Robert A. Pearce

Specification of motoneurons from human embryonic stem cells

An understanding of how mammalian stem cells produce specific neuronal subtypes remains elusive. Here we show that human embryonic stem cells generated early neuroectodermal cells, which organized into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube–like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro–generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage.

Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells

How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC‐derived Sox1+neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)–positive neurons that were mostly bipolar cells, coexpression with γ‐aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro–generated DA neurons were electrophysiologically active and released DA in an activity‐dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system.

Diversity of inhibitory neurotransmission through GABAA receptors

In the brain, highly connected and heterogeneous GABAergic cells are crucial in controling the activity of neuronal networks. They accomplish this task by communicating through remarkably diverse sets of inhibitory processes, the complexity of which is reflected by the variety of interneuron classification schemes proposed in recent years. It is now becoming clear that the subcellular localization and intrinsic properties of heteropentameric GABA(A) receptors themselves also constitute major sources of diversity in GABA-mediated signaling. This review summarizes some of the factors underlying this diversity, including GABA(A) receptor subunit composition, localization, activation, number and phosphorylation states, variance of GABA concentration in the synaptic cleft, and some of the presynaptic factors regulating GABA release.

Breakdown in cortical effective connectivity during midazolam-induced loss of consciousness

By employing transcranial magnetic stimulation (TMS) in combination with high-density electroencephalography (EEG), we recently reported that cortical effective connectivity is disrupted during early non-rapid eye movement (NREM) sleep. This is a time when subjects, if awakened, may report little or no conscious content. We hypothesized that a similar breakdown of cortical effective connectivity may underlie loss of consciousness (LOC) induced by pharmacologic agents. Here, we tested this hypothesis by comparing EEG responses to TMS during wakefulness and LOC induced by the benzodiazepine midazolam. Unlike spontaneous sleep states, a subject’s level of vigilance can be monitored repeatedly during pharmacological LOC. We found that, unlike during wakefulness, wherein TMS triggered responses in multiple cortical areas lasting for >300 ms, during midazolam-induced LOC, TMS-evoked activity was local and of shorter duration. Furthermore, a measure of the propagation of evoked cortical currents (significant current scattering, SCS) could reliably discriminate between consciousness and LOC. These results resemble those observed in early NREM sleep and suggest that a breakdown of cortical effective connectivity may be a common feature of conditions characterized by LOC. Moreover, these results suggest that it might be possible to use TMS-EEG to assess consciousness during anesthesia and in pathological conditions, such as coma, vegetative state, and minimally conscious state.

Physiological evidence for two distinct GABAA responses in rat hippocampus

The gamma-aminobutyric acid(A) (GABAA) receptor is a ligand-gated ionophore involved in synaptic inhibition. Biochemical and molecular biological studies indicate that considerable receptor heterogeneity exists, but physiological differences between inhibitory GABAA synaptic responses have not been identified in the brain. The present report describes two anatomically segregated GABAA-mediated synaptic currents in the hippocampal CA1 region that have distinct physiological, pharmacological, and functional properties. GABAA,fast enters at or near the cell body, decays rapidly (3-8 ms), is blocked by furosemide, and rapidly curtails the excitatory response. GABAA,slow enters far from the cell body, decays slowly (30-70 ms), is not blocked by furosemide, and underlies the conventionally recognized early inhibitory postsynaptic potential. The receptors producing these responses may represent subtypes of the GABAA receptor.

Functional Neural Development from Human Embryonic Stem Cells: Accelerated Synaptic Activity via Astrocyte Coculture

How a naive human neuroepithelial cell becomes an electrophysiologically active neuron remains unknown. Here, we describe the early physiological development of neurons differentiating from naive human embryonic stem (hES) cells. We found that differentiating neuronal cells progressively decrease their resting membrane potential, gain characteristic Na+ and K+ currents, and fire mature action potentials by 7 weeks of differentiation. This is similar to the maturation pattern observed in animals, albeit on a greatly expanded time scale. An additional 3 weeks of differentiation resulted in neurons that could fire repetitive trains of action potentials in response to depolarizing current pulses. The onset of spontaneous synaptic activity also occurred after 7 weeks of differentiation, in association with the differentiation of astrocytes within the culture. Cocultures of hES cell-derived neuroepithelial cells with exogenous astrocytes significantly accelerated the onset of synaptic currents but did not alter action potential generation. These findings suggest that the development of membrane characteristics and action potentials depend on the intrinsic maturation of Na+ and K+ currents, whereas synaptic transmission is enhanced by astrocytes, which may be achieved independently of the maturation of action potentials. Furthermore, we found that although astrocyte-conditioned medium accelerated synaptic protein localization, it did not increase synaptic activity, suggesting a contact-dependant mechanism by which astrocytes augment synaptic activity. These results lay the foundation for future studies examining the functional development of human neurons and provide support for the potential application of human cells in restorative neuronal therapies.

Interactions between Distinct GABAA Circuits in Hippocampus

Synchronous activity among synaptically connected interneurons is thought to organize temporal patterns such as gamma and theta rhythms in cortical circuits. Interactions between distinct interneuron circuits may underlie more complex patterns, such as nested rhythms. Here, we demonstrate such an interaction between two groups of CA1 interneurons, GABA(A,slow) and GABA(A,fast) cells, that may contribute to theta and gamma rhythms, respectively. Stratum lacunosum-moleculare (SL-M) stimuli that activate GABA(A,slow) inhibitory postsynaptic currents (IPSCs) in pyramidal cells simultaneously depress the rate and amplitude of spontaneous GABA(A,fast) IPSCs for several hundred milliseconds. This suppression has a similar pharmacological profile to GABA(A,slow) IPSCs, and SL-M stimuli elicit GABA(A,slow) IPSCs in interneurons. We conclude that GABA(A,slow) cells inhibit both pyramidal cells and GABA(A,fast) interneurons and postulate that this interaction contributes to nested theta/gamma rhythms in hippocampus.

GABAA,slow: causes and consequences

GABA(A) receptors in the CNS mediate both fast synaptic and tonic inhibition. Over the past decade a phasic current with features intermediate between fast synaptic and tonic inhibition, termed GABA(A,slow), has received increasing attention. This has coincided with an ever-growing appreciation for GABAergic cell type diversity. Compared with classical fast synaptic inhibition, GABA(A,slow) is slower by an order of magnitude. In this review, we summarize recent studies that have enhanced our understanding of GABA(A,slow). These include the discovery of specialized interneuron types from which this current originates, the factors that could underlie its characteristically slow kinetics, its contribution to specific aspects of integrative function and network oscillations, and its potential usefulness as a novel drug target for modulating inhibitory synaptic transmission in the CNS.

Effect of Volatile Anesthetics on Synaptic Transmission in the Rat Hippocampus

The synaptic effects of halothane, isoflurane, and enflurane were examined in the rat hippocampus in vivo and compared with the effects of ketamine and urethane. Actions of the agents on excitatory amino acid-mediated neurotransmission were studied by observing evoked responses and long-term potentiation in the stratum pyramidale of CA1 with stimulation of the contralateral CA3 region. Long-term potentiation is a long-lasting increase in synaptic efficacy, which follows a brief stimulus train. It has been shown to be established through activation of the NMDA subclass of excitatory amino acid receptors and is thought to be involved in memory processing. Volatile anesthetics had no effect on evoked excitatory responses or on long-term potentiation. Actions of the anesthetics on inhibitory processes in the hippocampus were studied by pairing stimuli at a range of interpulse intervals. The first stimulus activated inhibitory processes that caused the response to the second stimulus to be smaller than the initial response, a phenomenon termed paired pulse depression. Paired pulse depression was significantly prolonged by the volatile anesthetics compared with that under urethane or ketamine. These results indicate that the mechanism of action of the volatile anesthetics at the hippocampal CA1 synapse does not involve amino acid-mediated excitation but does involve enhancement of inhibition.

GABAA Receptor α5 Subunits Contribute to GABAA,slow Synaptic Inhibition in Mouse Hippocampus

gamma-Aminobutyric acid type A (GABA(A)) receptor alpha5 subunits, which are heavily expressed in the hippocampus, are potential drug targets for improving cognitive function. They are found at synaptic and extrasynaptic sites and have been shown to mediate tonic inhibition in pyramidal neurons. We tested the hypothesis that alpha5 subunits also contribute to synaptic inhibition by measuring the effect of diazepam (DZ) on spontaneous and stimulus-evoked inhibitory postsynaptic currents (IPSCs) in genetically modified mice carrying a point mutation in the alpha5 subunit (alpha5-H105R) that renders those receptors insensitive to benzodiazepines. In wild type mice, DZ (1 microM) increased the amplitude of spontaneous IPSCs (sIPSCs) and stimulus-evoked GABA(A,slow) IPSCs (eIPSCs) and prolonged the decay of GABA(A,fast) sIPSCs. In alpha5-mutant mice, DZ increased the amplitude of a small-amplitude subset of sIPSCs (<50 pA) and eIPSCs (<300 pA) GABA(A,slow) and prolonged the decay of GABA(A,fast) sIPSCs, but failed to increase the amplitude of larger sIPSCs and eIPSCs GABA(A,slow). These results indicate that alpha5 subunits contribute to a large-amplitude subset of GABA(A,slow) synapses and implicate these synapses in modulation of cognitive function by drugs that target alpha5 subunits.