James L. Olds

Lithium Decreases Membrane-Associated Protein Kinase C in Hippocampus: Selectivity for the α Isozyme

We investigated the effects of lithium on alterations in the amount and distribution of protein kinase C (PKC) in discrete areas of rat brain by using [3H]phorbol 12, 13‐dibutyrate quantitative autoradiography as well as western blotting. Chronic administration of lithium resulted in a significant decrease in membrane‐associated PKC in several hippocampal structures, most notably the subiculum and the CA1 region. In contrast, only modest changes in [3H]phorbol 12, 13‐dibutyrate binding were observed in the various other cortical and subcortical structures examined. Immunoblotting using monoclonal anti‐PKC antibodies revealed an isozyme‐specific 30% decrease in hippocampal membrane‐associated PKC α, in the absence of any changes in the labeling of either the β(I/II) or γ isozymes. These changes were observed only after chronic (4 week) treatment with lithium, and not after acute (5 days) treatment, suggesting potential clinical relevance. Given the critical role of PKC in regulating neuronal signal transduction, lithiums effects on PKC in the limbic system represent an attractive molecular mechanism for its efficacy in treating both poles of manic‐depressive illness. In addition, the decreased hippocampal membrane‐associated PKC observed in the present study offers a possible explanation for lithium‐induced memory impairment.

Discrimination learning alters the distribution of protein kinase C in the hippocampus of rats

Protein kinase C (PKC), an enzyme that plays an essential role in eukaryotic cell regulation (Nishizuka, 1988; Huang et al., 1989), is critical to memory storage processes both in the marine snail Hermissenda crassicornis and in the rabbit (Alkon et al., 1988; Bank et al., 1988; Olds et al., 1989). Specifically, activation of PKC mimics neurobiological correlates of classical conditioning in both Hermissenda and the rabbit, and the distribution of the enzyme within the rabbit hippocampus changes after Pavlovian conditioning. Here, we report that the amount of PKC, as assayed by specific binding of 3H- phorbol-12,13-dibutyrate (3H-PDBU), decreased significantly within the hippocampal CA3 cell region in rats trained to solve a water maze task either by cognitive mapping or by visual discrimination strategies, but not in control rats. Furthermore, hippocampal lesions interfered with acquisition of both of these tasks. We interpret these findings to support the conclusion that distributional changes of PKC within the mammalian hippocampus play a crucial role in memory storage processes.

Cell Specificity of Molecular Changes During Memory Storage

Abstract: The aeolid nudibranch, Hermissenda crassicornis, exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C (PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B‐cell photoreceptor. The present study was designed to further examine learning‐specific PKC involvement in identified cellular areas, particularly those in the visual‐vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [3H]phorbol‐12,13‐dibutyrate ([3H]‐PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [3H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [3H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.

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 α-helix and one-fifth in β-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 α-helix and a decrease in β-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.

A sequential double-label autoradiographic method that quantifies altered rates of regional glucose metabolism

An autoradiographic sequential double-label variant of the deoxyglucose method for measurement of local glucose utilization has been developed. This technique takes advantage of the short half-life of the positron emitter, 18F (t 1/2 = 110 min) relative to that of 14C. Sequential injection of [18F]fluorodeoxyglucose (FDG) and 14C-labeled FDG allows the production of two separable autoradiograms, each of which represents the same 20-micron brain slice, but under potentially different cerebral metabolic states. We have used this technique to demonstrate that ibotenic acid-lesioned rat striatum is selectively refractory to the depressing effects of barbiturate anesthesia upon brain glucose utilization. The described method has applicability to the analysis of small changes in regional cerebral metabolism in localized brain regions and represents a solution to the problem of intersubject variability inherent in conventional approaches to the deoxyglucose method.

Protein kinase C in the hippocampus is altered by spatial but not cued discriminations: A component task analysis

The exact role of the mammalian hippocampus in memory formation remains essentially as an unanswered question for cognitive neuroscience. Experiments with humans and with animals indicate that some types of mnemonic associative processes involve hippocampal function while others do not. Support for the spatial processing hypothesis of hippocampal function has stemmed from the impaired performance of rats with hippocampal lesions in tasks that require spatial discriminations, but not cued discriminations. Previous procedures, however, have confounded the interpretation of spatial versus cued discrimination learning with the number and kinds of irrelevant stimuli present in the discrimination. An empirical set of data describing a role of protein kinase C (PKC) in different mnemonic processes is similarly being developed. Recent work has implicated the activation of this serine-threonine kinase in a variety of learning paradigms, as well as long-term potentiation (LTP), a model system for synaptic plasticity which may subserve some types of learning. The present study employs the principles of component task analysis to examine the role of membrane-associated PKC (mPKC) in hippocampal-dependent memory when all factors other than the type of learning were equivalent. The results indicate that hippocampal mPKC is altered by performance in hippocampally-dependent spatial discriminations, but not hippocampally-independent cued discriminations and provide a general experimental procedure to relate neural changes to specific behavioral changes.

Partial reinforcement across trials impairs escape performance but spares place learning in the water maze

We studied the effects of partial reinforcement on escape performance and place learning in the water maze. Rats given 50% reinforcement across trials (i.e. the escape platform was present only on odd trials) were compared to controls given 100% reinforcement (platform present on all trials). Control groups either received 8 or 4 trials per day, which was equal to either the total number of trials (100%-8) or reinforced escapes (100%-4) of the 50% group. Analysis of escape performance (latency) revealed that the 50% group was impaired relative to the 100%-8 group, but not the 100%-4 group, during the first 5 days of acquisition. The 50% group was impaired relative to both control groups on days 6-10 of overtraining. However, analyses of within-trial behavior (target annulus preference and thigmotaxis) on nonreinforced trials suggest that the 50% group did learn the location of the hidden platform (place information), in addition to a wall-based thigmotactic response. By dividing the 60s nonreinforced trials into three 20-s time bins, we were able to detect a significant preference for the target annulus early in the trial (bin 1 of trial 40 and bins 1-2 of trial 80). Further, there was a significant increase in time spent in the periphery of the pool, near the wall, in the last time bin of trial 40. Because the platform was in the middle zone, this behavior competed with a place response. We conclude that across-trial partial reinforcement procedures may promote response competition and mask evidence of place learning in addition to weakening escape performance late in training.

Theta phase precession emerges from a hybrid computational model of a CA3 place cell

The origins and functional significance of theta phase precession in the hippocampus remain obscure, in part, because of the difficulty of reproducing hippocampal place cell firing in experimental settings where the biophysical underpinnings can be examined in detail. The present study concerns a neurobiologically based computational model of the emergence of theta phase precession in which the responses of a single model CA3 pyramidal cell are examined in the context of stimulation by realistic afferent spike trains including those of place cells in entorhinal cortex, dentate gyrus, and other CA3 pyramidal cells. Spike-timing dependent plasticity in the model CA3 pyramidal cell leads to a spatially correlated associational synaptic drive that subsequently creates a spatially asymmetric expansion of the model cell’s place field. Following an initial training period, theta phase precession can be seen in the firing patterns of the model CA3 pyramidal cell. Through selective manipulations of the model it is possible to decompose theta phase precession in CA3 into the separate contributing factors of inheritance from upstream afferents in the dentate gyrus and entorhinal cortex, the interaction of synaptically controlled increasing afferent drive with phasic inhibition, and the theta phase difference between dentate gyrus granule cell and CA3 pyramidal cell activity. In the context of a single CA3 pyramidal cell, the model shows that each of these factors plays a role in theta phase precession within CA3 and suggests that no one single factor offers a complete explanation of the phenomenon. The model also shows parallels between theta phase encoding and pattern completion within the CA3 autoassociative network.

Lateralization of membrane-associated protein kinase C in rat piriform cortex: Specific to operant training cues in the olfactory modality

Rats were trained on an olfactory and a control modality (auditory or visual) discrimination task and brain membrane-associated protein kinase C (mPKC) was subsequently assessed using quantitative autoradiography of radiolabelled phorbol ester binding. In rats which received olfactory-cued training, mPKC showed a highly significant lateralization in the piriform cortex but not in the hippocampus. Both olfactory-trained rats and control modality rats showed a significant increase in mPKC in the hippocampus when compared to naive rats. Thus, while behavioral training procedures appeared to result in a hippocampal increase in the activated state of this enzyme as has been reported elsewhere, only olfactory learning produced an piriform cortex lateralization in the activated state of the enzyme. While the functional significance of such a change in the distribution of protein kinase C is still unclear, it does suggest that the monitoring of this enzymes activational state may prove to be a useful tool in the study of memory formation in a wide variety of behavioral contexts.

The Interfluence of Neuroscience, Neuroethics, and Legal and Social Issues: The Need for (N)ELSI

until recently. But upstream research may be a way for social science or ethics investigations to have more policy-relevant impact. The National Science Foundation-funded Center for Nanotechnology in Society at Arizona State University, for example, emphasizes the integration of social science, humanities, and bioethics research methods and questions in the earliest stages of nanotech research and development (Fisher 2005). In this way, the societal end of nanotechnology development is also the beginning of the scientific process, and is built into the nature of the final product itself. This sort of integration depends on the willingness of scientists to collaborate with nonscientists, but funding mechanisms can provide incentives to scientists to participate in such collaborations. In neuroscience there is already goodwill toward research on societal impact, given the work of the US Neuroethics Society and public engagement activity funders such as the Dana Center. How should ELSI-L Neuroscience be managed and structured? I don’t see any obvious reason why there should be one centralized mechanism, such as the ELSI HGP, to oversee this program of research. To ensure that there is interaction across the four streams of research, and to support collaborations between scientists and nonscientists, I like the idea of multidisciplinary centers, in which principal investigators build a research group and agenda around each stream, ensuring a balance of methodological and theoretical orientations. A center model does not guarantee interdisciplinarity or good outcomes, but here too we can learn from the genomics precedent. Genomics centers, at least in the United Kingdom, have not delivered the high level of research and impact that was hoped for. This is due in part to the nature of the genomics beast, which has had far less societal impact than was expected when the genomics centers were set up. There have also been the perennial problems of translating social science-speak into policy-speak, and building productive, mutually beneficial, multidisciplinary collaborations. These are not easy tasks, but they can be fruitfully addressed now that we know so much more about the pitfalls of the collaborative process. My own vision for ELSI-L Neuroscience is that it integrate the political and the public health dimensions of neuroscience within a global field, thereby radically growing the scope of neuroethics research and interest. The ELSI-L agenda should include investigating the possibilities that neuroscience creates for tackling pernicious social and ethical problems across diverse national contexts and social conditions.