Loss of Postnatal Arx Transcriptional Activity in Parvalbumin Interneurons Reveals Non-cell Autonomous Disturbances in CA1 Pyramidal Cells.

Maintenance of proper electrophysiological and connectivity profiles in the adult brain may be a perturbation point in neurodevelopmental disorders (NDDs). How these profiles are maintained within mature circuits is unclear. We recently demonstrated that postnatal ablation of the Aristaless (Arx) homeobox gene in parvalbumin interneurons (PVIs) alone led to dysregulation of their transcriptome and alterations in their functional as well as network properties in the hippocampal cornu Ammoni first region (CA1). Here, we characterized CA1 pyramidal cells (PCs) responses in this conditional knockout (CKO) mouse to further understand the circuit mechanisms by which postnatal Arx expression regulates mature CA1 circuits. Field recordings of network excitability showed that CA1 PC ensembles were less excitable in response to unpaired stimulations but exhibited enhanced excitability in response to paired-pulse stimulations. Whole-cell voltage clamp recordings revealed a significant increase in the frequency of spontaneous inhibitory postsynaptic currents onto PCs. In contrast, excitatory drive from evoked synaptic transmission was reduced while that of inhibitory synaptic transmission was increased. Current clamp recordings showed increase excitability in several sub- and threshold membrane properties that correlated with an increase in the conductance of Na+ current. Our data suggest that, in addition to cell-autonomous disruption in PVIs, loss of Arx postnatal transcriptional activity in PVIs led to complex dysfunctions in PCs in CA1 microcircuits. These non-cell autonomous effects are likely the product of breakdown in feedback and/or feedforward processes and should be considered as fundamental contributors to the circuit mechanisms of NDDs such as Arx-linked early-onset epileptic encephalopathies.

Protocol for isolating young adult parvalbumin interneurons from the mouse brain for extraction of high-quality RNA.

Dysfunction in the parvalbumin (PV) subclass of GABAergic interneurons is implicated in several neurodevelopmental disorders that evolve in severity with postnatal developmental stages. Understanding the molecular underpinnings of the postnatal changes in the function of PV interneurons has been limited by the difficulty in the isolation of pure adult PV interneurons and high-quality RNA. Here, we describe our protocol for the isolation of pure young adult PV interneurons and preparation of high-quality RNA from these cells. For complete details on the use and execution of this protocol, please refer to Joseph et al. (2021).

Islet1 Precursors Contribute to Mature Interneuron Subtypes in Mouse Neocortex.

Cortical interneurons (GABAergic cells) arise during embryogenesis primarily from the medial and caudal ganglionic eminences (MGE and CGE, respectively) with a small population generated from the preoptic area (POA). Progenitors from the lateral ganglionic eminence (LGE) are thought to only generate GABAergic medium spiny neurons that populate the striatum and project to the globus pallidus. Here, we report evidence that neuronal precursors that express the LGE-specific transcription factor Islet1 (Isl1) can give rise to a small population of cortical interneurons. Lineage tracing and homozygous deletion of Nkx2.1 in Isl1 fate-mapped mice showed that neighboring MGE/POA-specific Nkx2.1 cells and LGE-specific Isl1 cells make both common and distinct lineal contributions towards cortical interneuron fate. Although the majority of cells had overlapping transcriptional domains between Nkx2.1 and Isl1, a population of Isl1-only derived cells also contributed to the adult cerebral cortex. The data indicate that Isl1-derived cells may originate from both the LGE and the adjacent LGE/MGE boundary regions to generate diverse neuronal progeny. Thus, a small population of neocortical interneurons appear to originate from Isl-1-positive precursors.

Postnatal Arx transcriptional activity regulates functional properties of PV interneurons.

The transcription factor Aristaless-related X-linked gene (Arx) is a monogenic factor in early onset epileptic encephalopathies (EOEEs) and a fundamental regulator of early stages of brain development. However, Arx expression persists in mature GABAergic neurons with an unknown role. To address this issue, we generated a conditional knockout (CKO) mouse in which postnatal Arx was ablated in parvalbumin interneurons (PVIs). Electroencephalogram (EEG) recordings in CKO mice revealed an increase in theta oscillations and the occurrence of occasional seizures. Behavioral analysis uncovered an increase in anxiety. Genome-wide sequencing of fluorescence activated cell sorted (FACS) PVIs revealed that Arx impinged on network excitability via genes primarily associated with synaptic and extracellular matrix pathways. Whole-cell recordings revealed prominent hypoexcitability of various intrinsic and synaptic properties. These results revealed important roles for postnatal Arx expression in PVIs in the control of neural circuits and that dysfunction in those roles alone can cause EOEE-like network abnormalities.

Isoflurane mediated neuropathological and cognitive impairments in the triple transgenic Alzheimer's mouse model are associated with hippocampal synaptic deficits in an age-dependent manner.

Many in vivo studies suggest that inhalational anesthetics can accelerate or prevent the progression of neuropathology and cognitive impairments in Alzheimer Disease (AD), but the synaptic mechanisms mediating these ambiguous effects are unclear. Here, we show that repeated exposures of neonatal and old triple transgenic AD (3xTg) and non-transgenic (NonTg) mice to isoflurane (Iso) distinctly increased neurodegeneration as measured by S100ß levels, intracellular Aß, Tau oligomerization, and apoptotic markers. Spatial cognition measured by reference and working memory testing in the Morris Water Maze (MWM) were altered in young NonTg and 3xTg. Field recordings in the cornu ammonis 1 (CA1) hippocampus showed that neonatal control 3xTg mice exhibited hypo-excitable synaptic transmission, reduced paired-pulse facilitation (PPF), and normal long-term potentiation (LTP) compared to NonTg controls. By contrast, the old control 3xTg mice exhibited hyper-excitable synaptic transmission, enhanced PPF, and unstable LTP compared to NonTg controls. Repeated Iso exposures reduced synaptic transmission and PPF in neonatal NonTg and old 3xTg mice. LTP was normalized in old 3xTg mice, but reduced in neonates. By contrast, LTP was reduced in old but not neonatal NonTg mice. Our results indicate that Iso-mediated neuropathologic and cognitive defects in AD mice are associated with synaptic pathologies in an age-dependent manner. Based on these findings, the extent of this association with age and, possibly, treatment paradigms warrant further study.

Anesthetic preconditioning inhibits isoflurane-mediated apoptosis in the developing rat brain.

BACKGROUND: We hypothesized that preconditioning (PC) with a short exposure to isoflurane (ISO) would reduce neurodegeneration induced by prolonged exposure to ISO in neonatal rats, as previously shown in neuronal cell culture. METHODS: We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO. The control group was exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to carrier gas again for 6 hours the following day. The 1.5% ISO group was exposed to carrier gas for 30 minutes and then to 1.5% ISO for 6 hours the following day. The PC + 1.5% ISO group was preconditioned with a 30-minute 1.5% ISO exposure and then exposed to 1.5% ISO for 6 hours the following day. Blood and brain samples were collected 2 hours after the exposures for determination of neurodegenerative biomarkers, including caspase-3, S100ß, caspase-12, and an autophagy biomarker Beclin-1. RESULTS: Prolonged exposure to ISO significantly increased cleaved caspase-3 expression in the cerebral cortex of 7-day-old rats compared with the group preconditioned with ISO and the controls using Western blot assays. However, significant differences were not detected for other markers of neuronal injury. CONCLUSIONS: The ISO-mediated increase in cleaved caspase-3 in the postnatal day 7 rat brain is ameliorated by PC with a brief anesthetic exposure, and differences were not detected in other markers of neuronal injury.

Dual effects of isoflurane on proliferation, differentiation, and survival in human neuroprogenitor cells.

BACKGROUND: Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors. METHODS: Using the ReNcell CX (Millipore, Billerica, MA) human neural progenitor cell line, the authors investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, the authors assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of inositol-1,4,5-trisphosphate or ryanodine receptor activation to these processes. RESULTS: Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate and promoted glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with inositol-1,4,5-trisphosphate or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane-preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration. CONCLUSION: The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation, and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized inositol-1,4,5-trisphosphate and/or ryanodine receptors.

Dantrolene ameliorates cognitive decline and neuropathology in Alzheimer triple transgenic mice.

Disruption of intracellular calcium homeostasis via abnormal and excessive activation of ryanodine receptors plays an important role in the neuropathology of Alzheimer's disease. We investigated the therapeutic effect of dantrolene, a ryanodine receptor antagonist, on cognitive dysfunction and neuropathology in the triple transgenic Alzheimer mouse model (3xTg-AD). 3xTg-AD mice were treated with dantrolene from 2 to 13 months of age. Learning and memory were measured with the Morris Water Maze at 6, 10, and 13 months of age. Amyloid and tau neuropathology and biomarkers for synaptic dysfunction and neurodegeneration were examined in the brain using immunoblotting or immunohistochemistry. Dantrolene treatment for 11 months significantly reduced both memory deficits and amyloid plaque load in the hippocampus in 13-month-old 3xTg-AD mice. Dantrolene treatment, however, had no effect on phosphorylated tau, phosphorylated or total GSK-3ß, synaptic markers, or mitochondrial or cytosolic cytochrome C. Our results suggest that dantrolene significantly improves cognition in a murine model of Alzheimer's disease and is associated with a reduction in amyloid plaque burden, forming the basis for a novel therapeutic approach for Alzheimer's disease.

Modulation of neurite outgrowth by activation of calcium-permeable kainate receptors expressed by rat nociceptive-like dorsal root ganglion neurons.

Neurite outgrowth is a fundamental step in establishing proper neuronal connections in the developing central nervous system. Dynamic control of outgrowth has been attributed to changes in growth cone Ca2+ levels in response to extracellular cues. Here we have investigated a possible role for Ca2+ permeable kainate (KA) receptors in regulating neurite outgrowth of nociceptive-like dorsal root ganglion (DRG) neurons. To identify KA receptor subunits likely to be involved, we used quantitative RT-PCR on acutely dissociated DRG and dorsal horn neurons. DRG neurons expressed more GluK1, particularly the GluK1b spice variant, than dorsal horn neurons. Conversely, dorsal horn neurons expressed more GluK2, particularly GluK2a, than DRG neurons. Further, an RNA editing assay indicated that the majority of GluK1 and GluK2 mRNA transcripts in DRG were unedited. Imaging Ca2+ transients following application of a KA receptor agonist to DRG and dorsal horn co-cultures revealed increases in intracellular Ca2+ in the growth cones of DRG neurons. In the majority of cases, this increase in Ca2+ was partly or completely blocked by Joro spider toxin (JSTX), an antagonist for Ca2+-permeable AMPA and KA receptors. Treatment of DRG/dorsal horn co-cultures with KA for 18 hours suppressed neurite outgrowth while application of the rapidly desensitizing KA receptor agonist SYM 2081, the competitive AMPA/KA receptor antagonist, CNQX, and JSTX or philanthotoxin enhanced neurite outgrowth and prevented KA effects on neurite outgrowth. Thus, Ca2+ entry through KA receptors at the growth cone of DRG neurons may be an important regulator of neurite outgrowth.

An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons.

Synapses between nociceptive dorsal root ganglion (DRG) neurons and spinal cord dorsal horn neurons represent the first loci for transmission of painful stimuli. Our knowledge of the molecular organization and development of these synapses is sparse due, partly, to a lack of a reliable model system that reconstitutes synaptogenesis between these two neuronal populations. To address this issue, we have established an in vitro assay system consisting of separately purified DRG neurons and dorsal horn neurons on astrocyte microislands. Using immunocytochemistry, we have found that 97%, 93%, 98%, 96%, and 94% of DRG neurons on these microislands express markers often associated with nociceptive neurons including Substance P, TRPV1, calcitonin-gene related peptide (CGRP), TrKA, and peripherin, respectively. Triple labeling with these nociceptive-like markers, synaptic vesicle marker Vglut2 and using MAP2 as a dendritic marker revealed the presence of nociceptive-like markers at synaptic terminals. Using this immunocytochemical approach, we counted contact points as overlapping MAP2/Vglut2 puncta and showed that they increased with time in culture. Single and dual patch-clamp recordings showed that overlapping Vglut2/MAP2 puncta observed after a few days in culture are likely to be functional synapses between DRG and dorsal horn neurons in our in vitro assay system. Taken together, these data suggest our co-culture microisland model system consists of mostly nociceptive-like DRG neurons that express presynaptic markers and form functional synapses with their dorsal horn partners. Thus, this model system may have direct application for studies on factors regulating development of nociceptive DRG/dorsal horn synapses.

Anesthetic-induced neurodegeneration mediated via inositol 1,4,5-trisphosphate receptors.

The commonly used general anesthetic isoflurane induces widespread neurodegeneration in the developing mammalian brain through poorly understood mechanisms. We have investigated whether excessive Ca2+ release from the endoplasmic reticulum via overactivation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) is a contributing factor in such neurodegeneration in rodent primary cultured neurons and developing rat brain. We also investigated the correlation between isoflurane exposure and cognitive decline in rats at 1 month of age. Our results show that isoflurane increases cytosolic calcium in the primary cortical neurons through release from the endoplasmic reticulum and influx from the extracellular space. Pharmacological inhibition of InsP3R activity and knockdown of its expression nearly abolishes the isoflurane-mediated elevation of the cytosolic calcium concentration and cell death in rodent primary cortical and hippocampal neurons. Inhibition of InsP3R activity by its antagonist xestospongin C significantly inhibits neurodegeneration induced by isoflurane at clinically used concentration in the developing brain of postnatal day 7 rats. Moreover, our results show that isoflurane activates beta-site amyloid beta precursor protein-cleaving enzyme via activation of the InsP3R. We also noted that mice exposed to isoflurane during early postnatal development showed transient memory and learning impairments, which did not correlate well with the noted neuropathological defects. Taken together, our results suggest that Ca2+ dysregulation through overactivation of the InsP3R may be a contributing factor in the mechanism of isoflurane-induced neurodegeneration in rodent neuronal cell culture and during brain development.

Differential effects of AMPA receptor activation on survival and neurite integrity during neuronal development.

While neuronal cultures are an established model for analyzing excitotoxic brain injury in the adult, in vitro systems have not been extensively employed to study how developing neurons respond to levels of excitatory compounds that are lethal to mature neurons. Recently, we reported that the in vivo differentiation programs of cerebellar granule cells (CGNs) are recapitulated in purified CGN cultures [Manzini M.C., Ward M.S., Zhang Q., Lieberman M.D., Mason C.A. (2006) The stop-signal revised: immature cerebellar granule neurons in the external germinal layer arrest pontine mossy fiber growth. J. Neurosci. 26:6040-6051]. Here, we have used this model system to compare the response of immature and mature neurons to excitotoxic compounds. We found that immature CGNs are less sensitive to AMPA receptor (AMPA-R) activation than mature cells and that levels of AMPA-R expression on the plasma membrane are critical in regulating the balance between death and survival during maturation of these neurons. However, the majority of immature cells that survive excitotoxic treatment bear a degenerating neurite, suggesting that AMPA-R activation can still cause damage in the absence of cell death.

Cell type-specific interleukin-1beta signaling in the CNS.

Interleukin-1beta (IL-1beta) is a potent and pleiotropic inflammatory cytokine that is highly produced in the CNS under conditions of damage, disease, or stress. This cytokine acts on CNS glia to effect inflammatory responses, mediated in part via activation of the nuclear factor-kappaB (NF-kappaB) transcription factor, and consequent induction of numerous cytokines. Neurons as well as astrocytes in the hippocampus also express the type 1 IL-1 receptor, indicating that this cytokine can influence neuronal function directly, yet IL-1beta does not induce production of cytokines in neurons as it does in glia. In contrast, IL-1beta regulates synaptic function of hippocampal neurons. Here we demonstrate that different signaling pathways mediate IL-1beta actions in neurons as compared with astrocytes. IL-1beta activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway and induces the activation of CREB in hippocampal neurons, in contrast to the activation of NF-kappaB in hippocampal astrocytes, demonstrating cell type-specific signaling responses to IL-1 in the brain and yielding distinct functional responses.

Functional expression of AMPA receptors on central terminals of rat dorsal root ganglion neurons and presynaptic inhibition of glutamate release.

No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA(A) receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.