Reaction Time Task Performance in Concussed Athletes over a 30-Day Period: An Observational Study.

OBJECTIVE: Reaction time is a common deficit following concussion, making its evaluation critical during return-to-play protocol. Without proper evaluation, an athlete may return-to-play prematurely, putting them at risk of further injury. Although often assessed, we propose that current clinical testing may not be challenging enough to detect lingering deficits. Thus, the aim of this study was to examine reaction time in concussed individuals three times over a 30-day period through the use of a novel reaction time device consisting of simple, complex, and go/no-go reaction time tasks. METHODS: Twenty-three concussed subjects completed simple, complex, and go/no-go reaction time tests at three different timepoints: within 7-, 14-, and 30-days of injury, and 21 healthy controls completed the three reaction time tasks during a single session. RESULTS: Independent t-tests revealed that for the simple reaction time task, concussed participants were only significantly slower at session 1 (p = .002) when compared to controls. Complex reaction time task results showed concussed participants to be significantly slower at session 1 (p = .0002), session 2 (p = .001), and session 3 (p = .002). Go/no-go results showed concussed participants to be significantly slower than controls at session 1 (p = .003), session 2 (p = .001), and session 3 (p = .001). CONCLUSIONS: Concussed individuals display prolonged reaction time deficits beyond the acute phase of injury, illustrated using increasingly complex tasks.

Thermodynamics of coaxially stacked helixes with GA and CC mismatches.

The thermodynamics of RNA helix-helix interfaces with intervening single- and tandem-GA or single-CC mismatches were studied by UV melting experiments. The model system consists of a hairpin with a four- or five-nucleotide 5' overhang which is bound by a short oligomer, creating the helical interface. Single GA interfaces are found to have favorable free energy increments of about 2 kcal/mol. This is similar to those reported for coaxially stacked flush interfaces of AU base pairs [Walter A. E., & Turner, D. H. (1994) Biochemistry 33, 12715-12719]. The free energy increment of the GA mismatches depends little on the sequence of the closing base pairs of the helixes, whether the break in the phosphate backbone is 5' or 3' with respect to the mismatch or whether the chains are extended beyond the helix-helix interface. Surprisingly, interfaces with single-CC mismatches have favorable free energy increments similar to those of GA interfaces, even though CC mismatches in coaxial stacks occur much less frequently in known RNA secondary structures. The results provide experimental support for the assumption that a bonus free energy is required for coaxially stacked helixes with intervening GA mismatches when free energy minimization is used to predict RNA secondary structures.

Sequence dependence of stability for coaxial stacking of RNA helixes with Watson-Crick base paired interfaces.

Thermodynamic parameters from UV melting studies are reported for the helix-helix interfaces of coaxially stacked helixes in RNA. The model system consists of a short oligomer binding to a four-nucleotide overhang at the end of a hairpin stem, creating the helix-helix interface. Interfaces containing Watson-Crick base pairs are approximately 1 kcal/mol more stable than the corresponding nearest neighbor interaction in an uninterrupted helix. Thus the sequence dependence of stability for coaxially stacked interfaces is similar to that for regular helixes. This provides experimental evidence for an assumption that has been shown to improve predictions of RNA secondary structure [Walter, A. E., Turner, D. H., Kim, J., Lyttle, M. H., Muller, P., Mathews, D. H., & Zuker, M. (1994) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The results should also be useful for modeling three-dimensional structures of RNA.

The stability and structure of tandem GA mismatches in RNA depend on closing base pairs.

UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal loops of sequence 5'XGAY3'3'YAGXS' and 5'XAGY3'3'YGAX5' in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chemical shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' [SantaLucia, J., Jr., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819] is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.

Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.

Effect of a 5'-phosphate on the stability of triple helix.

An effect of 5'-phosphorylation on the stability of triple helical DNA containing pyrimidine:purine:pyrimidine strands has been demonstrated by both gel electrophoresis and UV melting. A 5'-phosphate on the purine-rich middle strand of a triple helix lowers the stability of triple helix formation by approximately 1 kcal/mol at 25 degrees C. The middle strand is involved in both Watson-Crick and Hoogsteen base pairing. In contrast, a 5'-phosphate on the pyrimidine-rich strands, which are involved in either Watson-Crick or Hoogsteen base pairing, has a smaller effect on the stability of triple helix. The order of stability is: no phosphate on either strand > phosphate on both pyrimidine strands > phosphate on purine strand > phosphate on all three strands. Differential stability of triple helix species is postulated to stem from an increase in rigidity due to steric hindrance from the 5'-phosphate. This result indicates that labelling with 32P affect equilibrium in triplex formation.

Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text].

Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest-neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence.

Low molecular weight mRNA encodes a protein that controls serotonin 5-HT1c and acetylcholine M1 receptor sensitivity in Xenopus oocytes.

Serotonin 5-HT1c and acetylcholine M1 receptors activate phosphoinositidase, resulting in an increased formation of IP3 and 1,2 diacylglycerol. In Xenopus oocytes injected with mRNA encoding either of these receptors, Ca2+ released from intracellular stores in response to IP3 then opens Ca(2+)-gated Cl-channels. In the present experiments, oocytes expressing a transcript from a cloned mouse serotonin 5-HT1c receptor were exposed to identical 15-s pulses of agonist, administered 2 min apart; the second current response was two to three times that of the first. However, in those oocytes coinjected with the 5-HT1c receptor transcript and a low molecular weight fraction (0.3-1.5 kb) of rat brain mRNA, the second current response was approximately 50% of the first. Thus, the low molecular weight RNA encodes a protein (or proteins) that causes desensitization. Experiments using fura-2 or a Ca(2+)-free superfusate indicated that desensitization of the 5-HT1c receptor response does not result from a sustained elevation of intracellular Ca2+ level or require the entry of extracellular Ca2+. Photolysis of caged IP3 demonstrated that an increase in IP3 and a subsequent rise in Ca2+ do not produce desensitization of either the IP3 or 5-HT1c peak current responses. Furthermore, in oocytes coinjected with the low molecular weight RNA and a transcript from the rat M1 acetylcholine receptor, the M1 current response was greatly attenuated. Our data suggest that the proteins involved in attenuation of the M1 current response and desensitization of the 5-HT1c current response may be the same.

Modulation of a cloned mouse brain potassium channel.

The mouse brain K+ channel (MBK), previously cloned by others, has been independently cloned and shown to express in Xenopus oocytes. This K+ current (IK) inactivated over a time course of seconds and was sensitive to the K+ channel-blocking reagent tetraethylammonium. When the K+ channel was coexpressed with a cloned mouse brain serotonin receptor (5HT1c) in oocytes, activation of the 5HT1c receptor by a brief application of serotonin resulted in a suppression of the IK amplitude over the next 20 min. IK could also be suppressed by activation of G proteins. Suppression was also caused by intracellular Ca2+ injections and was blocked by intracellular injection of EGTA. Calmodulin antagonists block the IK suppression, but a known protein kinase inhibitor did not block suppression. The 5HT1c suppression was reversible; recovery from suppression was blocked by the protein kinase inhibitor H-7. These data suggest that the IK suppression occurs through a novel mechanism independent of A- or C-type protein kinases; suppression is best explained as being due to the action of a Ca2+/calmodulin-activated phosphatase; recovery from suppression is due to the action of a protein kinase.

Identification of a peanut agglutinin-binding protein from human retina.

With the use of an antiserum raised to peanut agglutinin (PNA)-binding protein(s) from human retinas, we have identified a glycoprotein that has the same subunit molecular weight (135,000) as human interphotoreceptor retinol-binding protein (IRBP) on human retinal protein transblots, and, like IRBP, is localized in the interphotoreceptor matrix surrounding both rods and cones. We show that this PNA-binding protein is not IRBP by comparing the binding patterns of antiserum to the PNA-binding protein to binding patterns obtained with antisera to bovine and monkey IRBP.

Light-induced phosphorylation of proteins from the all-cone retina of the lizard, Anolis carolinensis.

Proteins from the all-cone retina of the lizard Anolis carolinensis were phosphorylated using [gamma 32P] ATP, separated by SDS-PAGE and detected by autoradiography. Several proteins incorporated 32P. Exposure of the retinal homogenates to light brought about a dramatic increase in phosphorylation of the protein(s) with a molecular weight nearly identical to that of rat rhodopsin. It is likely that these proteins are the cone visual pigments, and that they incorporate phosphate when bleached by light. Increasing the time of the phosphorylation reaction from 1 to 30 min led to an increase in the amount of incorporation of labeled phosphate by the putative cone visual pigments, but changing the temperature from 4 degrees C to 20 degrees C decreased it. The amount of phosphate incorporation was substantially increased by NaF, a phosphatase inhibitor. This latter finding, along with the changes in incorporation of 32P with increased temperature, suggest that a phosphoprotein phosphatase is active in the lizard retina. The cation requirements, as well as the effects of cyclic nucleotides on light-induced phosphorylation of retinal lizard proteins, were also investigated.

The effect of lead on photoreceptor response amplitude--influence of removing external calcium and bleaching rhodopsin.

The relationship between the effect of lead on the amplitude of the photoreceptor response to light and the ability of the photoreceptor response to be enhanced by a light stimulus was studied in the excised, superfused, bullfrog retina. Photoreceptor responses were isolated by treatment of the retina with sodium aspartate and were recorded with extracellular electrodes. Rod and cone responses were monitored separately by employing a stimulus regimen which took advantage of differing rates of rapid dark adaptation. When the ability of the rod response to undergo enhancement by light was severely reduced by bleaching 80% of the rhodopsin in the retina, the usual depressive effect of 12.5 microM lead was not observed. When light enhancement of the rod response was completely eliminated by removing calcium from the superfusing solution, the effect of 12.5 microM lead was actually to increase rather than decrease rod response amplitude. We conclude from this and previous studies that the effect of lead on the rod photoreceptor is complex, that it can involve both inhibitory and excitatory components, and that the inhibitory effect is exerted on the same mechanism which is responsible for the ability of the rod response to be enhanced by exposure of the retina to light. The cone response never exhibited enhancement by light and was never depressed by exposure of the retina to lead, but the ability of lead to increase cone response amplitude appeared to be greater in the absence of calcium.

Barium removes the ouabain-induced increase in the rod response to light.

The mass receptor potential of the excised, superfused retina of the bullfrog was isolated with aspartate. Rods were selectively stimulated by using very dim flashes of light. In the presence of 0.1 mM ouabain, the amplitude of the receptor response was found first to increase transiently and, subsequently, to decrease progressively. The ouabain-induced transient increase in receptor response was completely eliminated by 0.4 mM barium chloride. However, barium did not affect the rate at which the response decayed in the presence of ouabain. The ability of barium to remove the ouabain-induced transient increase in the amplitude of the receptor response is discussed in terms of reducing the coupling ratio of the postulated electrogenic sodium-potassium pump of rods.

The effect of lead on photoreceptor response amplitude--influence of the light stimulus.

The mass receptor potential of the excised, superfused retina of the bullfrog was studied. Photoreceptor responses were isolated by addition of sodium aspartate to the Ringer solutions. Responses of the cones were monitored independently from responses of the rods by employing a two-flash method of stimulation which took advantage of the very different rates of rapid dark adaptation of rods and of cones. Stimulation with paired flashes of white light at regular intervals caused enhancement of rod response amplitude in that the response grew larger with subsequent flashes until reaching a stable plateau. The degree of enhancement was directly proportional to the amount of light exposure and increased with either increasing stimulus intensity or decreasing stimulus interval. Only the rod response was affected; the cone response was not enhanced by continued stimulation. The effects of 12.5 microM PbCl2 on rod response amplitude were complex. There was a small (less than 10%) but consistent depression of rod response amplitude even when the rods were in the unenhanced state. However, the most striking effect of lead was on the enhanced response, where treatment with 12.5 microM PbCl2 led to a depression of about 33%. When added prior to light stimulation, lead significantly decreased the degree to which the rod response could be enhanced, but never prevented enhancement entirely. Removal of lead resulted in a very large increase in the degree to which the rod response was enhanced by light, even when compared to the first, lead-free control. The cone response was unaffected by 12.5 microM PbCl2.

Dichotomous effects of cadmium on the cone photoreceptor response to light.

The effects of cadmium (1.0 to 400.0 microM CdCl2) on the cone photoreceptor's response to light were studied in the superfused bullfrog retina. The effects of cadmium were complex, characterized by a purely excitatory component at the lower levels and an inhibitory component that became evident with 75.0 microM CdCl2. The maximum increase in cone response amplitude due to the excitatory component was about 60% at 100.0 microM CdCl2. At 400.0 microM CdCl2, the highest concentration examined in this study, cone response amplitude was suppressed by about 35%. Both components were dose-dependent. The excitatory component was reversible only at CdCl2 concentrations below 50.0 microM, and irreversible at levels at and above 50.0 microM. The inhibitory component was always reversible. The slope of the dose-response curve describing the excitatory component (0.60) was about twice that describing the inhibitory component (0.31). The effects of cadmium on cone response amplitude are discussed in terms of the most probable sensitive sites.

The effects of lead and of cadmium on the mass photoreceptor potential: the dose-response relationship.

The effects of micromolar (1.0-60.0 microM) amounts of lead and of cadmium on the light response of photoreceptors were studied using the isolated, perfused bullfrog retina. The effect of lead in depressing rod activity is readily and fully reversible. Lead is very effective in depressing the rod response at concentrations as low as 1.0 microM, but the effect of lead saturates at about 25.0 microM with about 34% depression of the rod response. At the higher concentrations some spontaneous recovery of rod response amplitude is observed shortly after exposure of the retina to lead begins. The cone response is affected by lead only rarely and then only at the higher concentrations. When affected, the cone response is enhanced rather than depressed. The effects of cadmium are generally similar to those of lead. However, saturating concentrations of cadmium depress the rod response to a greater degree than lead and the cadmium effects are not as readily reversible as the effects of lead at the higher concentrations. Cones are much more sensitive to cadmium than they are to lead and 60.0 microM cadmium always enhances cone response amplitude. The results of experiments in which lead and cadmium were both added to the retinal perfusate indicate to us that cadmium and lead both affect the same sensitive site or sites responsible for generating the rod response, but that cadmium affects an additional site that is not sensitive to lead. The depressive effects of lead and of cadmium on the rod response are discussed in terms of the postulated mechanisms of transduction in the vertebrate photoreceptor.

Rapid dark adaptation of bullfrog rods is delayed by barium.

With use of sodium aspartate, the late receptor potential of the excised, perfused bullfrog retina was isolated. By means of a two-flash technique, rapid dark adaptation of rods was monitored. As in cones, barium ions were found to delay the onset of rapid dark adaptation of rods, but the rate of recovery, once begun, was virtually unaffected. The effect of barium on the amplitude-intensity relationship of rods was also determined. Unlike its ability to dramatically increase the amplitude of the receptor potential of rods, barium had no effect on the absolute threshold of rods. We propose that barium ions act on the enzyme system postulated to govern the onset of rapid dark adaptation of rods and suggest that a reduction in the activity of an ATP protein kinase might be the basis for this effect.

The influence of calcium on the aspartate-isolated mass receptor potential of bullfrog cones.

The aspartate-isolated receptor potential was studied in the excised, perfused bullfrog retina. Cones were monitored without interference from rods by employing conditioning and test stimuli in a manner previously described (23,24). Lowering extracellular calcium by switching from a perfusate containing 0.4mM CaCl2 to one having no added calcium resulted in an increase in cone response amplitude. Conversely, elevating extracellular calcium by perfusing with a Ringer containing 0.8mM CaCl2 resulted in a decrease in cone response amplitude. These changes were sustained and fully reversible. In contrast, perfusing the retina with a Ringer solution containing EGTA resulted in a transient increase in cone response amplitude. Decreasing external calcium by simple depletion also shortened the delay prior to onset of rapid dark adaptation of the cones, thereby hastening the entire process of recovery. Increasing external calcium had little effect on rapid dark adaptation. Decreasing external calcium with EGTA led to extremely rapid response recovery, but the effect was not reversible. In no case did EGTA lead to a complete suppression of the response. The results of this study are interpreted as being inconsistent with the view that calcium is the internal transmitter responsible for the generation of the receptor potential in cones. They are consistent with the view that calcium functions to modulate recovery of the cones' ability to generate a response following a stimulus, perhaps by affecting the activity of a cyclic nucleotide.

Barium delays the onset of rapid dark adaptation in bullfrog cones.

The late receptor potential of the excised, perfused bullfrog retina was isolated with sodium aspartate. By employing a three-flash technique, cone responses were monitored without interference from rods. In cones barium ions were found to delay the onset of rapid dark adaptation, but the rate of recovery, once begun, was unaffected. We propose that barium ions act directly upon the enzyme system postulated to govern the onset of rapid dark adaptation of cones. In addition, barium was found to affect the amplitude of the rod receptor potential differently from that of cones, increasing the former but decreasing the latter. The effect of barium upon photoreceptor potential amplitude is discussed in terms of a reduction in the potassium conductance of the photoreceptors and the mechanisms postulated for photoreceptor excitation and rapid dark adaptation.