Brain Advance Access published online on July 29, 2007
Brain, doi:10.1093/brain/awm164
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Mild in vitro trauma induces rapid Glur2 endocytosis, robustly augments calcium permeability and enhances susceptibility to secondary excitotoxic insult in cultured Purkinje cells
1Cara Phelan Center for Trauma Research, St Michaels Hospital, Toronto and 2Institute for Medical Science, University of Toronto, Ontario, Canada
Correspondence to:
Andrew J. Baker, MD, FRCPC, Director, Traumatic Brain Injury Laboratory, Cara Phelan Centre for Trauma Research, St Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1W8, Canada E-mail: bakera{at}smh.toronto.on.ca
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Mild brain trauma results in a wide range of neurological symptoms that are not easily explained by the primary pathology. Purkinje neurons of the cerebellum are selectively vulnerable to brain trauma, including indirect remote trauma to the forebrain. This vulnerability manifests itself as a selective and delayed cell loss, for which the underlying mechanisms are poorly understood. Alterations to the surface expression of calcium impermeable AMPA receptors (GluR2-containing) may mediate post-traumatic calcium overload, and initiate biochemical cascades that ultimately cause progressive cell death. Our current study examined this hypothesis using an in vitro model of mild Purkinje trauma, delivered by an elastic stretch at 2.5–2.9 pounds per square inch (psi). This mild trauma alone did not increase cell loss as measured by propidium iodide (PI) uptake (at 20 h) compared to uninjured controls. However, there was a marked increase in cell loss, when cells following mild trauma, were exposed to 10 µM AMPA for 1 h compared to either mild trauma or AMPA exposure alone. Mild injury rendered Purkinje neurons significantly more permeable to AMPA-stimulated (4 µM) calcium influx at 15 min post-injury, including a sustained calcium plateau. This effect was eliminated by inhibiting protein kinase C-dependent GluR2 endocytosis with 2 µM Go6976 or blocking the calcium pore of GluR1/3 containing AMPARs with 500 nM 1-naphthylacetyl spermine (Naspm). Nifedipine (2 µM) eliminated the calcium plateau following mild injury but not the initial spike of Ca2+ increase. These results suggest that mild injuries resulted in a rapid AMPA receptor subtype switch (GluR2 was replaced by GluR1/3), which in turn resulted in an enhanced Ca2+ permeability. We further confirmed this by immunocytochemistry. Dendritic GluR2 co-localization with the pre-synaptic marker synaptophysin was markedly down-regulated at 15 min following mild stretch (P < 0.01), indicative of a rapid decrease in the synaptic expression of receptors containing this subunit. Carboxyfluorescence (CBF) assays revealed that mild stretch did not alter membrane integrity. Finally, we demonstrated that the combination of 500 nM Naspm and 5 nM Go6976 conferred a powerful neuroprotective effect on Purkinje cells by effectively eliminating the effects of mild stretch combined with AMPA in 95% of cells. These results represent a newly described mechanism rendering neurons susceptible to secondary injuries following trauma. Prevention of GluR2 endocytosis may be critical in the development of pharmacotherapies aimed at mild, seemingly inconsequential trauma, to avoid ensuing secondary damage.
Key Words: GluR2; AMPA; stretch; traumatic brain injury; cerebellum; Purkinje cells; calcium; endocytosis
Abbreviations: ABP, AMPA binding protein; CBF, carboxyfluorescence; EAA, excitatory amino acid; FDA, fluorescein diacetate; HBSS, Hank's balanced salt solution; Naspm, 1-naphthylacetyl spermine; OGD, oxygen–glucose deprivation; PI, propidium iodide; PICK1, protein interacting with C kinase 1; psi, per square inch; TBI, traumatic brain injury
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Received December 20, 2006. Revised May 9, 2007. Accepted June 27, 2007.
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In the developed world, traumatic brain injury (TBI) is a leading cause of morbidity and mortality, frequently resulting in debilitating cognitive, motor or autonomic CNS impairment (Luchter and Walz, 1995
; Kersel et al., 2001; Blackman et al., 2004; Fujimoto et al., 2004). The majority of work implementing experimental models of neurotrauma has focused on hippocampal and cortical neuronal response to insult (for a review of neuroprotection and TBI, see Faden, 2002), with cerebellar injury not often considered when examining the anatomical distribution of damage to the brain following impact. Cerebellar damage should not be overlooked in trauma, as this area of the brain unquestionably plays a crucial role in motor coordination (reviewed by Middleton and Strick, 1998, 2000; Manni and Petrosini, 2004) and as evidenced more recently, higher neurological function (Middleton and Strick, 1994, 1998, 2001; Petrosini et al., 1998; Lalonde and Strazielle, 2003). Previous work from our laboratory (Park et al., 2006) has demonstrated a marked vulnerability of Purkinje cells to both mild and severe in vivo models of forebrain trauma. Posterior regions of the cerebellar vermis, the gyrus of the horizontal fissure and gyrus of lobules II and IV exhibit significant delayed Purkinje neuron cell death following cortical trauma (Park et al., 2006).
The formidable challenge in TBI research is that primary damage is essentially complete by the time any intervention is feasible. However, with adequate therapy, secondary vulnerability to subsequent insults or progressive degeneration over time could be prevented, particularly in mild trauma (Teasdale and Graham, 1998; Amar and Levy, 1999; Arundine et al., 2004). There is evidence from in vitro models of trauma to suggest that primary mechanical deformation to central cortical neurons in mild injuries affects the vulnerability of these cells to subsequent excitotoxic challenge (Arundine et al., 2003, 2004; Geddes-Klein et al., 2006). Despite the presence of preserved membrane integrity, viability and normal electrophysiological capacity, mild stretch injuries (also termed sublethal) can result in delayed overproduction of reactive oxygen species, mitochondrial dysfunction and increases in DNA fragmentation (Arundine et al., 2004; Geddes-Klein et al., 2006), all tell-tale signs of calcium overload in central neurons. As a result, further investigation is needed to examine alterations to intracellular signaling following mild trauma—and to elucidate how these changes contribute to enhanced calcium permeability and vulnerability to secondary excitotoxicity.
There is a plethora of evidence to suggest that adult Purkinje cells of the cerebellum do not express entirely functional NMDA receptors (i.e. receptors that open ion channels), attributed primarily to a developmental down-regulation of the NMDA glutamatergic response over the course of Purkinje cell development (Llano et al., 1988; Joels et al., 1989; Audinat et al., 1990). Though developing Purkinje neurons in slice cultures are thought to possess distinct, low-conductance, native NMDA receptors early in development (Momiyama et al., 1996), the functional sensitivity of these channels to both glutamate and NMDA is transient. That is, the response of Purkinje neurons to NMDA is markedly diminished in primary cultures by 14 days in vitro (DIV) (Yuzaki et al., 1996), and entirely eliminated by post-natal day 12 in slice cultures (Momiyama et al., 1996). Therefore, we hypothesize that calcium-permeable AMPA receptors (i.e. AMPARs lacking the GluR2 subunit, but containing the GluR1/3 subunits) may play a role in mediating post-injury calcium overload.
AMPA receptors are heterologous and assembled from a pool of four subunits—namely, GluR1-4 (reviewed by Groc and Choquet, 2006). Receptors containing the GluR2 subunit are calcium impermeable, due to RNA editing of the M2 transmembrane domain of this subunit. The M2 domain of the GluR2 subunit undergoes a glutamine (Q) to a charged arginine (R) residue switch at the channel pore region (position 607) that inhibits the passage of calcium ions through the receptor (Hume et al., 1991; Burnashev et al., 1992; Greger et al., 2002). This charged arginine is not observed in the mRNA of GluR1, GluR3 or GluR4 subunits. Thus, only receptors lacking the GluR2 subunit (e.g. heteromers of GluR1/3) are calcium permeable.
There is a large body of information detailing the expression of AMPAR subunit mRNA in Purkinje neurons. Using both in situ hybridization (Keinenen et al., 1990; Sommer et al., 1990; Monyer et al., 1991) and single cell RT-PCR (Lambolez et al., 1992; Tempia et al., 1996) a number of groups indeed identify the presence of all four AMPA receptor (i.e. GluR1-4) subunits in Purkinje neurons. Some evidence suggests that the majority of AMPA receptors on Purkinje cells are GluR2 containing receptors that are Ca2+ non-permeable (Linden et al., 1993; Tempia et al., 1996), while other groups report large calcium accumulation and excitotoxicity after AMPAR stimulation (Brorson et al., 1992, 1994; Sorimachi, 1993). Interestingly, some groups have demonstrated that the calcium permeability of cells in the cerebellum can be altered either by synaptic activities or pathological stimuli that change the composition of the receptors (Brorson et al., 1994, 1995; Liu and Cull-Candy, 2000). Accordingly, we conjectured that excitotoxic input following mechanical trauma is capable of changing the composition of synaptic AMPA receptors, thereby increasing their calcium permeability and perpetuating ensuing damage.
In supporting our hypothesis, recent work from Liu et al. (2006) suggests that mild in vitro ischaemia can alter GluR2 trafficking immediately following insult. Namely, mild oxygen-glucose deprivation (OGD) results in rapid GluR2 internalization by associating GluR2 with protein interacting with C kinase 1 (PICK1) and perturbing GluR2/AMPA binding protein (ABP) interactions in a PKC
dependent process. Subsequently, these cells underwent GluR1/3 exocytosis, thereby inserting calcium permeable receptors into the plasma membrane, in place of the impermeable GluR2 containing receptors (Liu et al., 2006). Given the common pathophysiology between ischaemic and mechanical cell death (see Arundine and Tymianski, 2003 for a review of excitotoxic mechanisms of cell death in ischaemia and trauma), we sought to examine whether there are similar alterations to the surface expression of GluR2 subunit containing AMPARs following mild in vitro trauma, and whether this results in an increased permeability to extracellular calcium, conferring a heightened susceptibility to subsequent glutamatergic challenge.
Accordingly, in the present study, we examined the secondary vulnerability of mildly stretched Purkinje neurons to excitotoxic insult. We have investigated changes to the surface expression of GluR2-containing AMPA receptors immediately following the mild injury via immunocytochemistry, and any concurrent changes to calcium permeability (evidenced through ratiometric Fura-2 analysis), controlling for changes in membrane integrity with a carboxyflourescein assay. Moreover, we sought to identify any neuroprotective effects of inhibiting PKC-dependent clathrin-mediated GluR2 endocytosis, or blocking the calcium permeability of newly inserted GluR1/3 containing receptors. It was the goal of this work to identify a mechanism responsible for enhancing secondary Purkinje cell vulnerability, thereby providing insight into why this area of the brain exhibits profound delayed cell loss with mild cortical insults.
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Isolation and dissociation of Purkinje cell cultures
All procedures described here were approved by the Animal Care Committee, St Michael's Hospital and complied with regulations of Canadian Council on Animal Care. Purkinje cell culture was performed as previously described (Slemmer et al., 2004
). Cultures were prepared on 6-well BioFlex culture plates (FlexCell, Hillsborough, NC, USA). The bottom of the plate wells was made of flexible SilasticTM silicone substrates of 0.02 in. thick with a growth area of 57.75 cm2. Wells were coated overnight with poly-L-lysine (5 µg/ml; Sigma). Purkinje cells were isolated from embryonic day 16–17 Wistar rats (Charles River Laboratories, Wilmington, MA, USA). Pregnant animals were anaesthetized with isofluorane and sacrificed via decapitation. Embryos were surgically removed, isolated from the amniotic sac and decapitated. Embryo heads were placed in 20 ml 1 x Hank's Balanced Salt Solution (HBSS, Invitrogen Corp. Carlsbad, CA, USA). Brains were removed and placed in a separate dish containing 20 ml supplemented HBSS. Cerebella were dissected from whole brains using microdissection forceps, and then were incubated in 2 ml of 0.1% trypsin (Sigma–Aldrich, St Louis, MO, USA) at 33–35°C for 10 min, and placed in 2 ml HBSS again. Tissue was triturated by glass pipette 10–20 times, and seeding medium Dulbecco's modified eagle's medium (DMEM) /F-12 containing 10% Horse serum, Invitrogen) was added. Purkinje cells were centrifuged for 5 min. Cell counts were done by loading phosphate-buffered saline (PBS), Typan Blue (Sigma–Aldrich) and 50 µl of cell suspension into a haemocytometer. Cell suspension was seeded in a plating medium (neurobasal medium containing 2% B-27 supplement, 1% fetal bovine serum, 0.5 mM L-glutamine, 25 µM glutamic acid and 100 nM L-thyroxine, Invitrogen) at a density of 1 x 106 cells/well. After 96 h of isolation, cells were fed with fresh maintenance medium (neurobasal medium containing 2% B-27 supplement, 0.5 mM L-Glutamine, 100 nM L-thyroxine, Invitrogen) containing 10 µM FDU (5 mM uridine, 5 mM (+)-5-Fluor-2'-deoxyuridine, Invitrogen) and left to incubate for 48 h. Cells were fed with maintenance medium every 3–4 days until stretch assays. We used the cells for experiments 11–14 days after isolation to stay consistent with previous in vitro stretch assays (Zhang et al., 1996; Goforth et al., 1999; Weber et al., 1999; Arundine et al., 2003; Lusardi et al., 2004). Immunocytochemistry confirmed the presence of strictly neuronal, Purkinje cell cultures (Fig. 1).
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In Vitro stretch protocol
Prior to stretch, the culture medium was replaced with 2 ml HEPES buffered Saline (concentrations in mM: 121 NaCl, 5 KCl, 20 glucose, 10 HEPES acid, 7 HEPES–Na salt, 3 NaHCO3, 1 Na-pyruvate, 1.8 CaCl2 and 0.01 glycine, adjusted to pH 7.4 with NaOH). Purkinje cell monolayer cultures were subjected to rapid stretch-induced injury as established by Ellis et al. (1995
), using a cell injury controller II (Custom Design and Fabrication, Virginia Commonwealth University, Richmond, VA, USA). Our system is equipped with a feedback system that regulated the maximum pressure exerted on the plate bottom, minimizing the variation between trials. In brief, a controlled pulse of nitrogen gas induced a physiologically rapid deformation of the aforementioned BioFlex's SilasticTM bottom resulting in a quantifiable biaxial stretch of the Purkinje cells adhered to the SilasticTM surface, without detachment. Transient pulse duration was set to 50 ms and the stretch magnitudes ranged in pressure from 2.5–7.5 pounds per square inch (psi), representative of what would likely occur in humans after rotational acceleration/deceleration injury (Slemmer et al., 2002).
Secondary AMPA toxicity, determination of cell death and Go6976/NASPM neuroprotection assays
In order to establish the in vitro model, dose–response (injury–cell death) experiments were performed on the culture cells. Purkinje cells underwent varying levels of stretch (2.5–7.5 psi) in 2 ml HEPES buffer as described earlier. Wells were subsequently loaded with 10 µg/ml PI (warmed in 37°C water bath). The quantitative measurements of PI fluorescence were used as a determination of the prevalence of cell death using a Victor3V multiwell plate fluorescence scanner (PerkinElmer, Wellesley, MA, USA) controlled by Workout software (Dazdaq, Finland). All parameters including the size and number of scanning area, the duration of scanning, etc. were kept constant by using the same protocol for all groups. A second dye, fluorescein diacetate (FDA) was used as a marker of healthy, viable cells, as observed by us and others (McKinney et al., 1996; Weber et al., 1999; Pike et al., 2000; Zhao et al., 2000). It has been reported that damaged membranes lose their capacity to retain FDA, and thus will not fluoresce (Slemmer et al., 2002). In brief, immediately following stretch, baseline PI and FDA fluorescence readings were taken, cells were incubated at 37°C in the absence of CO2 and a subsequent reading was taken 20 h later. Cell death along the continuum of mechanical deformation was normalized to unstretched wells exposed to 1 mM glutamate for 1 h (Glu). This exposure routinely produced nearly 100% cell death in our observations, and that of others (Bruno et al., 1994; David et al., 1996; Sattler et al., 1997; Arundine et al., 2003), and thus PI fluorescence for each condition was normalized to these wells. Cell death was calculated according to the formula: Fraction dead = F20 – F0/F20GLU – F0GLU, where F20 = PI fluorescence 20 h post-stretch, F0 = initial PI fluorescence, F20GLU = PI fluorescence of cells 20 h post-exposure of 1 mM Glu for 1 h, F0GLU = initial PI fluorescence of 1 mM Glu exposed wells. Cells exposed to 1 mM Glu were identical cultures from the same dissection.
To examine the vulnerability of mildly stretched cells to secondary AMPA toxicity, cells were subjected to mild stretch injury (2.5–2.9 psi), immediately followed by a challenge of 10 µM, or 20 µM AMPA for 60 min at 37°C (no CO2). Wells were washed three times with HEPES buffer at the end of the incubation to remove AMPA, and PI readings were taken immediately and at 20 h post-AMPA challenge.
In our investigation into the neuroprotective effects of the PKC inhibitor 12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go6976, Calbiochem), or the GluR1/3 calcium pore blocker 1-naphthylacetyl spermine, (Naspm, Sigma) against secondary AMPA excitotoxicity of 20 µM, varying concentrations of drugs were loaded into the wells 30 min prior to mild stretch. Naspm concentrations tested were 100 nM, 300 nM and 500 nM, and Go6976 concentrations ranged from 1 nM to 500 nM. Drugs were diluted and dissolved in HEPES buffered saline, sterile filtered and warmed in 37°C water bath prior to bath application. All the toxicity assays were done in duplicate, and three or more dissections were used for each tested treatment.
[Ca2+] measurement
The membrane permeable calcium sensitive fluorescent dye, Fura-2AM (Molecular Probes, Eugene, OR, USA), was used to determine AMPA-induced alterations in intracellular calcium concentration in both mildly stretched neurons and matched controls. Purkinje cells were incubated with 5 µM Fura-2 AM for 40 min at 37°C, and then washed three times with HEPES buffered saline. Circular selections of membranes (0.75 in. diametre) were then removed from the well using a hole cutter, and placed in HEPES buffer. Cells were excited alternately at 340 and 380 nm at 1 s intervals, and an image from each excitation wavelength was captured using a high performance cooled CCD camera (Sensicam, Cooke, Eugene, OR, USA). In both conditions (15 min post-mild stretch and control) 20 pre-stimulation images were recorded (i.e. 20 s of baseline data was taken). AMPA of 4 µM was then applied via pipette pulse to the surrounding buffer solution, and calcium was monitored continuously for another 20–30 epochs. Glutamate of 50 µM was subsequently applied, to measure any immediate changes to calcium permeability in response. No changes in the responses to glutamate were observed in our initial five experiments. Given the sharp initial spike and subsequent plateau in intracellular free calcium in response to AMPA, it is likely that the ionic gradient would not favour influx of Ca2+ in response to glutamate immediately following AMPA pulse. Thus, glutamate stimulation was discontinued, with only AMPA induced calcium changes post-processed to calculate changes in permeability. Regions of interest were selected using Slidebook 4.0 software (Intelligent Imaging Innovations, Denver, CO, USA). All cells within the field of view were analysed for each experiment, with an average of 12 cells per reading (±2) and a range from 10 to 15. Three to four experimental wells from separate Purkinje cell culture were used for each experimental condition. The role of GluR2 endocytosis, and contribution of L-type voltage gated calcium channels was investigated through the application of various inhibitors, all loaded with Fura-2 AM (i.e. 40 min prior to stimulation). Protein kinase C inhibition (specifically PKC) was accomplished with 2 µM Go6976 (Calbiochem). GluR1/3 containing AMPA receptors were blocked with the application of 2 µM Naspm trihidrochloride (Sigma–Aldrich), and voltage-gated calcium channels were antagonized with 3 µM Nifedipine (Sigma). Go6976 and Naspm were diluted to their final concentrations in sterile HEPES buffer; Nifedipine was first dissolved in 10–20 mM DMSO and further diluted to its final concentration in sterile HEPES buffer.
Calcium analysis
Normalized ratiometric values (340 nm/380 nm) representing the changes of intracellular calcium were calculated by taking the average ratio at each time point (i.e. 1–60, for each of the 12 cells, with the 20th epoch representing stimulation induced changes). Baseline calcium was then averaged as readings 1 through 19. Each epoch was normalized to the calculated average baseline and plotted. This was repeated for three to four wells in all conditions, with representative traces of the average normalized readings across all trials.
Detections of alterations to plasma membrane permeability
Plasma membrane permeability following mild mechanical stretch was assessed by evaluating uptake of the ordinarily impermeant fluorescent molecule, CBF (MW = 380 Da, radius = 0.5 nm; Sigma). We adopted this technique (established by Geddes-Klein et al., 2003, 2006) for use in stretch-induced alterations to cell permeability. The technique however, has also previously been implemented to detect permeability changes in electroporated cells (Bartoletti et al., 1989; Gift and Weaver, 2000). Immediately prior to injury, cells were treated with 100 µM CBF, and nuclei were stained with Hoechst 33 342 (20 µg/ml; Molecular Probes, Eugene, OR, USA). Purkinje cells were stretched in the presence of CBF and incubated at 37°C, 5% CO2 for 10 min to maximize diffusion of the dye into cells (Geddes-Klein et al., 2006). Cells were then rinsed with buffer to ensure the removal of extracellular CBF. Sections of membranes were detached (0.75 in.), placed in HEPES buffer, and fluorescent images were taken from five different areas per section of membrane. Cells positively stained with CBF were later counted and normalized to the total number of Hoescht-positive nuclei. This procedure was repeated 3–4 times in each condition, across separate Purkinje cell isolations.
Immunocytochemistry and quantification of synaptic GluR2
In order to label total GluR2 expression, cells were fixed at 15 min post 2.9 psi in 4% paraformaldehyde for 10 min, washed 3 times for 5 min each with PBS, and blocked with 10% normal goat serum at room temperature for 1 h. Primary polyclonal rabbit anti-GluR2 (Chemicon, Temecula, CA, USA) antibodies (1 : 100) and primary monoclonal mouse anti-synaptophysin antibodies (1 : 300) were added and cells were incubated overnight at 4°C. Concurrently with primary antibody application, membranes were permeabilized with 0.3% Triton x-100 (Chemicon) for 10 min. This allowed for all GluR2 expression to be accounted for, as unpermeabilized membranes would only have labelled surface receptor subunits. Cells were washed with PBS thoroughly, and secondary antibodies (anti-rabbit, Alexa Fluor 488-conjugated, 1 : 1000, and anti-mouse, 1 : 1000, Cy3, Invitrogen) were diluted and applied with 3% normal goat serum. Cells were incubated at room temperature for 1 h in the dark. To minimize non-specific reactivity, cells were washed thoroughly again, membranes were cut and removed and mounted on slides.
During our data acquisition and analysis, neurons were selected randomly under brightfield optics, and the investigator was blind to the treatment condition.
Florescence images were captured using a confocal microscope (Nikon Eclipse E100 equipped with a Radiance 2100, laser scanning system, Bio-Rad, Hercules, CA, USA) controlled by a software, Lasersharp 2000. To reduce the chance of photo bleaching, we used epi-fluorescence optics to locate and observe the area of interest. All parameters for capturing in each channel were kept constant among images (capture speed, laser intensity, number of passes and optical filtering). We used a 100x oil lens (digitally zoomed to 2.5x for dendritic processes). As an assessment of synaptic localization of GluR2 subunit, GluR2 and synaptophysin images of the same field were merged, and two to three dendrites per cell were captured and analyzed for yellow puncta. For each condition, we analysed fluorescent signal in regions of interest by taking the total area occupied by yellow puncta. Co-localized yellow masks were generated separately for each cell using Image Pro Plus 4.0 analysis software, and total area (in pixels) corresponding to each mask was calculated. Total puncta area was analysed for every 50 µm length of process, with dendrites not exceeding 2 µm in width. Identical procedures were followed to quantify total GluR2 and synaptophysin expression. We analysed 10 cells per well in each condition, and repeated this 3–4 times (across separate cultures) in both control and mildly stretched neurons.
Statistical analysis
Fura-2 data and toxicity data are presented as means ± standard error of the mean (SEM). Immunohistochemical, carboxyfluorescein, are all presented as means + SEM. Unpaired two-tailed Students t-tests or one-way analysis of variance (ANOVA) were used for statistical analysis.
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Stretch injury characterization and secondary excitotoxic vulnerability
To establish the in vitro model for Purkinje cell injury, we first validated the cell culture system using immunocytochemistry. We used MAP2, GFAP and calbindin D-28K as markers for neurons, astrocytes and Purkinje neurons, respectively. We found that in our optimized cultures, there were <5% GFAP positive cells. More than 95% overlapping of MAP2 and calbindin 28 k positive staining were found in the culture, suggesting the majority of the cells in our culture were Purkinje neurons (Fig. 1).
To validate our stretch paradigm as an experimental model of delayed Purkinje cell death, we examined cell death at 20 h post-injury along the continuum of stretch amplitudes as assessed by PI uptake. A robust gradient was observed in PI uptake from magnitudes ranging between 3.5 and 7.5 psi (e.g. cell death averaged 23.2 ± 2.45%, n = 3 for 3.5 psi versus 70.1 ± 6.5%, n = 3 for 7.5 psi, Fig. 2A). Each magnitude tested resulted in significantly greater PI uptake than the lower pressure tested (P < 0.05–0.001, Fig. 2A). However, stretch at 2.5 psi did not alter PI uptake relative to controls (P > 0.05). Both conditions averaged approximately 11.5 (±0.85% for control, ±1.73% for 2.5 psi, n = 3 for both conditions) of the PI uptake relative to wells treated with 1 mM Glu (Fig. 2A). Mildly stretched neurons stained brightly with FDA, whereas severely injured neurons did not (data not quantified, see Fig. 2B). This would suggest insult at 2.5 psi is rather mild and does not confer delayed cell death on its own.
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Relative to both control neurons and 2.5 psi treated neurons, application of 10 µM AMPA to uninjured cells resulted in significant uptake of PI at 20 h (20.2 ± 2.85%, n = 3 versus 11.9 ± 0.85%, n = 3, P < 0.01, Fig. 2C). However, combining mild stretch with 10 µM AMPA induced markedly higher PI uptake relative to cells treated with AMPA alone (32.6 ± 2.32%, n = 3 for the combination versus 20.2 ± 2.85%, n = 3 for 10 µM AMPA alone, P < 0.01). Similarly, when exposed to 20 µM AMPA, the mildly injured cells had significantly greater PI uptake than non-injured cells (38.8 ± 1.71%, n = 3 for the combination versus 30.1 ± 2.4%, n = 3 for 20 µM AMPA alone, P < 0.05, Fig. 2C). Subsequent experiments were designed to identify the mechanisms underlying the increased vulnerability of Purkinje cells to AMPA toxicity following mild injury.
Calcium influx in response to AMPA stimulation following mild stretch
We next sought to measure the acute cellular calcium response to application of 4 µM AMPA both in control Purkinje cells, and those exposed to 2.5–2.9 psi, 15 min following injury. It should be noted that previous studies have determined that FlexCell SilasticTM membranes contribute no background fluorescence in acetoxymethyl ester (Fura-2AM) imaging experiments (Lusardi et al., 2004). Prior to AMPA application, we observed no detectable changes in free cytosolic calcium in response to control pulse of HEPES buffer in either mildly injured or control Purkinje cells (data not shown), demonstrating that the responses we later observed were in fact, ionotropic cellular responses, and not artifacts of the pipette pulse. Interestingly, we also observed no difference in the Fura-2 AM ratios of baseline emission at 340/380 nm between stretched and control neurons (respectively: 0.21 ± 0.04, n = 3 separate cultures and 36 total cells; 0.19 ± 0.04, n = 3 separate cultures and 33 total cells, P = 0.34) indicating that the mild mechanical deformation itself had no effect on cytosolic free Ca2+.
We did, however, observe remarkably different responses to application of 4 µM AMPA between mildly injured and control Purkinje cultures. AMPA application in control cells induced a small transient rise in cytosolic Ca2+, a response that quickly returned to baseline (Fig. 3A). Control cells appeared to demonstrate intact extrusion capacities, as evidenced by the transient nature of the increase in free calcium. Intracellular calcium, when normalized across all trials, increased on average to approximately 152.1 (±7.3%, n = 3) of baseline in response to 4 µM AMPA. Conversely, in mildly injured neurons 15 min post-insult, an identical application of 4 µM AMPA induced markedly greater Ca2+ influx, calculated to equal approximately 331.8 (±47.6%, n = 3) of baseline values (P < 0.001 as compared to the response in non-injured control cells, Fig. 3B). Interestingly, the acute calcium response of mildly injured neurons in response to AMPA also contained a plateau that was observed following the initial rise in free Ca2+ (Fig. 3B). This plateau (30th epoch used for statistical analysis) was steady at approximately 240.4 (±54.3%, n = 3) of baseline calcium levels (P < 0.01, Fig. 3D). The residual high Ca2+ levels are indicative of a stage of Ca2+ overload, which reflects either an impaired ability of the cells to extrude free calcium or an excessive amount of Ca2+ loading that exceeds the cell's extruding capacity. Either way, the overload of Ca2+ most likely was responsible for the lethality observed in the above 2.5 psi plus AMPA toxicity experiments.
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We sought to examine the contribution of L-type calcium channels to the AMPA stimulated increase in free Ca2+. The calcium plateau was Nifedipine-sensitive, suggesting that the initial depolarization caused by AMPA in mildly stretched neurons was large enough to activate voltage-gated calcium channels, in turn sustaining free intracellular Ca2+ at a high level after the initial influx through AMPA receptors. Nifedipine treated cells responded with a statistically significant initial increase in Ca2+ (
262.1 ± 19.1%, n = 3, P < 0.01), however this increase rapidly returned to baseline levels in injured Purkinje neurons (i.e. no plateau was seen, Fig. 3B). While the initial large Ca2+ influx appears to reflect ionotropic AMPA channel activity, the increase in intracellular calcium to 330% of baseline, as well as the plateau, appear to both be the result of L-type channel activation. We hypothesized that the endocytosis of GluR2-containing AMPARs was responsible for the initial increase in AMPA stimulated Ca2+ permeability, and sought to disrupt the cascade that leads to the internalization of GluR2 subunits. We were successful in reducing the calcium response of injured Purkinje cells to AMPA stimulation by inhibiting PKC-dependent clathrin mediated endocytosis of GluR2 with the use of 2 µM Go6976 (calcium response on average = 156.4 of baseline ±13.9%, n = 4, P < 0.01 compared to 2.5 psi) or by inhibiting the Ca2+ pore of GluR1/3 containing AMPA receptors with 2 µM Naspm (calcium response on average = 148.4 of baseline ±21.3%, n = 3, P < 0.01 compared to 2.5 psi). The Ca2+ influx returned to that of control cells in both conditions (Fig. 3B). Each drug had no measurable effects when used to treat control cells (2 µM Go6976 = 142.8 ± 11.6%, n = 3, P > 0.05 compared to control, 2 µM Naspm = 189.1 ± 17.2%, n = 3, P > 0.05 compared to control, 3 µM Nifedipine = 158.5 ± 12.2%, n = 3, P > 0.05 compared to control, Fig. 3A).
Immunocytochemistry—Mild stretch reduces co-localization of GluR2 containing AMPA receptors with the pre-synaptic marker synaptophysin
To examine the impact of mild mechanical deformation on synaptic localization of GluR2, we juxtaposed dendritic immunocytochemical labelling of GluR2 with the pre-synaptic marker synaptophysin. Synaptophysin is currently the most widely used marker of nerve terminals and has been used extensively to characterize glutamate subunit expression as synaptic (Burette et al., 2001; Ogoshi and Weiss, 2003; Liu et al., 2006). In control neurons, a large proportion of dendritic GluR2 clusters (Fig. 4A, green fluorescence) co-localized to synaptic sites with synaptophysin (Fig. 4B, red fluorescence), resulting in large yellow puncta (Fig. 4C). In contrast, as assessed 15 min following mild mechanical stretch, injury significantly reduced the total overlying yellow area, and presumably the number of dendritic GluR2-containing AMPARs (Figs 4F and 5B, n = 10 cells, repeated over three cultures, P < 0.01). Permeabilizing the membrane with 0.3% Triton x-100 allowed us to quantify total GluR2 expression (including endocytosed protein) and total synaptophysin expression. There were no significant differences between total GluR2 protein expression (P > 0.05, Fig. 5A), or total synaptophysin expression (data not shown), suggesting mild insult does not effect GluR2 mRNA levels or transcriptional regulation of GluR2, but rather favours the hypothesis that the protein is internalized from synapses. Highly consistent with recent work examining GluR2 endocytosis following mild ischaemia (Liu et al., 2006), on average, 53.4% of control GluR2 was synaptic (±10.1%), versus 15.3% (±11.6%) in mildly stretched Purkinje cells (P < 0.001, Fig. 5C).
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Mild mechanical stretch does not alter membrane integrity as assessed by CBF uptake
There were no observable changes to neuronal morphology immediately following mild stretch, as evidenced through the differential interference contrast (DIC) micrographs in Fig. 6B. However, it is possible that stretched cells may exhibit enhanced calcium permeability in response to AMPA stimulation as a result of changes to membrane permeability. We sought to verify that free cytosolic Ca2+ was not rising as a result of entry through non-specific holes or tears in the membrane. Applications of CBF enabled us to image uptake of an ordinarily impermeable fluorescent molecule and as a result determine immediate, post-stretch alterations to plasma membrane permeability in mildly stretched Purkinje cells. Representative CBF micrographs are shown in Fig. 6C for control cultures (Fig. 6C1, C2 and C3), mildly stretched cultures (Fig. 6C4, C5 and C6, 2.5 psi) and severely stretched cultures (Fig. 6C7, C8 and C9, 6.5 psi). We observed almost no CBF uptake (denoted by bright green staining) in both control and mildly stretched cultures (quantified at
6.5 ± 1.31% and 5.6 ± 1.91% CBF positive neurons, respectively, n = 3 for both conditions, Fig. 6A). CBF uptake was significantly higher in severely stretched cultures (33.6 ± 4.03%, n = 3, P < 0.001, Fig. 6A). Thus, CBF uptake was a function of the pressure exerted on cultures, and did not increase in mildly stretched neurons relative to controls. It should be noted, however, that changes to permeability that would have occurred more than 10 min post-stretch would not have been accounted for. However, recent work by Geddes-Klein et al. (2006) suggests that plasma membrane permeability changes are transient and repaired rapidly following stretch.
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Neuroprotection of Purkinje cells by Go6976 and Naspm
Finally, we tested the neuroprotective effects of the PKC
inhibitor Go6976 and the GluR1/3 calcium pore blocker Naspm against the secondary AMPA toxicity (20 µM) we observed initially following mild injury. We hypothesized that inhibiting PKC dependent clathrin-mediated GluR2 endocytosis with Go6976, or blocking the Ca2+ permeability of exocytosed GluR1/3 containing AMPARs would rescue cells otherwise destined to die from excitotoxicity. Fraction dead in Fig. 7A was calculated identically to initial toxicity protocols (see Materials and Methods section).
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Fig. 7A illustrates the neuroprotective effects of the various doses of Naspm, tested and the only neuroprotective dose of Go6976 (5 nM). We were successful at reducing cell death to control levels, by combining the most effective doses of both drugs (i.e. 500 nM Naspm and 5 nM Go6976, Fig. 7A). Naspm doses (100 nM, 300 nM, 500 nM) on their own corresponded on average (n = 3 trials for each dose) to 34.7 ± 4.4%, 28.4 ± 3.1%, 23.1 ± 5.2% cell death, respectively, of which the latter two were significant reductions from untreated injured cells (41 ± 4.9%, P < .05 for 300 nM Naspm, P < .01 for 500 nM Naspm, Fig 7A). Remarkably, cells treated with the combination of 500 nM Naspm and 5 nM Go6976 demonstrated on average only 16.7 cell death (±3.3% n = 3, P < 0.001, Fig. 7A), which was at the same level as in non-injured controls (16.43 ± 4.5%,)
As a more effective way to present this data we sought also to represent our findings as the percentage of cells saved relative to injury. Given that control cells exhibited on average 16.4% cell death at 20 h, (Figs 2A and C and 7A), the maximum possible percentage of surviving cells relative to stretched neurons was 83.6% of the entire well. It was, therefore, necessary to normalize the cytoprotective data relative to the total surviving cells under control conditions. The percentage of cells saved was calculated as:
 |
Using the above formula, on an average we were able to eliminate the lethal effects of mild stretch +20 µM AMPA in 95.2 (±3.3%) of cells by pre-treating them with 500 nM Naspm and 5 nM Go6976 (Fig. 7B). The percentages of cells saved under other treatments are summarized in Fig. 7B.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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The contribution of AMPA receptor trafficking to synaptic reorganization under pathological conditions is not well established, and represents an interesting hypothesis in the neurobiology of disease states. Through immunocytochemistry, we show that mild trauma, in the absence of conferring delayed cell death, reduces synaptic GluR2 as early as 15 min post-injury in cultured Purkinje neurons of the cerebellum, thereby robustly enhancing both calcium permeability and the susceptibility of these cells to secondary excitotoxic challenge. These changes occurred in the absence of any observable changes to morphology or damage to membrane integrity.
We demonstrate, here, as well the sensitivity of the calcium response of injured neurons to inhibitors of GluR1/3 containing AMPA receptors (Naspm), as well as Go6976, a PKC inhibitor intended to disrupt clathrin-mediated GluR2 endocytosis. Interestingly, our data suggest that mild trauma renders Purkinje cells hyper-excitable when stimulated with exogenous transmitter, possibly allowing for the lethal, delayed activation of voltage gated calcium channels, inducing a Ca2+ plateau that overrides the extrusion capacities of these cells. We further identify the subsequent novel neuroprotective effects of both Go6976 and Naspm in our model of mild trauma coupled with 1 h of excitotoxic challenge, hypothesized to be related to the prevention of GluR2 subunit internalization after mild injury. Taken together, our findings implicate AMPAR trafficking in mediating post-trauma cerebellar neuropathology and identify potential targets of post-trauma therapeutics.
Relevance of cerebellar injury to forebrain TBI
There exists substantially less data on the pathophysiological changes to the cerebellum after forebrain TBI relative to cortical or hippocampal regions. However, clinical cases of forebrain TBI have noted diffuse cerebellar axonal injury, cerebellar atrophy and various degenerative changes as indicated by MRI (Gale et al., 1995; Soto-Ares et al., 2001; Gorrie et al., 2002) Accordingly, we propose that clinical manifestations of supratentorial TBI might be due, in part, to delayed changes at the cellular level in the cerebellum (including those that are unidentified by conventional imaging techniques), in addition to primary mechanical injury of the cerebrum. We have demonstrated experimentally that Purkinje cells display a marked delayed vulnerability to mild and severe forebrain head trauma in the rat (Park et al., 2006), and this data is supported by other groups who have noted a similar phenomenon (Fukuda et al., 1996; Mautes et al., 1996; Igarashi et al., 2007). Given that many studies have attributed higher-order cognitive processing independent of motor function to the cerebellum (Schmahmann, 1991; Joyal et al., 1996; Petrosini et al., 1998; Leggio et al., 2000), we propose that cerebellar injury or dysfunction after TBI could play an important role in the progression of neurologic and motor impairments, despite going unidentified during post-TBI diagnostics. Based on the data we have collected previously in our laboratory, and on the clinical findings of other groups, we suggest that the importance of cerebellar injury to the overall pathophysiology of supratentorial TBI needs to be addressed at both the anatomic and cellular levels. This study begins to address this need.
Calcium overload in Purkinje cells following mild trauma
Increases in intracellular calcium concentrations have been shown previously in a number of in vitro models of TBI (LaPlaca et al., 1997; Weber et al., 1999; Geddes and Cargill, 2001; Lusardi et al., 2004). However, our data are not suggestive of immediate trauma-induced changes to calcium concentrations, as baseline Fura-2AM ratios did not differ between mildly injured and control cells. Rather, our findings demonstrates a pathologically high calcium permeability in response to exogenous excitatory amino-acid (EAA) stimulation, and a possible form of pathological synaptic remodelling. With limited pre-synaptic neurotransmission in our in vitro preparation (i.e. the absence of climbing fibres, parallel fibres), exogenous AMPA-induced stimulation served to mimic in vivo afferents that would ordinarily connect to this population of cells in the intact brain.
It is certainly possible that the injured Purkinje cells display more depolarized membrane potentials upon a whole cell clamp, and we are currently investigating this possibility. However, because we have not done the electrophysiology in this study, we do not know if the injured cells are more depolarized immediately after the insult. We have seen in vivo that Purkinje neurons in slice preparations taken from injured animals have more depolarized resting potentials after injury relative to control neurons (Ai et al., 2007), but we do not know if this transfers to our primary culture/stretch injury preparation. From the data we have collected, we can suggest that the calcium response of these injured cells is, in part, mediated by the endocytosis of GluR2-containing receptors. The pharmacological manipulations we have employed inhibit the kinase responsible for GluR2 serine 880 phosphorylation, and it is well established that this modification is essential to the internalization of these receptors via PICK1 (reviewed by Groc and Choquet, 2006, in Cell and Tissue Research). Given that PKCa blockade significantly blunts the calcium response of the cultured cells to AMPA, we believe the phosphorylation (and therefore the internalization) of GluR2 plays a substantial role in the ionotropic response we are seeing. As well as evidenced by our immunostaining, there is in fact a high level of somatic GluR2 immunoreactivity, due to the large numbers of GluR2-containing extrasynaptic AMPARs present in Purkinje cells (Momiyama et al., 2003). Thus, a large contingent of the AMPA receptors being activated in our calcium experiments might be extrasynaptic. However, it is unlikely that this would influence the applicability of our findings to an intact brain preparation (where these receptors might receive considerably less activation), due to the evidence that extrasynaptic and synaptic AMPA receptors possess very similar electrophysiological properties [i.e. linear current–voltage relationships, as well as low single-channel conductances (Momiyama et al., 2003)].
This study, therefore, identifies a dysfunction of Purkinje cell post-synaptic responsivity following mild trauma. It is possible that our model of mild injury did not induce delayed cell death in Purkinje cells because of the absence of pre-synaptic fibres and associated EAA spillage that is responsible for excitotoxicity following traumatic brain injury (reviewed by Arundine and Tymianski, 2004). It should be noted that distal cortical trauma has been shown to result in mild cerebellar impact, (Fukuda et al., 1996; Mautes et al., 1996; Park et al., 2006). Our results suggest that this mild impact may induce Purkinje cell GluR2 endocytosis, and allow for EAA spillage from forebrain afferents to cause dangerously high levels of Purkinje cell cytosolic [Ca2+]. Forebrain trauma might induce intracellular modifications in areas as distal as the cerebellum, thereby priming this area of the brain for indirect delayed cell death mediated by excess [Ca2+].
Excitotoxic transmission from forebrain afferents to cerebellum following trauma
Indeed, we have previously demonstrated pathophysiological changes in the cerebellum in an animal model of forebrain TBI (fluid percussion injury) (Park et al., 2006), but the intracellular modifications responsible for these changes were not fully addressed. We observed increased calbindin expression in surviving cells, suggesting a neuroprotective role for calcium buffering capacities, but did not examine the possibility of forebrain-cerebellar synaptic remodelling. Coupling these in vitro findings to what we observed in animal models suggests that the cerebellum is highly vulnerable to even mild trauma induced by an impact that is primarily cortical. Thus, cerebellar synaptic modifications could play an important role in the progression of neurological and motor impairments in patients who suffer forebrain trauma. Our results suggest that preventing Purkinje cell GluR2 internalization through the use of PKC inhibitors or blockade of GluR1/3 containing AMPARs may prevent delayed cerebellar neuronal death in animal models of cortical impact.
Pathological synaptic remodelling as a recurring theme
Previous work from our laboratory has shown other insult-induced changes to in vitro synaptic transmission. We recently demonstrated a form of pre-synaptic pathological LTP at CA3-CA1 synapses following a few minutes of anoxia and aglycaemia in hippocampal slices, attributed primarily to hyperexcitability of afferent fibres following ischaemia (Ai and Baker, 2006). However, any post-synaptic modifications that may have occurred were not accounted for. Very interestingly, recently published work from Liu et al. (2006) showed immediate GluR2 endocytosis in cultured hippocampal cells following brief, mild OGD. The results from our slice preparation (Ai and Baker, 2006) and the work presented here are highly consistent with Liu et al.'s observations, perhaps identifying a post-synaptic modification that perpetuates excitotoxic transmission in both ischaemia and trauma.
Our study demonstrates mechanically-induced modifications to neuronal AMPA responsivity and possibly highlights a form of pathological synaptic remodelling. There is a growing body of evidence that suggests Ca2+ permeable AMPA channels might be crucial contributors to neuronal injury in stroke, trauma and amyotrophic lateral sclerosis, particularly in cell populations that lack functional NMDA receptors (Brorson et al., 1994, 1995; Weiss and Sensi, 2000; Tanaka et al., 2005; Kwak and Weiss, 2006). In contributing to this evidence, our findings implicate AMPAR trafficking in mild cerebellar trauma, and identify concomitant changes to calcium permeability that may underlie delayed cerebellar cell death that worsens symptoms in patients who have suffered traumatic cortical impact. It is likely that the complexity and dispersion of neuronal damage following brain impact is vast, and is reflected in this vulnerability of Purkinje cells to secondary excitotoxic challenge following mild insult.
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Conclusions |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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The present study shows that Purkinje cells of the cerebellum are left increasingly vulnerable to excitotoxic injury following mild mechanical deformation. While the nature of the insult does not induce structural or morphological abnormalities, we demonstrate changes to intracellular receptor endocytosis as early as 15 min post-injury; namely, that of the Ca2+-impermeable AMPA receptor subunit GluR2. Additionally, it appears as though L-type calcium channels trigger a lethal calcium plateau in response to AMPA stimulation only following mild deformations, and not in control conditions. Abolishing this plateau, or circumventing lethal calcium influx in response to AMPA through PKC
inhibition or blocking the calcium pore of GluR1/3 containing receptors confers a profound mode of neuroprotection in TBI research. The rapid alterations to AMPA receptor trafficking and pathological synaptic remodelling demonstrated here may underlie the susceptibility of cells of the cerebellum to delayed neuronal death that we and others have previously observed with cortical trauma (Fukuda et al., 1996; Mautes et al., 1996; Park et al., 2006).
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Footnotes |
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* These authors contributed equally to this work.
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Acknowledgements |
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Funding to pay the Open Access publication charges for this article was provided by the Cara Phelan Endowment, St. Michael's Hospital, Toronto.
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References |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
Ai J, Baker A. Long-term potentiation of evoked presynaptic response at CA3-CA1 synapses by transient oxygen-glucose deprivation in rat brain slices. Exp Brain Res (2006) 169:126–9.[CrossRef][Web of Science][Medline]
Ai J, Liu E, Park E, Baker AJ. Structural and functional alterations of cerebellum following fluid percussion injury in rats. Exp Brain Res (2007) 177:95–112.[CrossRef][Web of Science][Medline]
Amar AP, Levy ML. Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery (1999) 44:1027–39.[CrossRef][Web of Science][Medline]
Arundine M, Tymianski M. Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci (2004) 61:657–68.[CrossRef][Web of Science][Medline]
Arundine M, Chopra GK, Wrong AW, Lei S, Aarts M, MacDonald JF, et al. Enhanced vulnerability to NMDA toxicity in sublethal traumatic neuronal injury in-vitro. J Neurotrauma (2003) 20:1377–95.[CrossRef][Web of Science][Medline]
Arundine M, Tymianski M. Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium (2003) 34:325–37.[CrossRef][Web of Science][Medline]
Arundine M, Aarts M, Lau A, Tymianski M. Vulnerability of central neurons to secondary insults after in vitro mechanical stretch. J Neurosci (2004) 24:8106–23.
Audinat E, Knöpfel T, Gähwiler BH. Responses to excitatory amino acids of Purkinje cells and neurones of the deep nuclei in cerebellar slice cultures. J Physiol (1990) 430:297–313.
Bartoletti DC, Harrison GI, Weaver JC. The number of molecules taken up by electroporated cells: quantitative determination. FEBS Lett (1989) 256:4–10.[CrossRef][Web of Science][Medline]
Blackman JA, Patrick PD, Buck ML, Rust RS Jr. Paroxysmal autonomic instability with dystonia after brain injury. Arch Neurol (2004) 61:321–8.
Brorson JR, Bleakman D, Chard PS, Miller RJ. Calcium directly permeates kainate/-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured cerebellar Purkinje neurons. Mol Pharmacol (1992) 41:603–8.[Abstract]
Brorson JR, Manzolillo PA, Miller RJ. Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells. J Neurosci (1994) 14:187–97.[Abstract]
Brorson JR, Manzolillo PA, Gibbons SJ, Miller RJ. AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity. J Neurosci (1995) 15:4515–24.[Abstract]
Bruno VMG, Goldberg MP, Dugan LL, Giffard RG, Choi DW. Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen-glucose deprivation or excitatory amino acids. J Neurochem (1994) 63:1398–1406.[Web of Science][Medline]
Burette A, Khatri L, Wyszynski M, Sheng M, Ziff EB, Weinberg RJ. Differential cellular and subcellular localization of AMPA receptor-binding protein and glutamate receptor-interacting protein. J Neurosci (2001) 21:495–503.
Burnashev N, Monyer H, Seeburg PH, Sakmann B. Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron (1992) 8:189–98.[CrossRef][Web of Science][Medline]
David JC, Yamada KA, Bagwe MR, Goldberg MP. AMPA receptor activation is rapidly toxic to cortical astrocytes when desensitization is blocked. J Neurosci (1996) 16:200–209.
Ellis EF, McKinney JS, Willoughby KA, Liang S, Povlishock JT. A New model for rapid stretch-induced injury of cells in culture: characterization of the model using astrocytes. J Neurotrauma (1995) 12:325–39.[Web of Science][Medline]
Faden AI. Neuroprotection and traumatic brain injury: theoretical option or realistic proposition. Curr Opin Neurol (2002) 15:707–12.[CrossRef][Web of Science][Medline]
Fukuda K, Aihara N, Sagar SM, et al. Purkinje cell vulnerability to mild traumatic brain injury. J Neurotrauma (1996) 13:255–66.[Web of Science][Medline]
Fujimoto ST, Longhi L, Saatman KE, Conte V, Stocchetti N, McIntosh TK. Motor and cognitive function evaluation following experimental traumatic brain injury. Neurosci Biobehav Rev (2004) 28:365–78.[CrossRef][Web of Science][Medline]
Gale SD, Johnson SC, Bigler ED, Blatter DD. Trauma-induced degenerative changes in brain injury: a morphometric analysis of three patients with preinjury and postinjury MR scans. J Neurotrauma (1995) 12:151–8.[Web of Science][Medline]
Geddes DM, Cargill RS. An in vitro model of neurotrauma: device characterization and calcium response to mechanical stretch. J Biomech Eng (2001) 123:247–55.[CrossRef][Web of Science][Medline]
Geddes DM, Cargill RS, LaPlaca MC. Mechanical stretch to neurons results in a strain rate and magnitude-dependent increase in plasma membrane permeability. J Neurotrauma (2003) 20:1039–49.[CrossRef][Web of Science][Medline]
Geddes-Klein DM, Schiffman KB, Meaney DF. Mechanisms and consequences of neuronal stretch injury in vitro differ with the model of trauma. J Neurotrauma (2006) 23:193–204.[CrossRef][Web of Science][Medline]
Gift EA, Weaver JC. Simultaneous quantitative determination of electroporated molecular uptake and subsequent cell survival using gel microdrops and flow cytometry. Cytometry (2000) 9:243–9.
Goforth PB, Ellis EF, Satin LS. Enhancement of AMPA-mediated current after traumatic injury in cortical neurons. J Neurosci (1999) 19:7367–74.
Gorrie C, Oakes S, Duflou J, Blumbergs P, Waite PM. Axonal injury in children after motor vehicle crashes: extent, distribution, and size of axonal swellings using beta-APP immunohistochemistry. J Neurotrauma (2002) 19:1171–82.[CrossRef][Web of Science][Medline]
Greger IH, Khatri L, Ziff EB. RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum. Neuron (2002) 34:759–72.[CrossRef][Web of Science][Medline]
Groc L, Choquet D. AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse. Cell Tissue Res (2006) 326:423–38.[CrossRef][Web of Science][Medline]
Hume RI, Dingledine R, Heinemann SF. Identification of a site in glutamate receptor subunits that controls calcium permeability. Science (1991) 253:1028–31.
Igarashi T, Potts MB, Noble-Haeusslein LJ. Injury severity determines Purkinje cell loss and microglial activation in the cerebellum after cortical contusion injury. Exp Neurol (2007) 203:258–68.[CrossRef][Web of Science][Medline]
Joels M, Yool AJ, Gruol DL. Unique properties of non-N-methyl-d-aspartate excitatory responses in cultured Purkinje cells. Proc Natl Acad Sci USA (1989) 86:3404–8.
Joyal CC, Meyer C, Jacquart G, Mahler P, Caston J, Lalonde R. Effects of midline and lateral cerebellar lesions on motor coordination and spatial orientation. Brain Res (1996) 739:1–11.[CrossRef][Web of Science][Medline]
Keinenen K, Wisden W, Sommer B, Werner P, Herb A, Verdoorn TA, et al. A family of AMPA-selective glutamate receptors. Science (1990) 249:556–60.
Kersel DA, Marsh NV, Havill JH, Sleigh JW. Neuropsychological functioning during the year following severe traumatic brain injury. Brain Inj (2001a) 15:283–96.[Web of Science][Medline]
Kersel DA, Marsh NV, Havill JH, Sleigh JW. Psychosocial functioning during the year following severe traumatic brain injury. Brain Inj (2001b) 15:683–96.[Web of Science][Medline]
Kwak S, Weiss JH. Calcium-permeable AMPA channels in neurodegenerative disease and ischemia. Curr Opin Neurobiol (2006) 16:281–7.[CrossRef][Web of Science][Medline]
Lalonde R, Strazielle C. The effects of cerebellar damage on maze learning in animals. Cerebellum (2003) 2:300–9.[CrossRef][Web of Science][Medline]
Lambolez B, Audinat E, Bochet P, Crtpel F, Rossier J. AMPA receptor subunits expressed by single Purkinje cells. Neuron (1992) 9:247–58.[CrossRef][Web of Science][Medline]
LaPlaca MC, Lee VM, Thibault LE. An in vitro model of traumatic neuronal injury: loading rate-dependent changes in acute cytosolic calcium and lactate dehydrogenase release. J Neurotrauma (1997) 14:355–68.[Web of Science][Medline]
Leggio MG, Molinari M, Neri P, Graziano A, Mandolesi L, Petrosini L. Representation of actions in rats: the role of cerebellum in learning spatial performances by observation. Proc Natl Acad Sci USA (2000) 97:2320–5.
Linden DJ, Smeyne M, Connor JA. Induction of cerebellar long.term depression in culture requires postsynaptic action of sodium ions. Neuron (1993) 11:1093–100.[CrossRef][Web of Science][Medline]
Liu SQ, Cull-Candy SG. Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype. Nature (2000) 405:454–8.[CrossRef][Medline]
Liu B, Liao M, Mielke JG, Ning K, Chen Y, Li L, et al. Ischemic insults direct glutamate receptor subunit 2-lacking AMPA receptors to synaptic sites. J Neurosci (2006) 26:5309–19.
Llano I, Marty A, Johnson JW, Ascher P, Gahwiler BH. Patch-clamp recording of amino acid-activated responses in "organotypic" slice-cultures. Proc Natl Acad Sci USA (1988) 85:3221–5.
Luchter S, Walz MC. Long-term consequences of head injury. J Neurotrauma (1995) 12:517–26.[Web of Science][Medline]
Lusardi TA, Wolf JA, Putt ME, Smith DH, Meaney DF. Effect of acute calcium influx after mechanical stretch injury in vitro on the viability of hippocampal neurons. J Neurotrauma (2004) 21:61–72.[CrossRef][Web of Science][Medline]
Manni E, Petrosini L. A century of cerebellar somatotopy: a debated representation. Nat Rev Neurosci (2004) 5:241–9.[Web of Science][Medline]
Mautes AE, Fukuda K, Noble LJ. Cellular response in the cerebellum after midline traumatic brain injury in the rat. Neurosci Lett (1996) 214:95–8.[CrossRef][Web of Science][Medline]
McKinney JS, Willoughby KA, Liang S, Ellis EF. Stretch-induced injury of cultured neuronal, glial, and endothelial cells. Effect of polyethylene glycol-conjugated superoxide dismutase. Stroke (1996) 27:934–40.
Middleton FA, Strick PL. Cerebellar projections to the prefrontal cortex of the primate. J Neurosci (2001) 15:700–12.
Middleton FA, Strick PL. Basal ganglia and cerebellar loops: motor and cognitive circuits. Brain Res Brain Res Rev (2000) 31:236–50.[CrossRef][Medline]
Middleton FA, Strick PL. The cerebellum: an overview. Trends Neurosci (1998) 21:367–9.[CrossRef][Web of Science][Medline]
Middleton FA, Strick PL. Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function. Science (1994) 266:458–61.
Momiyama A, Feldmeyer D, Cull-Candy SG. Identification of a native low-conductance NMDA channel with reduced sensitivity to Mg2+ in rat central neurones. J Physiol (1996) 494:479–92.
Momiyama A, Silver RA, Hausser M, Notomi T, Wu Y, Shigemoto R, et al. The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats. J Physiol (2003) 549:75–92.
Monyer H, Seeburg PH, Wisden W. Glutamate-operated channels: developmentally early and mature forms arise by alternative splicing. Neuron (1991) 6:799–810.[CrossRef][Web of Science][Medline]
Ogoshi F, Weiss JH. Heterogeneity of Ca2+-permeable AMPA/kainate channel expression in hippocampal pyramidal neurons: fluorescence imaging and immunocytochemical assessment. J Neurosci (2003) 23:10521–30.
Park E, McKnight S, Ai J, Baker AJ. Purkinje cell vulnerability to mild and severe forebrain head trauma. J Neuropathol Exp Neurol (2006) 65:226–34.[Web of Science][Medline]
Petrosini L, Leggio MG, Molinari M. The cerebellum in the spatial problem solving: a co-star or a guest star? Prog Neurobiol (1998) 56:191–210.[CrossRef][Web of Science][Medline]
Pike BR, Zhao X, Newcomb JK, Glenn CC, Anderson DK, Hayes RL. Stretch injury causes calpain and caspase-3 activation and necrotic and apoptotic cell death in septo-hippocampal cell cultures. J Neurotrauma (2000) 17:283–98.[Web of Science][Medline]
Sattler R, Charlton MP, Hafner M, Tymianski M. Determination of the time-course and extent of neurotoxicity at defined temperatures in cultured neurons using a modified multi-well plate fluorescence scanner. J Cereb Blood Flow Metab (1997) 17:455–63.[CrossRef][Web of Science][Medline]
Schmahmann JD. An emerging concept. The cerebellar contribution to higher function. Arch Neurol (1991) 48:1178–87.
Slemmer JE, Matser EJ, De Zeeuw CI, Weber JT. Repeated mild injury causes cumulative damage to hippocampal cells. Brain (2002) 125:2699–709.
Slemmer JE, Weber JT, De Zeeuw CI. Cell death, glial protein alterations and elevated S-100 beta release in cerebellar cell cultures following mechanically induced trauma. Neurobiol Dis (2004) 15:563–72.[CrossRef][Web of Science][Medline]
Sommer B, Keinanen K, Verdoorn TA, Wisden W, Burnashev N, Herb A, et al. Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science (1990) 249:1580–5.
Sorimachi M. Calcium permeability of non-N-methyl-D-aspartate receptor channels in immature Purkinje cells: studies using Fura- microfluorometry. J Neurochem (1993) 60:1236–43.[CrossRef][Web of Science][Medline]
Soto-Ares G, Vinchon M, Delmaire C, Abeciden E, Dhellemes P, Pruvo JP. Cerebellar atrophy after severe traumatic head injury in children. Childs Nerv Syst (2001) 17:263–69.[CrossRef][Web of Science][Medline]
Tanaka J, Matsuzaki M, Tarusawa E, Momiyama A, Molnar E, Kasai H, et al. Number and density of AMPA receptors in single synapses in immature cerebellum. J Neurosci (2005) 25:799–807.
Teasdale GM, Graham DI. Craniocerebral trauma: protection and retrieval of the neuronal population after injury. Neurosurgery (1998) 43:723–37.[Web of Science][Medline]
Tempia F, Kano M, Schneggenburger R, Schirra C, Garaschuk O, Plant T, et al. Fractional calcium current through neuronal AMPA-receptor channels with a low calcium permeability. J Neurosci (1996) 16:456–66.
Weber JT, Rzigalinski BA, Willoughby KA, Moore SF, Ellis EF. Alterations in calcium-mediated signal transduction after traumatic injury of cortical neurons. Cell Calcium (1999) 26:289–99.[CrossRef][Web of Science][Medline]
Weiss JH, Sensi SL. Ca2+-Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration. Trends Neurosci (2000) 23:365–71.[CrossRef][Web of Science][Medline]
Yuzaki M, Forrest D, Verselis LM, Sun SC, Curran T, Connor JA. Functional NMDA receptors are transiently active and support the survival of Purkinje cells in culture. J Neurosci (1996) 16:4651–61.
Zhang L, Rzigalinski BA, Ellis EF, Satin LS. Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons. Science (1996) 274:1921–3.
Zhao X, Newcomb JK, Pike BR, Wang KK, d’Avella D, Hayes RL. Novel characteristics of glutamate-induced cell death in primary septohippocampal cultures: relationship to calpain and caspase-3 protease activation. J Cereb Blood Flow Metab (2000) 20:550–62.[Web of Science][Medline]
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