Motor Sequence Learning in the Brain: The Long and Short of It.

Motor sequence learning involves predictive processing that results in the anticipation of each component of a sequence of actions. In smooth pursuit, this predictive processing is required to decrease tracking errors between the eye and the stimulus. Current models for motor sequence learning suggest parallel mechanisms in the brain for acquiring sequences of differing complexity. We examined this model by comparing shorter versus longer sequences of pursuit eye movements during fMRI. In this way we were able to identify overlapping and distinct brain areas involved in simple versus more complex oculomotor learning. Participants revealed predictive pursuit eye movements from the second presentation of the stimulus in both short and long sequences. Brain imaging results indicated activation of parallel brain areas for the different sequence lengths that consisted of the Inferior Occipital Gyrus and the Cingulate as areas in common. In addition, distinct activation was found in more working memory related brain regions for the shorter sequences (e.g. the middle frontal cortex and dorsolateral prefrontal cortex), and higher activation in the frontal eye fields, supplementary motor cortex and motor cortex for the longer sequences, independent on the number of repetitions. These findings provide new evidence that there are parallel brain areas that involve working memory circuitry for short sequences, and more motoric areas when the sequence is longer and more cognitively demanding. Additionally, our findings are the first to show that the parallel brain regions involved in sequence learning in pursuit are independent of the number of repetitions, but contingent on sequence complexity.

Dissociation of the rostral and dorsolateral prefrontal cortex during sequence learning in saccades: a TMS investigation.

This experiment sought to find whether differences exist between the dorsolateral prefrontal cortex (DLPFC) and the medial rostral prefrontal cortex (MRPFC) for performing stimulus-independent and stimulus-oriented tasks, respectively. To find a causal relationship in these areas, we employed the use of trans-cranial magnetic stimulation (TMS). Prefrontal areas were stimulated whilst participants performed random or predictable sequence learning tasks at stimulus onset (1st presentation of the sequence only for both Random and Predictable), or during the inter-sequence interval. Overall, we found that during the predictable task a significant decrease in saccade latency, gain and duration was found when compared to the randomised conditions, as expected and observed previously. However, TMS stimulation in DLPFC during the delay in the predictive sequence learning task reduced this predictive ability by delaying the saccadic onset and generating abnormal reductions in saccadic gains during prediction. In contrast, we found that stimulation during a delay in MRPFC reversed the normal effects on peak velocity of the task with the predictive task revealing higher peak velocity than the randomised task. These findings provide causal evidence for independent functions of DLPFC and MRPFC in performing stimulus-independent processing during sequence learning in saccades.

The contribution of the right supra-marginal gyrus to sequence learning in eye movements.

We investigated the role of the human right Supra-Marginal Gyrus (SMG) in the generation of learned eye movement sequences. Using MRI-guided transcranial magnetic stimulation (TMS) we disrupted neural activity in the SMG whilst human observers performed saccadic eye movements to multiple presentations of either predictable or random target sequences. For the predictable sequences we observed shorter saccadic latencies from the second presentation of the sequence. However, these anticipatory improvements in performance were significantly reduced when TMS was delivered to the right SMG during the inter-trial retention periods. No deficits were induced when TMS was delivered concurrently with the onset of the target visual stimuli. For the random version of the task, neither delivery of TMS to the SMG during the inter-trial period nor during the presentation of the target visual stimuli produced any deficit in performance that was significantly different from the no-TMS or control conditions. These findings demonstrate that neural activity within the right SMG is causally linked to the ability to perform short latency predictive saccades resulting from sequence learning. We conclude that neural activity in rSMG constitutes an instruction set with spatial and temporal directives that are retained and subsequently released for predictive motor planning and responses.

The brain uses efference copy information to optimise spatial memory.

Does a motor response to a target improve the subsequent recall of the target position or can we simply use peripheral position information to guide an accurate response? We suggest that a motor plan of the hand can be enhanced with actual motor and efference copy feedback (GoGo trials), which is absent in the passive observation of a stimulus (NoGo trials). To investigate this effect during eye and hand coordination movements, we presented stimuli in two formats (memory guided or visually guided) under three modality conditions (eyes only, hands only (with eyes fixated), or eyes and hand together). We found that during coordinated movements, both the eye and hand response times were facilitated when efference feedback of the movement was provided. Furthermore, both eye and hand movements to remembered locations were significantly more accurate in the GoGo than in the NoGo trial types. These results reveal that an efference copy of a motor plan enhances memory for a location that is not only observed in eye movements, but also translated downstream into a hand movement. These results have significant implications on how we plan, code and guide behavioural responses, and how we can optimise accuracy and timing to a given target.

Is tracing or copying better when learning to reproduce a pattern?

Learning to write requires the repeated manual production of spatial patterns. It remains unclear whether tracing or copying provides better training: tracing provides accurate and immediate performance feedback, whereas copying may require greater use of memory and recall during training. We asked sixteen adults to copy or trace novel patterns then reproduce these from memory using a stylus and tablet PC. A week later, a retention test was performed. Sophisticated analyses indexed the extent to which participants had learned the dimensions and shape of patterns. We found that participants: (a) showed better shape and dimensional accuracy when tracing; (b) had better shape and dimensional retention immediately after tracing; (c) showed no differences between copying and tracing in their ability to redraw the pattern (shape or dimensions) 1 week later. Our methods provide a useful starting point for examining training and feedback on the generation and recall of spatial patterns.

Effect of retinal and/or extra-retinal information on age in memory-guided saccades.

The aim of the study was to determine whether the addition of retinal or extra-retinal information could be used to improve memory-guided saccadic performance in healthy participants. Furthermore, we included two age groups in our study; healthy young adult subjects (mean age 20 years) and healthy middle-aged adult subjects (mean age 52 years). All subjects performed a novel task that incorporated a Go/NoGo task with a memory-guided saccade paradigm to investigate whether extra-retinal information (making a saccade towards the visible target) or retinal (a visible frame-of-reference) has any affect on the accuracy or variability of the response. We found all subjects made slight hypometric responses to the memory-guided targets. Both younger and middle-aged subjects revealed an increase in accuracy in the Go task compared with the NoGo task and the framed condition compared to the frameless condition, respectively. The frame also revealed a significant decrease in variability in the memory-guided saccades. A positive correlation in errors between the 1st and 2nd saccade in the Go task was revealed for all subjects; however, the older subjects revealed a greater correlation than younger subjects. The results presented indicate that younger and middle-aged perform highly similar patterns of errors during eye movements to remembered locations. However, middle-aged subjects show a greater tendency to use extra-retinal and retinal feedback to guide the response.

Anticipatory eye movements evoked after active following versus passive observation of a predictable motion stimulus.

We used passive and active following of a predictable smooth pursuit stimulus in order to establish if predictive eye movement responses are equivalent under both passive and active conditions. The smooth pursuit stimulus was presented in pairs that were either 'predictable' in which both presentations were matched in timing and velocity, or 'randomized' in which each presentation in the pair was varied in both timing and velocity. A visual cue signaled the type of response required from the subject; a green cue indicated the subject should follow both the target presentations (Go-Go), a pink cue indicated that the subject should passively observe the 1st target and follow the 2nd target (NoGo-Go), and finally a green cue with a black cross revealed a randomized (Rnd) trial in which the subject should follow both presentations. The results revealed better prediction in the Go-Go trials than in the NoGo-Go trials, as indicated by higher anticipatory velocity and earlier eye movement onset (latency). We conclude that velocity and timing information stored from passive observation of a moving target is diminished when compared to active following of the target. This study has significant consequences for understanding how visuomotor memory is generated, stored and subsequently released from short-term memory.

Brain and behavior: a task-dependent eye movement study.

Recent electrophysiological and behavioral studies have found similarities in the neurology of pursuit and saccadic eye movements. In a previous study on eye movements using closely matched paradigms for pursuit and saccades, we revealed that both exhibit bimodal distributions of latency to predictable (PRD) and randomized (RND) stimuli; however, the latency to each type of stimulus was different, and there was more segregation of latencies in saccades than pursuit (Burke MR, Barnes GR. 2006. Quantitative differences in smooth pursuit and saccadic eye movements in humans. Exp Brain Res. 175(4):596-608). To investigate the brain areas involved in these tasks, and to search for correlates of behavior, we used functional magnetic resonance imaging during equivalent PRD and RND target presentations. In the contrast pursuit > saccades, which reflects velocity-dependent versus position-dependent activities, respectively, we found higher activation in the dorsolateral prefrontal cortex (DLPFC) for pursuit and in the frontopolar region for saccades. In the contrast RND > PRD, which principally reflects activation related to visually driven versus memory-driven responses, respectively, we found a higher sustained level of activation in the frontal eye fields during visually guided eye movements. The reverse contrast revealed higher activity for the memory-guided responses in the supplementary eye fields and the superior parietal lobe. In addition, we found learning-related activation during the PRD condition in visual area V5, the DLPFC, and the cerebellum.

Quantitative differences in smooth pursuit and saccadic eye movements.

Recently it has been suggested that smooth pursuit (SP) and saccadic (SAC) eye movements share many common brain substrates in the planning and control of eye movements (Krauzlis in J Neurophysiol 91:591-603, 2004). Evidence is mounting that these two types of eye movements may also share similar mechanisms used to drive both reactive and predictive eye movement responses (Missal and Keller in J Neurophysiol 88:1880-1892, 2002, Keller and Missal in Ann NY Acad Sci 1004:29-39, 2003). The objective of this study was to quantify these similarities by establishing whether the behavioural response properties of human eye movements to predictive (PRD) and randomized (RND) conditions are quantitatively similar for both SP and SAC in directly comparable paradigms. Two previous studies have attempted to evaluate the coordination and motor preparation time of SP and saccadic eye movements (Erkelens in Vis Res 46:163-170, 2006; Joiner and Shelhamer in Exp Brain Res, Epub ahead of print, 2006). However, no previous study has quantitatively evaluated PRD and RND conditions to discretely presented SP and SAC tasks. We used simple SAC and SP paradigms in blocks of PRD and RND presentations, with eye movements monitored throughout using an IR-limbus eye-tracking system (Skalar). Twelve normal subjects (aged between 20 and 39 years) participated in the study which took place over two recording sessions, on two separate days. Data were analysed for two main comparable descriptive statistics: latency and eye velocity/displacement gain. The results presented here support the notion that SP and SAC share common brain substrates/mechanisms in the generation of responses to PRD and RND visual targets but differ in the movement preparation time.

Touch responses made to remembered and visual target locations in the dark: a human psychophysical study.

UNLABELLED: Saccadic eye movements made to remembered locations in the dark show a distinct up-shift in macaque monkey, and slight upward bias in humans (Gnadt et al. 1991). This upward bias created in the visual spatial mapping of a saccade may be translated downstream in a hand/touch movement. This error could possibly reveal (a) information about the frames of reference used in each scenario and (b) the sources of this error within the brain. This would suggest an early planning stage if they are shared, or a later stage if the errors are distinct. METHODS: Eight human subjects performed touch responses to a touch screen monitor to both visual and remembered target locations. The subjects used a high-resolution touch-screen monitor, a bite bar and chin-rest for restricting head movements during responses. All target locations were 20 degrees vectors from the central starting position in horizontal, vertical and oblique planes of motion. RESULTS: Subjects were accurate to both visual and remembered target locations with little variance. Subject means showed no significant differences between control and memory trials; however, a distinct asymmetry was observed between cardinal and oblique planes during memory trials. Subjects consistently made errors to oblique locations during touches made to the remembered location that was not evident in control conditions. This error pattern revealed a strong hypermetric tendency for oblique planes of touches made to a remembered location.

Subunit-subunit interactions in trimeric arginase. Generation of active monomers by mutation of a single amino acid.

The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-A resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554-557). A key feature of this structure is a novel S-shaped oligomerization motif at the carboxyl terminus of the protein that mediates approximately 54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10(5). These monomeric arginase variants are catalytically active, with k(cat)/K(m) values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6-65.5 degrees C, while the corresponding value for the wild-type enzyme is 70 degrees C. The three-dimensional structure of the R308K variant has been determined at 3-A resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4.

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase.

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

Self-activation of guanosine triphosphatase activity by oligomerization of the bacterial cell division protein FtsZ.

The essential bacterial cell division protein FtsZ (filamentation temperature-sensitive protein Z) is a distant homologue to the eukaryotic cytoskeletal protein tubulin. We have examined the GTP hydrolytic activity of Escherichia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations greater than 1 microM. At 5 microM FtsZ, we have determined a K(m) of 82 microM GTP and a V(max) of 490 nmol of P(i) min(-1) (mg of protein)(-1). Hydrolysis of GTP requires Mg(2+) and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation experiments show that FtsZ exists as 68% dimer and 13% trimer at 2 microM total protein concentration. Chemical cross-linking of FtsZ also shows that monomer, dimer, trimer, and tetramer species are present at higher (>2 microM) FtsZ concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization. We also monitored the effect of nucleotide and metal ion on the secondary structure of FtsZ; nucleotide yielded no evidence of structural changes in FtsZ, but both Mg(2+) and Ca(2+) had significant effects on secondary structure. Taken together, our results support the hypothesis that Mg(2+)-dependent GTP hydrolysis by FtsZ requires oligomerization of FtsZ. On the basis of these results and structural comparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.

The scientific rationale and development of an optimized stannous fluoride dentifrice, Part 1.

Stannous fluoride has been recognized as an effective anticavity therapeutic agent since the early 1950s. There has recently been a resurgence in activity to discover ways to fully exploit its documented antimicrobial activity. Through the use of targeted in vitro methodology to predict in vivo efficacy, a highly optimized stannous fluoride dentifrice has been developed. Careful selection of stabilizing agents formulated into a unique system has resulted in a Colgate Optimized Stannous Fluoride (COSF) dentifrice that has been proven to simultaneously help control supragingival plaque, gingivitis, supragingival calculus, and caries. Furthermore, the COSF dentifrice has been clinically shown not to cause the traditional stannous fluoride staining of dentition.

Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography.

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.