Peter B. Guthrie

Interactions between entorhinal axons and target hippocampal neurons: A role for glutamate in the development of hippocampal circuitry

A coculture system consisting of input axons from entorhinal cortex explants and target hippocampal pyramidal neurons was used to demonstrate that glutamate, released spontaneously from afferent axons, can influence both dendritic geometry of target neurons and formation of presumptive synaptic sites. Dendritic outgrowth was reduced in hippocampal neurons growing on entorhinal axons when compared with neurons growing off the axons. Presumptive presynaptic sites were observed in association with hippocampal neuron dendrites and somas. HPLC analysis showed that glutamate was released from the explants in an activity- and Ca2(+)-dependent manner. The general glutamate receptor antagonist D-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca2+ also promoted dendritic outgrowth and reduced the formation of presumptive synaptic sites. The results suggest that the neurotransmitter glutamate may play important roles in the development of hippocampal circuitry.

Calcium-induced neuronal degeneration: a normal growth cone regulating signal gone awry (?).

The neuronal growth cone is involved in neurite elongation, directional pathfinding, and target recognition. These activities are essential for proper assembly of functional circuits within the developing nervous system, for regeneration of functional circuitry following damage, and also, perhaps, for remodeling of the nervous system in response to environmental stimuli. Our studies of both molluscan and mammalian neurons in culture have shown that neurite outgrowth can only proceed when intracellular calcium levels lie within a specific outgrowth-permissive range. Cessation of outgrowth can be induced by a variety of signals normally used for communication within the adult nervous system, including neurotransmitters, and action potentials; all of these signals elevate levels of intracellular calcium above the outgrowth-permissive range. For example, glutamate, whether added to the medium or released from co-cultured entorhinal explants, can selectively inhibit dendritic outgrowth. Conversely, inhibitory neurotransmitters can block the outgrowth-inhibitory effects of glutamate and actually promote expansion of dendritic arbors. Dendritic outgrowth is therefore regulated by a balance between excitatory and inhibitory neurotransmitter activity. Extreme excitatory imbalance in neurotransmitter input to pyramidal neurons causes cell death. Each of these changes in neuroarchitecture is mediated by changes in levels of intracellular calcium. We therefore put forward the hypothesis that key mechanisms which normally control the development and plasticity of neural circuitry, are also involved in neurodegeneration. Local, moderate elevations in calcium result in dendritic pruning. Higher, global elevations in calcium result in cell death. This cell death may serve an important function during normal development; aging may result in the same mechanism being employed pathologically. When intracellular calcium levels are not regulated within normal limits, as may occur in aging, neurodegeneration may occur.

Localized calcium influx orients axon formation in embryonic hippocampal pyramidal neurons

A fundamental property of neurons is their polarization into distinct axonal and dendritic compartments which have characteristic structural and functional properties. The mechanisms regulating the formation of neuronal polarity are unknown. We used cultured embryonic rat hippocampal pyramidal neurons to test the hypothesis that a localized calcium influx can orient axon formation, and thereby direct the establishment of neuronal polarity. Transection of an initial axon, or focal application of A23187 or K+ to the initial axon, caused a new axon to form at a site distant from the initial axon. Fura-2 measurements of intracellular calcium revealed a localized calcium influx at the site of axon transection or focal application of A23187 or K+, and a calcium gradient spreading into the soma. New axon formation was inhibited when axons were transected in medium lacking calcium or containing calcium-elevating agents (conditions which prevented the formation of a calcium gradient). When calcium ionophore A23187 was applied focally to neurons which had not yet established an axon, the axon always formed at a site distant from the site of ionophore application; bath exposure to A23187 prevented axon formation. Taken together, these data demonstrate that a localized influx of calcium can suppress axon formation at the site of influx, and can thereby influence where the axon forms. These data suggest that gradients of intracellular calcium may be involved in orienting neuronal polarity.

Self-Recognition: A Constraint on the Formation of Electrical Coupling in Neurons

Electrical coupling between specific neurons is important for proper function of many neuronal circuits. Identified cultured neurons from the snail Helisoma show a strong correlation between electrical coupling and presence of gap junction plaques in freeze-fracture replicas. Gap junction plaques, however, were never seen between overlapping neurites from a single neuron, even though those same neurites formed gap junctions with neurites from another essentially identical identified neuron. This observation suggests that a form of self-recognition inhibits reflexive gap junction formation between sibling neurites. When one or both of those growth cones had been physically isolated from the neuronal cell body, both electrical coupling and gap junction plaques, between growth cones from the same neuron, were observed to form rapidly (within 30 min). Thus, inhibition of electrical coupling between sibling neurites apparently depends on cytoplasmic continuity between neurites, and not the molecular composition of neurite membrane. The formation of gap junctions is not likely due to the isolation process; rather, the physical isolation appears to release an inhibition of reflexive gap junction formation. These data demonstrate the existence of a previously unknown constraint on the formation of electrical synapses.

Spatial gradients of cytosolic calcium concentration in neurones during paradoxical activation by calcium

4-Br-A23187 caused a calcium influx into chick sensory neurones and raised cytosolic calcium from a rest level of 97 +/- 7 nM to a peak of 296 +/- 30 nM. Despite the continued presence of ionophore, however, cytosolic calcium concentrations then fell. After 30 min in ionophore, cytosolic calcium concentration had returned to 105 +/- 5 nM, not significantly different from the value before ionophore addition. The permeability of the plasmalemma to divalent cations, as estimated by the manganese quench technique, was no lower at 30 min than at the peak of the cytosolic calcium transient. Thus the fall of calcium from its peak was not due to a slowing of calcium influx, but was due to an upregulation of mechanisms that remove calcium from the cytosol- an upregulation that persists even though cytosolic calcium has apparently returned to pre-stimulus levels. We used a novel fixed slit confocal microscope to examine the calcium concentration profile close to the plasmalemma. We found that after 25-30 min ionophore treatment, calcium concentration was elevated only in the cytoplasm within 1 micron of the plasmalemma. A maintained, elevated calcium under the plasmalemma can help explain the phenomenon of paradoxical activation seen in this and other cell types.