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Studies the role of behavioral and environmental interactions in the emergence, development and adjustment of complex cortical response. Essentials for the development of translation invariance; Characteristics of visual responses; Enhancement of the neuronal responses.

Recent evidence suggests that neurons in primary sensory cortex arrange into competitive groups, representing stimuli by their joint activity rather than as independent feature analysers. A possible explanation for these results is that sensory cortex implements attractor dynamics, although this proposal remains controversial. Here we report that fast attractor dynamics emerge naturally in a computational model of a patch of primary visual cortex endowed with realistic plasticity (at both feedforward and lateral synapses) and mutual inhibition. When exposed to natural images (but not random pixels), the model spontaneously arranges into competitive groups of reciprocally connected, similarly tuned neurons, while developing realistic, orientation-selective receptive fields. Importantly, the same groups are observed in both stimulus-evoked and spontaneous (stimulus-absent) activity. The resulting network is inhibition-stabilized and exhibits fast, non-persistent attractor dynamics. Our results suggest that realistic plasticity, mutual inhibition and natural stimuli are jointly necessary and sufficient to generate attractor dynamics in primary sensory cortex.

Mental imagery occurs "when a representation of the type created during the initial phases of perception is present but the stimulus is not actually being perceived." How does the capability to perform mental imagery arise? Extending the idea that imagery arises from learned associations, we propose that mental rotation, a specific form of imagery, could arise through the mechanism of sequence learning-that is, by learning to regenerate the sequence of mental images perceived while passively observing a rotating object. To demonstrate the feasibility of this proposal, we constructed a simulated nervous system and embedded it within a behaving humanoid robot. By observing a rotating object, the system learns the sequence of neural activity patterns generated by the visual system in response to the object. After learning, it can internally regenerate a similar sequence of neural activations upon briefly viewing the static object. This system learns to perform a mental rotation task in which the subject must determine whether two objects are identical despite differences in orientation. As with human subjects, the time taken to respond is proportional to the angular difference between the two stimuli. Moreover, as reported in humans, the system fills in intermediate angles during the task, and this putative mental rotation activates the same pathways that are activated when the system views physical rotation. This work supports the proposal that mental rotation arises through sequence learning and the idea that mental imagery aids perception through learned associations, and suggests testable predictions for biological experiments., Competing Interests: The authors have declared that no competing interests exist.

Reentry in nervous systems is the ongoing bidirectional exchange of signals along reciprocal axonal fibers linking two or more brain areas. The hypothesis that reentrant signaling serves as a general mechanism to couple the functioning of multiple areas of the cerebral cortex and thalamus was first proposed in 1977 and 1978 (Edelman, 1978). A review of the amount and diversity of supporting experimental evidence accumulated since then suggests that reentry is among the most important integrative mechanisms in vertebrate brains (Edelman, 1993). Moreover, these data prompt testable hypotheses regarding mechanisms that favor the development and evolution of reentrant neural architectures.

Animal behavior often involves a temporally ordered sequence of actions learned from experience. Here we describe simulations of interconnected networks of spiking neurons that learn to generate patterns of activity in correct temporal order. The simulation consists of large-scale networks of thousands of excitatory and inhibitory neurons that exhibit short-term synaptic plasticity and spike-timing dependent synaptic plasticity. The neural architecture within each area is arranged to evoke winner-take-all (WTA) patterns of neural activity that persist for tens of milliseconds. In order to generate and switch between consecutive firing patterns in correct temporal order, a reentrant exchange of signals between these areas was necessary. To demonstrate the capacity of this arrangement, we used the simulation to train a brain-based device responding to visual input by autonomously generating temporal sequences of motor actions.

In Alzheimer disease, elevated levels of the BACE1 enzyme are correlated with increased production of amyloid peptides and disease pathology. The increase in BACE1 levels is post-transcriptional and may involve altered translation efficiency. Earlier studies have indicated that translation of BACE1 mRNA is cap-dependent. As ribosomal subunits move from the cap-structure to the initiation codon, they fail to recognize several AUG codons in the 5' leader. In this study, we looked for physical evidence of the mechanism underlying ribosomal scanning or shunting along the BACE1 5' leader by investigating structural stability in the 5' leaders of endogenous mRNAs in vivo. To perform this analysis, we probed RNAs using lead(II) acetate, a cell-permeable chemical that induces cleavage of unpaired nucleotides having conformational flexibility. The data revealed that the ≈440-nt 5' leader was generally resistant to cleavage except for a region upstream of the initiation codon. Cleavage continued into the coding region, consistent with destabilization of secondary structures by translating ribosomes. Evidence that a large segment of the BACE1 5' leader was not cleaved indicates that this region is structurally stable and suggests that it is not scanned. The data support a mechanism of translation initiation in which ribosomal subunits bypass (shunt) part of the BACE1 5' leader to reach the initiation codon. We suggest that a nucleotide bias in the 5' leader may predispose the initiation codon to be more accessible than other AUG codons in the 5' leader, leading to an increase in its relative utilization.

We describe simulations of large-scale networks of excitatory and inhibitory spiking neurons that can generate dynamically stable winner-take-all (WTA) behavior. The network connectivity is a variant of center-surround architecture that we call center-annular-surround (CAS). In this architecture each neuron is excited by nearby neighbors and inhibited by more distant neighbors in an annular-surround region. The neural units of these networks simulate conductance-based spiking neurons that interact via mechanisms susceptible to both short-term synaptic plasticity and STDP. We show that such CAS networks display robust WTA behavior unlike the center-surround networks and other control architectures that we have studied. We find that a large-scale network of spiking neurons with separate populations of excitatory and inhibitory neurons can give rise to smooth maps of sensory input. In addition, we show that a humanoid brain-based-device (BBD) under the control of a spiking WTA neural network can learn to reach to target positions in its visual field, thus demonstrating the acquisition of sensorimotor coordination.

The Dynamic Core and Global Workspace hypotheses were independently put forward to provide mechanistic and biologically plausible accounts of how brains generate conscious mental content. The Dynamic Core proposes that reentrant neural activity in the thalamocortical system gives rise to conscious experience. Global Workspace reconciles the limited capacity of momentary conscious content with the vast repertoire of long-term memory. In this paper we show the close relationship between the two hypotheses. This relationship allows for a strictly biological account of phenomenal experience and subjectivity that is consistent with mounting experimental evidence. We examine the constraints on causal analyses of consciousness and suggest that there is now sufficient evidence to consider the design and construction of a conscious artifact.

mRNA-binding proteins are critical regulators of protein synthesis during neural development. We demonstrated previously that the cold-inducible mRNA-binding protein 3 (RBM3) is present within euthermic neurons and that it enhances translation. Other studies have attributed anti-apoptotic and proliferative functions to RBM3. Here we characterize the developmental expression of RBM3 in rat brain. RBM3 is expressed widely during early brain development, peaking in the first to second postnatal weeks. This is followed by a decline in most brain regions and a shift from a nuclear to a more somatodendritic distribution by approximately P13. The highest levels of RBM3 in adult brain were observed in the cerebellum, olfactory bulb, proliferating cell fields and other regions reported to have high translation rates. RBM3 was expressed in glutamatergic and GABAergic cells, subtypes of which exhibited strong dendritic labeling for RBM3 mRNA and protein. Expression of RBM3 was also high in newly formed and migrating neurons marked by Ki67, nestin, and doublecortin, such as those in the subventricular zone and rostral migratory stream. These results indicate that expression of RBM3, a cold stress-responsive mRNA-binding protein, is dynamically regulated in the developing brain and suggest that it contributes to translation-dependent processes underlying proliferation, differentiation, and plasticity.

Epidemiological studies indicate that caffeine consumption reduces the risk of Parkinson's disease (PD) in men, and antagonists of the adenosine 2A receptor ameliorate the motor symptoms of PD. These findings motivated us to identify proteins whose expression is regulated by caffeine in a sexually dimorphic manner. Using mass spectroscopy, we found that Cox7c, a nuclear-encoded subunit of the mitochondrial enzyme cytochrome oxidase, is up-regulated in the striatum of male but not female mice after receiving a single dose of caffeine. The expression of two other Cox subunits, Cox1 and Cox4, was also stimulated by caffeine in a male-specific fashion. This up-regulation of Cox subunits by caffeine was accompanied by an increase in Cox enzyme activity in the male striatum. Caffeine-induced stimulation of Cox expression and activity were reproduced using the adenosine 2A receptor (A2AR)-specific antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-epsilon]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261), and coadministration of the A2AR-specific agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) counteracted the elevation of Cox expression and activity by caffeine. Caffeine also increased Cox activity in PC-12 cells. In contrast, small interfering RNA (siRNA) knockdown of Cox7c expression in PC-12 cells blunted Cox activity, and this was counteracted by caffeine treatment. Caffeine was also found to increase Cox7c mRNA expression in the striatum and in PC-12 cells. This occurred at the level of transcription and was mediated by a segment of the Cox7c promoter. Overall, these findings indicate that cytochrome oxidase is a metabolic target of caffeine and that stimulation of Cox activity by caffeine via blockade of A2AR signaling may be an important mechanism underlying the therapeutic benefits of caffeine in PD.

In order to respond appropriately to environmental stimuli, organisms must integrate over time spatiotemporal signals that reflect object motion and self-movement. One possible mechanism to achieve this spatiotemporal transformation is to delay or lag neural responses. This paper reviews our recent modeling work testing the sufficiency of delayed responses in the nervous system in two different behavioral tasks: (1) Categorizing spatiotemporal tactile cues with thalamic "lag" cells and downstream coincidence detectors, and (2) Predictive motor control was achieved by the cerebellum through a delayed eligibility trace rule at cerebellar synapses. Since the timing of these neural signals must closely match real-world dynamics, we tested these ideas using the brain based device (BBD) approach in which a simulated nervous system is embodied in a robotic device. In both tasks, biologically inspired neural simulations with delayed neural responses were critical for successful behavior by the device.

The understanding of the structural and dynamic complexity of mammalian brains is greatly facilitated by computer simulations. We present here a detailed large-scale thalamocortical model based on experimental measures in several mammalian species. The model spans three anatomical scales. (i) It is based on global (white-matter) thalamocortical anatomy obtained by means of diffusion tensor imaging (DTI) of a human brain. (ii) It includes multiple thalamic nuclei and six-layered cortical microcircuitry based on in vitro labeling and three-dimensional reconstruction of single neurons of cat visual cortex. (iii) It has 22 basic types of neurons with appropriate laminar distribution of their branching dendritic trees. The model simulates one million multicompartmental spiking neurons calibrated to reproduce known types of responses recorded in vitro in rats. It has almost half a billion synapses with appropriate receptor kinetics, short-term plasticity, and long-term dendritic spike-timing-dependent synaptic plasticity (dendritic STDP). The model exhibits behavioral regimes of normal brain activity that were not explicitly built-in but emerged spontaneously as the result of interactions among anatomical and dynamic processes. We describe spontaneous activity, sensitivity to changes in individual neurons, emergence of waves and rhythms, and functional connectivity on different scales.

Biologically based mobile devices have been constructed that differ from robots based on artificial intelligence. These brain-based devices (BBDs) contain simulated brains that autonomously categorize signals from the environment without a priori instruction. Two such BBDs, Darwin VII and Darwin X, are described here. Darwin VII recognizes objects and links categories to behavior through instrumental conditioning. Darwin X puts together the "what,""when," and "where" from cues in the environment into an episodic memory that allows it to find a desired target. Although these BBDs are designed to provide insights into how the brain works, their principles may find uses in building hybrid machines. These machines would combine the learning ability of BBDs with explicitly programmed control systems.

The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it.

We describe a theoretical network analysis that can distinguish statistically causal interactions in population neural activity leading to a specific output. We introduce the concept of a causal core to refer to the set of neuronal interactions that are causally significant for the output, as assessed by Granger causality. Because our approach requires extensive knowledge of neuronal connectivity and dynamics, an illustrative example is provided by analysis of Darwin X, a brain-based device that allows precise recording of the activity of neuronal units during behavior. In Darwin X, a simulated neuronal model of the hippocampus and surrounding cortical areas supports learning of a spatial navigation task in a real environment. Analysis of Darwin X reveals that large repertoires of neuronal interactions contain comparatively small causal cores and that these causal cores become smaller during learning, a finding that may reflect the selection of specific causal pathways from diverse neuronal repertoires.

The brain-derived neurotrophic factor (BDNF) plays an important role in neuronal development, and in the formation and plasticity of synaptic connections. These effects of BDNF are at least partially due to the ability of the neurotrophin to increase protein synthesis both globally and locally. However, only a few proteins have been shown to be up-regulated at the synapse by BDNF. Using multidimensional protein identification technology (MudPIT) and relative quantification by spectra counting, we found that several hundred proteins are up-regulated in a synaptoneurosome preparation derived from cultured cortical neurons that were treated with BDNF. These proteins fall into diverse functional categories, including those involved in synaptic vesicle formation and movement, maintenance or remodeling of synaptic structure, mRNA processing, transcription, and translation. A number of translation factors, ribosomal proteins, and tRNA synthetases were rapidly up-regulated by BDNF. This up-regulation of translation components was sensitive to protein synthesis inhibitors and dependent on the activation of the mammalian target of rapamycin (mTOR), a regulator of cap-dependent mRNA translation. The presence of a subset of these proteins and their mRNAs in neuronal processes was corroborated by immunocytochemistry and in situ hybridization, and their up-regulation was confirmed by Western blotting. The data demonstrate that BDNF increases the synthesis of a wide variety of synaptic proteins and suggest that the neurotrophin may enhance the translational capacity of synapses.

Recent recordings of place field activity in rodent hippocampus have revealed correlates of current, recent past, and imminent future events in spatial memory tasks. To analyze these properties, we used a brain-based device, Darwin XI, that incorporated a detailed model of medial temporal structures shaped by experience-dependent synaptic activity. Darwin XI was tested on a plus maze in which it approached a goal arm from different start arms. In the task, a journey corresponded to the route from a particular starting point to a particular goal. During maze navigation, the device developed place-dependent responses in its simulated hippocampus. Journey-dependent place fields, whose activity differed in different journeys through the same maze arm, were found in the recordings of simulated CA1 neuronal units. We also found an approximately equal number of journey-independent place fields. The journey-dependent responses were either retrospective, where activity was present in the goal arm, or prospective, where activity was present in the start arm. Detailed analysis of network dynamics of the neural simulation during behavior revealed that many different neural pathways could stimulate any single CA1 unit. That analysis also revealed that place activity was driven more by hippocampal and entorhinal cortical influences than by sensory cortical input. Moreover, journey-dependent activity was driven more strongly by hippocampal influence than journey-independent activity.

Eukaryotic mRNAs often recruit ribosomal subunits some distance upstream of the initiation codon; however, the mechanisms by which they reach the initiation codon remain to be fully elucidated. Although scanning is a widely accepted model, evidence for alternative mechanisms has accumulated. We previously suggested that this process may involve tethering of ribosomal complexes to the mRNA, in which the intervening mRNA is bypassed, or clustering, in which the initiation codon is reached by dynamic binding and release of ribosomal subunits at internal sites. The present studies tested the feasibility of these ideas by using model mRNAs and revealed that translation efficiency varied with the distance between the site of ribosomal recruitment and the initiation codon. The present studies also showed that translation could initiate efficiently at AUG codons located upstream of an internal site. These observations are consistent with ribosomal tethering at the cap structure and clustering at internal sites.

We studied whether magnetoencephalography (MEG) could detect deceptive responses on a single-subject, trial-by-trial basis. To elicit spontaneous, ecologically valid deception, we developed a paradigm in which subjects in a simulated customs setting were presented with a series of pictures of items which might be in their baggage, and for each item, they decided whether to "declare" (tell the truth) or "smuggle" (lie). Telling the truth involved a small but certain monetary penalty, whereas lying involved both greater monetary risk and greater potential reward. Most subjects showed decreased signal power in the 8-12 Hz (alpha) range during deceptive responses as compared to truthful responses. In a cross-validation analysis, we were able to use alpha power to classify truthful and deceptive responses on a trial-by-trial basis, with significantly greater predictive accuracy than that achieved using simultaneously recorded skin conductance signals. Average predictive accuracy for spontaneous deception was greater than 78%, and for some subjects, predictive accuracy exceeded 90%. Our results raise the possibility that alpha power modulation during deception may reflect risk management and/or cognitive control.

A recent theoretical emphasis on complex interactions within neural systems underlying consciousness has been accompanied by proposals for the quantitative characterization of these interactions. In this article, we distinguish key aspects of consciousness that are amenable to quantitative measurement from those that are not. We carry out a formal analysis of the strengths and limitations of three quantitative measures of dynamical complexity in the neural systems underlying consciousness: neural complexity, information integration, and causal density. We find that no single measure fully captures the multidimensional complexity of these systems, and all of these measures have practical limitations. Our analysis suggests guidelines for the specification of alternative measures which, in combination, may improve the quantitative characterization of conscious neural systems. Given that some aspects of consciousness are likely to resist quantification altogether, we conclude that a satisfactory theory is likely to be one that combines both qualitative and quantitative elements.

In eukaryotes, 40S ribosomal subunits move from their recruitment site on the mRNA to the initiation codon by an as yet poorly understood process. One postulated mechanism involves ribosomal shunting, in which ribosomal subunits completely bypass regions of the 5' leader. For some mRNAs, shunting has been shown to require various mRNA elements, some of which are thought to base pair to 18S rRNA; however, the role of base pairing has not yet been tested directly. In earlier studies, we demonstrated that a short mRNA element in the 5' leader of the Gtx homeodomain mRNA functioned as a ribosomal recruitment site by base pairing to the 18S rRNA. Using a model system to assess translation in transfected cells, we now show that this intermolecular interaction also facilitates ribosomal shunting across two types of obstacles: an upstream AUG codon in excellent context or a stable hairpin structure. Highly efficient shunting occurred when multiple Gtx elements were present upstream of the obstacles, and a single Gtx element was present downstream. Shunting was less efficient, however, when the multiple Gtx elements were present only upstream of the obstacles. In addition, control experiments with mRNAs lacking the upstream elements showed that these results could not be attributed to recruitment by the single downstream element. Experiments in yeast in which the mRNA elements and 18S rRNA sequences were both mutated indicated that shunting required an intact complementary match. The data obtained by this model system provide direct evidence that ribosomal shunting can be mediated by mRNA-rRNA base pairing, a finding that may have general implications for mechanisms of ribosome movement.

The cerebellum is known to be critical for accurate adaptive control and motor learning. We propose here a mechanism by which the cerebellum may replace reflex control with predictive control. This mechanism is embedded in a learning rule (the delayed eligibility trace rule) in which synapses onto a Purkinje cell or onto a cell in the deep cerebellar nuclei become eligible for plasticity only after a fixed delay from the onset of suprathreshold presynaptic activity. To investigate the proposal that the cerebellum is a general-purpose predictive controller guided by a delayed eligibility trace rule, a computer model based on the anatomy and dynamics of the cerebellum was constructed. It contained components simulating cerebellar cortex and deep cerebellar nuclei, and it received input from a middle temporal visual area and the inferior olive. The model was incorporated in a real-world brain-based device (BBD) built on a Segway robotic platform that learned to traverse curved paths. The BBD learned which visual motion cues predicted impending collisions and used this experience to avoid path boundaries. During learning, the BBD adapted its velocity and turning rate to successfully traverse various curved paths. By examining neuronal activity and synaptic changes during this behavior, we found that the cerebellar circuit selectively responded to motion cues in specific receptive fields of simulated middle temporal visual areas. The system described here prompts several hypotheses about the relationship between perception and motor control and may be useful in the development of general-purpose motor learning systems for machines.

Fragile X syndrome (FXS) is caused by the transcriptional silencing of the Fmr1 gene, which encodes a protein (FMRP) that can act as a translational suppressor in dendrites, and is characterized by a preponderance of abnormally long, thin and tortuous dendritic spines. According to a current theory of FXS, the loss of FMRP expression leads to an exaggeration of translation responses linked to group I metabotropic glutamate receptors. Such responses are involved in the consolidation of a form of long-term depression that is enhanced in Fmr1 knockout mice and in the elongation of dendritic spines, resembling synaptic phenotypes over-represented in fragile X brain. These observations place fragile X research at the heart of a long-standing issue in neuroscience. The consolidation of memory, and several distinct forms of synaptic plasticity considered to be substrates of memory, requires mRNA translation and is associated with changes in spine morphology. A recent convergence of research on FXS and on the involvement of translation in various forms of synaptic plasticity has been very informative on this issue and on mechanisms underlying FXS. Evidence suggests a general relationship in which the receptors that induce distinct forms of efficacy change differentially regulate translation to produce unique spine shapes involved in their consolidation. We discuss several potential mechanisms for differential translation and the notion that FXS represents an exaggeration of one 'channel' in a set of translation-dependent consolidation responses.

In earlier studies, we identified short (6- to 22-nt) sequences that functioned as internal ribosome entry sites (IRESes) and enhanced translation. The size of these IRES elements suggested that they might be prevalent within the messenger population and that individual elements might affect the translation of different groups of mRNAs. To begin to assess the number of different IRES elements in mammalian cells, we have developed a powerful method that uses a positive feedback mechanism to amplify the activities of individual IRES elements. This method uses a vector that encodes a dicistronic mRNA with a reporter gene (Renilla luciferase or the EGFP) as the first cistron and the yeast Gal4/viral protein 16 (VP16) transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing four copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4/VP16, which binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4/VP16. A corresponding increase in the translation of the first cistron (luciferase or EGFP) is monitored either by measuring luciferase activity or by using FACS. The latter enables IRES-positive cells to be isolated. We present tests of the feedback mechanism by using an IRES module from Gtx homeodomain mRNA and an IRES from hepatitis C virus and demonstrate the utility of this vector system for the screening, identification, and analysis of IRES elements.

Analyzing neural dynamics underlying complex behavior is a major challenge in systems neurobiology. To meet this challenge through computational neuroscience, we have constructed a brain-based device (Darwin X) that interacts with a real environment, and whose behavior is guided by a simulated nervous system incorporating detailed aspects of the anatomy and physiology of the hippocampus and its surrounding regions. Darwin X integrates cues from its environment to solve a spatial memory task. Place-specific units, similar to place cells in the rodent, emerged by integrating visual and self-movement cues during exploration without prior assumptions in the model about environmental inputs. Because synthetic neural modeling using brain-based devices allows recording from all elements of the simulated nervous system during behavior, we were able to identify different functional hippocampal pathways. We did this by tracing back from reference neuronal units in the CA1 region of the simulated hippocampus to all of the synaptically connected units that were coactive during a particular exploratory behavior. Our analysis identified a number of different functional pathways within the simulated hippocampus that incorporate either the perforant path or the trisynaptic loop. Place fields, which were activated by the trisynaptic circuit, tended to be more selective and informative. However, place units that were activated by the perforant path were prevalent in the model and were crucial for generating appropriate exploratory behavior. Thus, in the model, different functional pathways influence place field activity and, hence, behavior during navigation.

The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

Fragile X syndrome results from the transcriptional silencing of a gene, Fmr1, that codes for an mRNA-binding protein (fragile X mental retardation protein, FMRP) present in neuronal dendrites. FMRP can act as a translational suppressor, and its own translation in dendrites is regulated by group I metabotropic glutamate receptors (mGluRs). Multiple lines of evidence suggest that mGluR-induced translation is exaggerated in Fragile X syndrome because of a lack of translational inhibition normally provided by FMRP. We characterized the role of FMRP in the regulation of mRNA granules, which sediment as a heavy peak after polysomes on sucrose gradients. In WT mouse brain, FMRP distributed with polysomes and granules. EM and biochemical analyses suggested that the granule fraction itself contained clusters of polysomes. In Fmr1 knockout brain, we observed a significant decrease in the amount of mRNA granules relative to WT mice. This difference appeared to be due to a role of FMRP in regulating the activation of granules during mGluR-induced translation; in vivo administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine increased granule content in Fmr1 knockout mouse brain to levels comparable with those seen in WT brain. In accord with a role of mGluR5 in the regulation of ongoing translation in vivo, we observed that the phosphorylation of several initiation factors in response to application of the mGluR1/5 agonist S-3,5-dihydroxyphenylglycine in vitro was blocked by methyl-6-(phenylethynyl)pyridine. Together, these data suggest that although large, polysome-containing granules can form in the absence of FMRP, their use in response to mGluR-induced translation is exaggerated.

We describe Darwin X, a physical device that interacts with a real environment, whose behavior is guided by a simulated nervous system incorporating aspects of the detailed anatomy and physiology of the hippocampus and its surrounding regions. This brain-based device integrates cues from its environment and solves a spatial memory task. The responses of simulated neuronal units in the hippocampal areas during its exploratory behavior are comparable to place cells in the rodent hippocampus and emerged by associating sensory cues during exploration. To identify different functional hippocampal pathways and their influence on behavior, we employed a time series analysis that distinguishes causal interactions within and between simulated hippocampal and neocortical regions while the device is engaged in a spatial memory task. Our analysis identified different functional pathways within the neural simulation and prompts novel predictions about the influence of the perforant path, the trisynaptic loop and hippocampal-cortical interactions on place cell activity and behavior during navigation. Moreover, this causal time series analysis may be useful in analyzing networks in general.

The simultaneous study of brain function at all levels of organization is difficult to undertake with current experimental tools. Present day electrophysiology only allows the recording of at most hundreds of neurons while an animal is performing a behavioral task. Because of this limitation and the sheer complexity of the nervous system, computational modeling has become essential in developing theories of brain function. Accordingly, our group has constructed a series of brain-based devices (BBDs), that is, physical devices with simulated nervous systems that guide behavior, to serve as a heuristic for testing theories of brain function. Unlike animal models, BBDs permit analysis of activity at all levels of the nervous system as the device behaves in its environment. Although the principal focus of developing BBDs has been to test theories of brain function, this type of modeling may also provide a basis for robotic design and practical applications.

The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.

Effective visual object recognition requires mechanisms to bind object features (e.g. color, shape and motion) while distinguishing distinct objects. Synchronously active neuronal circuits among reentrantly connected cortical areas may provide a basis for visual binding. To assess the potential of this mechanism, we have constructed a mobile brain-based device, Darwin VIII, which is guided by simulated analogues of cortical and sub-cortical areas required for visual processing, decision-making, reward and motor responses. These simulated areas are reentrantly connected and each area contains neuronal units representing both the mean activity level and the relative timing of the activity of groups of neurons. Darwin VIII learns to discriminate among multiple objects with shared visual features and associates 'target' objects with innately preferred auditory cues. We observed the co-activation of globally distributed neuronal circuits that corresponded to distinct objects in Darwin VIII's visual field. These circuits, which are constrained by a reentrant neuroanatomy and modulated by behavior and synaptic plasticity, are necessary for successful discrimination. By situating Darwin VIII in a rich real-world environment involving continual changes in the size and location of visual stimuli due to self-generated movement, and by recording its behavioral and neuronal responses in detail, we were able to show that reentrant connectivity and dynamic synchronization provide an effective mechanism for binding the features of visual objects.

A neuronal network inspired by the anatomy of the cerebral cortex was simulated to study the self-organization of spiking neurons into neuronal groups. The network consisted of 100 000 reentrantly interconnected neurons exhibiting known types of cortical firing patterns, receptor kinetics, short-term plasticity and long-term spike-timing-dependent plasticity (STDP), as well as a distribution of axonal conduction delays. The dynamics of the network allowed us to study the fine temporal structure of emerging firing patterns with millisecond resolution. We found that the interplay between STDP and conduction delays gave rise to the spontaneous formation of neuronal groups--sets of strongly connected neurons capable of firing time-locked, although not necessarily synchronous, spikes. Despite the noise present in the model, such groups repeatedly generated patterns of activity with millisecond spike-timing precision. Exploration of the model allowed us to characterize various group properties, including spatial distribution, size, growth, rate of birth, lifespan, and persistence in the presence of synaptic turnover. Localized coherent input resulted in shifts of receptive and projective fields in the model similar to those observed in vivo.

Sleep is a ubiquitous component of animal life, and prolonged sleep deprivation is fatal in both vertebrates and invertebrates. The physiologic function of sleep, however, is not known. We propose here that sleep provides a period of time necessary to reapportion resources within neurons and neural systems that become sub-optimally distributed during active waking. Three specific examples of such reapportionment during sleep are suggested: (1) the return of the neurotransmitter, glutamate, to synaptic vesicles at presynaptic sites most active during waking, (2) the intracellular movement of mitochondria from neuronal processes to the cells soma where mitochondrial replication can occur, and (3) the readjustment of the level and distribution of neurotransmitters within the brainstem modulatory systems and elsewhere that must function in an integrated fashion during waking. Experimental approaches that might be utilized to test these hypotheses are suggested.

The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.

We previously identified an internal ribosome entry site (IRES) within the 5' leader of the mRNA encoding the Gtx homeodomain protein and showed that shorter nonoverlapping segments of this 5' leader could enhance the translation of a second cistron in a dicistronic mRNA. One of these segments was 9 nt in length, and when multiple copies of this IRES module were linked together, IRES activity was greatly enhanced. To further expand the potential uses of these synthetic constructs and facilitate analyses of the mechanism by which they affect translation, we show here that an IRES containing five linked copies of the 9-nt sequence can also enhance translation in the 5' leader of a monocistronic mRNA. Moreover, a search for interactions of the IRES module with cellular factors revealed specific binding to 40S ribosomal subunits but not to other cellular components. Based on the results of earlier studies suggesting that this sequence could bind to a complementary segment of 18S rRNA, we tested various sequences for possible links between the length of the complementary match, their binding to ribosomes, and their influence on translational efficiency. We found that the length of the complementary match was directly correlated with the ability of RNA probes to bind to ribosomes. In addition, translation was maximally enhanced ( approximately 8-fold) by a 7-nt segment of the 9-nt element; the enhancement declined progressively as the complementary stretches became progressively longer or shorter. The results suggest that the Gtx 9-nt sequence affects translation efficiency by a mechanism that involves base pairing to 18S rRNA.

We previously reported that the 5' leader of the mRNA-encoding initiation factor eIF4G in Saccharomyces cerevisiae can function as a translational enhancer and as an internal ribosome entry site (IRES) when tested in cells. However, Verge and colleagues recently suggested that this sequence does not facilitate translation initiation, but inhibits translation in vitro and has promoter activity when tested in cells. We disagree with these conclusions and respond by showing that the data are most consistent with an internal initiation mechanism.

beta-Secretase [also known as the beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1)] is an enzyme involved in the production of A beta-amyloid plaques in the brains of patients with Alzheimer's disease. The enhanced production of this enzyme occurs without corresponding changes in BACE1 mRNA levels. The complex 5' leader of BACE1 mRNA contains three upstream ORFs (uORFs) preceding the BACE1 initiation codon. In this study, we investigated how this 5' leader affects translation efficiency as a first step in understanding the enhanced production of the enzyme in the disease. Using reporter constructs in transfected mammalian cell lines and cell-free lysates, we showed that the translation mediated by the BACE1 5' leader is cap-dependent and inhibited by cis-acting segments contained within the 5' leader. Disruption of the uORFs had no effect on translation in B104 cells, which was surprising because the first two AUGs reside in contexts able to function as initiation codons. Possible mechanisms to explain how ribosomes bypass the uORFs, including reinitiation, leaky scanning, and internal initiation of translation were found to be inconsistent with the data. The data are most consistent with a model in which ribosomes shunt uORF-containing segments of the 5' leader as the ribosomes move from the 5' end of the mRNA to the initiation codon. In PC12 cells, however, the second uORF appears to be translated. We hypothesize that the translation efficiency of the BACE1 initiation codon may be increased in patients with Alzheimer's disease by molecular mechanisms that enhance shunting or increase the relative accessibility the BACE1 initiation codon.