Stanley B. Kater

Neurotransmitter regulation of neuronal outgrowth, plasticity and survival

Molecules used for communication in mature nervous systems also play important roles in development, maintenance and plasticity of individual neurons. This paper reviews the evidence that neurotransmitters, in addition to their mediation of trans-synaptic information coding, can induce a spectrum of effects on neuronal cytoarchitecture, ranging from neurite sprouting to dendritic pruning and even cell death. Such profound alterations may well constitute a part of the normal functioning and structuring of the nervous system as well as contribute to severe pathological processes.

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.

Excitatory and inhibitory neurotransmitters in the generation and degeneration of hippocampal neuroarchitecture.

The possibility that excitatory and inhibitory inputs to neurons can affect the generation and degeneration of neuroarchitecture was examined in hippocampal pyramidal neurons in isolated cell culture. Dendritic outgrowth and cell survival were directly monitored in neurons exposed to: the excitatory neurotransmitter glutamate, the inhibitory transmitter GABA, anticonvulsants or combinations of these agents. Glutamate caused a graded series of changes in pyramidal neuron cytoarchitecture: a selective inhibition in dendritic outgrowth and dendritic pruning was observed with subtoxic levels of glutamate while cell death was induced by higher levels. Low levels of GABA alone or in combination with diazepam, carbamazepine, phenobarbital or phenytoin were without effect on dendrite outgrowth while higher levels caused moderate reductions in outgrowth. Neither GABA nor the anticonvulsants affected cell survival. GABA plus diazepam, phenobarbital, carbamazepine and phenytoin each significantly reduced the dendritic regression and cell death normally caused by glutamate. Elevation of extracellular K+ to 50 mM caused dendritic regression and 100 mM K+ caused cell death; these effects were greatly reduced by GABA and anticonvulsants. The calcium channel blocker Co2+ prevented the dendritic regression and cell death caused by both glutamate and K+ indicating that calcium influx was required for the neuroarchitectural responses. Taken together, these results demonstrate that neurotransmitters and neuromodulatory drugs can have direct and interactive effects on both neurite outgrowth and cell survival. Such neurotransmitter actions may play roles in both the formation and degeneration of the neuronal circuits in which they participate in information coding.

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.

Myelin-associated glycoprotein inhibits neurite/axon growth and causes growth cone collapse

We have previously shown that myelin‐associated glycoprotein (MAG) inhibits neurite growth from a neuronal cell line. In this study we show that 60% of axonal growth cones of postnatal day 1 hippocampal neurons collapsed when they encountered polystyrene beads coated with recombinant MAG (rMAG). Such collapse was not observed with denatured rMAG. Neurite growth from rat embryonic hippocampal and neonatal cerebellar neurons was also inhibited about 80% on tissue culture substrates coated with rMAG. To investigate further the inhibitory activity of MAG in myelin, we purified myelin from MAG‐deficient mice and separated octylglucoside extracts of myelin by diethylaminoethyl (DEAE) ion‐exchange chromatography. Although there was no significant difference in neurite growth on myelin purified from MAG‐/‐ and MAG+/+ mice, differences were observed in the fractionated material. The major inhibitory peak that is associated with MAG in normal mice was significantly reduced in MAG‐deficient mice. These results suggest that although MAG contributes significantly to axon growth inhibition associated with myelin, its lack in MAG‐deficient mice is masked by other non‐MAG inhibitors. Axon regeneration in these mice was also examined after thoracic lesions of the corticospinal tracts. A very small number of anterogradely labeled axons extended up to 13.2 mm past the lesion in MAG‐/‐ mice. Although there is some enhancement of axon generation, the poor growth after spinal cord injury in MAG‐/‐ mice may be due to the presence of other non‐MAG inhibitors. The in vitro studies, however, provide the first evidence that MAG modulates growth cone behavior and inhibits neurite growth by causing growth cone collapse.

Expression and function of the neurotransmitter serotonin during development of the Helisoma nervous system

Exogenous serotonin has been shown to evoke a neuron-selective inhibition of neurite outgrowth and synaptogenesis in identified Helisoma neurons in vitro. We demonstrate here that serotonin is present in the embryonic nervous system of Helisoma and can act as a regulator of neuronal development in vivo. Serotonin-like immunoreactivity was first observed in neurons at an early stage of nervous system development (E20). Throughout embryogenesis, the number of serotonin-immunoreactive neurons increased in a stereotypic pattern that was unique for each type of ganglion. Strikingly, the number of serotonin-immunoreactive neurons continued to increase throughout adult life. Transient perturbation of endogenous serotonin levels during embryogenesis had profound effects on the development of specific identified neurons. Embryos treated with 5,7-dihydroxytryptamine and raised to maturity showed aberrations in neuronal morphology, neuronal dye coupling, and strength of electrical synaptic connections. These effects were restricted to neurons known to be sensitive to the growth-inhibitory effects of serotonin in vitro. These results support the hypothesis that neurotransmitters are an important class of regulatory factors during normal development of the nervous system.

LAMININ AND FIBRONECTIN GUIDEPOSTS SIGNAL SUSTAINED BUT OPPOSITE EFFECTS TO PASSING GROWTH CONES

Guidepost cells are known to alter the behavior of growth cones in vivo, yet the nature of communication and the type of signals employed are largely undefined. The present study demonstrates that model guideposts, composed of a single molecular species, are sufficient to change the navigation and the behavior of advancing growth cones well beyond the time of contact. Laminin on model guideposts caused a sustained increase in growth cone velocity, whereas fibronectin led to a sustained decrease. A spatially discrete array of multiple laminin-model guideposts maintained increased growth rates on fibronectin, as expected for homogeneous laminin, and also provided unambiguous directional guidance information. Laminin-evoked growth cone responses required activation of protein kinase C-dependent intracellular signalling mechanisms.

Isolated hippocampal neurons in cryopreserved long-term cultures: Development of neuroarchitecture and sensitivity to NMDA

Isolated neurons in long‐term culture provide a unique opportunity to address important problems in neuronal development. In the present study we established conditions for cryopreservation and long‐term primary culture of isolated embryonic hippocampal neurons. This culture system was then used for initial characterizations of the development of neuroarchitecture and neurotransmitter response systems. Cryoprotection with 8% dimethylsulfoxide, slow freezing, and rapid thawing provided high‐yield cultures which appeared normal in terms of cell types, mitotic ability, axonal and dendritic outgrowth, and sensitivity to glutamate neurotoxicity. A reduced medium volume and moderate elevation in extracellular K+ to 20 mM promoted survival of isolated neurons through 3 weeks of culture. The outgrowth of axons and dendrites in pyramidal‐like neurons was found to differ over a 3‐week culture period such that axons continued to grow at a relatively constant rate while dendritic outgrowth slowed during the second week and ceased by the end of week 3. Developmental changes were also observed in the sensitivity of pyramidal neurons to glutamate neurotoxicity; functional kainate/ quisqualate receptors were present during the first week of culture, while responses to N‐methyl‐d‐aspartic acid (NMDA) did not appear until the second week. The technologies for cryopreservation and long‐term culture of isolated hippocampal neurons reported here provide a useful system in which to address a variety of problems in developmental neuroscience.

Local increases in intracellular calcium elicit local filopodial responses in helisoma neuronal growth cages

Highly localized changes in intracellular Ca2+ concentration ([Ca2+]i) can be evoked in neuronal growth cones; these are followed by local changes in filopodia. Focally applied electric fields evoked spatially restricted, high magnitude increases in growth cone [Ca2+]i. The earliest and greatest increases were localized to small regions within a growth cone. Such fields also produced characteristic changes in the disposition of filopodia: both filopodial length and number were significantly increased on the cathode side of growth cones. The requirement for extracellular Ca2+ and the strong correlation between the evoked rise in [Ca2+]i and the changes in filopodia (r = 0.98) indicate that cathode stimulation results in local Ca2+ influx, leading to locally increased [Ca2+]i and local changes in filopodial behavior.

The sensory-motor role of growth cone filopodia

Neuronal growth cones, the motile tips of elongating axons and dendrites, respond very precisely to cues encountered during pathfinding. During the past year, our knowledge about their sensory function, their integrative properties, and their motor function has advanced significantly. In particular, growth cone filopodia are currently being recognized for their prominent roles as sensors, transducers, and autonomous motor structures important for growth cone steering.