Static couch non-coplanar arc selection optimization for lung SBRT treatment planning.

Objective. Non-coplanar arc geometry optimizations that take advantage of beam's eye view (BEV) geometric overlap information have been proven to reduce dose to healthy organs-at-risk (OARs). Recently, a metric called mean arc distance (MAD) has been developed that quantifies the arc geometry sampling of 4πspace. The purpose of this research is to combine improved BEV overlap information with MAD to generate static couch lung stereotactic body radiotherapy (SBRT) treatment plans deliverable on a C-arm linear accelerator.Approach. An algorithm utilizing the Moller-Trumbore ray-triangle intersection method was employed to compute a cost surrogate for dose to overlapping OARs using distances interpolated onto a PDD. Cost was combined with MAD for 100 000 random combinations of arc trajectories. A pathfinding algorithm for arc selection was created, balancing the contributions of MAD and 4πcost for the final trajectory. This methodology was evaluated for 18 lung SBRT patients. Cases were also planned with arcs from a clinical treatment template protocol for dosimetric and plan quality comparison. Results were evaluated using dose constraints in the context of RTOG0915.Main results. Five of six OARs had maximum dose reductions when planned with the arc trajectory optimization algorithm. Significant maximum dose reductions were found for esophagus (7.41 ± 0.91 Gy,p= 0.00019), trachea (5.56 ± 1.55 Gy,p= 0.0025), spinal cord (2.87 ± 1.13 Gy,p= 0.039), large bronchus (3.47 ± 1.49 Gy,p= 0.0075), and aorta (3.13 ± 0.99 Gy,p= 0.012). Mean dose to contralateral lung was also significantly reduced (0.50 ± 0.06 Gy,p= 0.00019). There were two significant increases in OAR doses: mean dose to ipsilateral lung (0.40 ± 0.09,p= 0.00086) and V5Gyto ipsilateral lung (1.95 ± 0.70%,p= 0.011). Paddick conformity index increased by 0.03 ± 0.02 (p= 0.14), remaining below a limit of 1.2 for both techniques.Significance. Static couch non-coplanar optimization yielded maximum dose reductions to OARs while maintaining target conformity for lung SBRT.

A First Look at Equity, Diversity, and Inclusion of Canadian Medical Physicists: Results From the 2021 COMP EDI Climate Survey.

PURPOSE: In 2021, the Canadian Organization of Medical Physicists (COMP) conducted its first equity, diversity, and inclusion Climate Survey. The membership's experiences of inclusion, belonging, professional opportunities, discrimination, microaggressions, racism, and harassment in their professional lives are presented. METHODS AND MATERIALS: The ethics-reviewed survey was distributed in English and French to full members of COMP. Participants responded to questions covering demographics and professional climate. Simple descriptive statistics were used to measure frequency of responses. Data pertaining to impressions on the climate within the profession were compared using nonparametric statistical tests. RESULTS: The survey was distributed to 649 eligible members; 243 (37%) responded, and 214 (33%) provided full response sets. From the full response sets, findings showed that in general, age, highest academic degree, and racial and ethnic distribution trends of medical physicists were comparable with previously collected data and/or the Canadian population. The experiences of respondents relating to harassment in the workplace and perception of climate are reported and provide a useful benchmark for future assessments of interventions or training programs. In the workplace, fewer women (58%) reported having professional opportunities compared with men (70%). The survey also found that 17% of respondents (most of whom were women) directly or indirectly experienced sexual harassment in the workplace within the past 5 years. Finding that 23% of survey respondents identified as having a disability is a valuable reminder that accommodations in the workplace are necessary for more than 1 in every 5 medical physicists working in clinics. CONCLUSIONS: This study provided insight into the diversity and experiences of medical physicists in Canada. The majority of respondents had positive perceptions about their professional environment. However, equity-lacking groups were identified, such as women, underrepresented minorities, Indigenous peoples, and people with visible and invisible disabilities.

Intra-arc binary collimation with dynamic axes trajectory optimization for the SRS treatment of multiple metastases with multiple prescriptions.

PURPOSE: This work generates multi-metastases cranial stereotactic radiosurgery/radiotherapy (SRS/SRT) plans using a novel treatment planning technique in which dynamic couch, collimator, and gantry trajectories are used with periodic binary target collimation. The performance of this planning technique is evaluated against conventional volumetric arc therapy (VMAT) planning in terms of various dose and plan quality metrics. METHODS: A 3D cost space (referred to herein as the combined optimization of dynamic axes or CODA cube) was calculated based on an overlap between targets and organs-at-risk (OARs) and uncollimated areas between targets (island blocking) for all combinations of couch, gantry, and collimator angles. Gradient descent through the cube was applied to determine dynamic trajectories. At each control point (CP), each target can either be conformally treated or blocked by the multi-leaf collimator (referred to as intra-arc binary collimation, iABC). Simulated annealing was used to optimize the collimation patterns throughout the arcs as well as the monitor units (MUs) delivered at each CP. Seven previously treated VMAT plans were selected for comparison against the CODA-iABC planning technique. Two CODA-iABC plans were developed: a single gantry arc plan and a plan with one gantry arc and two couch arcs. Plan quality comparison metrics included maximum and mean dose to OARs (brainstem, chiasm, optic nerves, eyes, and lenses), the volume of normal brain receiving 12 Gy (V12Gy), total MUs, target conformity, and dose-gradient index. RESULTS: Treatment plans generated with 1-arc CODA-iABC plans delivered an average of 21% and 30% higher maximum and mean doses to brainstem, respectively, when compared to VMAT plans. Treatment plans generated with 3-arc CODA-iABC used an average of 24% fewer MUs and resulted in an average reduction of 48% maximum dose and 50% mean dose to the OARs, when compared to VMAT. Target conformity values were worse in both CODA-iABC plans than VMAT by an average of 35% and 15%, respectively. There are no significant differences in V12Gy for all three planning techniques; however, 3-arc CODA-iABC is more effective at reducing dose to normal brain in the low-dose region (<12 Gy). CONCLUSION: CODA-iABC is a novel planning technique that has been developed to automatically generate patient-specific multi-axis trajectories for multiple metastases cranial SRS/SRT. This work has demonstrated the feasibility of planning with this novel method. The 1-arc CODA-iABC planning technique is slightly dosimetric inferior to VMAT. With an increased sampling of a three-dimensional CODA cube by using a 3-arc CODA-iABC technique, there was improved total dose sparing to all the OARs and increased MU efficiency, but with a cost in target conformity, when compared to VMAT.

Determining Planning Priorities for SABR for Oligometastatic Disease: A Secondary Analysis of the SABR-COMET Phase II Randomized Trial.

PURPOSE: SABR may improve survival in patients with oligometastases, but for some lesions, safe delivery of SABR may require a reduction in delivered dose or target coverage. This study assessed the association between target coverage compromise and oncologic and survival outcomes. METHODS AND MATERIALS: Patients with a controlled primary malignancy and 1 to 5 oligometastases were randomized (1:2) between standard of care (SOC) treatment and SOC plus SABR. In patients receiving SABR, the target dose coverage was reduced to meet organ at risk (OAR) constraints, if necessary. The D99 value (minimum dose received by the hottest 99% of the planning target volume [PTV]) was used as a measure of PTV coverage for each treatment plan, and the relationship between the coverage compromise index (CCI, defined as D99/prescription dose) and patient outcomes was assessed. RESULTS: Sixty-two patients in the SABR arm had dosimetric information available and a total of 109 lesions were evaluated. The mean CCI per lesion was 0.96 (95% CI, 0.56-1.61). Of the 109 lesions evaluated, 29.4% (n = 32) required coverage compromise (CCI <0.9). Adrenal metastases required coverage compromise in 100% of analyzed lesions (n = 7). CCI was not significantly associated with lesional control, adverse events, overall survival (OS), or progression-free survival (PFS). CONCLUSIONS: Target compromise was required in a substantial minority of cases, but PTV coverage was not associated with OS, progression-free survival, or lesional control. This suggests that OAR constraints used for SABR treatments in the oligometastatic setting should continue to be prioritized during planning.

Patient-specific collision zones for 4π trajectory optimized radiation therapy.

PURPOSE: The 4π methodology determines optimized noncoplanar subarcs for stereotactic radiation therapy that minimize dose to organs-at-risk. Every combination of treatment angle is examined, but some angles are not appropriate as a collision would occur between the gantry and the couch or the gantry and the patient. Those combinations of couch and gantry angles are referred to as collision zones. A major barrier to applying 4π to stereotactic body radiation therapy (SBRT) is the unknown shape of the collision zones, which are significant as patients take up a large volume within the 4π sphere. This study presents a system that determines patient-specific collision zones, without additional clinical steps, to enable safe and deliverable noncoplanar treatment trajectories for SBRT patients. METHODS: To augment patient's computed tomography (CT) scan, full body scans of patients in treatment position were acquired using an optical scanner. A library of a priori scans (N = 25) was created. Based on the patients' treatment position and their body dimensions, a library scan is selected and registered to the CT scan of the patient. Next, a model of the couch and immobilization equipment is added to the patient model. This results in a patient model that is then aligned with a model of the treatment LINAC in a "virtual treatment room," where both components can be rotated to test for collisions. To test the collision detection algorithm, an end-to-end test was performed using a cranial phantom. The registration algorithm was tested by comparing the registered patient collision zones to those generated by using the patient's matching scan. RESULTS: The collision detection algorithm was found to have a 97.80% accuracy, a 99.99% sensitivity, and a 99.99% negative predictive value (NPV). Analysis of the registration algorithm determined that a 6 cm buffer was required to achieve a 99.65% mean sensitivity, where a sensitivity of unity is considered to be a requirement for safe treatment delivery. With a 6 cm buffer, the mean accuracy was 86.70% and the mean NPV was 99.33%. CONCLUSIONS: Our method of determining patient-specific collision zones can be accomplished with minimal user intervention based on an a priori library of body surface scans, thus enabling the safe application of 4π SBRT.

SBRT of ventricular tachycardia using 4pi optimized trajectories.

PURPOSE: To investigate the possible advantages of using 4pi-optimized arc trajectories in stereotactic body radiation therapy of ventricular tachycardia (VT-SBRT) to minimize exposure of healthy tissues. METHODS AND MATERIALS: Thorax computed tomography (CT) data for 15 patients were used for contouring organs at risk (OARs) and defining realistic planning target volumes (PTVs). A conventional trajectory plan, defined as two full coplanar arcs was compared to an optimized-trajectory plan provided by a 4pi algorithm that penalizes geometric overlap of PTV and OARs in the beam's-eye-view. A single fraction of 25 Gy was prescribed to the PTV in both plans and a comparison of dose sparing to OARs was performed based on comparisons of maximum, mean, and median dose. RESULTS: A significant average reduction in maximum dose was observed for esophagus (18%), spinal cord (26%), and trachea (22%) when using 4pi-optimized trajectories. Mean doses were also found to decrease for esophagus (19%), spinal cord (33%), skin (18%), liver (59%), lungs (19%), trachea (43%), aorta (11%), inferior vena cava (25%), superior vena cava (33%), and pulmonary trunk (26%). A median dose reduction was observed for esophagus (40%), spinal cord (48%), skin (36%), liver (72%), lungs (41%), stomach (45%), trachea (53%), aorta (45%), superior vena cava (38%), pulmonary veins (32%), and pulmonary trunk (39%). No significant difference was observed for maximum dose (p = 0.650) and homogeneity index (p = 0.156) for the PTV. Average values of conformity number were 0.86 ± 0.05 and 0.77 ± 0.09 for the conventional and 4pi optimized plans respectively. CONCLUSIONS: 4pi optimized trajectories provided significant reduction to mean and median doses to cardiac structures close to the target but did not decrease maximum dose. Significant improvement in maximum, mean and median doses for noncardiac OARs makes 4pi optimized trajectories a suitable delivery technique for treating VT.

Toward the combined optimization of dynamic axes (CODA) for stereotactic radiotherapy and radiosurgery using fixed couch trajectories.

PURPOSE: To develop a novel system for patient-specific combined optimization of couch, collimator, and gantry angles for use in volumetric modulated arc therapy (VMAT) treatment planning. The system was designed to produce highly compact dose distributions by extensively sampling the 4π space. Automated fixed couch trajectory planning was used to reduce normal tissue doses by avoiding beams-eye-view (BEV) overlap with organs-at-risk (OARs) and improve monitor unit (MU) efficiency through collimator angle optimization. METHODS: By merging distinct BEV objective functions used to optimize the couch rotation angle and collimator angle, a three-dimensional (3D) cost space (the CODA cube) was constructed with axes of gantry, couch, and collimator rotation angles. At each voxel in this CODA cube, the cost of implementing this combination of axes positions in fixed couch trajectories was quantified. The CODA cube was sampled and explored using a modified constrained Bellman-Ford algorithm to suggest low-cost fixed candidate arcs on each plane of the space, from which 10-arcs are chosen throughout the 3D space using a k-means clustering algorithm. These fixed couch trajectories were then imported into the Eclipse treatment planning system (v.11) and inverse-optimized according to clinical standards. Eight artificial cranial targets were contoured in a test-patient anatomy, and seven treatment plans were generated from combinations of three and four targets. The CODA cube optimized plans were compared to standard 4-arc VMAT plans for cranial stereotactic radiotherapy/surgery that were optimized for the same sets of targets; maximum dose to each OAR, V12Gy to normal brain, conformity, and total MUs were compared. Both planning methods were inverse-optimized with identical dosimetric objectives. RESULTS: CODA plans resulted in a reduction in maximum dose to OARs of 20.6% (P < 0.01), with maximum brainstem dose decreased by 2.63 Gy (P = 0.031) on average when compared to the standard arc arrangement. The mean reduction in total MU was 8.6% (P = 0.156), the mean increase in the inverse of the van't Riet conformation number was 0.1%, (P = 0.67) and the mean decrease in normal brain tissue receiving 12 Gy or higher was 3.9% (P = 0.16), when compared to the standard VMAT arc configuration (n = 7). CONCLUSIONS: The optimization of couch, collimator, and gantry angles simultaneously using a 3D optimization space achieved improvement on multiple clinical metrics when compared to the standard VMAT arc configuration. A statistically significant sparing to OAR maximum doses was seen. Combining these optimizations may yield superior results to independent optimization.

Intra-arc binary collimation algorithm for the optimization of stereotactic radiotherapy treatment of multiple metastases with multiple prescriptions.

PURPOSE: To design and implement a novel treatment planning algorithm based on a modification of dynamic conformal arc (DCA) therapy for the treatment of multiple cranial metastases with variable prescription doses. METHODS: A workflow was developed in which separate dose matrices were calculated for each target at each control point (i.e., the multileaf collimator (MLC) was fit conformally to that single target). A cost function was used to quantify the relative contributions of each dose matrix in the plan to the overall plan objectives. Simulated annealing was used to allow for the inclusion or exclusion of individual dose matrices at each control point. The exclusion of individual targets at a given control point is termed intra-arc binary collimation (iABC) in this work and is accomplished by closing the MLCs over the target for a duration specified by simulated annealing optimization. Dynamic collimator motions were employed to minimize the variation between the idealized dose matrices (i.e., perfectly collimated targets) and actual dose matrices (i.e., MLC apertures that include quantities of nontarget tissue due to the relative orientations of targets in the field). An additional simulated annealing optimization was performed to weight the relative contributions of dose at each control point [referred to as the monitor unit distribution (MUD)] to improve compliance with plan objectives. The algorithm was tested on seven previously treated multiple metastases patients and plans were compared to the clinically treated VMAT plans. RESULTS: Treatment plans generated with iABC used an average of 2716 (34%) fewer MU in the total plan than VMAT (P = 0.01). All normal tissue metrics for all plans and all patients were clinically acceptable. There were no statistically significant differences in any normal tissue dose metrics. Normalized prescription target coverage accuracy for all targets was 3% better on average for VMAT plans when compared to iABC (P = 0.07), and 14% better on average for iABC when compared to optimized DCA (P = 0.03). CONCLUSION: A novel method of aperture and dose distribution design has been developed to significantly increase the MU efficiency of single isocenter treatment of multiple metastases with variable prescription doses when compared to VMAT, and which improves target coverage accuracy significantly when compared to optimized DCA. By applying a DCA approach to subsets of targets across control points, a hybrid method of treatment delivery has been developed that combines the efficiency of dynamic conformal treatments and the dosimetric flexibility of VMAT.

Dynamic collimator trajectory algorithm for multiple metastases dynamic conformal arc treatment planning.

PURPOSE: To develop an algorithm for dynamic collimator positioning to optimize beam's eye view (BEV) fitting of targets in dynamic conformal arc (DCA)-based radiotherapy procedures, of particular use in multiple metastases stereotactic radiosurgery procedures. METHODS: A trajectory algorithm was developed to dynamically modify the angle of the collimator as a function of arc-based control point to provide optimized collimation of target volume(s). Central to this algorithm is a concept denoted herein as "whitespace" defined as any nontarget area in the BEV that is not covered by any collimation system and is open to exposure from the radiation beam. Calculating whitespace at all collimator angles and every control point, a two-dimensional topographical map depicting the tightness-of-fit of the MLC was generated. A bidirectional gradient trajectory algorithm identified a number of candidate trajectories of continuous collimator motion. Minimization of integrated whitespace was used to identify an optimal solution for the navigation of the parameter space. Plans with dynamic collimator trajectories were designed for multiple metastases targets and were compared with fixed collimator angle dynamic conformal arc (DCA) plans and standard VMAT plans. RESULTS: Algorithm validation was performed on simple test cases with known solutions. The whitespace metric showed a strong correlation (R2 = 0.90) with mean dose to proximal normal tissue. Seventeen cases were studied using our algorithm to generate dynamic conformal arc (DCA) plans with optimized collimator trajectories for three and four target SRS patients and comparing them to DCA plans generated with optimized fixed collimator angles per arc and standard VMAT plans generated via template. Optimized collimator trajectories were found to produce a reduction in monitor units of up to 49.7 ± 5.1% when compared to VMAT across 17 patients, and all organ-at-risk and normal brain metrics were found to be superior or comparable. CONCLUSION: Dynamic collimator trajectories have the potential to improve DCA deliveries through increased efficiency, especially in the treatment of multiple cranial metastases. Implementation of this technology should not be hindered by mechanical safety considerations as collimator motions do not modify or introduce any new risks of collisions with patients.

Overlap-guided fixed-patient support positioning optimization for cranial SRT.

PURPOSE: To investigate potential dosimetric improvements through the optimization of fixed-couch rotational position in cranial cancer stereotactic treatments. METHODS: Using previously delivered cranial stereotactic radiotherapy plans treated at the Nova Scotia Health Authority (NSHA), we have redesigned the treatment arrangement to find the optimal couch rotation positions based on the reduction of overlap between organs-at-risk of exposure (OARs) and target volume (PTV). Maintaining the gantry arrangements from the delivered treatment, the couch positions were determined based on a cost function analysis of accumulation of overlap score from an equation developed by Yang et al. and refined by MacDonald et al. The algorithm incorporates factors for radiation dose sensitivities of each OAR, depth of both OARs and target (PTV) volumes, and orthogonality of the 3D vector between OAR and PTV in the case of proximal OAR position. RESULTS: The plan evaluation was conducted on 16 acoustic neuroma patients treated with stereotactic radiotherapy plans at the NSHA. Maximum and mean doses to the OARs were reduced by approximately 14.30% ± 2.86% and 19.25% ± 2.10%, respectively, with application of this optimization technique as compared to the delivered treatment plans. In addition, PTV conformity and homogeneity were improved with application of this optimization technique. CONCLUSION: This variation of the existing delivery techniques with guidance from a PTV-OAR overlap cost function analysis technique can yield significant dosimetric improvements with no increase to delivery or planning time.

Dynamic trajectory-based couch motion for improvement of radiation therapy trajectories in cranial SRT.

PURPOSE: To investigate potential improvement in external beam stereotactic radiation therapy plan quality for cranial cases using an optimized dynamic gantry and patient support couch motion trajectory, which could minimize exposure to sensitive healthy tissue. METHODS: Anonymized patient anatomy and treatment plans of cranial cancer patients were used to quantify the geometric overlap between planning target volumes and organs-at-risk (OARs) based on their two-dimensional projection from source to a plane at isocenter as a function of gantry and couch angle. Published dose constraints were then used as weighting factors for the OARs to generate a map of couch-gantry coordinate space, indicating degree of overlap at each point in space. A couch-gantry collision space was generated by direct measurement on a linear accelerator and couch using an anthropomorphic solid-water phantom. A dynamic, fully customizable algorithm was written to generate a navigable ideal trajectory for the patient specific couch-gantry space. The advanced algorithm can be used to balance the implementation of absolute minimum values of overlap with the clinical practicality of large-scale couch motion and delivery time. Optimized cranial cancer treatment trajectories were compared to conventional treatment trajectories. RESULTS: Comparison of optimized treatment trajectories with conventional treatment trajectories indicated an average decrease in mean dose to the OARs of 19% and an average decrease in maximum dose to the OARs of 12%. Degradation was seen for homogeneity index (6.14% ± 0.67%-5.48% ± 0.76%) and conformation number (0.82 ± 0.02-0.79 ± 0.02), but neither was statistically significant. Removal of OAR constraints from volumetric modulated arc therapy optimization reveals that reduction in dose to OARs is almost exclusively due to the optimized trajectory and not the OAR constraints. CONCLUSIONS: The authors' study indicated that simultaneous couch and gantry motion during radiation therapy to minimize the geometrical overlap in the beams-eye-view of target volumes and the organs-at-risk can have an appreciable dose reduction to organs-at-risk.

The relative roles of diffusion and uptake in clearing synaptically released glutamate change during early postnatal development.

Glutamate uptake by transporters expressed in astrocytes combines with synaptic structure to regulate the time that synaptically released glutamate remains in the extracellular space and, consequently, the duration and location of postsynaptic receptor activation. Both factors change greatly in the rodent hippocampus during the second postnatal week when most synapses become established and begin to mature, processes that are influenced by synaptically released glutamate. Transporter expression increases, potentially speeding removal of synaptically released glutamate, whereas extracellular space decreases, thereby slowing dilution. We investigated whether these competing changes influence the glutamate concentration time course and postsynaptic responses in the CA1 region of the mouse hippocampus during this critical period of synaptic development. Our results suggest that the glutamate concentration time course remains relatively consistent over this period, although the primary mechanisms regulating glutamate clearance change. Before the second postnatal week, clearance of synaptically released glutamate depends primarily on diffusion into large extracellular spaces, whereas later in development it relies more on increased uptake capacity. Thus, increased transporter expression during this period accompanies structural changes in the neuropil, preserving a relatively consistent glutamate concentration time course and ensuring that postsynaptic receptor activation remains brief and primarily localized to receptors close to release sites.

Antirestenotic effects of a novel polymer-coated d-24851 eluting stent. Experimental data in a rabbit iliac artery model.

Experimental and clinical data suggest that stents eluting antiproliferative agents can be used for the prevention of in-stent restenosis. Here we investigate in vitro the antiproliferative and apoptotic effect of D-24851 and evaluate the safety and efficacy of D-24851-eluting polymer-coated stents in a rabbit restenosis model (n = 53). Uncoated stents (n = 6), poly (DL: -lactide-co-glycolide) (PLGA)-coated stents (n = 7), and PLGA-coated stents loaded with 0.08 +/- 0.0025 microM (31 +/- 1 mug; low dose; n = 7), 0.55 +/- 0.02 microM (216 +/- 8 mug; high dose; n = 6), and 4.55 +/- 0.1 microM (1774 +/- 39 mug; extreme dose; n = 5) of D-24851 were randomly implanted in New Zealand rabbit right iliac arteries and the animals were sacrificed after 28 days for histomorphometric analysis. For the assessment of endothelial regrowth in 90 days, 12 rabbits were subjected to PLGA-coated (n = 3), low-dose (n = 3), high-dose (n = 3), and extreme-dose (n = 3) stent implantation. In vitro studies revealed that D-24851 exerts its growth inhibitory effects via inhibition of proliferation and induction of apoptosis without increasing the expression of heat shock protein-70, a cytoprotective and antiapoptotic protein. Treatment with low-dose D-24851 stents was associated with a significant reduction in neointimal area and percentage stenosis only compared with bare metal stents (38% [P = 0.029] and 35% [P = 0.003] reduction, respectively). Suboptimal healing, however, was observed in all groups of D-24851-loaded stents in 90 days in comparison with PLGA-coated stents. We conclude that low-dose D-24851-eluting polymer-coated stents significantly inhibit neointimal hyperplasia at 28 days through inhibition of proliferation and enhancement of apoptosis. In view of the suboptimal re-endothelialization, longer-term studies are needed in order to establish whether the inhibition of intimal growth is maintained.

Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons.

Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 +/- 2.4% [6 days in vitro (DIV)] to 54.9 +/- 2.6% (8 DIV), before reaching a plateau in the second week (42.5 +/- 2%, 12-19 DIV). In NR2A-/- neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; > or =12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A(-/-) neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.

Probing N-methyl-D-aspartate receptor desensitization with the substituted-cysteine accessibility method.

Several forms of macroscopic N-methyl-D-aspartate (NMDA) receptor desensitization affect the amplitude and duration of postsynaptic responses. In addition to its functional significance, desensitization provides one means to examine the conformational coupling of ligand binding to channel gating. Segments flanking the ligand binding domain in the extracellular N terminus of the NMDA receptor NR2 subunit influence the glycine-independent form of desensitization. The NR2A pre-M1 region, the linker between the glutamate binding domain and the channel pore, plays a critical role in desensitization. Thus, we used the substituted-cysteine accessibility method to scan the accessibility of residues in the pre-M1 region and the first transmembrane domain (M1) of NR2A. Cysteine mutants were expressed with NR1 in human embryonic kidney 293 cells and were assayed by whole-cell recording. With activation of the receptor by glutamate and glycine, only a single mutant, V557C, which is located at the beginning of M1, led to irreversible inhibition by the methanethiosulfonate derivative methanethiosulfonate ethyltrimethylammonium (MTSET). The NR2 ligand glutamate was insufficient on its own to induce modification of V557C by MTSET, suggesting that the change in accessibility required channel gating. The rate of MTSET modification of the homologous residue on NR1 (NR1-1a(L562C)/NR2A) was much slower than V557C. We also substituted cysteine in the V557 site of mutant subunits that exhibit either enhanced or reduced desensitization. Modification by MTSET correlated with the degree of desensitization for these subunits, suggesting that V557C is a sensitive detector of desensitization gating.

Vitamin C transiently arrests cancer cell cycle progression in S phase and G2/M boundary by modulating the kinetics of activation and the subcellular localization of Cdc25C phosphatase.

Regulation of cell cycle progression involves redox (oxidation-reduction)-dependent modification of proteins including the mitosis-inducing phosphatase Cdc25C. The role of vitamin C (ascorbic acid, ASC), a known modulator of the cellular redox status, in regulating mitotic entry was investigated in this study. We demonstrated that vitamin C inhibits DNA synthesis in HeLa cells and, mainly the form of dehydroascorbic acid (DHA), delays the entry of p53-deficient synchronized HeLa and T98G cancer cells into mitosis. High concentrations of Vitamin C caused transient S and G2 arrest in both cell lines by delaying the activation of the M-phase promoting factor (MPF), Cdc2/cyclin-B complex. Although vitamin C did not inhibit the accumulation of cyclin-B1, it may have increased the level of Cdc2 inhibitory phosphorylation. This was achieved by transiently maintaining Cdc25C, the activator of Cdc2, both in low levels and in a phosphorylated on Ser216 inactive form that binds to 14-3-3 proteins contributing thus to the nuclear exclusion of Cdc25C. As expected, vitamin C prevented the nuclear accumulation of Cdc25C in both cell lines. In conclusion, it seems that vitamin C induces transient cell cycle arrest, at least in part, by delaying the accumulation and the activation of Cdc25C.

Learning-related fMRI activation associated with a rotational visuo-motor transformation.

The unique ability to learn transformed or altered visuo-motor relationships during motor learning (visuo-motor transformation learning) has engaged researchers for over a century. Compared to other forms of motor learning (e.g., sequence learning), little is known about plasticity in the cortical and/or subcortical systems involved. We used fMRI to isolate region-specific activation changes during the learning of a visuo-motor (joystick) task under a simple transformation (90 degree rotation of visual feedback). Distributed brain systems were engaged in the learning process. In particular, we found evidence of a learning-dependent transition from early activation of the posterior parietal cortex to later distributed cortico-subcortical-cerebellar responses (in the temporal and occipital cortices, basal ganglia, cerebellum and thalamus). The role of the posterior parietal cortex may relate specifically to the acquisition of the transformation, while that of the fusiform and superior temporal gyri may reflect higher level visual and visuo-spatial processing underlying consolidation. Learning-related increases in cerebellar responses are consistent with its proposed role in the acquisition of internal models of the motor apparatus. These learning-related changes suggest a role for interacting neural systems involving the co-operation of cortico-cortico, cortico-cerebellar and cortico-basal ganglia loops during visuo-motor transformation learning.

Direct interaction of myosin regulatory light chain with the NMDA receptor.

NMDA receptors interact with a variety of intracellular proteins at excitatory synapses. In this paper we show that myosin regulatory light chain (RLC) isolated from mouse brain is a NMDA receptor-interacting protein. Myosin RLC bound directly to the C-termini of both NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) subunits, rendering the interaction of myosin RLC with NMDA receptors distinct from that of calmodulin which is considered a NR1-interacting protein. Myosin RLC co-localized with NR1 in the dendritic spines of isolated hippocampal neurons, and was co-immunoprecipitated from brain extracts in a complex with NR1, NR2A, NR2B, PSD-95, Adaptor protein-2 and myosin II heavy chain. The C0 region of NR1 was necessary and sufficient for binding myosin RLC. Ca2+/calmodulin, but not calmodulin alone, displaced recombinant myosin RLC from the carboxy tail of NR1 indicating that myosin RLC and Ca2+/calmodulin can compete for a common binding site on NR1 in vitro. Myosin RLC is the only known substrate for myosin regulatory light chain kinase, which has recently been shown to modulate NMDA receptor function in isolated hippocampal neurons. Our results suggest that an additional level of NMDA receptor regulation may be mediated via a direct interaction with a light chain of myosin II. Thus, myosin RLC-NMDA receptor interactions may contribute to the contractile and motile forces that are placed upon NMDA receptor subunits during changes associated with synaptic plasticity and neural morphogenesis.

Flexible retinotopy: motion-dependent position coding in the visual cortex.

Although the visual cortex is organized retinotopically, it is not clear whether the cortical representation of position necessarily reflects perceived position. Using functional magnetic resonance imaging (fMRI), we show that the retinotopic representation of a stationary object in the cortex was systematically shifted when visual motion was present in the scene. Whereas the object could appear shifted in the direction of the visual motion, the representation of the object in the visual cortex was always shifted in the opposite direction. The results show that the representation of position in the primary visual cortex, as revealed by fMRI, can be dissociated from perceived location.

Noise reduction in BOLD-based fMRI using component analysis.

Principle Component Analysis (PCA) and Independent Component Analysis (ICA) were used to decompose the fMRI time series signal and separate the BOLD signal change from the structured and random noise. Rather than using component analysis to identify spatial patterns of activation and noise, the approach we took was to identify PCA or ICA components contributing primarily to the noise. These noise components were identified using an unsupervised algorithm that examines the Fourier decomposition of each component time series. Noise components were then removed before subsequent reconstruction of the time series data. The BOLD contrast sensitivity (CS(BOLD)), defined as the ability to detect a BOLD signal change in the presence of physiological and scanner noise, was then calculated for all voxels. There was an increase in CS(BOLD) values of activated voxels after noise reduction as a result of decreased image-to-image variability in the time series of each voxel. A comparison of PCA and ICA revealed significant differences in their treatment of both structured and random noise. ICA proved better for isolation and removal of structured noise, while PCA was superior for isolation and removal of random noise. This provides a framework for using and evaluating component analysis techniques for noise reduction in fMRI.