evoked by single stimuli led to a rapid and persistent suppression of cortical activity, mediated by mAChRs expressed in layer 4 and to a lesser extent, by nAChRs in layers 1-3. In agreement, we found that cholinergic inputs to layer 4 evoked short-latency and long-lasting mAChR-dependent inhibition of the large majority of excitatory neurons, whereas inputs to layers 1-3 primarily evoked nAChR-dependent excitation of different classes of interneurons. Our results indicate that the rapid cholinergic control of cortical network dynamics is mediated by both nAChRs and mAChRs-dependent mechanisms, which are expressed in distinct cortical layers and cell types.SIGNIFICANCE STATEMENT Acetylcholine (ACh) release from basal forebrain (BF) afferents to cortex influences a variety of cognitive functions including attention, sensory processing, and learning. Cholinergic control occurs on the time scale of seconds and is mediated by BF neurons that generate action potentials at low rates, indicating that ACh acts as a point-to-point neurotransmitter. Our findings highlight that even brief activation of cholinergic afferents can recruit both nicotinic and muscarinic ACh receptors expressed in several cell types, leading to modulation of cortical activity on distinct time scales. Furthermore, they indicate that the initial stages of cortical sensory processing are under direct cholinergic control.

Fluidic Microactuation of Flexible Electrodes for Neural Recording.

Soft and conductive nanomaterials like carbon nanotubes, graphene, and nanowire scaffolds have expanded the family of ultraflexible microelectrodes that can bend and flex with the natural movement of the brain, reduce the inflammatory response, and improve the stability of long-term neural recordings. However, current methods to implant these highly flexible electrodes rely on temporary stiffening agents that temporarily increase the electrode size and stiffness thus aggravating neural damage during implantation, which can lead to cell loss and glial activation that persists even after the stiffening agents are removed or dissolve. A method to deliver thin, ultraflexible electrodes deep into neural tissue without increasing the stiffness or size of the electrodes will enable minimally invasive electrical recordings from within the brain. Here we show that specially designed microfluidic devices can apply a tension force to ultraflexible electrodes that prevents buckling without increasing the thickness or stiffness of the electrode during implantation. Additionally, these "fluidic microdrives" allow us to precisely actuate the electrode position with micron-scale accuracy. To demonstrate the efficacy of our fluidic microdrives, we used them to actuate highly flexible carbon nanotube fiber (CNTf) microelectrodes for electrophysiology. We used this approach in three proof-of-concept experiments. First, we recorded compound action potentials in a soft model organism, the small cnidarian Hydra. Second, we targeted electrodes precisely to the thalamic reticular nucleus in brain slices and recorded spontaneous and optogenetically evoked extracellular action potentials. Finally, we inserted electrodes more than 4 mm deep into the brain of rats and detected spontaneous individual unit activity in both cortical and subcortical regions. Compared to syringe injection, fluidic microdrives do not penetrate the brain and prevent changes in intracranial pressure by diverting fluid away from the implantation site during insertion and actuation. Overall, the fluidic microdrive technology provides a robust new method to implant and actuate ultraflexible neural electrodes.

Examining Cholinergic Synaptic Signaling in the Thalamic Reticular Nucleus.

This protocol describes how to obtain monosynaptic cholinergic responses in neurons of the thalamic reticular nucleus (TRN) by making use of extracellular stimulation techniques. These methods are easy to implement and allow for the study of various forms of cholinergic synaptic plasticity and modulation. For many synapses throughout the mammalian brain, short-term plasticity is mediated by endocannabinoids released from postsynaptic neurons that activate presynaptic type I cannabinoid receptors (CB1Rs), resulting in the inhibition of presynaptic Ca2+ channels and a reduction of release probability. Neurons in the TRN are known to liberate endocannabinoids that can control transmitter release at GABAergic terminals. However, expression of CB1Rs on cholinergic terminals contacting the TRN has not been demonstrated. Here we outline strategies aimed to record stable postsynaptic responses and to quantify changes in cholinergic synaptic strength, using presynaptic modulation of acetylcholine (ACh) release by a CB1R agonist as an illustrative example.

mGluR1 and mGluR5 Synergistically Control Cholinergic Synaptic Transmission in the Thalamic Reticular Nucleus.

UNLABELLED: Acetylcholine (ACh) signaling is involved in a wide range of processes, including arousal, attention, and learning. An increasing number of studies indicate that cholinergic control of these functions is highly deterministic, mediated by synaptic afferents that generate reliable and precise responses in postsynaptic neurons. However, mechanisms that govern plastic changes of cholinergic synaptic strength are poorly understood, even though they are likely critical in shaping the impact of cholinergic inputs on neuronal networks. We have recently shown that in the thalamic reticular nucleus (TRN), synaptic release of ACh generates excitatory-inhibitory biphasic postsynaptic responses, mediated by the activation of α4ß2 nicotinic (nAChRs) and M2 muscarinic receptors (mAChRs), respectively. Here, using voltage-clamp recordings from TRN neurons in thalamocortical slices of mice, we demonstrate that the activation of Group I metabotropic glutamate receptors (mGluRs) by ambient or synaptically released glutamate evokes transient increases of nicotinic EPSCs. Additionally, we find that the selective Group I mGluR agonist DHPG [(S)-3,5-dihydroxyphenylglycine] evokes long-term potentiation of nicotinic EPSCs (mGluR-nLTP), dependent on increases in postsynaptic Ca(2+) concentration and the activation of phospholipase C. Both the induction and the maintenance of mGluR-nLTP require synergistic activation of mGluR1 and mGluR5. Together, our results show that postsynaptic Group I mGluRs are critically involved in the regulation of cholinergic synaptic strength on different time scales, suggesting that cholinergic control of local thalamic circuits is highly context-dependent and regulated by the overall levels of glutamatergic afferent activity. SIGNIFICANCE STATEMENT: Cholinergic signaling controls information processing and plasticity in neuronal circuits, but the mechanisms underlying the regulation of cholinergic synaptic strength on different time scales are unknown. Here we identify mGluR1 and mGluR5 as key elements in the dynamic regulation of cholinergic synaptic inputs onto neurons of the TRN. Our findings highlight potential mechanisms that regulate cholinergic signaling in the mammalian brain.

Noradrenaline-mediated facilitation of inhibitory synaptic transmission in the dorsal horn of the rat spinal cord involves interlaminar communications.

In the dorsal horn of the spinal cord (DH), noradrenaline (NA) is released by axons originating from the locus coeruleus and induces spinal analgesia, the mechanisms of which are poorly understood. Here, the effects of NA on synaptic transmission in the deep laminae (III-V) of the DH were characterized. It was shown that exogenously applied, as well as endogenously released, NA facilitated inhibitory [γ-aminobutyric acid (GABA)ergic and glycinergic] synaptic transmission in laminae III-IV of the DH by activating α1-, α2- and ß-adrenoceptors (ARs). In contrast, NA had no effect on excitatory (glutamatergic) synaptic transmission. Physical interruption of communications between deep and more superficial laminae (by a mechanical transection between laminae IV and V) totally blocked the effects of α2-AR agonists and strongly reduced the effects of α1-AR agonists on inhibitory synaptic transmission in laminae III-IV without directly impairing synaptic release of GABA or glycine from neurons. Short-term pretreatment of intact spinal cord slices with the glial cell metabolism inhibitor fluorocitrate or pharmacological blockade of ionotropic glutamate and ATP receptors mimicked the consequences of a mechanical transection between laminae IV and V. Taken together, the current results indicate that the facilitation of inhibitory synaptic transmission in laminae III-IV of the DH by NA requires functional interlaminar connections between deep and more superficial laminae, and might strongly depend on glia to neuron interactions. These interlaminar connections and glia to neuron interactions could represent interesting targets for analgesic strategies.