Molecular specificity of synaptic changes responsible for associative memory.
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Alzheimer Disease↗
Biomedical subjects
D L Alkon
Publications and source records attributed to D L Alkon.
Thermal imaging of receptor-activated heat production in single cells.
Changes in enthalpy (i.e., heat content) occur during the diverse intracellular chemical and biophysical interactions that take place in the life cycle of biological cells. Such changes have previously been measured for cell suspensions or cell-free biochemical extracts by using microcalorimetry, thermocouples, or pyroelectric films, all of which afford minimal spatial or temporal resolution. Here we present a novel thermal imaging method that combines both diffraction-limited spatial (approximately 300 nm) and sampling-rate-limited time resolution, using the temperature-dependent phosphorescence intensity of the rare earth chelate Eu-TTA (europium (III) thenoyltrifluoro-acetonate). With this thermosensitive dye, we imaged intracellular heat waves evoked in Chinese hamster ovary cells after activation of the metabotropic m1-muscarinic receptor. Fast application of acetylcholine onto the cells evoked a biphasic heat wave that was blocked by atropine, and after a brief delay was followed by a calcium wave. Atropine applied by itself produced a monophasic heat wave in the cells, suggesting that its interactions with the receptor activate some intracellular metabolic pathways. The thermal imaging technique introduced here should provide new insights into cellular functions by resolving the location, kinetics, and quantity of intracellular heat production.
Animals↗
Identification of ion channel regulating proteins by patch-clamp analysis.
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Animals↗
Time domains of neuronal Ca2+ signaling and associative memory: steps through a calexcitin, ryanodine receptor, K+ channel cascade.
Synaptic changes that underlie associative learning and memory begin with temporally related activity of two or more independent synaptic inputs to common postsynaptic targets. In turn, temporally related molecular events regulate cytosolic Ca2+ during progressively longer-lasting time domains. Associative learning behaviors of living animals have been correlated with changes of neuronal voltage-dependent K+ currents, protein kinase C-mediated phosphorylation and synthesis of the Ca2+ and GTP-binding protein, calexcitin (CE),and increased expression of the Ca2+-releasing ryanodine receptor (type II). These molecular events, some of which have been found to be dysfunctional in Alzheimer's disease, provide means of altering dendritic excitability and thus synaptic efficacy during induction, consolidation and storage of associative memory. Apparently, such stages of behavioral learning correspond to sequential differences of Ca2+ signaling that could occur in spatially segregated dendritic compartments distributed across brain structures, such as the hippocampus.
Animals↗
Evolution of adaptive neural networks: the role of voltage-dependent K+ channels.
The vestibular pathway of the mollusk Hermissenda crassicornis mediates a reflexive, unconditioned response to disorientation, clinging, that has been conserved during evolution even to the emergence of our own species. This response becomes associated with a visual stimulus (mediated by a precisely ordered visual-vestibular synaptic network) according to principles of Pavlovian conditioning that are also followed in human learning. It is not entirely surprising therefore that molecular and biophysical cascades responsible for this associative learning appear to function in both mollusks and mammals. In brief, combinational elevation of (Ca2+)i, diacylglycerol, and arachidonic acid activates protein kinase C to phosphorylate the Ca2+ and guanosine triphosphate-binding protein, cp20 (now called calexcitin (Nelson T, et al. Proc Natl Acad Sci USA 1996;93:13808-13)), which potently inactivates postsynaptic voltage-dependent K+ currents and thereby increases synaptic weight. Longer term changes included rearrangement of synaptic terminals and modified protein synthesis. This cascade has also been implicated in other associative-learning paradigms (e.g., spatial maze, olfactory discrimination) and as a pathophysiologic target in early Alzheimer's disease. Recent molecular biologic experiments also demonstrate the dependence of associative memory (but not long-term potentiation) on voltage-dependent K+ currents. Theoretic learning models based on these findings focus on dendritic spine clusters and yield computer implementations with powerful pattern-recognition capabilities.
Adaptation, Physiological↗
Lead, learning, and calexcitin in Hermissenda.
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Animals↗
Use of CD and FT-IR to determine the secondary structure of purified proteins in the low-microgram range.
The spectroscopic characterization of protein secondary structure is often partially unreliable when samples are not extremely pure and abundant. This problem may be overcome by the combination of circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR). We used these methods to characterize the secondary structure of two proteins of neurobiological interest, calexcitin (CE) and the cellular isoform of prion protein (PrPC). Both proteins were purified with multiple chromatographic steps and were obtained in buffer with high purity (> 95%) and in low amount (approximately 2 micrograms). The samples were analyzed by circular dichroism (down to 184 or 182 nm), recovered, and deposited on films for infrared analysis. The spectral deconvolution from the two methods yielded secondary structures in good agreement with each other as well as with theoretical predictions based on amino acid sequence. The conformation of CE was found to be dependent on its concentration and on calcium binding. The secondary structure of cellular native PrP varied dramatically with the detergent used. In conclusion, the combination of CD and FT-IR analysis is suitable for the characterization of the conformational changes induced by ligand binding and/or by different solvent conditions when the protein of interest is only scarcely available. The methods used here provide valuable insights into the putative correlation between protein structure and activity.
Algorithms↗
Pattern matching in a model of dendritic spines.
Pattern matching, the ability to recognize and maximally respond to an input pattern that is similar to a previously learned pattern, is an essential step in any learning process. To investigate the properties of pattern matching in biological neurons, and in particular the role of a calcium-dependent potassium conductance, a circuit model of a small area of dendritic membrane with a number of dendritic spines is developed. Circuit model simulations show that dendritic membrane depolarization is greater in response to a previously learned pattern of synaptic inputs than in response to a novel pattern of synaptic inputs. These simulations, in combination with an analysis of the circuit model equations, reveal that when a synaptic input pattern is similar to the learned pattern of synaptic inputs, the total dendritic depolarization is a linear combination of dendritic depolarization contributed by individual spines. When at least one synaptic input differs markedly from the learned value, dendritic depolarization is a nonlinear combination of individual spine depolarizations. These principles of spine interactions are captured in a computationally simple set of 'similarity measure' equations which are shown to reproduce the response surface of the circuit model output. Thus, these similarity measure equations not only describe a biologically plausible model of pattern matching, they also satisfy computational requirements for use in artificial neural networks.
Animals↗
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.
Amino Acid Sequence↗
Late memory-related genes in the hippocampus revealed by RNA fingerprinting.
Although long-term memory is thought to require a cellular program of gene expression and increased protein synthesis, the identity of proteins critical for associative memory is largely unknown. We used RNA fingerprinting to identify candidate memory-related genes (MRGs), which were up-regulated in the hippocampus of water maze-trained rats, a brain area that is critically involved in spatial learning. Two of the original 10 candidate genes implicated by RNA fingerprinting, the rat homolog of the ryanodine receptor type-2 and glutamate dehydrogenase (EC 1.4.1.3), were further investigated by Northern blot analysis, reverse transcription-PCR, and in situ hybridization and confirmed as MRGs with distinct temporal and regional expression. Successive RNA screening as illustrated here may help to reveal a spectrum of MRGs as they appear in distinct domains of memory storage.
Animals↗
Time-resolved imaging of protein kinase C activation during sea urchin egg fertilization.
To study protein kinase C (PKC) activation during sea urchin egg fertilization we used three different fluorescent probes specific for PKC, namely, fim-1, which recognizes the catalytic site of the enzyme, and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. We were able to follow PKC activation during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increased and clustered in the cortical zone of the cell. The process was Ca2+ dependent and was inhibited in the presence of staurosporine, a PKC inhibitor. According to our in vitro probe characterization, this signal increase is due to PKC activation. These findings were further confirmed by Western blot analysis. This initial phase was followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca2(+)-dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization.
Animals↗
Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat.
Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic "knockouts".
Animals↗
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.
Animals↗
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+.
Amino Acid Sequence↗
Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity.
The present study examines the synergistic activation of PKC by arachidonic acid and diacylglycerols in phospholipid vesicles and demonstrates that this combination of activators leads to the formation of a constitutively active, phospholipid-bound form of the enzyme. Activation of PKC was almost entirely calcium-dependent with vesicles containing dioleoylglycerol alone. In contrast, considerable calcium-independent activity was observed when vesicles contained both a diacylglycerol and free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reversible. However, addition of arachidonic acid to diacylglycerol-containing vesicles resulted in irreversible PKC binding in the absence of calcium. Immunoblot analysis indicated that the calcium-independent binding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly calcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the subsequent activity was almost entirely resistant to calcium chelation. This vesicle-associated form of the enzyme, when detergent extracted and recombined with phospholipid vesicles, maintained significant 'constitutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a model of the physiological regulation of PKC in which the combined actions of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC.
Animals↗
Soluble beta-amyloid induces Alzheimer's disease features in human fibroblasts and in neuronal tissues.
It has been shown that K+ channels, Cp20 (a 20kD GTP-binding protein), and intracellular calcium release, play a key role in associative memory storage. These same elements have been shown to be altered in fibroblasts from Alzheimer's Disease (AD) patients. In addition, it has been shown that PKC, also implicated in memory storage and closely related to the above mentioned components, is also altered in AD fibroblasts. Moreover, beta-amyloid was capable of inducing an AD-like phenotype for K+ channels and Cp20 in otherwise normal fibroblasts, providing additional evidence for the potential involvement of these components in AD and suggesting a possible pathological consequence of soluble beta-amyloid elevation in AD. Preliminary evidence shows that comparable changes in potassium channel function are also present in human olfactory neuroblasts from AD patients. These results indicate that the observed changes not only occur in peripheral tissues such as fibroblasts, but also in neural tissue, the primary site of AD pathology.
Alzheimer Disease↗
Orange juice classification with a biologically based neural network.
Dystal, an artificial neural network, was used to classify orange juice products. Nine varieties of oranges collected from six geographical regions were processed into single-strength, reconstituted or frozen concentrated orange juice. The data set represented 240 authentic and 173 adulterated samples of juices; 16 variables [8 flavone and flavanone glycoside concentrations measured by high-performance liquid chromatography (HPLC) and 8 trace element concentrations measured by inductively coupled plasma spectroscopy] were selected to characterize each juice and were used as input to Dystal. Dystal correctly classified 89.8% of the juices as authentic or adulterated. Classification performance increased monotonically as the percentage of pulpwash in the sample increased. Dystal correctly identified 92.5% of the juices by variety (Valencia vs non-Valencia).
Algorithms↗
Pairing-specific long-term depression of Purkinje cell excitatory postsynaptic potentials results from a classical conditioning procedure in the rabbit cerebellar slice.
1. Using a rabbit cerebellar slice preparation, we stimulated a classical conditioning procedure by stimulating parallel fiber inputs to Purkinje cells with the use of a brief, high-frequency train of eight constant-current pulses 80 ms before climbing fiber inputs to the same Purkinje cell were stimulated with the use of a brief, lower frequency train of three constant-current pulses. In all experiments, we assessed the effects of stimulation by measuring the peak amplitude of Purkinje cell excitatory postsynaptic potentials (EPSPs) to single parallel fiber test pulses. 2. Intradendritically recorded Purkinje cell EPSPs underwent a long-term (> 20 min) reduction in peak amplitude (30%) after paired stimulation of the parallel and climbing fibers but not after unpaired or parallel fiber alone stimulation. We call this phenomenon pairing-specific long-term depression (PSD). 3. Facilitation of the peak amplitude of a second EPSP elicited by a parallel fiber train occurred both before and after paired stimulation suggesting that the locus of depression was not presynaptic. Depression of the peak amplitude of a depolarizing response to focal application of glutamate following pairings of parallel and climbing fiber stimulation added support to a suggested postsynaptic locus of the PSD effect. 4. The application of aniracetam potentiated EPSP peak amplitude by 40%, but these values returned to baseline as a result of pairings. With the removal of aniracetam from the bath 20 min after pairings, normal levels of pairing-specific EPSP depression were observed, indicating that the effect did not result from direct desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptors. 5. Incubation of slices in the protein kinase inhibitor H-7 potentiated EPSP peak amplitudes slightly (9%), but peak amplitudes returned to baseline levels after pairings. The net reduction in EPSP peak amplitude of < 10% after pairings suggested that H-7 partially blocked PSD and that, in turn, PSD involved protein kinases. 6. The means of induction and the specificity of those means suggest that the phenomenology of PSD is fundamentally different from that of long-term depression. PSD only occurs with pairings of trains of parallel fiber and climbing fiber stimulation; it occurs without the need for bicuculline; and it can overcome the blocking effects of aniracetam. 7. Nevertheless, the involvement of protein kinases and the potential role of calcium suggest that the mechanisms involved in the induction of PSD and long-term depression have a number of features in common. 8. Because of the pairing-specific nature of the long-term synaptic depression observed in these experiments, PSD provides a mechanism that may contribute to the role of the cerebellar cortex in classical conditioning.
Animals↗