John A. Connor

Calcium regulation of the neuronal growth cone

Abstract The growth cone behaviors that are involved in the generation of neuronal cytoarchitecture are apparently regulated in quite specific ways by Ca 2+ . Neurotransmitters and electrical activity, well known for their roles in information coding, have recently been shown to affect growth cone motility by mechanisms linked to Ca 2+ . Ca 2+ may therefore act as a common integrator of environmental cues that influence neurite outgrowth and synaptogenesis, and in this way may play a key role in the establishment and modulation of brain circuitry.

TARGETED DISRUPTION OF NMDA RECEPTOR 1 GENE ABOLISHES NMDA RESPONSE AND RESULTS IN NEONATAL DEATH

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

A Long-Term Depression of AMPA Currents in Cultured Cerebellar Purkinje Neurons

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.

Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns

In primary dissociated cultures of mouse cerebellum a number of Purkinje cell-specific marker proteins and characteristic ionic currents appear at the appropriate developmental time. During the first week after plating, Purkinje cell dendrites elongate, but as electrical activity emerges the dendrites stop growing and branch. If endogenous electrical activity is inhibited by chronic tetrodotoxin or high magnesium treatment, dendrites continue to elongate, as if they were still immature. At the time that branching begins, intracellular calcium levels become sensitive to tetrodotoxin, suggesting that this cation may be involved in dendrite growth. Even apparently mature Purkinje cells alter their dendritic growth in response to changes in activity, suggesting long-term plasticity.

Cellular mechanisms of long-term depression in the cerebellum

Cerebellar long-term depression is a persistent, input-specific attenuation of the parallel fiber-Purkinje neuron synapse induced by co-activation of parallel fibers and climbing fibers. This phenomenon endows the Purkinje neuron with a powerful associative computational ability. Recent investigations have provided strong evidence that two mechanisms, Ca2+ influx via voltage-gated channels, and stimulation of protein kinase C via metabotropic receptor activation, are required for induction of long-term depression. In addition, two other mechanisms, Na+ influx via AMPA receptors, and stimulation of a nitric oxide/cGMP cascade may also be involved in this process.

Micromolar Ca2+ transients in dendritic spines of hippocampal pyramidal neurons in brain slice

The magnitude and dynamics of [Ca2+] changes in spines and dendrites of hippocampal CA1 pyramidal neurons have been characterized using a low affinity fluorescent indicator, mag-Fura 5, that is sensitive to Ca2+ in the micromolar range. During tetanic stimulation (1 s), we observed progressive [Ca2+] increases in distal CA1 spines to as much as 20-40 microM, both in organotypic slice culture and acute slice. Similar accumulations were reached during continuous depolarization (+10 mV, 1 s) when K+ channels had been blocked, but not with spike trains driven by postsynaptic current injection. The large [Ca2+] increases due to tetanic stimulation were blocked by APV, indicating that NMDA receptor-dependent influx was critical for the large responses. These findings have significant implications for low affinity Ca(2+)-dependent biochemical processes and show a new upper limit for [Ca2+] changes measured in these neurons during stimulation.

The role of Ca2+ entry via synaptically activated NMDA receptors in the induction of long-term potentiation

Influx of Ca2+ through the NMDA subtype of glutamate receptor is widely accepted as a trigger for many forms of neural plasticity. However, direct support for this model has been elusive, since indirect activation of dendritic voltage-sensitive Ca2+ channels is difficult to exclude. We have optically measured synaptically induced changes in cytoplasmic free Ca2+ concentration in pyramidal cell dendrites in hippocampal slices. Steady postsynaptic depolarization to the synaptic reversal potential eliminated the effect of voltage-sensitive Ca2+ channels. Under these conditions, synaptically induced Ca2+ transients were observed, which were blocked by the NMDA receptor antagonist APV. In addition, the magnitude of LTP was diminished when induced with the postsynaptic membrane held at progressively more positive potentials. LTP could be completely suppressed at potentials near +100 mV. These results provide important experimental support for a role for Ca2+ influx through NMDA receptors in synaptic plasticity.

Long-term depression of glutamate currents in cultured cerebellar Purkinje neurons does not require nitric oxide signalling

Cerebellar long‐term depression (LTD) is produced when conjunctive stimulation of parallel fibre (PF) and climbing fibre (CF) inputs to a Purkinje neuron (PN) results in a prolonged decrease in the strength of the PF‐PN synapse. In cultured PNs, LTD may be induced by substituting depolarization of the PN and iontophoretic glutamate pulses for CF and PF stimulation, respectively, allowing an unambiguous analysis of post‐synaptic processes (Linden et al., Neuron, 7, 81–89, 1991). Recent studies have suggested that release of the newly described second messenger, nitric oxide, in the cerebellar molecular layer, is necessary for LTD produced in the slice preparation by PF/CF conjunctive stimulation (Shibuki and Okada, Nature, 349, 326–328, 1991) or PF/depolarization conjunctive stimulation (Crepel and Jaillard, NeuroReport, 1, 133–136, 1990). We report that LTD of glutamate currents produced without synaptic stimulation in cultured PNs is unaffected by reagents that stimulate (sodium nitroprusside) or inhibit (haemoglobin, NG‐nitro‐l‐arginine) nitric oxide signalling.

Interactive Effects of Serotonin and Acetylcholine on Neurite Elongation

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.

Induction of cerebellar long-term depression in culture requires postsynaptic action of Sodium Ions

Cerebellar long-term depression (LTD) is a persistent attenuation of the parallel fiber-Purkinje neuron (PF-PN) synapse induced by conjunctive stimulation of PF and climbing fiber (CF) inputs. A similar phenomenon is seen in the voltage-clamped PN in tissue culture when iontophoretic quisqualate application and PN depolarization are substituted for PF and CF stimulation, respectively. In this model, LTD induction requires activation of both AMPA and metabotropic receptors, together with PN depolarization. We have sought to determine the role of the AMPA receptor in LTD induction. The AMPA receptor does not appear to exert its effect by directly gating Ca2+ influx. Replacement of external Na+ during quisqualate/depolarization conjunction with permeant ions caused a blockade of LTD induction, suggesting that Na+ influx through the AMPA-associated channel is necessary for this process. Similarly, pairing quisqualate pulses with depolarizing steps near ENa also failed to induce LTD. The present results indicate that postsynaptic Na+ influx is necessary for LTD induction. While a portion of the relevant Na+ influx is provided by voltage-gated channels, the AMPA-associated ion channel is most important in this regard.