Showing papers by "Mark P. Mattson published in 1988"

Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons

Abstract: The present study examined the effects of glutamate on the outgrowth of dendrites and axons in isolated hippocampal pyramidal-like neurons in cell culture During the first day of culture the survival and outgrowth of these neurons was unaffected by high concentrations (up to 1 nM) of glutamate, quisqualic acid (QA), kainic acid (KA), and N- methyl-D-aspartic acid Beginning on day 2 of culture high levels of glutamate, KA and QA were toxic to the majority of pyramidal neurons, while subtoxic levels of these agents caused a well-defined, dose- dependent, sequence of effects on dendritic outgrowth At increasing concentrations of glutamate, QA, and KA, the following events were observed: (1) dendritic outgrowth rates were reduced, while axonal elongation rates were unaffected; (2) dendritic length was reduced, while axons continued to grow; (3) dendrites regressed dramatically, and axonal outgrowth rate was reduced These dendrite-specific effects of glutamate were apparently mediated at the growth cones since focal application of glutamate to individual dendritic growth cones resulted in suppression of growth cone activity and a regression of the dendrite; axons were unaffected by focal glutamate application Pharmacological tests using glutamate receptor agonists and antagonists demonstrated that receptors of the KA/QA type mediated the glutamate effects on outgrowth and survival The calcium channel blocker Co2+ prevented both glutamate neurotoxicity and glutamate-induced dendritic regression Ionophore A23187 and elevations in extracellular K+ levels each caused a dose-dependent series of outgrowth and survival responses similar to those caused by glutamate Taken together, these results indicate that activation of glutamate receptors leads to the opening of voltage-dependent calcium channels; the resulting increases in calcium influx lead to the observed alterations in dendritic outgrowth and neuronal survival

Neurite outgrowth in individual neurons of a neuronal population is differentially regulated by calcium and cyclic AMP

Abstract: In identified Helisoma neurons, intracellular calcium can regulate neurite elongation and growth cone motility. Neurotransmitters such as 5-HT suppress both neurite elongation and the filopodial and lamellipodial movements of growth cones by causing increases in intracellular calcium (Haydon et al., 1984; Cohan et al., 1987; Mattson and Kater, 1987). Since an additional second messenger, cyclic AMP (cAMP), is known to mediate many physiological effects of neurotransmitters, we tested (1) the possible involvement of cAMP in the regulation of neurite outgrowth from Helisoma buccal neurons and (2) calcium-cAMP interrelationships in the regulation of outgrowth. The cAMP-elevating agents forskolin (5 x 10(-6)-10(-4) M) and dibutyryl cAMP (dbcAMP; 5 x 10(-3)-10(-2) M) suppressed neurite elongation and growth cone movements in identified neurons B19 (5-HT sensitive) and B5 (5-HT insensitive); the suppression was reversible. Exposure of these particular identified neurons to the calcium channel blocker La3+ (10(- 5) M) or a culture medium with reduced calcium prevented and reversed the suppressive effects of forskolin and dbcAMP. In order to determine if the results on neurons B5 and B19 were representative of all neurons or only a subset, we examined a larger population of neurons. Calcium ionophore A23187 suppressed outgrowth from all neurons in mass dissociate cultures of buccal neurons, while forskolin or dbcAMP plus IBMX suppressed outgrowth from only one-half of buccal neurons. Finally, we found that 2 subpopulations exist among the neurons whose outgrowth is suppressed by cAMP: One subpopulation requires calcium influx for cAMP to act, while the other does not. Thus, even within the relatively small population of neuronal types comprising the buccal ganglion of Helisoma, second messengers within different neurons can act and interact in different ways to regulate outgrowth.

Intracellular messengers in the generation and degeneration of hippocampal neuroarchitecture.

Abstract: The actions and interactions of the neurotransmitter glutamate and the intracellular messengers calcium, cyclic AMP, and protein kinase C (PKC) in the regulation of neurite outgrowth and cell survival were examined in hippocampal pyramidal-like neurons in isolated cell culture. Low, subtoxic levels of glutamate (10-100 microM) caused the regression of dendrites but not axons; millimolar levels caused cell death. Calcium ionophore A23187 (50-100 nM) and the PKC activator phorbol-12-myristate-13-acetate (PMA; 10-50 nM) caused the regression of both axons and dendrites, whereas the adenylate cyclase activator forskolin enhanced outgrowth rates in both axons and dendrites. The effects of glutamate, A23187, PMA, and forskolin on outgrowth were mediated locally at the growth cones; dendrites were more sensitive than axons to each of these agents. High levels of A23187 (1 microM) or PMA (100 nM) significantly reduced cell survival. Co2+ and trifluoperazine each significantly reduced glutamate-induced dendritic regression and neurotoxicity suggesting that calcium influx and/or PKC activation mediated glutamate's actions. Fura-2 measurements showed that glutamate caused a rapid rise in intracellular calcium levels; this rise was prevented by Co2+. PMA and forskolin did not alter intracellular calcium levels, nor did these agents affect glutamate-induced calcium rises. Taken together, the results indicate that parallel intracellular messenger pathways that influence neurite outgrowth and cell survival are operative in hippocampal neurons; these messengers may play roles in the formation and modification of neuronal circuitry.

Components of neurite outgrowth that determine neuronal cytoarchitecture: influence of calcium and the growth substrate.

Abstract: The present study examined the cellular mechanisms underlying the generation of neuroarchitecture. Identified Helisoma neurons in isolated cell culture normally require factors present in brain-conditioned medium (CM) in order to display the different components of neurite outgrowth (sprouting, elongation, branching, and growth cone motility), which ultimately determine their overall architecture. We report here that cell calcium and cell-substrate interactions can play quite specific roles in the regulation of these different components of neuronal outgrowth. CM-induced neurite outgrowth was inhibited by calcium ionophore A23187. In the absence of CM the calcium channel blocker La3+ (10 microM) reduced intracellular calcium levels and induced neurite sprouting and elongation; growth cone motility and branching were greatly reduced in the La3+-induced neurites. Neurons plated into an environment containing La3+ and a fibronectin substrate exhibited all of the components of neuronal outgrowth normally seen in response to CM. Fibronectin alone had little outgrowth-promoting activity. Neurite elongation rates and branching were increased by exposure to La3+ in neurons on either a CM or fibronectin substrate. The neurons growing on CM or fibronectin whose outgrowth was accelerated by La3+ elaborated neuritic arbors that differed from those of neurons grown in response to CM; differences were seen in neurite length, area of outgrowth, branching frequency, and varicosity numbers. Taken together, these results indicate that 1) calcium and the growth substrate can exert specific effects on neurite sprouting, elongation, growth cone motility, and branching; 2) appropriate levels of activation of these two systems can elicit neurite outgrowth that closely resembles that induced by endogenous growth factors; 3) both the differential expression of the separate components of outgrowth and the kinetics of outgrowth determine a neuron's morphology.

Fibronectin‐like immunoreactivity in Helisoma buccal ganglia: Evidence that an endogenous fibronectin‐like molecule promotes neurite outgrowth

Abstract: We examined the distribution of fibronectin-like (FNL) immunoreactivity associated with intact buccal ganglia, cell-cultured buccal ganglia neurons and nonneuronal cells, and brain-conditioned medium from the snail Helisoma. In addition, the possible roles of fibronectin in the regulation of neurite outgrowth were studied. Immunofluorescent staining for FNL antigens revealed intense staining in patches and fibrous arrays over the connective tissue sheaths of buccal ganglia and nerve trunks. Within the ganglia, heavy staining was seen surrounding neurons and in track-like arrangements. In cell cultures, specific staining was associated with nonneuronal cell surfaces and to a lesser degree with the surface of identified neurons. In addition, a noncellular, substrate-bound component of brain-conditioned medium displayed FNL immunoreactivity. Since cultured Helisoma neurons require a substrate-associated, brain-derived conditioning factor (CF) in order to elaborate neurites with motile growth cones, we tested whether the FNL immunoreactive substance might act as a neuritotropic agent. Fibronectin antiserum suppressed, in a dose-dependent manner, the CF-induced sprouting of identified neurons in isolated cell culture. When added at increasing concentrations to neurons already growing in response to CF, fibronectin antiserum exerted a biphasic effect on neurite elongation; outgrowth was accelerated at low, but inhibited at high, antiserum concentrations. In contrast, growth cone structures associated with motility (filopodia and lamellipodia) were progressively reduced by increasing levels of antiserum. A short peptide derived from fibronectin's cell-binding domain (Arg-Gly-Asp-Ser) also greatly reduced neurite outgrowth. The combined results of this study indicate an abundance of FNL immunoreactive molecules within the CNS of Helisoma, their probable production by nonneuronal cells, and their function as a substrate-associated component of CF which promotes growth cone filopodial and lamellipodial activity.

Actions Glutamate in Isolated Hippocampal

Abstract: The present study examined the effects of glutamate on the outgrowth of dendrites and axons in isolated hippocampal pyramidal-like neurons in cell culture. During the first day of culture the survival and outgrowth of these neurons was unaffected by high concentrations (up to 1 mu) of glutamate, quisqualic acid (QA), kainic acid (KA), and Kmethyl-D-aspartic acid. Beginning on day 2 of culture high levels of glutamate, KA and QA were toxic to the majority of pyramidal neurons, while subtoxic levels of these agents caused a welldefined, dose-dependent, sequence of effects on dendritic outgrowth. At increasing concentrations of glutamate, QA, and KA, the following events were observed: (1) dendritic outgrowth rates were reduced, while axonal elongation rates were unaffected; (2) dendritic length was reduced, while axons continued to grow; (3) dendrites regressed dramatically, and axonal outgrowth rate was reduced. These dendritespecific effects of glutamate were apparently mediated at the growth cones since focal application of glutamate to individual dendritic growth cones resulted in suppression of growth cone activity and a regression of the dendrite; axons were unaffected by focal glutamate application. Pharmacological tests using glutamate receptor agonists and antagonists demonstrated that receptors of the KAIQA type mediated the glutamate effects on outgrowth and survival. The calcium channel blocker Co2+ prevented both glutamate neurotoxicity and glutamate-induced dendritic regression. lonophore A23187 and elevations in extracellular K+ levels each caused a dose-dependent series of outgrowth and survival responses similar to those caused by glutamate. Taken together, these results indicate that activation of glutamate receptors leads to the opening of voltage-dependent calcium channels; the resulting increases in calcium influx lead to the observed alterations in dendritic outgrowth and neuronal survival.