Mitogen-activated protein kinase regulates early phosphorylation and delayed expression of Ca2+/calmodulin-dependent protein kinase II in long-term potentiation.

Activation of mitogen-activated protein kinase (MAPK) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). We induced LTP in rat hippocampal area CA1 using theta-pulse stimulation (TPS) paired with beta-adrenergic receptor activation [isoproterenol (ISO)], a protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation resulted in a transient phosphorylation of p42 MAPK, and the resulting LTP was MAPK dependent. In addition, CaMKII was regulated in two, temporally distinct ways after TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and CaMKII phosphorylation were strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed expression of CaMKII were prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may contribute to both post-translational modification and increased gene expression.

Long-term potentiation induced by theta frequency stimulation is regulated by a protein phosphatase-1-operated gate.

Long-term potentiation (LTP) can be induced in the Schaffer collateral-->CA1 synapse of hippocampus by stimulation in the theta frequency range (5-12 Hz), an effect that depends on activation of the cAMP pathway. We investigated the mechanisms of the cAMP contribution to this form of LTP in the rat hippocampal slice preparation. theta pulse stimulation (TPS; 150 stimuli at 10 Hz) by itself did not induce LTP, but the addition of either the beta-adrenergic agonist isoproterenol or the cAMP analog 8-bromo-cAMP (8-Br-cAMP) enabled TPS-induced LTP. The isoproterenol effect was blocked by postsynaptic inhibition of cAMP-dependent protein kinase. Several lines of evidence indicated that cAMP enabled LTP by blocking postsynaptic protein phosphatase-1 (PP1). Activators of the cAMP pathway reduced PP1 activity in the CA1 region and increased the active form of inhibitor-1, an endogenous inhibitor of PP1. Postsynaptic injection of activated inhibitor-1 mimicked the LTP-enabling effect of cAMP pathway stimulation. TPS evoked complex spiking when isoproterenol was present. However, complex spiking was not sufficient to enable TPS-induced LTP, which additionally required the inhibition of postsynaptic PP1. PP1 inhibition seems to promote the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), because (1) a CaMKII inhibitor blocked the induction of LTP by TPS paired with either isoproterenol or activated inhibitor-1 and (2) CaMKII in area CA1 was activated by the combination of TPS and 8-Br-cAMP but not by either stimulus alone. These results indicate that the cAMP pathway enables TPS-induced LTP by inhibiting PP1, thereby enhancing Ca(2+)-independent CaMKII activity.

Amyloid beta peptides activate the phosphoinositide signaling pathway in oocytes expressing rat brain RNA.

Amyloid beta peptides (Abetas) of 39-43 amino acids constitute the major protein component of the amyloid plaques found in Alzheimer's disease brain. The generation of Abetas is regulated by the phosphoinositide (PI) pathway, which commonly couples to transmitter receptors. This study reports evidence for the activation of the PI pathway by Abetas in Xenopus oocytes expressing rat brain RNA. The naturally occurring peptides Abeta1-40 and Abeta1-42 were both active, whereas the cytotoxic fragment Abeta25-35 and the reverse peptide Abeta40-1 did not stimulate the PI pathway. Abetas rapidly lost potency in solution, suggesting that they were active only in their non-aggregated form. The Abeta response was saturable and not reduced by a substance P antagonist. This pharmacology excludes the participation of known Abeta binding proteins. The results indicate that a PI coupled receptor for non-aggregated Abeta may be present in brain.

Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP.

Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways. Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1), substituted for cAMP pathway activation in LTP. Stimulation that induced LTP triggered cAMP-dependent phosphorylation of endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increased phosphorylation of CaMKII at Thr286 and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor was not overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP.

Metabotropic glutamate receptors limit adenylyl cyclase-mediated effects in rat hippocampus via protein kinase C.

Glutamate receptors of the metabotropic type (mGluRs) activate protein kinase C in hippocampus, but few physiological functions of this pathway are known. The present data show that mGluRs utilize protein kinase C to inhibit another second messenger system, the adenylyl cyclase pathway, in neurons of the CA1 area of hippocampus. Activation of mGluRs prevented beta-adrenergic receptors, which couple to adenylyl cyclase, from blocking the slow Ca2+-dependent afterhyperpolarization (AHP). Since the afterhyperpolarization modulates neuronal responsiveness, crosstalk between protein kinase C and the adenylyl cyclase pathway is likely to have physiological consequences. Moreover, mGluRs themselves block the afterhyperpolarization, so the observed interference with the beta-adrenergic response constitutes a hierarchical relationship in which mGluRs are dominant over beta-adrenergic receptors.

Synthesis and structure-activity relationships of N-propyl-N-(4-pyridinyl)-1H-indol-1-amine (besipirdine) and related analogs as potential therapeutic agents for Alzheimer's disease.

A series of novel N-(4-pyridinyl)-1H-indol-1-amines and other heteroaryl analogs was synthesized and evaluated in tests to determine potential utility for the treatment of Alzheimer's disease. From these compounds, N-propyl-N-(4-pyridinyl)-1H-indol-1-amine (besipirdine, 4c) was selected for clinical development based on in-depth biological evaluation. In addition to cholinomimetic properties based initially on in vitro inhibition of [3H]quinuclidinyl benzilate binding, in vivo reversal of scopolamine-induced behavioral deficits, and subsequently on other results, 4c also displayed enhancement of adrenergic mechanisms as evidenced in vitro by inhibition of [3H] clonidine binding and synaptosomal biogenic amine uptake, and in vivo by reversal of tetrabenazine-induced ptosis. The synthesis, structure-activity relationships for this series, and the biological profile of 4c are reported.

Postsynaptic cAMP pathway gates early LTP in hippocampal CA1 region.

The role of the cAMP pathway in LTP was studied in the CA1 region of hippocampus. Widely spaced trains of high frequency stimulation generated cAMP postsynaptically via NMDA receptors and calmodulin, consistent with the Ca2+/calmodulin-mediated stimulation of postsynaptic adenylyl cyclase. The early phase of LTP produced by the same pattern of high frequency stimulation was dependent on postsynaptic cAMP. However, synaptic transmission was not increased by postsynaptic application of cAMP. Early LTP became cAMP-independent when protein phosphatase inhibitors were injected postsynaptically. These observations indicate that in early LTP the cAMP signaling pathway, instead of transmitting signals for the generation of LTP, gates LTP through postsynaptic protein phosphatases.

The cholinergic inhibition of afterhyperpolarization in rat hippocampus is independent of cAMP-dependent protein kinase.

The possible involvement of protein kinase A (PKA) in the muscarinic inhibition of the slow afterhyperpolarizing current (IAHP) was investigated in rat hippocampal pyramidal cells. IAHP was recorded using the whole cell method in hippocampal slices, and Rp-cAMPS, a PKA antagonist, was applied intracellularly. The inhibition of IAHP by carbachol was not affected by Rp-cAMPS. In contrast, Rp-cAMPS reduced the cAMP-dependent inhibition of IAHP by norepinephrine. The results show that phosphorylation by PKA does not contribute to the muscarinic effect on IAHP.

Receptor-evoked Cl- current in Xenopus oocytes is mediated through a beta-type phospholipase C. Cloning of a new form of the enzyme.

Xenopus oocytes exhibit a receptor-evoked Cl- current that is mediated through the activation of phospholipase C (PLC) and release of intracellular Ca2+. The identity of PLC(s) mediating this effect is unknown. We have cloned cDNAs encoding a new form of PLC-beta from a Xenopus oocyte cDNA library. The Xenopus PLC-beta has substantial (33-64%) homology with mammalian beta 1, beta 2, beta 3, and beta 4 phospholipase C and is closest to PLC-beta 3, with 64% identity and 80% similarity. Injection of antisense oligonucleotides to a specific region of Xenopus PLC-beta results in degradation of its mRNA and significantly reduces Cl- currents evoked by both endogenous angiotensin receptors and expressed mammalian alpha 1b-adrenergic receptors and M1-muscarinic receptors as compared to responses in sense oligonucleotide-injected oocytes. Inhibition of the M1-muscarinic response by antisense oligonucleotides was nonadditive with pertussis toxin inhibition. PLC antisense oligonucleotide-injected oocytes show Cl- current responses to IP3 that are indistinguishable from sense oligonucleotide-injected oocytes. Since the receptor responses are pertussis toxin-sensitive, we conclude that we have isolated a new form of PLC-beta involved in the pertussis toxin-sensitive receptor stimulation of the Ca2+ activated Cl- current in Xenopus oocytes.

Coupling of the expressed alpha 1B-adrenergic receptor to the phospholipase C pathway in Xenopus oocytes. The role of Go.

alpha 1B-Adrenergic receptor mRNA was injected into Xenopus oocytes, resulting in a norepinephrine-evoked Cl- current. The response was proportional to norepinephrine concentration, blocked by prazosin, and dependent on intracellular Ca2+ derived from inositol trisphosphate-sensitive stores. Oocytes treated with 2 micrograms/ml pertussis toxin showed a time-dependent decrease of the norepinephrine response, taking up to 72 h to show an 80% decrease. Overnight treatment with 10 micrograms/ml pertussis toxin also resulted in 80% reduction. Responses to two other cloned receptors (M1-muscarinic and serotonin-1c) expressed in oocytes were also reduced 50% or more by 72 h of pertussis toxin treatment. Pertussis toxin labeling of the cloned Xenopus alpha o-subunit translated in vitro showed that it was a significantly poorer substrate for pertussis toxin than the two mammalian alpha o-subunits expressed and assayed under identical conditions. This unexpected biochemical behavior of the Xenopus alpha o-subunit is in agreement with the rather unusual treatment conditions required to observe the effects of pertussis toxin on the receptor-evoked Cl- current in the oocyte. Injection of mammalian heterotrimeric G(o) but not Gi3 significantly enhanced the norepinephrine-evoked Cl- current in oocytes. Injection of mixtures of anti-sense oligonucleotides to the Xenopus alpha o-subunit reduced the norepinephrine-evoked Cl- current by 60% within 24 h, compared with oocytes injected with the oligonucleotides encoding sense sequences. These studies indicate that the expressed alpha 1B-adrenergic receptor, like the native muscarinic receptor, utilizes G(o) to couple to the phospholipase C-mediated Cl- current in Xenopus oocytes.

Nifedipine blocks calcium-dependent cholinergic depolarization in the guinea pig hippocampus.

The possibility that cholinergic stimulation might directly activate a receptor-operated Ca2+ channel was investigated in the CA1 region of guinea pig hippocampus using intracellular recording techniques. Two cholinergic responses were studied: (1) the plateau depolarization evoked by cholinergic stimulation in the presence of Ba2+; and (2) the Ca2(+)-dependent component of membrane depolarization. Both of these responses were blocked by 1-5 microM of nifedipine, a blocker of voltage-dependent L-type Ca2+ channels. In addition, the plateau response was mimicked by direct postsynaptic depolarization in the presence of Ba2+. We conclude that cholinergic stimulation does not directly activate a Ca2+ conductance in these neurons, but rather leads to the indirect activation of L channels which may be located both pre- and postsynaptically.

Long-term potentiation in rat hippocampus is inhibited by low concentrations of ethanol.

Acute ethanol ingestion impairs memory in humans at concentrations associated with mild intoxication. A possible neurophysiological correlate of this effect is the suppression by ethanol of long-tem potentiation (LTP), a persistent increase in synaptic efficiency which has been proposed as a substrate for memory. However, in previous studies ethanol has been shown to impair LTP only at very high concentrations, near the lethal level in humans. We now report that ethanol can significantly reduce LTP in rat hippocampus at concentrations as low as 5 mM, a level attainable following ingestion of a single alcoholic drink. We also demonstrate that the potency of ethanol in depressing LTP correlates well with its potency in inhibiting the response to N-methyl-D-aspartate, an agonist at the glutamate receptors implicated in LTP induction. The influence of low ethanol concentrations on LTP may contribute to the memory impairment associated with its use in humans.

Cholinergic stimulation enhances long-term potentiation in the CA1 region of rat hippocampus.

The effect of the cholinergic agonist carbachol on a putative substrate for memory (long-term potentiation; LTP) was investigated in slices of rat hippocampus (CA1 region). Carbachol (5 microM) increased LTP when the presynaptic depression of the EPSP was controlled. The results indicate that carbachol enhances the effectiveness of the tetanus, probably through postsynaptic mechanisms. This effect may have implications for the role of acetylcholine in memory and the use of cholinergics in memory disorders.

Ethanol suppresses hippocampal cell firing through a calcium and cyclic AMP-sensitive mechanism.

The effects of ethanol were studied intracellularly in hippocampal pyramidal cells in vitro. Ethanol, 50-100 mM, produced a marked suppression of neuronal firing. This effect was blocked by treating the cell with cyclic 3', 5'-adenosine monophosphate (cAMP) or cadmium ions. Ethanol had no effect on the after-hyperpolarizing current. It is concluded that the ethanol-induced reduction of firing rate is due to a calcium-dependent process, and modulated by cAMP.

Functional muscarinic supersensitivity in denervated rat hippocampus.

The effects of carbachol (CCh), a cholinergic agonist, were compared in voltage-clamped hippocampal pyramidal neurons in vitro, obtained from normal and fimbria-fornix-lesioned rats. A substantial increase in sensitivity to the effects of CCh was seen in denervated neurons. The supersensitivity was demonstrated on both the inward leak current and the calcium-dependent potassium current, IAHP. These findings provide convincing evidence for cholinergic denervation supersensitivity in the hippocampus.

An analysis of the depolarization produced in guinea-pig hippocampus by cholinergic receptor stimulation.

1. The effects of carbachol on hippocampal pyramidal neurones were studied in tissue slices in vitro with intracellular microelectrodes, employing current clamp and voltage clamp methods. 2. The calcium-dependent potassium current, IAHP, and the voltage-dependent potassium current, IM, were both reversibly blocked by the application of carbachol (5-10 microM). 3. Carbachol (1-10 microM) induced a steady inward current under circumstances in which both IAHP and IM were inactive. This inward current was sometimes difficult to reverse upon carbachol wash-out, an effect possibly related to receptor desensitization. 4. The depolarizing effect of carbachol was reversed by 0.1 microM-atropine, and exhibited an apparent dissociation coefficient of 1.2 microM for carbachol and 18 nM for pirenzepine, indicating that it is mediated by activation of an M1 muscarinic receptor. 5. The depolarizing effect or inward current induced by carbachol was completely blocked by the potassium channel blockers caesium, tetraethylammonium and barium. 6. The slope of the current-voltage (I-V) plots in carbachol was reduced in the majority of cells, and crossed the control I-V plots at a negative membrane potential. The reversal potentials in carbachol shifted in a positive direction when bathing potassium concentration was increased. 7. In a number of cells, the I-V curves in carbachol were parallel to or converged positively with the control I-V curves. 8. The effects of carbachol were compared to those of serotonin, which increases a 'pure' potassium conductance. Serotonin (10 microM) produced an increase in the slope of the I-V curve, with a reversal potential sensitive to changes in bathing potassium concentration. The carbachol reversal potential values were negative to those of serotonin at 5 and 10 mM-potassium. The equilibrium potentials for carbachol and serotonin were equal at 25 mM-potassium. 9. The negative values of the reversal potential at 5 and 10 mM-potassium and the occurrence of non-crossing I-V characteristics in carbachol could be explained by postulating a second effect of carbachol: namely, a non-specific conductance increase in the dendrites. 10. It is concluded that carbachol depolarizes pyramidal cells in the hippocampus by blocking a voltage-insensitive potassium leak channel and does so by activating M1 muscarinic receptors. In addition, carbachol may also activate a second conductance in the dendrites, which could account for the anomalous I-V characteristics sometimes seen in response to carbachol in these cells.

Functional expression of brain cholecystokinin and bombesin receptors in Xenopus oocytes.

Total RNA was extracted from 15-day-old whole rat brains. Microinjection of the RNA into Xenopus laevis oocytes induced electrophysiological responsiveness to cholecystokinin-8 (CCK) and bombesin (BBS) but not to corticotropin-releasing factor (CRF) or somatostatin. The responses to CCK and BBS were similar in shape, time course, and reversal potential to that induced by receptor mediated phospholipid breakdown and that which is induced by intracellular injection of IP3. These responses were not blocked by atropine or by mianserin, did not require extracellular Ca2+ and were completely suppressed by intracellular injection of EGTA.

Characterization of the bupropion cue in the rat: lack of evidence for a dopaminergic mechanism.

Using a two-lever operant task rats were trained to discriminate 40 mg/kg IP of bupropion from saline. Despite bupropion's established dopaminergic activity in vitro and in vivo, it was found that the bupropion cue was neither mimicked by the dopaminergic drugs L-DOPA and bromocriptine nor blocked by a variety of neuroleptics (haloperidol, thioridazine, and thiothixene). In addition, bupropion was active in attenuating the behavior-suppressing effects of haloperidol, unlike amphetamine and the atypical antidepressants, nomifensine and viloxazine. The bupropion cue was not mimicked or disrupted by adrenergic or serotonergic drugs, but it did generalize to some stimulants (amphetamine, cocaine and caffeine) as well as to nomifensine and viloxazine. The generalizations were blocked by neuroleptics. These data indicate that bupropion's cue properties may not be based on its ability to modulate dopaminergic receptor activity. The possible involvement of phenylethylamine in the bupropion cue is also discussed.