BDNF-TrkB Signaling in Single-Spine Structural Plasticity

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

Attenuation of inflammatory events in human intervertebral disc cells with a tumor necrosis factor antagonist.

STUDY DESIGN: The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro. OBJECTIVE: To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro. SUMMARY OF BACKGROUND DATA: TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown. METHODS: IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells. RESULTS: Across all patients, TNF-α induced large, statistically significant increases in NO, PGE₂, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P < 0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE₂ in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC₅₀ values for NO and PGE₂ were found to be 35.1 and 20.5 nmol/L, respectively. CONCLUSION: Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

Spatial vision in the purple sea urchin Strongylocentrotus purpuratus (Echinoidea).

Recent evidence that echinoids of the genus Echinometra have moderate visual acuity that appears to be mediated by their spines screening off-axis light suggests that the urchin Strongylocentrotus purpuratus, with its higher spine density, may have even more acute spatial vision. We analyzed the movements of 39 specimens of S. purpuratus after they were placed in the center of a featureless tank containing a round, black target that had an angular diameter of 6.5 deg. or 10 deg. (solid angles of 0.01 sr and 0.024 sr, respectively). An average orientation vector for each urchin was determined by testing the animal four times, with the target placed successively at bearings of 0 deg., 90 deg., 180 deg. and 270 deg. (relative to magnetic east). The urchins showed no significant unimodal or axial orientation relative to any non-target feature of the environment or relative to the changing position of the 6.5 deg. target. However, the urchins were strongly axially oriented relative to the changing position of the 10 deg. target (mean axis from -1 to 179 deg.; 95% confidence interval +/- 12 deg.; P<0.001, Moore's non-parametric Hotelling's test), with 10 of the 20 urchins tested against that target choosing an average bearing within 10 deg. of either the target center or its opposite direction (two would be expected by chance). In addition, the average length of the 20 target-normalized bearings for the 10 deg. target (each the vector sum of the bearings for the four trials) were far higher than would be expected by chance (P<10(-10); Monte Carlo simulation), showing that each urchin, whether it moved towards or away from the target, did so with high consistency. These results strongly suggest that S. purpuratus detected the 10 deg. target, responding either by approaching it or fleeing it. Given that the urchins did not appear to respond to the 6.5 deg. target, it is likely that the 10 deg. target was close to the minimum detectable size for this species. Interestingly, measurements of the spine density of the regions of the test that faced horizontally predicted a similar visual resolution (8.3+/-0.5 deg. for the interambulacrum and 11+/-0.54 deg. for the ambulacrum). The function of this relatively low, but functional, acuity - on par with that of the chambered Nautilus and the horseshoe crab - is unclear but, given the bimodal response, is likely to be related to both shelter seeking and predator avoidance.

Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance.

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [kappa opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the kappa agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.

OBJECT: Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. METHODS: Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups. RESULTS: The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2-3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1-2) with a predominantly heterogeneous staining pattern. CONCLUSIONS: This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

Measurements of Epidural Space Depth Using Preexisting CT Scans Correlate with Loss of Resistance Depth during Thoracic Epidural Catheter Placement.

Background. Thoracic epidural catheters provide the best quality postoperative pain relief for major abdominal and thoracic surgical procedures, but placement is one of the most challenging procedures in the repertoire of an anesthesiologist. Most patients presenting for a procedure that would benefit from a thoracic epidural catheter have already had high resolution imaging that may be useful to assist placement of a catheter. Methods. This retrospective study used data from 168 patients to examine the association and predictive power of epidural-skin distance (ESD) on computed tomography (CT) to determine loss of resistance depth acquired during epidural placement. Additionally, the ability of anesthesiologists to measure this distance was compared to a radiologist, who specializes in spine imaging. Results. There was a strong association between CT measurement and loss of resistance depth (P < 0.0001); the presence of morbid obesity (BMI > 35) changed this relationship (P = 0.007). The ability of anesthesiologists to make CT measurements was similar to a gold standard radiologist (all individual ICCs > 0.9). Conclusions. Overall, this study supports the examination of a recent CT scan to aid in the placement of a thoracic epidural catheter. Making use of these scans may lead to faster epidural placements, fewer accidental dural punctures, and better epidural blockade.

Sticky central limit theorems on open books

Given a probability distribution on an open book (a metric space obtained by gluing a disjoint union of copies of a half-space along their boundary hyperplanes), we define a precise concept of when the Fréchet mean (barycenter) is sticky. This nonclassical phenomenon is quantified by a law of large numbers (LLN) stating that the empirical mean eventually almost surely lies on the (codimension 1 and hence measure 0) spine that is the glued hyperplane, and a central limit theorem (CLT) stating that the limiting distribution is Gaussian and supported on the spine.We also state versions of the LLN and CLT for the cases where the mean is nonsticky (i.e., not lying on the spine) and partly sticky (i.e., is, on the spine but not sticky). © Institute of Mathematical Statistics, 2013.

Return to sport after open and microdiscectomy surgery versus conservative treatment for lumbar disc herniation: a systematic review with meta-analysis.

BACKGROUND: Lumbar disc herniation has a prevalence of up to 58% in the athletic population. Lumbar discectomy is a common surgical procedure to alleviate pain and disability in athletes. We systematically reviewed the current clinical evidence regarding athlete return to sport (RTS) following lumbar discectomy compared to conservative treatment. METHODS: A computer-assisted literature search of MEDLINE, CINAHL, Web of Science, PEDro, OVID and PubMed databases (from inception to August 2015) was utilised using keywords related to lumbar disc herniation and surgery. The design of this systematic review was developed using the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Methodological quality of individual studies was assessed using the Downs and Black scale (0-16 points). RESULTS: The search strategy revealed 14 articles. Downs and Black quality scores were generally low with no articles in this review earning a high-quality rating, only 5 articles earning a moderate quality rating and 9 of the 14 articles earning a low-quality rating. The pooled RTS for surgical intervention of all included studies was 81% (95% CI 76% to 86%) with significant heterogeneity (I(2)=63.4%, p<0.001) although pooled estimates report only 59% RTS at same level. Pooled analysis showed no difference in RTS rate between surgical (84% (95% CI 77% to 90%)) and conservative intervention (76% (95% CI 56% to 92%); p=0.33). CONCLUSIONS: Studies comparing surgical versus conservative treatment found no significant difference between groups regarding RTS. Not all athletes that RTS return at the level of participation they performed at prior to surgery. Owing to the heterogeneity and low methodological quality of included studies, rates of RTS cannot be accurately determined.