Amy B. MacDermott

Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses

Painful stimuli to the skin initiate action potentials in the peripheral terminals of dorsal root ganglion (DRG) neurons. These action potentials propagate to DRG central terminals in the dorsal horn of the spinal cord, evoking release of excitatory transmitters such as glutamate onto postsynaptic dorsal horn neurons. P2X receptors, a family of ligand-gated ion channels, activated by the endogenous ligand ATP, are highly expressed by DRG neurons. Immunoreactivity to P2X receptors has been identified in the dorsal horn superficial laminae associated with nociceptive DRG central terminals, suggesting the presence of presynaptic P2X receptors. Here we have used a DRG–dorsal horn co-culture system to show that P2X receptors are localized at presynaptic sites on DRG neurons; that activation of these receptors results in increased frequency of spontaneous glutamate release; and that activation of P2X receptors at or near presynaptic DRG nerve terminals elicits action potentials that cause evoked glutamate release. Thus activation of P2X receptors at DRG central terminals can modify sensory signal throughput, and might even initiate sensory signals at central synapses without direct peripheral input. This putative central modulation and generation of sensory signals may be associated with physiological and pathological pain sensation, making presynaptic P2X receptors a possible target for pain therapy.

A functionally characterized test set of human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.

Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons.

1. The mechanisms and effects of GABA‐ and glycine‐evoked depolarization were studied in cultured rat dorsal horn neurons using indo‐1 recordings of [Ca2+]i and patch clamp recordings in conventional whole‐cell or perforated‐patch mode. 2. Application of GABA to unclamped neurons caused [Ca2+]i increases that were dose dependent and exhibited GABAA receptor pharmacology. Calcium entered the neurons via high‐threshold voltage‐gated calcium channels (conotoxin and nimodipine sensitive). 3. In perforated‐patch recordings employing cation‐selective ionophores, GABAA receptor activation depolarized 123 of 132 cells to membrane potentials as depolarized as ‐33 mV (mean ‐50 mV in all 132 cells, +12 mV above resting potential). The ionic basis of the depolarization was determined by extracellular ion substitution; increased anionic conductance could account fully for the results. 4. Glycine, acting at a strychnine‐sensitive receptor, also caused Ca2+ entry into these neurons through voltage‐gated Ca2+ channels. Glycine and GABA both evoked [Ca2+]i responses in the same cells and the responses were highly correlated in amplitude. Glycine also depolarized all five cells tested with perforated recording. Each of the five cells was also depolarized by muscimol to a value similar to that obtained for glycine. 5. Both the depolarization and the increases in [Ca2+]i caused by GABA and glycine could potentially play a role in processes of development and differentiation and sensory transmission in the spinal cord dorsal horn.

A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition

DISC1 is a strong candidate susceptibility gene for schizophrenia, bipolar disorder, and depression. Using a mouse strain carrying an endogenous Disc1 orthologue engineered to model the putative effects of the disease-associated chromosomal translocation we demonstrate that impaired Disc1 function results in region-specific morphological alterations, including alterations in the organization of newly born and mature neurons of the dentate gyrus. Field recordings at CA3/CA1 synapses revealed a deficit in short-term plasticity. Using a battery of cognitive tests we found a selective impairment in working memory (WM), which may relate to deficits in WM and executive function observed in individuals with schizophrenia. Our results implicate malfunction of neural circuits within the hippocampus and medial prefrontal cortex and selective deficits in WM as contributing to the genetic risk conferred by this gene.

Characteristics and function of Ca2+ — and inositol 1,4,5-trisphosphate-releasable stores of Ca2+ in neurons

Molecular, biochemical and physiological evidence for the existence of releasable Ca2+ stores in neurons is strong. There are two separate molecules that function as release channels from those Ca2+ stores, the RyanR and InsP3R, and both have multiple regulatory sites for positive and negative control. Perhaps most intriguing is the biphasic, concentration-dependent action of cytosolic Ca2+ on both channels, first to stimulate release then, at higher concentration, to depress release. Whether the InsP3R and RyanR channels regulate Ca2+ release from different or identical functional compartments will need to be defined for each neuron type and perhaps even for each intracellular region within neurons since the evidence for functional separation of stores is mixed. The identification of Ca2+ storage and releasing capacity throughout all subcellular regions of neurons and the increasing evidence for a role for Ca2+ stores in neuronal plasticity suggests that the further characterization of the functional properties of Ca2+ stores will be an increasingly important and expanding area of interest in neurobiology.

Presynaptic ionotropic receptors and control of transmitter release

Presynaptic nerve terminals are dynamic structures that release vesicular packages of neurotransmitter, affecting the activity of postsynaptic cells. This release of transmitter occurs both spontaneously and after the arrival of an action potential at presynaptic terminals. How is the release process modulated? Although ionotropic receptors are commonly regarded as postsynaptic elements that mediate the effect of the released chemical signals, a wide variety of ionotropic receptors have also been found on presynaptic membranes near release sites, where they powerfully influence vesicle fusion. Here, we provide an overview of the presynaptic ionotropic receptors that modulate transmitter release, focusing on their proposed mechanisms of action.

Palmitoylation-dependent neurodevelopmental deficits in a mouse model of 22q11 microdeletion

Individuals with 22q11.2 microdeletions have cognitive deficits and a high risk of developing schizophrenia. Here we provide evidence that primary hippocampal neurons from a mouse model of 22q11.2 deletion (Df(16)A+/− mice) have decreased density of dendritic spines and glutamatergic synapses, as well as impaired dendritic growth. These deficits were prevented by introduction of the enzymatically active ZDHHC8 palmitoyltransferase encoded by a gene in the 22q11.2 locus, and they were also observed in primary cultures from Zdhhc8-deficient mice. Many of these deficits were also present in the hippocampi of adult Df(16)A+/− and Zdhhc8-deficient mice. Finally, we provide evidence that PSD95 is one of the substrates of ZDHHC8. Our analysis reveals that 22q11.2 microdeletion results in deficits in neuronal development and suggests that impaired neuronal protein palmitoylation contributes to many of these deficits.

Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex

Individuals with 22q11.2 microdeletions have cognitive and behavioral impairments and the highest known genetic risk for developing schizophrenia. One gene disrupted by the 22q11.2 microdeletion is DGCR8, a component of the “microprocessor” complex that is essential for microRNA production, resulting in abnormal processing of specific brain miRNAs and working memory deficits. Here, we determine the effect of Dgcr8 deficiency on the structure and function of cortical circuits by assessing their laminar organization, as well as the neuronal morphology, and intrinsic and synaptic properties of layer 5 pyramidal neurons in the prefrontal cortex of Dgcr8+/− mutant mice. We found that heterozygous Dgcr8 mutant mice have slightly fewer cortical layer 2/4 neurons and that the basal dendrites of layer 5 pyramidal neurons have slightly smaller spines. In addition to the modest structural changes, field potential and whole-cell electrophysiological recordings performed in layer 5 of the prefrontal cortex revealed greater short-term synaptic depression during brief stimulation trains applied at 50 Hz to superficial cortical layers. This finding was accompanied by a decrease in the initial phase of synaptic potentiation. Our results identify altered short-term plasticity as a neural substrate underlying the cognitive dysfunction and the increased risk for schizophrenia associated with the 22q11.2 microdeletions.

Presynaptic NMDA Receptors Modulate Glutamate Release from Primary Sensory Neurons in Rat Spinal Cord Dorsal Horn

NMDA receptors have the potential to produce complex activity-dependent regulation of transmitter release when localized presynaptically. In the somatosensory system, NMDA receptors have been immunocytochemically detected on presynaptic terminals of primary afferents, and these have been proposed to drive release of substance P from central terminals of a subset of nociceptors in the spinal cord dorsal horn. Here we report that functional NMDA receptors are indeed present at or near the central terminals of primary afferent fibers. Furthermore, we show that activation of these presynaptic receptors results in an inhibition of glutamate release from the terminals. Some of these NMDA receptors may be expressed in the preterminal axon and regulate the extent to which action potentials invade the extensive central arborizations of primary sensory neurons.

Developmental loss of GABA- and glycine-induced depolarization and Ca2+ transients in embryonic rat dorsal horn neurons in culture.

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca2+ concentration ([Ca2+]i) when maintained in culture for <1 week. This [Ca2+]i response has previously been shown to be due to depolarization and subsequent Ca2+ entry through voltage‐gated Ca2+ channels following activation of bicuculline‐sensitive GABAA receptors and strychnine‐sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca2+]i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca2+]i. The loss of the [Ca2+]i response was not due to a change in the abundance or the properties of voltage‐gated Ca2+ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca2+]i transient in response to 30 mM K+. Nor was the loss of the [Ca2+]i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca2+]i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.