Long-term synaptic depression.
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Animals↗
Biomedical subjects
D J Linden
Publications and source records attributed to D J Linden.
An evaluation of the nitric oxide/cGMP/cGMP-dependent protein kinase cascade in the induction of cerebellar long-term depression in culture.
Cerebellar long-term depression (LTD) is a model system of information storage in which a persistent attenuation of the parallel fiber-Purkinje neuron (PN) synapse is induced by conjunctive stimulation of parallel fiber and climbing fiber inputs at low frequency. As some studies have suggested that release of the gaseous second messenger, nitric oxide (NO), in the molecular layer and the consequent activation of soluble guanylate cyclase and cGMP-dependent protein kinase (PKG) in the PN, is necessary for LTD induction, we have further examined this hypothesis using a cell culture protocol. In cerebellar cultures made from transgenic mice in which the gene for neuronal nitric oxide synthase (nNOS) has been rendered null, LTD induced by glutamate/depolarization conjunctive stimulation was indistinguishable from that in cultures from wild-type mice in terms of amplitude, rate of onset, and duration. Bath application of cGMP analogs produced a large (80%), transient attenuation of glutamate-gated inward currents. However, application of an activator of soluble guanylate cyclase or an inhibitor of type V cGMP-phosphodiesterase did not mimic the effect of cGMP analogs, and inclusion of cGMP analogs in the patch pipette did not give rise to a slowly developing attenuation, suggesting that these compounds exert their effects at the cell surface. Free Ca was measured in the distal dendritic arbor of single PNs by fura-2 microfluorimetry.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate Ribose↗
Trans-ACPD, a metabotropic receptor agonist, produces calcium mobilization and an inward current in cultured cerebellar Purkinje neurons.
1. 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), a racemic mixture of 1-aminocyclopentane-1S,3R-dicarboxylic acid and 1-aminocyclopentane-1R,3S-dicarboxylic acid, a selective agonist of the metabotropic glutamate receptor, was applied to mouse Purkinje neurons (PNs) in culture. Measurements of free intracellular Ca2+ were made using fura-2 microfluorimetric imaging and of membrane current using perforated-patch voltage-clamp recording in separate experiments. 2. Brief pulses of t-ACPD (< or = 100 microM, 1-5 s) consistently produced a large (200-600 nM) increase in dendritic Ca2+ that was sometimes followed by a somatic increase. The dendrites typically returned to basal Ca2+ levels within 10-30 s. 3. Ca2+ increases produced by t-ACPD were measured in Ca(2+)-free external saline [0.5 mM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], suggesting that they result from intracellular mobilization rather than influx. In addition, Ca2+ increases were not attenuated by a mixture of DL-AP5 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [antagonists of N-methyl-D-aspartate (NMDA) and AMPA/kainate receptors, respectively], but were almost entirely eliminated by L-AP3 (100 microM), a putative metabotropic receptor antagonist or by preincubation of the cultures in pertussis toxin. 4. Brief pulses of t-ACPD (10 microM) produced a small inward current that was associated with an increase in membrane conductance. This current was reversibly blocked by L-AP3 but not by treatments that attenuate some voltage-gated K+ currents. Thus this current is unlikely to underlie the depolarization that is produced by metabotropic agonists in hippocampal pyramidal cells by K(+)-channel closure. 5. The t-ACPD induced inward current was attenuated by substitution of external Na+ with Li+ or choline, or by application of the membrane-permeable Ca2+ chelator, bis-(2-aminophenoxy)-N,N,N',N'- tetraacetic acid (BAPTA)/AM. One mechanism that could mediate this current is electrogenic Nao/Cai exchange, triggered by Ca2+ mobilization.
Animals↗
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.
Animals↗
Protoporphyrins modulate voltage-gated Ca current in AtT-20 pituitary cells.
1. Zinc-protoporphyrin-IX (ZnPP-IX) is an inhibitor of the enzyme heme-oxygenase-2 (HO-2) and consequently has been used to examine the role of carbon monoxide production in neural tissues. We have measured voltage-gated Ca current in AtT-20 pituitary cells using the whole-cell patch-clamp technique and have assessed the effects of extracellularly applied ZnPP-IX and related compounds. 2. Ca currents evoked by depolarizing steps from a holding potential of -90 mV were of the high-threshold, slowly inactivating type. Fifty-six percent of this current was blocked by 10 microM nifedipine and 16% by 3 microM omega-conotoxin with the remainder resistant to both drugs in combination, suggesting that the total Ca current was a mixture of L, N, and possibly P-type conductances. 3. Bath application of ZnPP-IX resulted in an irreversible, dose-dependent attenuation of Ca current. Five micromolar ZnPP-IX produced a 62% reduction of peak current amplitude with no shift in the current-voltage relation, 0.5 microM produced a 19% reduction, and 0.05 microM produced a variable response, either a small transient attenuation or potentiation. 4. The attenuation of Ca current by 5 microM ZnPP-IX could be nearly completely blocked by co-application of superoxide dismutase in the bath (90 U/ml) but not by addition of an inhibitor of cGMP-dependent protein kinase to the internal saline (KT5823, 1 microM). 5. Other inhibitors of heme-oxygenase with similar potency such as tin-protoporphyrin-IX (Sn-PP-IX) and Zn-deuteroporphyrin-bis-glycol (ZnBG) did not attenuate Ca current when applied at 5microM.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium↗
An electrophysiological correlate of protein kinase C isozyme distribution in cultured cerebellar neurons.
Protein kinase C (PKC) is a family of at least seven closely related molecules (isozymes) that vary in terms of their requirements for activation and their distribution among cells of the brain. A striking example of this differential distribution is seen in the cerebellum, where Purkinje cells express PKC-I, an isozyme that is strongly activated by both phorbol ester (PE), and low doses of cis-unsaturated fatty acid (c-UFA), while granule cells predominantly express PKC-II, an isozyme that is strongly activated by PE but not c-UFA. Both Purkinje and granule cells have large, easily recorded voltage-gated K currents. These currents are attenuated by PKC activators in several other varieties of neuron. We hypothesized that the effects of these two PKC activators would be predicted by the distribution of the relevant PKC isozyme, and that the delayed outward rectifier current, IK, would be attenuated by both PE and c-UFA in Purkinje cells, but only by PE in granule cells. This hypothesis was confirmed in perforated-patch recordings. The attenuation produced by both activators could be blocked by application of a specific PKC inhibitor, RO-31-8220, and could not be mimicked by inert forms of PE or c-UFA. To our knowledge, this study represents the first report of an electrophysiological correlate of PKC isozyme distribution.
Animals↗
Participation of postsynaptic PKC in cerebellar long-term depression in culture.
Long-term depression (LTD) in the intact cerebellum is a decrease in the efficacy of the parallel fiber-Purkinje neuron synapse induced by coactivation of climbing fiber and parallel fiber inputs. In cultured Purkinje neurons, a similar depression can be induced by iontophoretic glutamate pulses and Purkinje neuron depolarization. This form of LTD is expressed as a depression of alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA)-mediated current, and its induction is dependent on activation of metabotropic quisqualate receptors. The effect of inhibitors of protein kinase C (PKC) on LTD induction was studied. Inhibitors of PKC blocked LTD induction, while phorbol-12,13-diacetate (PDA), a PKC activator, mimicked LTD. These results suggest that PKC activation is necessary for the induction of cerebellar LTD.
Animals↗
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.
Amino Acids↗
Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two-dimensional gels.
Previous findings suggest: (1) that altering protein kinase C (PKC) activity alters the persistence of long-term potentiation (LTP) in the intact hippocampal formation; and (2) that PKC activity is directly correlated with persistence of LTP in vivo as measured by the in vitro phosphorylation of two major PKC substrates in adult hippocampus, protein F1 and 80k. Using quantitative analysis of two-dimensional gels, we report here two additional phosphoproteins of 72 and 55 kDa which were directly correlated to persistence of LTP induced in the intact dorsal hippocampal formation. The phosphorylation of both proteins in response to addition of different kinase stimulators was distinct from that of protein F1 and 80k. Moreover, neither protein was a substrate for exogenous PKC. The physicochemical properties of these phosphoproteins suggest they are identical to the previously described synaptic vesicle proteins IIIa and IIIb, and as such are immunologically indistinguishable. Because proteins IIIa and IIIb are known to be phosphorylated by a Ca2+/calmodulin (CaM)-stimulated kinase, and protein F1 is known to be a plasma membrane-associated protein (P-57) which releases bound CaM in response to phosphorylation by PKC, the present findings suggest a potential mechanism in which PKC-mediated changes in plasma membrane proteins produce CaM kinase-mediated changes in synaptic vesicle proteins through a phosphorylation cascade. These membrane/vesicle alterations are postulated to underlie the increased synaptic efficacy which marks persistent LTP.
Adaptation, Physiological↗
cis-Fatty acids, which activate protein kinase C, attenuate Na+ and Ca2+ currents in mouse neuroblastoma cells.
1. Activation of protein kinase C (PKC) by phorbol esters or diacylglycerols has been shown to modulate a number of ionic currents carried by Ca2+, K+ and Cl-. Recently, it has been demonstrated that PKC may be activated by cis-fatty acids in the absence of either phospholipid or Ca2+. We wished to determine if this new class of PKC-activating compound would also modulate ionic currents. To this end we applied the whole-cell voltage-clamp technique to N1E-115 neuroblastoma cells. 2. Analysis of families of currents evoked under voltage clamp by depolarizing steps from a holding potential of -85 mV during external application of 5 microM-oleate (a cis-fatty acid) showed a 36% reduction of the peak inward current with no shift in either the peak or the reversal potential of the current-voltage relation and no alteration of outward current. 3. External application of the cis-fatty acids oleate, linoleate and linolenate reversibly attenuated voltage-dependent Na+ current with approximate half-maximal dose values of 2, 3, and 10 microM respectively. Oleate was approximately 2 times more potent when applied internally (ED50 = 1 microM). Externally applied elaidate (a trans-isomer of oleate) and stearate (a saturated fatty acid) which do not activate PKC, had no effect. Since cis-fatty acids are known to fluidize membranes, as well as to activate PKC, we sought to dissociate these functions by applying compounds that fluidize membranes but do not activate PKC: methyloleate and lysophosphatidylcholine. Neither compound affected Na+ current when applied externally at concentrations of 1-50 microM. 4. In contrast to cis-fatty acids, three classical PKC activators, phorbol-12.13-dibutyrate (PDB), phorbol-12.13-diacetate (PDA), and 1.2-oleoylacetylglycerol (OAG) were found to have no effect on the voltage-dependent Na+ current when applied externally at 10 nM-1 microM (phorbol esters) or 1-150 microM (OAG) for incubation periods up to 1 h. 5. External application of the PKC inhibitors polymyxin B, H-7, sphingosine and staurosporine blocked the attenuation of the Na+ current by cis-fatty acid in a dose-dependent manner, with maximal inhibition occurring at doses of 50, 10, 200 and 0.1 microM, respectively. The cyclic nucleotide-dependent protein kinase inhibitor H-8 was much less effective in blocking the cis-fatty acid effect. Polymyxin B and staurosporine were more potent when applied internally. 6. Chronic (24 h) exposure to 1 microM phorbol-12-myristate-13-acetate (TPA) was employed to down-regulate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The two major phosphoproteins in growth cones are probably identical to two protein kinase C substrates correlated with persistence of long-term potentiation.
Regulation of neural protein kinase C (PKC) activity appears to directly affect the persistence of long-term potentiation (LTP; Akers and Routtenberg, 1985; Lovinger et al., 1985, 1986, 1987; Routtenberg et al., 1985, 1986; Akers et al., 1986; Linden et al., 1987), a model of neural plasticity (Bliss and Lomo, 1973). In addition, the in vitro phosphorylation of a brain-specific PKC substrate, protein F1 (Mr 47 kDa, pl 4.5), has been directly correlated with persistence of LTP (Lovinger et al., 1986). Because PKC has been implicated in neurite outgrowth and is present at high levels in growth cone-rich areas of fetal brain, we investigated and characterized PKC substrates in a preparation of isolated nerve growth cone fragments from fetal rat brain and compared them with PKC substrates found in adult rat hippocampus. Four major proteins in the growth cone preparation showed endogenous phosphorylation levels at least 10-fold greater than any other phosphoproteins. Three of these 4 phosphoproteins, termed pp40, pp46, and pp80 (Katz et al., 1985), were phosphorylated by exogenous PKC in a dose-dependent manner, indicating that PKC activity might be of particular importance relative to other kinases in growth cone function. The 2 most highly labeled PKC substrates, pp46 and pp80, comigrated on 2-dimensional gels with the adult hippocampal proteins F1 and "80k" (Mr 78-80 kDa, pl 4.0), respectively. In addition, similarities in charge heterogeneity, 2-dimensional phosphopeptide maps, and increased phosphorylation in the presence of exogenous PKC or PKC stimulators suggest that protein F1 and 80k are highly homologous to, if not identical to, pp46 and pp80, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
NMDA receptor blockade prevents the increase in protein kinase C substrate (protein F1) phosphorylation produced by long-term potentiation.
Recent evidence has implicated activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptor in the initiation of hippocampal long-term potentiation (LTP), an electrophysiological model of information storage in the brain. A separate line of evidence has suggested that activation of protein kinase C (PKC) and the consequent phosphorylation of its substrates is necessary for the maintenance of the LTP response. To determine if PKC activation is a consequence of NMDA receptor activation during LTP, we applied the NMDA receptor antagonist drug, DL-aminophosphonovalerate (APV) both immediately prior to and following high frequency stimulation, resulting in successful and unsuccessful blockade of LTP initiation, respectively. We then measured the phosphorylation of a PKC substrate (protein F1) in hippocampal tissue dissected from these animals. Only successful blockade of LTP initiation by prior application of APV was seen to block the LTP-associated increase in protein F1 phosphorylation measured in vitro (P less than 0.001 by ANOVA). This suggests that NMDA receptor-mediated initiation triggers maintenance processes that are, at least in part, mediated by protein F1 phosphorylation. These data provide the first evidence linking two mechanisms associated with LTP, NMDA receptor activation and PKC substrate phosphorylation.
2-Amino-5-phosphonovalerate↗
Enhancement of long-term potentiation by cis-unsaturated fatty acid: relation to protein kinase C and phospholipase A2.
Previous correlative and interventive work from this laboratory has suggested that activation of protein kinase C (PKC) is important for the maintenance of the hippocampal long-term potentiation (LTP) response. One such study demonstrated that application of the cis-unsaturated fatty acid, oleate, a newly discovered PKC activator, could prolong the time course of LTP. The present study explored the mechanism of cis-unsaturated fatty acid action on LTP produced by perforant path stimulation. First, neither oleate application nor high-frequency stimulation alone produced a persistent change in synaptic transmission, while the 2 in conjunction did so. This suggests that oleate acts synergistically with the consequences of this stimulation to produce an enhancement of the LTP response. Second, oleate enhancement of LTP was more potent when applied in the perforant path synaptic terminal zone than in the dentate hilus, implying that the site of oleate action is at the synapse (where PKC is reported to be enriched). Third, translocation of PKC activity to the membrane was significantly increased after oleate-enhanced LTP relative to vehicle controls. PKC translocation was found to be unaltered by oleate application alone. Fourth, mepacrine blockade of the Ca2+-dependent enzyme phospholipase A2, which releases endogenous oleate from membrane phospholipids, inhibited the time-course of a persistent LTP response. This inhibition was shown to be reversible with oleate application. We propose that high-frequency stimulation produces an elevation of intracellular Ca2+, which then triggers phospholipase A2-mediated oleate release. This free oleate then could act in synergy with processes that render PKC oleate-sensitive to produce a persistent activation of PKC, which is critical for and leads to the persistence of the LTP response.
Animals↗
A newly discovered protein kinase C activator (oleic acid) enhances long-term potentiation in the intact hippocampus.
Protein kinase C can be activated by oleate, an unsaturated fatty acid. Since protein kinase C is activated by long-term potentiation, we wished to determine whether iontophoretic ejection of oleate into the intact hippocampal dentate gyrus of urethane-anesthetized rats would cause an enhancement of the response potentiated by high frequency stimulation of the perforant path. Oleate ejection did significantly enhance the persistence of the potentiated response. Moreover, a growth of the response beyond the initial potentiation was seen. Arachidonate, which stimulates protein kinase C to a lesser degree, had a significant preservation effect, but no effect on growth of the response. After vehicle and elaidate (trans-stereoisomer of oleate) ejections, the potentiated response decayed to baseline values. In addition, the persistence of the potentiated response observed two hours after its induction was positively correlated with the ability of an unsaturated fatty acid to activate protein kinase C in vitro. The present results support the proposal that protein kinase C activation enhances synaptic strength. It is suggested that one mechanism for this activation may be PLA2-mediated release of oleate.
Animals↗
Translocation of protein kinase C activity may mediate hippocampal long-term potentiation.
Protein kinase C activity in rat hippocampal membranes and cytosol was determined 1 minute and 1 hour after induction of the synaptic plasticity of long-term potentiation. At 1 hour after long-term potentiation, but not at 1 minute, protein kinase C activity was increased twofold in membranes and decreased proportionately in cytosol, suggesting translocation of the activity. This time-dependent redistribution of enzyme activity was directly related to the persistence of synaptic plasticity, suggesting a novel mechanism regulating the strength of synaptic transmission.
Animals↗
The role of protein kinase C in long-term potentiation: a testable model.
With the use of appropriate reagents, LTP may be divided into at least two stages, induction and maintenance. Induction of LTP is dependent upon the activation of the NMDA receptor, and the consequent influx of calcium into the postsynaptic cell. Both correlational evidence (measures of PKC activity, protein F1 phosphorylation, and PI turnover) and interventive evidence (application of PKC inhibitors and activators) indicate that PKC activation is necessary for maintenance of the LTP response. An important regulatory pathway for PKC activation is the liberation of c-FAs from membrane phospholipids by PLA2. In LTP, activation of this pathway may stabilize PKC in an activated state, and thus contribute to maintenance of the potentiated response. LTP maintenance could result from presynaptic alteration (increased neurotransmitter release), postsynaptic alteration (increases in receptor number or sensitivity, or alterations of postsynaptic morphology), synapse addition, or any of these processes in combination. If LTP maintenance is mediated by presynaptic alteration, as has been indicated by measurement of glutamate release, then one must posit a signal that travels from the postsynaptic to the presynaptic membrane to activate presynaptic PKC. Alternatively, if LTP maintenance is mediated by postsynaptic alteration, a signal contained within the dendritic spine would suffice to activate postsynaptic PKC-mediated maintenance processes. We suggest that the contributions of presynaptic and postsynaptic processes to LTP maintenance may be determined by the differential distribution of PKC subtypes and substrates among hippocampal synaptic zones.
Action Potentials↗
Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.
Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1's possible role in age-related decline of hippocampal synaptic plasticity.
Aging↗