Postsynaptic Protein Kinase C Research Articles

Interferon-γ augments GABA release in the developing neocortex via nitric oxide synthase/soluble guanylate cyclase and constrains network activity.

Interferon-γ (IFN-γ), a cytokine with neuromodulatory properties, has been shown to enhance inhibitory transmission. Because early inhibitory neurotransmission sculpts functional neuronal circuits, its developmental alteration may have grave consequences. Here, we investigated the acute effects of IFN-γ on γ-amino-butyric acid (GABA)ergic currents in layer 5 pyramidal neurons of the somatosensory cortex of rats at the end of the first postnatal week, a period of GABA-dependent cortical maturation. IFN-γ acutely increased the frequency and amplitude of spontaneous/miniature inhibitory postsynaptic currents (s/mIPSC), and this could not be reversed within 30 min. Neither the increase in amplitude nor frequency of IPSCs was due to upregulated interneuron excitability as revealed by current clamp recordings of layer 5 interneurons labeled with VGAT-Venus in transgenic rats. As we previously reported in more mature animals, IPSC amplitude increase upon IFN-γ activity was dependent on postsynaptic protein kinase C (PKC), indicating a similar activating mechanism. Unlike augmented IPSC amplitude, however, we did not consistently observe an increased IPSC frequency in our previous studies on more mature animals. Focusing on increased IPSC frequency, we have now identified a different activating mechanism—one that is independent of postsynaptic PKC but is dependent on inducible nitric oxide synthase (iNOS) and soluble guanylate cyclase (sGC). In addition, IFN-γ shifted short-term synaptic plasticity toward facilitation as revealed by a paired-pulse paradigm. The latter change in presynaptic function was not reproduced by the application of a nitric oxide donor. Functionally, IFN-γ-mediated alterations in GABAergic transmission overall constrained early neocortical activity in a partly nitric oxide–dependent manner as revealed by microelectrode array field recordings in brain slices analyzed with a spike-sorting algorithm. In summary, with IFN-γ-induced, NO-dependent augmentation of spontaneous GABA release, we have here identified a mechanism by which inflammation in the central nervous system (CNS) plausibly modulates neuronal development.

FRETting over postsynaptic PKC signaling.

Protein kinases are key regulators of excitatory synapse plasticity. In this issue, using novel optical reporters of protein kinase C (PKC) activity, Colgan et al. identify PKCα as critical for integrating NMDA receptor and neurotrophin signaling to control dendritic spine structural plasticity, synaptic potentiation, and learning and memory.

Role of Protein Kinase C in the Induction and Maintenance of Serotonin-Dependent Enhancement of the Glutamate Response in Isolated Siphon Motor Neurons ofAplysia californica

Serotonin (5-HT) mediates learning-related facilitation of sensorimotor synapses in Aplysia californica. Under some circumstances 5-HT-dependent facilitation requires the activity of protein kinase C (PKC). One critical site of PKC's contribution to 5-HT-dependent synaptic facilitation is the presynaptic sensory neuron. Here, we provide evidence that postsynaptic PKC also contributes to synaptic facilitation. We investigated the contribution of PKC to enhancement of the glutamate-evoked potential (Glu-EP) in isolated siphon motor neurons in cell culture. A 10 min application of either 5-HT or phorbol ester, which activates PKC, produced persistent (> 50 min) enhancement of the Glu-EP. Chelerythrine and bisindolylmaleimide-1 (Bis), two inhibitors of PKC, both blocked the induction of 5-HT-dependent enhancement. An inhibitor of calpain, a calcium-dependent protease, also blocked 5-HT's effect. Interestingly, whereas chelerythrine blocked maintenance of the enhancement, Bis did not. Because Bis has greater selectivity for conventional and novel isoforms of PKC than for atypical isoforms, this result implicates an atypical isoform in the maintenance of 5-HT's effect. Although induction of enhancement of the Glu-EP requires protein synthesis (Villareal et al., 2007), we found that maintenance of the enhancement does not. Maintenance of 5-HT-dependent enhancement appears to be mediated by a PKM-type fragment generated by calpain-dependent proteolysis of atypical PKC. Together, our results suggest that 5-HT treatment triggers two phases of PKC activity within the motor neuron, an early phase that may involve conventional, novel or atypical isoforms of PKC, and a later phase that selectively involves an atypical isoform.

A Positive Feedback Signal Transduction Loop Determines Timing of Cerebellar Long-Term Depression

Synaptic activity produces short-lived second messengers that ultimately yield a long-term depression (LTD) of cerebellar Purkinje cells. Here, we test the hypothesis that these brief second messenger signals are translated into long-lasting biochemical signals by a positive feedback loop that includes protein kinase C (PKC) and mitogen-activated protein kinase. Histochemical "epistasis" experiments demonstrate the reciprocal activation of these kinases, and physiological experiments--including the use of a light-activated protein kinase--demonstrate that such reciprocal activation is required for LTD. Timed application of enzyme inhibitors reveals that this positive feedback loop causes PKC to be active for more than 20 min, allowing sufficient time for LTD expression. Such regenerative mechanisms may sustain other long-lasting forms of synaptic plasticity and could be a general mechanism for prolonging signal transduction networks.

Cell type- and region-specific expression of neurogranin mRNA in the cerebral cortex of the macaque monkey.

Neurogranin is a postsynaptic substrate for protein kinase C (PKC). It has been identified in the central nervous system, and the expression has been related to postsynaptic plasticity. Using non-radioactive in situ hybridization histochemistry, we investigated whether mRNA expression of neurogranin varied among the cerebral region and cell types. In most areas of the neocortex excluding area OC (the primary visual area), intense signals were observed in the pyramidal cells in layers III, V and VI. In area OC, intense signals were observed in layers IV as well as layers III and VI. We previously showed that intense signals for GAP-43, a presynaptic PKC substrate, were observed in relay neurons of the lateral geniculate nucleus. From this result and the present result in area OC, we conclude that both pre- and postsynaptic PKC substrates (GAP-43 and neurogranin) are abundant in the geniculocortical synapses. In the hippocampus, intense signals were observed in the pyramidal cells in the subiculum. Taken together with our previous study showing intense signals for GAP-43 in Ammon's horn, the result indicates that both PKC substrates are abundant in the connections between neurons in Ammon's horn and in the subiculum.

Participation of NMDA-mediated phosphorylation and oxidation of neurogranin in the regulation of Ca2+- and Ca2+/calmodulin-dependent neuronal signaling in the hippocampus.

Neurogranin/RC3 (Ng) is a postsynaptic protein kinase C (PKC) substrate and calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by Ca2+, phosphorylation and oxidation. Ng has been implicated in the modulation of postsynaptic signal transduction pathways and synaptic plasticity. Previously, we showed a severe deficit of spatial memory in Ng knockout (KO) mice. Activation of the NMDA receptor and its downstream signaling molecules are known to be involved in long-term memory formation. In the present study, using mouse hippocampal slices, we demonstrated that NMDA induced a rapid and transient phosphorylation and oxidation of Ng. NMDA also caused activation of PKC as evidenced by their phosphorylations, whereas, such activations were greatly reduced in the KO mice. A higher degree of phosphorylation of Ca2+/CaM-dependent kinase II and activation of cyclic AMP-dependent protein kinase were also evident in the WT compared to those of the KO mice. Phosphorylation of downstream targets, including mitogen-activated protein kinases and cAMP response element-binding protein, were significantly attenuated in the KO mice. These results suggest that by its Ca2+-sensitive CaM-binding feature, and through its phosphorylation and oxidation, Ng regulates the Ca2+- and Ca2+/CaM-dependent signaling pathways subsequent to the stimulation of NMDA receptor. These findings support the hypothesis that the derangement of hippocampal signal transduction cascades in Ng KO mice causes the deficits in synaptic plasticity, learning and memory that occur in these mice.

Postsynaptic kinase signaling underlies inhibitory synaptic plasticity in the lateral superior olive.

In the auditory system, inhibitory transmission from the medial nucleus of the trapezoid body (MNTB) to neurons of the lateral superior olivary nucleus (LSO) undergoes activity-dependent long-term depression, and may be associated with developmental elimination of these synapses [Sanes DH, Friauf E (2000). development and influence of inhibition in the laterial superior olivary nucleus. Hear Res 147:46-58]. Although GABA(B) receptor activation and postsynaptic free calcium are implicated in this depression, little is known about intracellular signaling mechanisms in this or other forms of inhibitory plasticity. In this study, we asked whether the calcium dependency of inhibitory depression was associated with the activation of calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and/or cAMP-dependent protein kinase A (PKA). Whole-cell voltage-clamp recordings were obtained from LSO neurons in a brain slice preparation, permitting for the selective pharmacologic manipulation of individual postsynaptic LSO neurons. Inclusion of a CaMKII antagonist (KN-62) in the internal pipet solution blocked inhibitory synaptic depression. A second CaMKII inhibitor (autocamtide peptide fragment) significantly decreased inhibitory depression. Inclusion of a specific antagonist of protein kinase C (PKC fragment 19-36) in the internal recording solution also blocked inhibitory depression. To test involvement of a cAMP-dependent intracellular cascade, two different manipulations were performed. Inclusion of PKA antagonists (Rp-cAMPS or a cAMP dependent protein kinase inhibitor peptide) prevented inhibitory depression. In contrast, when a nonhydrolyzable cAMP analog (Sp-cAMPS) was permitted to enter the postsynaptic cell, the MNTB-evoked IPSCs became depressed in the absence of low-frequency stimulation. Thus, three key postsynaptic kinases, CaMKII, PKC, and PKA, participate in the activity-dependent depression of inhibitory MNTB-LSO synapses during postnatal development.

Protein kinase C mediates potentiation of synaptic transmission by phorbol ester at parallel fibers in the dorsal cochlear nucleus

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.

Postsynaptic action of BDNF on GABAergic synaptic transmission in the superficial layers of the mouse superior colliculus.

The neurotrophin brain-derived neurotrophic factor (BDNF) is involved in numerous aspects of synapse development and plasticity. The present study was aimed at clarifying the significance of endogenous BDNF for the synaptically driven spontaneous network activity and GABAergic inhibition in the superficial layers of the mouse superior colliculus. In this structure neuron survival is unaffected by the absence of BDNF. Two experimental approaches were used: comparison of BDNF-deficient (-/-) and wild-type (+/+) mice and blockade of BDNF receptor signaling by the tyrosine kinase inhibitor K-252a. Patch-clamp recordings were performed on horizontal slices during postnatal days 15 and 16. The lack of BDNF in -/- mice caused a significant reduction of the spontaneous action potential frequency and an increase in the pharmacologically induced disinhibition of spike discharge. This change was accompanied by an increase in the amplitudes of GABAergic evoked, spontaneous, and miniature inhibitory postsynaptic currents (IPSCs). BDNF gene inactivation had no effect on the degree of paired-pulse facilitation or the frequency of miniature IPSCs. The increase of IPSC amplitudes by chronic BDNF deprivation was completely mimicked by acute exposure to K-252a in +/+ animals. The enhancement of GABAergic IPSCs in -/- animals was reversed by acute application of 100 ng/ml BDNF, but this rescue was completely prevented by blocking postsynaptic protein kinase C (PKC) activation with the PKC inhibitor peptide 19-31. From these results we conclude that BDNF increases spontaneous network activity by suppressing GABAergic inhibition, the site of action of BDNF is predominantly postsynaptic, BDNF-induced suppression of GABAergic synaptic transmission is caused by acute downregulation of GABA(A) receptors, and BDNF effects are mediated by its TrkB receptor and require PKC activation in the postsynaptic cell.

Activation of pre- and postsynaptic protein kinase C during tetraethylammonium-induced long-term potentiation in the CA1 field of the hippocampus

Tetraethylammonium (TEA) induces a form of long-term potentiation (LTP) that is independent on N-methyl- d-aspartate (NMDA) receptor activation (LTP K). LTP K may be a suitable chemical model to study molecular mechanisms underlying LTP. We monitored the phosphorylation state of two identified neural-specific protein kinase C (PKC) substrates (the presynaptic protein GAP-43/B-50 and postsynaptic protein RC3) after different chemical depolarisations. TEA induced a long-lasting increase in synaptic efficacy in the CA1 field of the hippocampus and increased the phosphorylation of both GAP-43/B-50 and RC3 (51 and 56.1%, respectively). These effects were blocked by the voltage-dependent calcium channel antagonist nifedipine, but not by the NMDA receptor antagonist AP5. These data show that in LTP K the in situ phosphorylation of pre-and postsynaptic PKC substrates is increased, indicating that NMDA receptor-dependent and NMDA receptor-independent LTP share common Ca 2+-dependent expression mechanisms, including activation of pre- and postsynaptic PKC.

Protein kinase C inhibitors block generation of anoxia-induced long-term potentiation

The aim of this study was to study the possible intracellular mechanisms underlying the anoxia-induced long-term potentiation (anoxic LTP) in the CA1 neurons of rat hippocampal slices using extra- and intracellular recording techniques. Superfusion of the hippocampal slices with the protein kinase C (PKC) inhibitors NPC-15437 (20 microM) or H-7 (20 microM) specifically prevented the induction of anoxic LTP. Moreover, the anoxic LTP was completely abolished in neurons intracellularly recorded with the selective PKC inhibitor PKCI 19-36 (50 microM). The specific cAMP-dependent protein kinase (PKA) inhibitor Rp-cyclic adenosine 3',5'-monophosphate (Rp-cAMPS, 25 microM) had no effect on the anoxic LTP. It is concluded that induction of anoxic LTP requires the activation of postsynaptic PKC.

Dendritic translocation of RC3/neurogranin mRNA in normal aging, Alzheimer disease and fronto-temporal dementia.

RC3/neurogranin is a postsynaptic protein kinase C (PKC)-/calmodulin-binding substrate implicated in long-term potentiation (LTP) forms of synaptic plasticity. Our previous digoxigenin in situ hybridization (DIG-ISH) studies detected RC3 mRNA in apical dendrites and cell bodies of neurons in the rat cerebral cortex and hippocampus. This observation suggested that RC3 mRNA is selectively translocated to dendrites, where it may be translated locally in response to synaptic activity. To test this hypothesis further, we isolated a full-length cDNA clone of the homologous human RC3 mRNA from a human cortex lambda GT11 library, determined its nucleotide and predicted amino acid sequences, and performed mRNA expression studies in cerebral cortex from normal human patients and from patients with Alzheimer disease (AD) and fronto-temporal dementia (FTD). The human cDNA clone detects a single approximately 1.3 kb mRNA whose nucleotide sequence is 73% similar to the rat nucleotide sequence and 96% similar to its amino acid sequence. DIG-ISH studies detect robust staining of RC3 mRNA in cell bodies of numerous neurons throughout Layers II-VI and in both apical and basal dendrites of pyramidal neurons in human neocortex (temporal/frontal). We conclude that dendritic targeting of RC3 mRNA is conserved in human brain. In AD neocortex tissue, there is little or no evidence for RC3 mRNA translocation to dendrites, while in FTD neocortex, targeting of RC3 mRNA to apical dendrites is preserved. Comparative studies in AD and FTD point to the potential importance of synapse integrity and the dendritic cytoskeleton in RC3 mRNA targeting in the human neocortex.

Block of induction and maintenance of calcium-induced LTP by inhibition of protein kinase C in postsynaptic neuron in hippocampal CA1 region

Calcium-induced LTP (Ca-LTP) refers to the long-lasting potentiation of synaptic transmission in hippocampal synapses that can be induced by a transient (7–10 min) and small increase (from 2 mM to 4 mM) in extracellular calcium concentration. In this paper the effects of protein kinase C (PKC) inhibitors, polymyxin B (PMB) and PKC(19–31), given intracellularly to the postsynaptic neuron in the CA1 region, on the induction and maintenance of Ca-LTP were studied and compared with those found in a similar study earlier made in this laboratory on tetanic stimulation-induced LTP (TS-LTP) [18]. When the intracellular delivery of the inhibitor(s) was made to begin 30 min before the exposure to increased [Ca 2+] 0, the development of Ca-LTP was completely blocked, leaving only a brief enhancement of EPSPs directly attributable to the brief increase in [Ca 2+] 0. When the intracellular delivery of either PKC(19–31) alone or PMB + PKC(19–31) was made to begin after the full establishment of Ca-LTP, it soon made the maintained potentiation begin to decline, the EPSP amplitude gradually returning to the control value before the exposure to increased calcium. Thus postsynaptic PKC inhibition blocks both the induction and the maintenance of Ca-LTP, just as it has been shown to do to TS-LTP. But quantitative differences exist: both the induction and the maintenance of Ca-LTP appeared to be more susceptible to block by postsynaptic PKC inhibition than those of TS-LTP.

Postsynaptic protein kinase C essential to induction and maintenance of long-term potentiation in the hippocampal CA1 region.

Previous studies on the effects of protein kinase C (PKC) inhibitors intracellularly introduced into the postsynaptic neuron on long-term potentiation (LTP) in the hippocampal CA1 region showed that given before the tetanic stimulation they only blocked the development of the maintenance phase of LTP and that given after the tetanus they did not affect the continued maintenance of established LTP. We now report different results in such experiments obtained by looking into the dose-effect relationship of the inhibitors given to the postsynaptic neuron and making use of a synergistic effect of two inhibitors given together. We used the following three PKC inhibitors: polymyxin B (PMB), PKC-(19-31), and H7. With the intracellular delivery of the inhibitor(s) beginning 30 min before the tetanus, PMB in adequate dosage or a combination of PMB and PKC-(19-31), each at a low dosage, could block the development of LTP completely including its initial induction phase. With the delivery beginning at the time of the tetanus, PKC-(19-31) or H7 slowly caused the established LTP to decline to the baseline; this decline was greatly accelerated when PMB and PKC-(19-31) or PMB and H7 were given together. PMB and PKC-(19-31) given together 75-90 min or even 3 h after the tetanus caused a decline of the maintained LTP similar to the decline observed when both inhibitors were given at the time of the tetanus. These results show that postsynaptic PKC is essentially involved in both the initial induction and the subsequent maintenance of LTP, contrary to current views on the subject.

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.

Inhibition of Postsynaptic PKC or CaMKII Blocks Induction But Not Expression of LTP

Long-term potentiation (LTP) of synaptic transmission is a widely studied cellular example of synaptic plasticity. However, the identity, localization, and interplay among the biochemical signals underlying LTP remain unclear. Intracellular microelectrodes have been used to record synaptic potentials and deliver protein kinase inhibitors to postsynaptic CA1 pyramidal cells. Induction of LTP is blocked by intracellular delivery of H-7, a general protein kinase inhibitor, or PKC(19-31), a selective protein kinase C (PKC) inhibitor, or CaMKII(273-302), a selective inhibitor of the multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII). After its establishment, LTP appears unresponsive to postsynaptic H-7, although it remains sensitive to externally applied H-7. Thus both postsynaptic PKC and CaMKII are required for the induction of LTP and a presynaptic protein kinase appears to be necessary for the expression of LTP.