Is persistent activity of calcium/calmodulin-dependent kinase required for the maintenance of LTP?

Calcium/calmodulin-dependent protein kinase II (CaMKII) is concentrated in the postsynaptic density (PSD) and plays an important role in the induction of long-term potentiation (LTP). Because this kinase is persistently activated after the induction, its activity could also be important for LTP maintenance. Experimental tests of this hypothesis, however, have given conflicting results. In this paper we further explore the role of postsynaptic CaMKII in induction and maintenance of LTP. Postsynaptic application of a CaMKII inhibitor [autocamtide-3 derived peptide inhibitor (AC3-I), 2 mM] blocked LTP induction but had no detectable affect on N-methyl-D-aspartate (NMDA)-mediated synaptic transmission, indicating that the primary function of CaMKII in LTP is downstream from NMDA channel function. We next explored various methodological factors that could account for conflicting results on the effect of CaMKII inhibitors on LTP maintenance. In contrast to our previous work, we now carried out experiments at higher temperature (33 degrees C), used slices from adult animals, and induced LTP using a tetanic stimulation. However, we still found that LTP maintenance was not affected by postsynaptic application of AC3-I. Furthermore the inhibitor did not block LTP maintenance under conditions designed to enhance the Ca(2+)-dependent activity of protein phosphatases 1 and 2B (elevated Ca(2+), calmodulin, and an inhibitor of protein kinase A). We also tested the possibility that CaMKII inhibitor might not be able to affect CaMKII once it was inserted into the PSD. In whole-brain extracts, AC3-I blocked autophosphorylation of both soluble and particulate/PSD CaMKII with similar potencies although the potency of the inhibitor toward other CaMKII substrates varied. Thus we were unable to demonstrate a functional role of persistent Ca(2+)-independent CaMKII activity in LTP maintenance. Possible explanations of the data are discussed.

Inhibition of the cAMP pathway decreases early long-term potentiation at CA1 hippocampal synapses.

Long-term potentiation (LTP) has several different phases, and there is general agreement that the late phase of LTP requires the activation of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). In contrast, several studies indicate that the early LTP is not affected by interfering with the cAMP pathway. We have further tested the role of the cAMP pathway in early LTP using several types of inhibitors. Bath application of the PKA inhibitor H89 suppressed the early LTP induced by a single tetanus. Similarly, the LTP induced by a pairing protocol was decreased by postsynaptic intracellular perfusion of the peptide PKA inhibitor PKI(6-22) amide. The decrease of LTP produced by these inhibitors was evident immediately after induction. These results indicate that PKA is important in early LTP, that its locus of action is postsynaptic, and that it does not act merely by enhancing the depolarization required for LTP induction. The failure of some other inhibitors of the cAMP pathway to affect the early phase of LTP might be attributable to the saturation of some step in the cAMP pathway during a tetanus. In agreement with this hypothesis we found that application of the AC inhibitor SQ 22536 by itself did not affect the early phase of LTP, but did produce a reduction if the cAMP pathway was already attenuated by the PKA inhibitor H89. Our analysis of the results of genetic modifications of the cAMP pathway, especially the work on AC knock-outs, indicates that the genetic data are generally consistent with the pharmacological results showing the importance of this pathway in early LTP.

Requirements for LTP induction by pairing in hippocampal CA1 pyramidal cells.

The induction of long-term potentiation (LTP) in the hippocampal CA1 region requires both presynaptic activity and large postsynaptic depolarization. A standard protocol for inducing LTP using whole-cell recording is to pair low-frequency synaptic stimulation (100-200 pulses, 1-2 Hz) with a depolarizing voltage-clamp pulse (1-3 min duration). In this standard protocol, a Cs(+)-based internal solution is used to improve the fidelity of the depolarization produced by voltage-clamp. In an attempt to induce LTP more rapidly, we tried to induce LTP by pairing high-frequency stimulation (200 pulses, 20-100 Hz) with a short depolarization ( approximately 15 s). Surprisingly, we found that this protocol failed to induce LTP, even though large LTP ( approximately 300% of baseline) could be induced by a subsequent standard protocol in the same cell. Pairing brief high-frequency stimulation at the beginning of a long depolarization (3 min) also did not induce LTP. However, the same high-frequency stimulation at the end of the long depolarization did induce LTP. When similar experiments were done with a K(+)-based internal solution, pairing high-frequency stimulation with a short depolarization did induce LTP. This indicates that the requirement for long depolarization is related to the use of Cs(+). We speculate that, when recording is made with Cs(+), a tetanus given at the beginning of depolarization initiates a process that inhibits N-methyl-D-aspartate (NMDA)-dependent LTP. This inhibitory process itself decays away during prolonged depolarization.

Postsynaptic inhibitors of calcium/calmodulin-dependent protein kinase type II block induction but not maintenance of pairing-induced long-term potentiation.

The role of postsynaptic kinases in the induction and maintenance of long-term potentiation (LTP) was studied in the CA1 region of the rat hippocampal slice. A peptide inhibitor for the catalytic domain of calcium/calmodulin-dependent protein kinase type II (CaM-kinase) was applied through a perfused patch pipette. The inhibitor completely blocked both the short-term potentiation and LTP induced by a pairing protocol. This indicates that the kinase or kinases affected by the peptide are downstream from depolarization in the LTP cascade. The ability to block LTP required that measures be taken to interfere with degradation of the peptide kinase inhibitor by endogenous proteases; either addition of protease inhibitors or modifications of the peptide itself greatly enhanced the effectiveness of the peptide. Protease inhibitors by themselves or control peptide did not block LTP induction. To study the effect of kinase inhibitor on LTP maintenance, we induced LTP in one pathway. Subsequent introduction of the kinase inhibitor blocked the induction of LTP in a second pathway, but it did not affect maintenance of LTP in the first. The implications for the role of kinases in LTP maintenance are discussed.

Visualizing hippocampal synaptic function by optical detection of Ca2+ entry through the N-methyl-D-aspartate channel.

Fura-2 and imaging technology were used to detect intracellular Ca2+ changes in CA1 pyramidal cells in hippocampal slices. During focal synaptic stimulation, one or more highly localized regions of Ca2+ elevation (hot spots) were detected in the dendrites. Ca2+ spread from the center of hot spots with properties consistent with diffusion. Several lines of evidence indicate that these hot spots were due to Ca2+ entry through N-methyl-D-aspartate synaptic channels. The spatial and temporal resolution of the method was sufficient to detect the response of single hot spots to single stimuli, thus providing a real-time method for monitoring local synaptic activity. Using this method, we show that synapses on the same dendrite differ in their probability of response and in their facilitation properties.

[The neuronal network of the hippocampus: a morphological analysis]. / Neironal'naia set' gippokampa: morfologicheskii analiz.

The use of new methods of morphologic analysis made it possible to detail significantly already known insights and to formulate the new ones concerning the hippocampal construction. This review discusses in detail the principles of structural organization of all intrahippocampal interneuronal connections and projections to the hippocampus from entorhinal cortex. Also, other afferent and efferent pathways and connections of polymorphic neurons of hippocampus are studied. Revision of the lamellar principle of hippocampal organization and involvement of inhibitory system in restricting an excitation propagation along the hippocampus is discussed.

Measuring the impact of probabilistic transmission on neuronal output.

We have investigated the impact of stochastic transmission on the input-output relations of neurons in hippocampal slices. A synaptic input that fires a cell has a significant trial-to-trial variability in amplitude, reflecting the probabilistic release of transmitter. By measuring miniature excitatory postsynaptic currents, we estimate that synchronous release of a few vesicles can fire a CA1 cell. The firing threshold and variability can be physiologically modulated. Different cell types have distinct firing thresholds and variabilities. Long-term potentiation (LTP) decreases trial-to-trial variability. If after LTP, the stimulus is reduced to produce a threshold response, the variability returns to that observed before LTP. Thus, for a threshold input, the trial-to-trial variability is maintained with LTP. This may be important for the proper functioning of a plastic nervous system.

[A comparative analysis of the functional stability (based on electrophysiological criteria) of slices of the suslik and guinea pig brains maintained under deep hypothermia]. / Sravnitel'nyi analiz funktsional'noi sokhrannosti (po élektrofiziologicheskim kriteriiam) srezov mozga suslikov i morskikh svinok pri soderzhanii v glubokoi gipotermii.

The effect of prolonged deep cooling has been investigated in hippocampal and septal slices from the brain of hibernating and active ground squirrels, as well as of the guinea pigs. The slices were kept at low temperatures (2-4 degrees C) for various periods of time (from several hours to 6 days) and were periodically tested in a warm (31 degrees C) incubation medium. Hippocampal field potentials (mainly of the field CA1) and spontaneous activity of single neurones of the medial septum were recorded. Significant differences were observed in the recovery of functional activity of the slices after their preparation as well as after cooling between experimental groups of animals. Slices from hibernating ground squirrels retained their activity for 7-9 days, those from active ones--for 6-7 days, whereas slices from guinea pigs did not recover their functional activity after cooling for more than 1-2 days.

Functional stability of the brain slices of ground squirrels, Citellus undulatus, kept in conditions of prolonged deep periodic hypothermia: electrophysiological criteria.

The brain of hibernating animals, controlling the physiological functions during the hibernation cycles, is itself subject to deep cooling during bouts of hibernation. This suggests its high tolerance to deep hypothermia. Effects of prolonged deep cooling were investigated in hippocampal and septal slices, taken from the brains of three groups of animals: hibernating ground squirrels, actively waking ground squirrels, and guinea-pigs. The slices were kept at a low temperature (2-4 degrees C) for various periods of time (from several hours up to six days) and periodically tested in warm (31 degrees C) incubation medium. The hippocampal field potentials (mainly of field CA1), as well as spontaneous activity of single neurons of hippocampus and medial septum were recorded. For comparative purposes mean amplitudes of population spikes and mean frequency of spontaneous neuronal discharge were used. Significant differences between the experimental groups were observed in recovery of functional activity of the slices after their dissection from the brain, as well as after deep cooling. In both cases re-establishment of neuronal activity in ground squirrels occurred more rapidly, than in guinea-pigs. The most dramatic was the difference in maximal time of survival of the slices under conditions of deep cooling. Independently of periodicity of the electrophysiological testing in warm medium, the slices taken from hibernating squirrels retained their activity for seven to nine days, the slices of waking ground squirrel hippocampus survived up to six to seven days, while those of guinea-pis did not recover their functional activity after cooling for more than one to two days.(ABSTRACT TRUNCATED AT 250 WORDS)

[Actin study of the synapses of surviving hippocampal slices during long-term potentiation]. / Izuchenie aktina sinapsov perezhivaiushchikh srezov gippokampa pri dolgovremennoi potentsiatsii.

A study was made of the synaptic actin ultrastructural localization in the hippocampal slices at long-lasting potentiation of area CA, using myosin subfragment-1 labeling. A specific qualitative ultrastructural sign of the potentiated hippocampal synapses was revealed for the first time - the formation in spines of rodlike bundles of actin filaments resembling the cilia. They penetrate the spine stalks to pass through the spine core towards the postsynaptic densities of active zones. The thinner bridges link the filament bundles with the actin cytoskeleton meshwork, with spine apparatus cisterns and with postsynaptic membranes of the active zones. Besides the increasing density of the presynaptic actin meshwork was shown. The changes in the actin cytoskeleton being taken into consideration, its contractile properties account for some morphofunctional features of the potentiated synapses known before and predict previously unknown ones.

[Action of mediators on pyramidal and nonpyramidal neurons in slices of the guinea pig hippocampus]. / Deistvie mediatorov na piramidnye i nepiramidnye neirony v srezakh gippokampa morskoi svinki.

Effects of acetylcholine (Ach), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) on the background activity were tested in the field CA3 in the guinea pig hippocampal slices. Three groups of neurons were investigated: nonpyramidal neurons of stratum radiatum-moleculare (NSR), neurons with single spike discharges of stratum pyramidale (SD-units) and neurons with complex spike discharges (CD-units) in the same stratum. Effects of Ach and NE were tested also on presumed interneurons of str. oriens-pyramidale (ISP). Similarity between NSR, ISP and SD units and their difference from CD units may be suggested on the basis of the drugs action. Activity of the CD units was suppressed by NE, 5-HT and GABA, while in a half of these cells Ach evoked inhibitory-activatory succession of effects. On the contrary, NSR, ISP and SD units were monophasically activated by NE and Ach. Though 5-HT and GABA suppressed the activity in some of NSR and SD units, many of them were activated. Excitatory influence of Ach, NE and 5-HT on NSR was preserved with the blockade of synaptic transmission, which suggests direct influence of the drugs on the investigated cells.

[Electrophysiologic study of non-pyramidal neurons of the stratum radiatum in slices of guinea pig hippocampus]. / Elektrofiziologicheskoe issledovanie nepiramidnykh neironov radial'nogo sloia v srezakh gippokampa morskoi svinki.

The activity of 67 nonpyramidal neurons of str. radiatum-moleculare (NSRM) and of 8 presumed interneurons of str. oriens-pyramiidale (NSOP) was recorded extracellularly in guinea pig hippocampal slices. In comparison with high frequency grouped discharges characteristic of NSOP, NSRM had low frequency background activity consisting of single (77%) and grouped (23%) spikes. The level of the background activity of NSRM decreased with an increase of the distance of their location from str. pyramidale. Electrical stimulation of dentate fascia usually evoked 1-2 spike discharges in NSRM, while bursts of spikes were evoked in NSOP. The thresholds of responses in NSRM were not different or higher than those of pyramidal neurons, while in NSOP they were significantly lower. The period of suppression of the spontaneous activity usually followed the evoked spike discharge in NSRM. During evoked synchronous epileptiform discharges of pyramidal neurons the simultaneous excitation of NSRM was observed. Excitatory influence of pyramidal neurons on NSRM and participation of the latter in dendritic inhibition of pyramidal neurons is suggested.

Effects of norepinephrine and serotonin upon spontaneous activity and responses to mossy fiber stimulation of CA3 neurons in hippocampal slices.

Effects of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) upon spontaneous activity and responses to mossy fiber stimulation (mfs) were tested in 192 units of the field CA3 in the guinea pig and rat hippocampal slices. The drugs were added to the incubating medium or ejected by pressure from a micropipette. After NE superfusion firing rate increased in 52% of the reactive units, while activity of 48% was suppressed. The direction of the effect strongly correlated with pattern of spontaneous activity: only the cells with 'complex discharges' (short bursts of 2-4 spikes with attenuation of amplitude) were suppressed by NE; the cells with single spikes increased the level of activity. Similar excitatory effects of NE were observed in all units (n = 14) with single spike activity recorded in the field CA1. 5-HT increased activity in 30% of the reactive units and suppressed it in 70% of them. Some of the cells which were suppressed by 5-HT, were excited by NE. In more than a half of the units tested, 5-HT led to prolonged (up to 30-40 min) increase of the level of background activity irrespective of the initial excitatory or suppressive action; periodic grouped discharges appeared in some units under the influence of 5-HT. The response to mfs usually changed in the same direction as the level of background activity during application of NE and 5-HT, though some exceptions were observed in both cases. Prolonged (up to 30-40 min) facilitation of responses to mfs was present after application of 5-HT.

[Effect of noradrenaline and serotonin on responses of field CA3 neurons of hippocampal slices evoked by electric stimulation of mossy fibers]. / Vliianie noradrenalina i serotonina na otvety neironov polia CA3 srezov gippokampa morskoi svinki, vyzvannye élektricheskoi stimuliatsiei mshistykh volokon.

Responses of the CA3 neurons to mossy fibre stimulation were investigated in incubated hippocampal slices in vitro under the influence of norepinephrine (NE) and 5-hydroxytryptamine (5HT). Both agents modulated the level of background activity (increased or decreased it), changed its pattern and influenced the responses to stimulation of nonaminergic input, facilitating or suppressing it. The changes in response level and pattern usually correlate with the corresponding changes of background activity. In some cases NE, lowering the frequency of background activity but increasing the proportion of "complex discharges" in it as well as the number of reduced spikes with in a complex discharge, increased the responses to stimulation. 5HT which evoked in some cells a periodic grouped activity, facilitated the occurrence of identical groups in response to stimulation. Unlike NF which evoked relatively short-term changes, 5HT produced prolonged (up to 40 min) augmentation of background activity and facilitation of responses.

[Effect of noradrenaline and serotonin on background activity of hippocampal CA3 field neurons in vitro]. / Issledovanie villianii noradrenalina i serotonina na fonovuiu aktivnost' neironov polia CA3 gippokampa in vitro.

Effect of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) upon the background activity of neurons in hippocampal slices (field CA3) was investigated during injection transmitter substances to the incubation medium. Both NE and 5-HT may suppress (32 and 49%, respectively) or activate (52 and 35%) the background activity. Activation was observed mainly in the cells without background discharges or with single spike irregular activity. Suppression of activity was observed in the units with "complex spikes". Activatory effects of NE, but not of 5-HT, could be imitated by NE against phenylephrine and blocked by alpha-adrenoreceptor blocker phentolamine. Effects of transmitter substances were maintained in the medium with increased Mg2+ concentration.

[Heterosynaptic interactions in fields CA1 and CA3 of the hippocampus in vitro]. / Issledovanie geterosinapticheskikh vzaimodeistvii v poliakh CA1 i CA3 gippokampa in vitro.

Heterosynaptic interactions between the perforant path and Schaffer's collaterals in the field CA1 and between the perforant path and dentate mossy fibres in the field CA3 were investigated in guinea pig hippocampal slices. Using the method of paired stimuli, space-time summation (for 20--50 ms) was observed in both systems with stimuli sub-threshold for spike generation. Spike responses of the neurons to testing stimulation of afferents synapsing upon the terminal parts of apical dendrites (perforant path) were depressed after spike discharge to conditioning stimulation of proximal afferents (for about 20 ms in CA1, and for about 300 ms in CA3). With inverse combination of the stimuli the period of suppression was much shorter (3--8 ms). Tetanization of the mossy fibres was followed by prolonged (2--30 min) depression of the CA3 responses to the perforant path stimulation. No other reliable long-lasting posttetanic heterosynaptic effects were observed.

[Comparative characteristics of the direct influences of the perforant pathway on neurons in hippocampal fields CA1 and CA3 in vitro]. / Sravnitel'nye kharakteristiki priamykh vliianii perforiruiushchego puti na neirony polei CA1 i CA3 gippokampa in vitro.

The effect of the perforant path stimulation on the CA1 and CA3 neurons was investigated in incubated slices of the guinea pig hippocampus. Spike generation was observed in both fields during stimulation of the perforant path. The majority of the CA1 neurons followed rhythmic stimulation up to 30-80/c. The CA3 neurons responded only to low-frequency stimulation (up to 5/c). The posttetanic potentiation of responses to the perforant path stimulation was observed in both hippocampal fields.