Intracellular correlates of spatial memory acquisition in hippocampal slices: long-term disinhibition of CA1 pyramidal cells.

Despite many advances in our understanding of synaptic models of memory such as long-term potentiation and depression, cellular mechanisms that correlate with and may underlie behavioral learning and memory have not yet been conclusively determined. We used multiple intracellular recordings to study learning-specific modifications of intrinsic membrane and synaptic responses of the CA1 pyramidal cells (PCs) in slices of the rat dorsal hippocampus prepared at different stages of the Morris water maze (WM) task acquisition. Schaffer collateral stimulation evoked complex postsynaptic potentials (PSP) consisting of the excitatory and inhibitory postsynaptic potentials (EPSP and IPSP, respectively). After rats had learned the WM task, our major learning-specific findings included reduction of the mean peak amplitude of the IPSPs, delays in the mean peak latencies of the EPSPs and IPSPs, and correlation of the depolarizing-shifted IPSP reversal potentials and reduced IPSP-evoked membrane conductance. In addition, detailed isochronal analyses revealed that amplitudes of both early and late IPSP phases were reduced in a subset of the CA1 PCs after WM training was completed. These reduced IPSPs were significantly correlated with decreased IPSP conductance and with depolarizing-shifted IPSP reversal potentials. Input-output relations and initial rising slopes of the EPSP phase did not indicate learning-related facilitation as compared with the swim and naïve controls. Another subset of WM-trained CA1 PCs had enhanced amplitudes of action potentials but no learning-specific synaptic changes. There were no WM training-specific modifications of other intrinsic membrane properties. These data suggest that long-term disinhibition in a subset of CA1 PCs may facilitate cell discharges that represent and record the spatial location of a hidden platform in a Morris WM.

Intracellular correlates of acquisition and long-term memory of classical conditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI.

Intradendritic recordings in Purkinje cells from a defined area in parasaggital slices of cerebellar lobule HVI, obtained after rabbits were given either paired (classical conditioning) or explicitly unpaired (control) presentations of tone and periorbital electrical stimulation, were used to assess the nature and duration of conditioning-specific changes in Purkinje cell dendritic membrane excitability. We found a strong relationship between the level of conditioning and Purkinje cell dendritic membrane excitability after initial acquisition of the conditioned response. Moreover, conditioning-specific increases in Purkinje cell excitability were still present 1 month after classical conditioning. Although dendritically recorded membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in cells from paired or control animals, the size of a potassium channel-mediated transient hyperpolarization was significantly smaller in cells from animals that received classical conditioning. In slices of lobule HVI obtained from naive rabbits, the conditioning-related increases in membrane excitability could be mimicked by application of potassium channel antagonist tetraethylammonium chloride, iberiotoxin, or 4-aminopyridine. However, only 4-aminopyridine was able to reduce the transient hyperpolarization. The pharmacological data suggest a role for potassium channels and, possibly, channels mediating an IA-like current, in learning-specific changes in membrane excitability. The conditioning-specific increase in Purkinje cell dendritic excitability produces an afterhyperpolarization, which is hypothesized to release the cerebellar deep nuclei from inhibition, allowing conditioned responses to be elicited via the red nucleus and accessory abducens motorneurons.

[The effect of atropine on the ultrastructural postsynaptic plasticity of the associative type in the rat neocortex]. / Vliianie atropina na ul'trastrukturnuiu postsinapticheskuiu plastichnost' assotsiativnogo tipa v neokortekse krys.

The effect of muscarinic antagonist atropine on thickness of postsynaptic density of axodendritic synapses was studied in the sensorimotor region of the brain cortex of rats during paired repeated microapplication of glutamate and acetylcholine. In the applied conditioning paradigm atropine significantly decreased morphological dimensions of the postsynaptic density, however, the control values were not reached. This finding testifies to participation of both muscarinic and nicotinic cholinoreceptors in associative postsynaptic plasticity.

Secondary structure and Ca2+-induced conformational change of calexcitin, a learning-associated protein.

Calexcitin/cp20 is a low molecular weight GTP- and Ca2+-binding protein, which is phosphorylated by protein kinase C during associative learning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons. Here, the secondary structure of cloned squid calexcitin was determined by circular dichroism in aqueous solution and by Fourier transform infrared spectroscopy both in solution and on dried films. The results obtained with the two techniques are in agreement with each other and coincide with the secondary structure computed from the amino acid sequence. In solution, calexcitin is one-third in alpha-helix and one-fifth in beta-sheet. The conformation of the protein in solid state depends on the concentration of the starting solution, suggesting the occurrence of surface aggregation. The secondary structure also depends on the binding of calcium, which causes an increase in alpha-helix and a decrease in beta-sheet, as estimated by circular dichroism. The conformation of calexcitin is independent of ionic strength, and the calcium-induced structural transition is slightly inhibited by Mg2+ and low pH, while favored by high pH. The switch of calexcitin's secondary structure upon calcium binding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physiologically meaningful range of Ca2+ concentration. The calcium-bound form is more globular than the apoprotein. Unlike other EF-hand proteins, calexcitin's overall lipophilicity is not affected by calcium binding, as assessed by hydrophobic liquid chromatography. Preliminary results from patch-clamp experiments indicated that calcium is necessary for calexcitin to inhibit potassium channels and thus to increase membrane excitability. Therefore the calcium-dependent conformational equilibrium of calexcitin could serve as a molecular switch for the short term modulation of neuronal activity following associative conditioning.

Dendritic excitability microzones and occluded long-term depression after classical conditioning of the rabbit's nictitating membrane response.

We made intradendritic recordings in Purkinje cells (n = 164) from parasaggital slices of cerebellar lobule HVI obtained from rabbits given paired presentations of tone and periorbital electrical stimulation (classical conditioning, n = 27) or explicitly unpaired presentations of tone and periorbital stimulation (control, n = 16). Purkinje cell dendritic membrane excitability, assessed by the current required to elicit local dendritic calcium spikes, increased significantly in slices from animals that received classical conditioning. In contrast, membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in slices from animals given paired or explicitly unpaired stimulus presentations. The location of cells with low thresholds for local dendritic calcium spikes suggested that there are specific sites for learning-related changes within lobule HVI. These areas may correspond to learning "microzones" and are consistent with locations of learning-related in vivo changes in Purkinje cell activity. Application of 4-aminopyridine, an antagonist of the rapidly inactivating potassium current IA, reduced the threshold for dendritic spikes in slices from naive animals to levels found in slices from trained animals. In cells where thresholds for eliciting parallel fiber-stimulated Purkinje cell excitatory postsynaptic potentials (EPSPs) were measured, levels of parallel fiber stimulation required to elicit a 6-mV EPSP as well as a 4-mV EPSP (n = 30) and a Purkinje cell spike (n = 56) were found to be significantly lower in slices from paired animals than unpaired controls. A classical conditioning procedure was simulated in slices of lobule HVI by pairing a brief, high-frequency train of parallel fiber stimulation (8 pulses, 100 Hz) with a brief, lower frequency train of climbing fiber stimulation (3 pulses, 20 Hz) to the same Purkinje cell. Following paired stimulation of the parallel and climbing fibers, Purkinje cell EPSPs underwent a long-term (> 20 min) reduction in peak amplitude (-24%) in cells (n = 12) from animals given unpaired stimulus presentations but to a far less extent (-9%) in cells (n = 20) from animals given in vivo paired training. Whereas 92% of cells from unpaired animals showed pairing-specific depression, 50% of cells from paired animals showed no depression and in several cases showed potentiation. Our data establish that there are localized learning-specific changes in membrane and synaptic excitability of Purkinje cells in rabbit lobule HVI that can be detected in slices 24 h after classical conditioning. Long-term changes within Purkinje cells that effect this enhanced excitability may occlude pairing-specific long-term depression.

Calexcitin: a signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability.

A previously uncharacterized 22-kDa Ca(2+)-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca2+, calexcitin bound GTP and possessed GTPase activity. Calexictin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca2+ and the absence of endogenous Ca2+/calmodulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 +/- 6 pS K+ channels. Calexcitin thus appears to directly regulate K+ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca(2+)-activated signaling molecule that increases cellular excitability, which would in turn increase Ca2+ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca2+.

Significance of the interstimulus time interval in repeating combined presentations of L-glutamate and acetylcholine for change in the reactivity of cortical neurons.

A comparative study of the probability, directionally, and intensity of the changes in the baseline and L-glutamate (Gl)- and acetylcholine(ACh)-induced average frequency of the impulse activity (BIA, GlIA, and AChIA, respectively) of individual neurons of the sensorimotor cortex of unanesthetized rats in the course of repeated isolated and combined presentations of Gl and ACh showed that the process of reduction in reactivity to Gl is slowed in the population of cells which experienced the combined action of Gl and ACh with a 1.5 second postponement of applications of ACh in comparison with the situation of its isolated and combined presentation with a 3 second postponement of ACh. The AChIA decreases to a greater degree in the case of a 3 second postponement of ACh, and the probability of an increase in BIA is less than with the isolated application of ACh. It is concluded that the character of the dynamics of the reactivity of neurons is determine by the temporal relationships of local neurochemical stimuli.

Dynamics of the reactivity of cortical neurons to the repeating isolated action of L-glutamate and acetylcholine.

A comparative analysis of the probability, directionality, and intensity of the changes in the average frequency of the baseline (BIA) and L-glutamate-induced (GlIA) and acetylcholine-induced (AChIA) impulse activity of individual neurons of the sensorimotor cortex of unanesthetized rats showed that the reactions to Gl are most variable; this is expressed in a significantly higher percent of neurons with potentiation of responses to Gl as compared with the proportion of cells which manifested an increase in the AChIA in the course of 20 applications of the mediators. As a result of 100 applications, the GlIA decreases most frequently; also, the degree of decrease in GlIA over the entire duration of the testing exceeds the degree of decrease in AChIA. It is more characteristic of ACh than of Gl to increase the level of BIA in the periods between the reactions to ACh, while in the case of repeated application of Gl, dynamics of decrease in BIA are characteristic. The results are discussed from the perspective of a possibly different functional role of the glutamatergic and cholinergic neuromediator systems in the cerebral cortex in the process of formation of the dynamics of neuronal activity during learning.

[The significance of the interstimulus time interval in repetitive paired applications of L-glutamate and acetylcholine for altering cortical neuronal reactivity]. / Znachenie mezhstimul'nogo vremennógo intervala v povtoriaiushchikhsia sochetannykh pred''iavleniiakh L-glutamata i atsetilkholina dlia izmeneniia reaktivnosti korkovykh neironov.

Comparative analysis of the probability, direction and magnitude of changes of the mean firing rates for the background and L-glutamate--(Glu) and acetylcholine--(ACh) induced activity of single sensorimotor cortical neurons in the course of repeated separate and paired presentations of Glu and ACh was carried out in unanesthetized rats. A decline of Glu-induced activity was shown to be slower in a population of neurons affected by paired actions of Glu-ACh with 1.5 s time delay of ACh comparing with a decline of Glu-induced activity under the separate and paired applications with 3 s intervals. In the latter case the decrease of ACh-induced activity was more pronounced and the probability of the increase of the background activity was lower than in the course of the isolated presentations of ACh. It is suggested that interstimulus time interval determines the type of changes of neuron responsiveness.

[The dynamic reactivity of cortical neurons to the repeated isolated action of L-glutamate and acetylcholine]. / Dinamika reaktivnosti korkovykh neironov k povtoriaiushchemusia izolirovannomu deistviiu L-glutamata i atsetilkholina.

Comparative analysis of the probability, direction and magnitude of changes of the mean firing rates for the background and L-glutamate--(Glu) and acetylcholine--(ACh) induced activity of single sensorimotor cortical neurons of unanesthetized rats showed that more significant changes occurred in reaction to the applications of Glu. Increases of Glu-induced activity were more frequent than increases of ACh-induced activity in the course of 20 microiontophoretic applications of the transmitters. Declines of Glu-induced activity were more frequent and profound than those of ACh-induced activity as a result of 100 presentations. Enhancements of the background activity occurred usually between the cellular responses to ACh, and its decreases were more specific during successive separate Glu presentations. The different functional roles of Glu and ACh in the dynamics of neuronal discharges in the course of learning are discussed.

Temporal specificity in the action of stimuli during the formation of associative ultrastructural reorganizations in neurons of the cerebral cortex.

A morphometric investigation of various components of the synapses of neurons of the sensorimotor region of the cerebral cortex of rats with different variants of combined and uncombined repeated microiontophoretic application of glutamate and acetylcholine has been carried out. A substantial dependence of the character and expressivity of the reorganizations of the thickness of the postsynaptic density (PSD), of the width of the synaptic cleft, and the length of the active zone of the synapses, on the temporal relationships in the action of mediators has been identified: significant changes in the thickness of the PSD appeared only with the combined applications of the stimuli (neuromediators); the maximum thickening of the PSD was induced by the combined action of glutamate and acetylcholine with a 3-second delay in the latter. A hypothesis is presented according to which the temporal specificity in the integration of associable signals arriving at neurons is determined by the kinetics of the various interacting biochemical regulator mechanisms.

[Temporal specificity in stimulus action during the formation of associative ultrastructural restructurings in cerebral cortex neurons]. / Vremennáia spetsifichnost' v deistvii razdrazhitelei pri formirovanii assotsiativnykh ul'trastrukturnykh perestroek v neironakh kory mozga.

By a method of electronic microscopy morphological characteristics were studied of various components of neurones synapses in the sensorimotor cortical zone of rats at different variants of combined and noncombined repeated microiontophoretic presentation of glutamate and acetylcholine. Significant dependence was found of the character and rate of reorganizations of postsynaptic thickness (PSTh), width of synaptic cleft and length of synapses active zone on temporal relations in transmitters action: significant changes of PSTh thickness appeared only in conditions of combined presentations of stimuli (neurotransmitters); maximum thickening of PsTh was caused by the combined action of glutamate and acetylcholine with 3 s delay of the latter. The hypothesis is suggested that temporal specificity during integration of associated signals action to neurones is determined by kinetics of interacting biochemical regulating mechanisms.

[Changes in the ultrastructure of cortical synapses during exposure to associated neurochemical stimuli]. / Izmeneniia ul'trastruktury kortikal'nykh sinapsov pri vozdeistvii assotsiirovannykh neirokhimicheskikh razdrazhitelei.

Dependence of changes in the postsynaptic density, active zone and synaptic cleft on time relations between the administered associated stimuli was established. Locally applied glutamate and acetylcholine were used as the stimuli.

[Reactions of neurons in the sensory-motor cortex on different amounts of administered acetylcholine]. / Reaktsii neironov sensomotornoi oblasti kory na raznoe kolichestvo applitsiruemogo atsetilkholina.

As a result of acetylcholine iontophoresis with different currents 3-fold increase of transmitter compared with the threshold one for reaction has been shown not to result in change of a type of reaction pattern more than in 80.3% of neurones. Such increase of action force is quite enough for the significant lengthening of the reaction excitatory components in the most of investigated neurones. After the following repeated application of smaller quantity of transmitter the number of neurones with growth of frequency of excited impulsive activity recovers as well as the level of firing frequency decrease in the course of repetitive administration of transmitter. The effect of large doses of transmitter results in aftereffect expressed by increasing probability of excitatory component reduction during the repetitive applications of acetylcholine.