neurite growth factor-CNS-conditioned media-molluscan cell culture Medium conditioned by tissue f... more neurite growth factor-CNS-conditioned media-molluscan cell culture Medium conditioned by tissue from the CNS of the snail, Helisoma, is capable of promoting neurite outgrowth in isolated neurons from adult central ganglia. The conditioning factor(s) (CF), contained in conditioned medium (CM), is produced only by central ganglionic rings and buccal ganglia and not by other tissues, including hemolymph. CF requires a minimum of 24 h to be produced or released into the medium. At 12 h growth-promoting activity was not detectable. CF binds tightly to the polylysine substratum and its activity is not mimicked by addition of various sera, NGF or fibronectin. CF activity is abolished by chymotrypsin, trypsin or heating to 100 °C, but is stable to DNase and RNase treatment. The percentage of cells exhibiting neurite outgrowth is approximately linear with the amount of neural tissue used to condition the medium up to 2 ganglionic rings/ml. Addition of more ganglia fails to stimulate a greater response. This apparent plateau of CM activity appears to be a function of production and/or release of CF, rather than a saturation effect on plated cells, since dose-response curves for dilutions of CM are approximately linear regardless of the number of ganglia used for conditioning. In addition, anisomycin inhibits 35% of CF appearance under conditions of over 90% protein synthesis inhibition in the ganglia used to produce the CM. Under these conditions anisomycin has no apparent effect on the maintenance of electrical excitability. The inhibitor data suggest that 65% of CF is derived from a pre-existing storage pool and that the remainder is synthesized during the 72 h conditioning period.
Advances in experimental medicine and biology, 1990
The generation and regeneration of neuronal form and connectivity both undoubtedly rely upon the ... more The generation and regeneration of neuronal form and connectivity both undoubtedly rely upon the integration of intrinsic and extrinsic information of many kinds. Our work has demonstrated that the concentration and spatial distribution of intracellular calcium is a key locus of integration of such information. Through a delicate balance of mechanisms that raise free calcium and mechanisms that lower free calcium, a steady state level is achieved that appears to have significant regulatory control over neuronal growth cone behavior. Cues, both internal and external, alter intracellular calcium levels, and consequently alter growth cone behavior. It is through the alteration of the various components of calcium homeostasis that we envision the complexities of neuronal architecture and connectivity may be fine-tuned throughout the life histories of neuronal ensembles.
The secretory cells of the salivary glands of the snail, Helisoma trivolvis, exhibit regenerative... more The secretory cells of the salivary glands of the snail, Helisoma trivolvis, exhibit regenerative, overshooting action potentials whose ampliture may exceed 100 mV. The salivary glands consist of paired, tubular, epithelial structures with acinar outpocketings. The secretory cells display extensive electrical coupling which allows action potentials to propagate along the glandular epithelium. Salivary glands from nine genera of gastropod molluscs were examined. The genera comprised one opisthobranch, one prosobranch, two terrestrial pulmonate slugs, one terrestrial pulmonate snail, and four aquatic pulmonate snails. Action potentials were recorded from all of the examined glands, suggesting that the production of action potentials in salivary glands is a general phenomenon among gastropods.
The electrically coupled buccal ganglion neurons 4R and 4L of the snail, Helisoma, display predic... more The electrically coupled buccal ganglion neurons 4R and 4L of the snail, Helisoma, display predictable plasticity. The strength of the electrotonic synapse between them increases significantly following axotomy. Synaptic strength was assayed by measurements of electrical coupling coefficients and by assessment of dye coupling (passage of dye into the uninjected neuron) following injection of Lucifer Yellow CH into one neuron. Within 3 to 5 days, axotomy induced an increase in electrical coupling coefficients between neurons 4R and 4L from 0.54-I 0.11 (n = 13) in normal preparations to 0.72 f 0.14 (n = 24). A parallel axotomy-induced increase in the probability of dye coupling occurred. Only 27% (n = 27) of normal neuron 4 pairs were dye coupled, compared with 87% (n = 15) of axotomized neuronal pairs. Irrespective of treatment, electrical and dye measurements in the same neuron 4 pairs showed a consistent correlation between the magnitude of the electrical coupling coefficients and the probability of detectable dye coupling. No dye coupling was observed in neuronal pairs with electrical coupling coefficients less than 0.50. Dye coupling always occurred when coupling coefficients were greater than 0.70. Sixty-seven percent of neuronal pairs with intermediate coupling (0.50 to 0.70) coefficients displayed dye coupling. The results show that axotomy evokes a predictable enhancement of communication at an "identified" electrotonic synapse and suggests that electrical and dye coupling are mediated by similar mechanisms.
Identified neurons of the snail, Helisoma, undergo extensive remodeling in response to axotomy, i... more Identified neurons of the snail, Helisoma, undergo extensive remodeling in response to axotomy, including the formation of specific sets of novel electrical connections. This communication addresses the question of why, under the conditions employed, some neurons readily form new connections with a single "test" neuron, whereas others do not. The present experiments are a test of the hypothesis that, for these adult neurons, competence to form electrical connections is restricted to pairs of neurons with interacting regions of active outgrowth. Morphological observations demonstrated profuse overlapping outgrowth from neurons which formed electrical connections, whereas neurons which did not connect displayed no simultaneous new outgrowth, although there could be regions of physical overlap or proximity. The causal relationship between growth and the ability to form new connections was tested more directly by two means: (1) Previously nonconnecting neurons were recruited into the connectivity pattern by axotomy-induced growth. (2) Previously connecting neurons did not connect when they were not induced to grow. Thus, growth or lack of growth is an effective discriminator for determining specific sets of interconnected neurons. Recent work from our laboratory has shown a significant potential for extensive remodeling of connections between identified neurons of the snail, Helisoma. Axotomy-evoked neuroplastic responses include restoration of broken connections (Murphy and Kater, 1978, 1980), changes in the efficacy of chemical and electrical connections (Bulloch et al., 1980; Murphy et al., 1983), and the formation of novel electrical synapses between previously unconnected neurons (Bulloch and Kater, 1981, 1982). This major reorganization involves every neuron thus far examined in the buccal ganglion and, on initial inspection, seems chaotic. When viewed from the perspective of a single identified neuron, a highly stereotyped and predictable set of changes becomes apparent. Many new connections are made, but only some are stabilized. For instance, neuron
Isolated neurons from adult central ganglia of the snail Helisoma were cultured in vitro in modif... more Isolated neurons from adult central ganglia of the snail Helisoma were cultured in vitro in modified Liebowitz L-15 medium. Such neurons displayed electrical excitability comparable to that in acutely dissected ganglia. Isolated neurons remained spherical in defined medium throughout culture durations up to 2 weeks. This static morphology was contrasted by the significant neuritic outgrowth which occurred from neurons maintained in medium with co-cultured intact Helisoma brains or in brain conditioned medium. A morphological sequence of growth cone formation and neurite extension occurred only in the presence of a conditioning factor(s) with a mode of action which included tight binding of the conditioning factor to the substratum. Under these conditions, the two primary neuronal phenotypes, electrical excitability and complex neuronal architecture, could be affected independently in adult molluscan neurons cultured in vitro.
During central nervous system development, neurons differentiate distinct axonal and dendritic pr... more During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since...
Progress in clinical and biological research, 1989
Selective degeneration of pyramidal neurons in regions CA1 and CA3 of the hippocampus is a common... more Selective degeneration of pyramidal neurons in regions CA1 and CA3 of the hippocampus is a common structural correlate of several neurodegenerative conditions including Alzheimer's disease, epilepsy and stroke. Several lines of evidence suggest that glutamate, an excitatory neurotransmitter intimately involved in learning and memory processes, may also be involved in hippocampal neurodegeneration. High levels of glutamate are toxic to select groups of pyramidal neurons both in vivo and in vitro and subtoxic levels of glutamate can cause the regression of pyramidal neuron dendrites. In order to determine the basis for this selective vulnerability we employed two rat hippocampal culture paradigms. The first paradigm consisted of neurons isolated from different hippocampal regions (CA1, CA2, CA3, dentate gyrus). Selective vulnerability in the isolated neurons mirrored the selective cell loss that occurs in situ. Dentate granule cells and CA2 pyramidal-like neurons were relatively r...
A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, ... more A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.
A coculture system consisting of input axons from entorhinal cortex explants and target hippocamp... more 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 activityand Ca2+-dependent manner. The general glutamate receptor antagonist o-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca*+ 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.
Although senile plaques represent a consistent neuropathological feature in Alzheimer's brains, i... more Although senile plaques represent a consistent neuropathological feature in Alzheimer's brains, it is not known what role plaques play in the etiology of the disease. Both growth-promoting and growth-inhibiting influences have been postulated. One of the major components in plaques, 13-amyloid, has been shown to affect neuron survival and neurite outgrowth in vitro. Because plaques consist of other components in addition to [3-amyloid, we undertook the present study to determine whether neuronal survival and neurite outgrowth are affected by the presence of a senile plaque. This was accomplished by using cryostat sections from the cerebral cortex of Alzheimer's patients as a substratum for cultured rat hippocampal neurons. Evaluation of these living neurons on Alzheimer's tissue demonstrated that senile plaques affect the amount, complexity, and direction of neurite outgrowth. In addition, neurons were more likely to extend processes away from plaques rather than toward a plaque. Although cell survival on plaques and in control regions was similar, cell survival was significantly reduced in the peri-plaque region. These observations suggest that senile plaques could have deleterious effects on neural organization in situ.
Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesio... more Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesion and neurite outgrowth. This phenomenon has been investigated most recently by culturing neurons on cryostat sections of the adult CNS. Employing this same technique, we have found, in accord with others, that neurons seldom adhere to or grow on central nervous system white matter but will attach and grow on gray matter. In the experiments presented here, embryonic rat hippocampal neurons were grown on cryostat sections from the adult rat CNS, in the presence of brain derived glial cocultures. It was found that the white matter in cryostat sections can be modified by interaction with medium conditioned by brain-derived glial cells. Neurons plated on sections pretreated by such media show significant increases in both attachment and neurite outgrowth. The activity contained in glial conditioned medium is likely complex in nature. While the majority of the activity can be eliminated by heat treatment and trypsinization, neural adhesion but not neurite initiation is affected by protease treatment. Therefore, cell attachment and neurite outgrowth may be regulated by different factors in the conditioned media.
By using cell-cultured identified neurons of the snail Helisoma, we demonstrate that the growth c... more By using cell-cultured identified neurons of the snail Helisoma, we demonstrate that the growth cones of different neurons are intrinsically different from one another in terms of their structure, behavior, and response to environmental signals. Structurally neuron 5 has a greater number of filopodia per growth cone, has shorter filopodia, and has a smaller interfilopodial distance than neuron 19. Behaviorally, the growth cones of neuron 5 advance over the substratum at a faster rate than those of neuron 19; and the growth cones of neuron 19, but not of neuron 5, respond to the presence of serotonin in their environment by retracting their filopodia. In addition to such intrinsic differences between the growth cones of different neurons, we also demonstrate that the separate growth cones of a single neuron, while having identical properties, can act independently of one another. Focal application of serotonin to a growth cone causes only a localized retraction of that growth cones' filopodia. Other growth cones that are attached to the same neuron but that are not exposed to serotonin retain their normal structural features.
Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(... more Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca(2+)-dependent effects is unclear. Since cytoskeletal proteins are rapidly degraded by Ca(2+)-dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca(2+)-induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors ethyl(+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butyl carbamoyl]-2 - oxiranecarboxylate (EST) and carbobenzoxyl-valyl-phenylalanyl-H (MDL 28170) to block the Ca2+ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 microM) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 microM) showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 microM) did not block the A23187-induced rise of [Ca2+]i as measured with fura-2. These results suggest that calpains play a role in Ca(2+)-induced pruning of neurites in isolated hippocampal pyramidal neurons.
Summary. Intracellular records were obtained from the identified neuron, Pal-B, in a relatively i... more Summary. Intracellular records were obtained from the identified neuron, Pal-B, in a relatively intact preparation of Helisoma trivolvis (Figs. 1 and 2). This neuron displayed a characteristic patterned activity. 1. The activity in Pal-B consists of bursts of action potentials alternating ...
neurite growth factor-CNS-conditioned media-molluscan cell culture Medium conditioned by tissue f... more neurite growth factor-CNS-conditioned media-molluscan cell culture Medium conditioned by tissue from the CNS of the snail, Helisoma, is capable of promoting neurite outgrowth in isolated neurons from adult central ganglia. The conditioning factor(s) (CF), contained in conditioned medium (CM), is produced only by central ganglionic rings and buccal ganglia and not by other tissues, including hemolymph. CF requires a minimum of 24 h to be produced or released into the medium. At 12 h growth-promoting activity was not detectable. CF binds tightly to the polylysine substratum and its activity is not mimicked by addition of various sera, NGF or fibronectin. CF activity is abolished by chymotrypsin, trypsin or heating to 100 °C, but is stable to DNase and RNase treatment. The percentage of cells exhibiting neurite outgrowth is approximately linear with the amount of neural tissue used to condition the medium up to 2 ganglionic rings/ml. Addition of more ganglia fails to stimulate a greater response. This apparent plateau of CM activity appears to be a function of production and/or release of CF, rather than a saturation effect on plated cells, since dose-response curves for dilutions of CM are approximately linear regardless of the number of ganglia used for conditioning. In addition, anisomycin inhibits 35% of CF appearance under conditions of over 90% protein synthesis inhibition in the ganglia used to produce the CM. Under these conditions anisomycin has no apparent effect on the maintenance of electrical excitability. The inhibitor data suggest that 65% of CF is derived from a pre-existing storage pool and that the remainder is synthesized during the 72 h conditioning period.
Advances in experimental medicine and biology, 1990
The generation and regeneration of neuronal form and connectivity both undoubtedly rely upon the ... more The generation and regeneration of neuronal form and connectivity both undoubtedly rely upon the integration of intrinsic and extrinsic information of many kinds. Our work has demonstrated that the concentration and spatial distribution of intracellular calcium is a key locus of integration of such information. Through a delicate balance of mechanisms that raise free calcium and mechanisms that lower free calcium, a steady state level is achieved that appears to have significant regulatory control over neuronal growth cone behavior. Cues, both internal and external, alter intracellular calcium levels, and consequently alter growth cone behavior. It is through the alteration of the various components of calcium homeostasis that we envision the complexities of neuronal architecture and connectivity may be fine-tuned throughout the life histories of neuronal ensembles.
The secretory cells of the salivary glands of the snail, Helisoma trivolvis, exhibit regenerative... more The secretory cells of the salivary glands of the snail, Helisoma trivolvis, exhibit regenerative, overshooting action potentials whose ampliture may exceed 100 mV. The salivary glands consist of paired, tubular, epithelial structures with acinar outpocketings. The secretory cells display extensive electrical coupling which allows action potentials to propagate along the glandular epithelium. Salivary glands from nine genera of gastropod molluscs were examined. The genera comprised one opisthobranch, one prosobranch, two terrestrial pulmonate slugs, one terrestrial pulmonate snail, and four aquatic pulmonate snails. Action potentials were recorded from all of the examined glands, suggesting that the production of action potentials in salivary glands is a general phenomenon among gastropods.
The electrically coupled buccal ganglion neurons 4R and 4L of the snail, Helisoma, display predic... more The electrically coupled buccal ganglion neurons 4R and 4L of the snail, Helisoma, display predictable plasticity. The strength of the electrotonic synapse between them increases significantly following axotomy. Synaptic strength was assayed by measurements of electrical coupling coefficients and by assessment of dye coupling (passage of dye into the uninjected neuron) following injection of Lucifer Yellow CH into one neuron. Within 3 to 5 days, axotomy induced an increase in electrical coupling coefficients between neurons 4R and 4L from 0.54-I 0.11 (n = 13) in normal preparations to 0.72 f 0.14 (n = 24). A parallel axotomy-induced increase in the probability of dye coupling occurred. Only 27% (n = 27) of normal neuron 4 pairs were dye coupled, compared with 87% (n = 15) of axotomized neuronal pairs. Irrespective of treatment, electrical and dye measurements in the same neuron 4 pairs showed a consistent correlation between the magnitude of the electrical coupling coefficients and the probability of detectable dye coupling. No dye coupling was observed in neuronal pairs with electrical coupling coefficients less than 0.50. Dye coupling always occurred when coupling coefficients were greater than 0.70. Sixty-seven percent of neuronal pairs with intermediate coupling (0.50 to 0.70) coefficients displayed dye coupling. The results show that axotomy evokes a predictable enhancement of communication at an "identified" electrotonic synapse and suggests that electrical and dye coupling are mediated by similar mechanisms.
Identified neurons of the snail, Helisoma, undergo extensive remodeling in response to axotomy, i... more Identified neurons of the snail, Helisoma, undergo extensive remodeling in response to axotomy, including the formation of specific sets of novel electrical connections. This communication addresses the question of why, under the conditions employed, some neurons readily form new connections with a single "test" neuron, whereas others do not. The present experiments are a test of the hypothesis that, for these adult neurons, competence to form electrical connections is restricted to pairs of neurons with interacting regions of active outgrowth. Morphological observations demonstrated profuse overlapping outgrowth from neurons which formed electrical connections, whereas neurons which did not connect displayed no simultaneous new outgrowth, although there could be regions of physical overlap or proximity. The causal relationship between growth and the ability to form new connections was tested more directly by two means: (1) Previously nonconnecting neurons were recruited into the connectivity pattern by axotomy-induced growth. (2) Previously connecting neurons did not connect when they were not induced to grow. Thus, growth or lack of growth is an effective discriminator for determining specific sets of interconnected neurons. Recent work from our laboratory has shown a significant potential for extensive remodeling of connections between identified neurons of the snail, Helisoma. Axotomy-evoked neuroplastic responses include restoration of broken connections (Murphy and Kater, 1978, 1980), changes in the efficacy of chemical and electrical connections (Bulloch et al., 1980; Murphy et al., 1983), and the formation of novel electrical synapses between previously unconnected neurons (Bulloch and Kater, 1981, 1982). This major reorganization involves every neuron thus far examined in the buccal ganglion and, on initial inspection, seems chaotic. When viewed from the perspective of a single identified neuron, a highly stereotyped and predictable set of changes becomes apparent. Many new connections are made, but only some are stabilized. For instance, neuron
Isolated neurons from adult central ganglia of the snail Helisoma were cultured in vitro in modif... more Isolated neurons from adult central ganglia of the snail Helisoma were cultured in vitro in modified Liebowitz L-15 medium. Such neurons displayed electrical excitability comparable to that in acutely dissected ganglia. Isolated neurons remained spherical in defined medium throughout culture durations up to 2 weeks. This static morphology was contrasted by the significant neuritic outgrowth which occurred from neurons maintained in medium with co-cultured intact Helisoma brains or in brain conditioned medium. A morphological sequence of growth cone formation and neurite extension occurred only in the presence of a conditioning factor(s) with a mode of action which included tight binding of the conditioning factor to the substratum. Under these conditions, the two primary neuronal phenotypes, electrical excitability and complex neuronal architecture, could be affected independently in adult molluscan neurons cultured in vitro.
During central nervous system development, neurons differentiate distinct axonal and dendritic pr... more During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since...
Progress in clinical and biological research, 1989
Selective degeneration of pyramidal neurons in regions CA1 and CA3 of the hippocampus is a common... more Selective degeneration of pyramidal neurons in regions CA1 and CA3 of the hippocampus is a common structural correlate of several neurodegenerative conditions including Alzheimer's disease, epilepsy and stroke. Several lines of evidence suggest that glutamate, an excitatory neurotransmitter intimately involved in learning and memory processes, may also be involved in hippocampal neurodegeneration. High levels of glutamate are toxic to select groups of pyramidal neurons both in vivo and in vitro and subtoxic levels of glutamate can cause the regression of pyramidal neuron dendrites. In order to determine the basis for this selective vulnerability we employed two rat hippocampal culture paradigms. The first paradigm consisted of neurons isolated from different hippocampal regions (CA1, CA2, CA3, dentate gyrus). Selective vulnerability in the isolated neurons mirrored the selective cell loss that occurs in situ. Dentate granule cells and CA2 pyramidal-like neurons were relatively r...
A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, ... more A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.
A coculture system consisting of input axons from entorhinal cortex explants and target hippocamp... more 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 activityand Ca2+-dependent manner. The general glutamate receptor antagonist o-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca*+ 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.
Although senile plaques represent a consistent neuropathological feature in Alzheimer's brains, i... more Although senile plaques represent a consistent neuropathological feature in Alzheimer's brains, it is not known what role plaques play in the etiology of the disease. Both growth-promoting and growth-inhibiting influences have been postulated. One of the major components in plaques, 13-amyloid, has been shown to affect neuron survival and neurite outgrowth in vitro. Because plaques consist of other components in addition to [3-amyloid, we undertook the present study to determine whether neuronal survival and neurite outgrowth are affected by the presence of a senile plaque. This was accomplished by using cryostat sections from the cerebral cortex of Alzheimer's patients as a substratum for cultured rat hippocampal neurons. Evaluation of these living neurons on Alzheimer's tissue demonstrated that senile plaques affect the amount, complexity, and direction of neurite outgrowth. In addition, neurons were more likely to extend processes away from plaques rather than toward a plaque. Although cell survival on plaques and in control regions was similar, cell survival was significantly reduced in the peri-plaque region. These observations suggest that senile plaques could have deleterious effects on neural organization in situ.
Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesio... more Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesion and neurite outgrowth. This phenomenon has been investigated most recently by culturing neurons on cryostat sections of the adult CNS. Employing this same technique, we have found, in accord with others, that neurons seldom adhere to or grow on central nervous system white matter but will attach and grow on gray matter. In the experiments presented here, embryonic rat hippocampal neurons were grown on cryostat sections from the adult rat CNS, in the presence of brain derived glial cocultures. It was found that the white matter in cryostat sections can be modified by interaction with medium conditioned by brain-derived glial cells. Neurons plated on sections pretreated by such media show significant increases in both attachment and neurite outgrowth. The activity contained in glial conditioned medium is likely complex in nature. While the majority of the activity can be eliminated by heat treatment and trypsinization, neural adhesion but not neurite initiation is affected by protease treatment. Therefore, cell attachment and neurite outgrowth may be regulated by different factors in the conditioned media.
By using cell-cultured identified neurons of the snail Helisoma, we demonstrate that the growth c... more By using cell-cultured identified neurons of the snail Helisoma, we demonstrate that the growth cones of different neurons are intrinsically different from one another in terms of their structure, behavior, and response to environmental signals. Structurally neuron 5 has a greater number of filopodia per growth cone, has shorter filopodia, and has a smaller interfilopodial distance than neuron 19. Behaviorally, the growth cones of neuron 5 advance over the substratum at a faster rate than those of neuron 19; and the growth cones of neuron 19, but not of neuron 5, respond to the presence of serotonin in their environment by retracting their filopodia. In addition to such intrinsic differences between the growth cones of different neurons, we also demonstrate that the separate growth cones of a single neuron, while having identical properties, can act independently of one another. Focal application of serotonin to a growth cone causes only a localized retraction of that growth cones' filopodia. Other growth cones that are attached to the same neuron but that are not exposed to serotonin retain their normal structural features.
Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(... more Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca(2+)-dependent effects is unclear. Since cytoskeletal proteins are rapidly degraded by Ca(2+)-dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca(2+)-induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors ethyl(+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butyl carbamoyl]-2 - oxiranecarboxylate (EST) and carbobenzoxyl-valyl-phenylalanyl-H (MDL 28170) to block the Ca2+ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 microM) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 microM) showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 microM) did not block the A23187-induced rise of [Ca2+]i as measured with fura-2. These results suggest that calpains play a role in Ca(2+)-induced pruning of neurites in isolated hippocampal pyramidal neurons.
Summary. Intracellular records were obtained from the identified neuron, Pal-B, in a relatively i... more Summary. Intracellular records were obtained from the identified neuron, Pal-B, in a relatively intact preparation of Helisoma trivolvis (Figs. 1 and 2). This neuron displayed a characteristic patterned activity. 1. The activity in Pal-B consists of bursts of action potentials alternating ...
Uploads
Papers by Ben Kater