Technical Reference #1647

1647.

AMPA Glutamate Receptor Subunits 1 and 2 Regulate Dendrite Complexity and Spine Motility in Neurons of the Developing Neocortex W. Chen; R. Prithviraj; A. Mahnke; K. Mcgloin; J. Tan; A. Gooch; F. Inglis, Tulane University, Neuroscience, 159(1647), (2009)

Abstract:
Abstract—Within neurons of several regions of the CNS mature dendrite architecture is attained via extensive reorganizationof arbor during the developmental period. Since dendritemorphology determines the firing patterns of the neuronmorphological

Keywords:
dendrite branching; activity-dependent development; filopodia; glutamate receptor; PSD95; calcium

Materials & Methods:
Animals All animal protocols used in this study were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For this study timed-pregnant Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington MA USA) and housed and maintained on a 12-h light/dark cycle with unlimited access to food and water. Every effort was made to minimize the number of animals used and their suffering. Cortical neuronal cultures Cortices were dissected from timed-pregnant embryonic day 17 (E17) Sprague Dawley rats and dissociated with trypsin (0.25%) for 15 min triturated via fire-polished Pasteur pipets and resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum 1% penicillin/streptomycin (10000 U/ml) and 2 mM glutamine. Cultures were plated (105 cells/coverslip) on glass-bottom culture dishes (12 mm diameter glass bottom area; MatTek Ashland MA USA) pretreated with poly-D-lysine (100 g/ml) and laminin (10 g/ml). Twenty-four hours later culture medium was replaced with Neurobasal medium supplemented with B27 1% penicillin/streptomycin and 0.5 mM glutamine. All tissue culture reagents were purchased from Invitrogen (Carlsbad CA USA) with the exception of poly-D-lysine (Sigma St. Louis MO USA). Transfection and imaging of cultured neurons To determine the effects of AMPA GluR1 and GluR2 subunits on parameters of cortical dendrite morphology cells were transfected with pcDNA3 expressing GluR1flip or pRK5 expressing GluR2flip (100 ng/coverslip) using Lipofectamine 2000 (Invitrogen) as previously described (Prithviraj et al. 2008). Neurons were visualized by the co-expression of green fluorescent protein (GFP) (50 ng/ coverslip); control neurons received 50 ng of GFP alone. These concentrations have been demonstrated to produce expression levels close to those of native subunits (Prithviraj et al. 2008; Robert et al. 2002) and confer predictable electrophysiological responses (Robert et al. 2002). Transfection of plasmids took place on the seventh day in vitro (DIV) so that the effects of glutamate receptor expression could be measured before dendritic trees are fully established. This time-point also corresponds to the normal time period during which AMPA receptors are likely to be inserted into synapses (Lu and Constantine-Paton 2006; Rumpel et al. 1998); we reasoned therefore that if AMPA receptor expression is an important determinant of dendrite morphology we would be most likely to observe morphological effects of AMPA receptor expression at this time. Twenty-four hours after transfection on DIV8 the medium within the culture dish was replaced with a dye-free buffered external medium (Robert et al. 2000) and the dish was placed on an inverted microscope (Zeiss Axiovert 200M) and maintained at 37 °C with plate and objective heaters. Time-lapse imaging of transfected cortical pyramidal neurons identified by their characteristic shape and the presence of numerous spines and filopodia was performed using Zeiss Axiovision image capture software. For each neuron a series of 25 images was captured at 5 s intervals (total 2 min) using a chargecoupled device (CCD) camera (Hamamatsu Orca; exposure 10 ms for each frame). These procedures were not observed to result in signs of injury such as swelling or blebbing suggesting that cells remained viable throughout. Quantitative analyses of dendrite morphology Following image capture indices of dendritic length and complexity were measured using Neurolucida (MBF Biosciences Williston VT USA) as described previously (Prithviraj et al. 2008). For each neuron the following parameters were calculated: the number of primary dendrites branch-points and tips per neuron; number of filopodia and spines per neuron; total amount of dendritic arbor per neuron; and average length of branch segments calculated by dividing the total amount of arbor by the number of branch segments (number of branch-points number of tips). Statistical variance among treatment groups was estimated using analysis of variance (ANOVA) (Statview 5.0). Pairwise comparisons between treatment groups were performed post hoc using Scheffé’s F test. To determine whether GluR1 or GluR2 expression resulted in intrinsic changes in geometry across the span of the dendritic tree dendrites were also analyzed according to branch order (Inglis et al. 2002; Prithviraj et al. 2008). For these analyses a dendritic process emerging from the cell body is considered a primary dendrite; once this bifurcates two secondary dendrites are formed and so on. Comparisons of the total amount of dendritic arbor per branch order and the number of segments of each branch order were made using repeated measures ANOVA. Pairwise comparisons of repeated measures were made between treatment groups using Scheffé’s post hoc F test. Quantification of filopodium and spine motility To determine whether the expression of AMPA receptor subunits promotes alterations in the stability of filopodia and spines the motility of each process was analyzed from time-lapse images using Metamorph software. For these analyses the position of the tip of each protrusion was recorded in the 25 successive frames captured during time-lapse imaging and the deflection from the point of origin was calculated for the duration of imaging. The average distance and movement speed was calculated for each protrusion and average motilities were calculated for each cell. Statistical analyses of the effects of treatment on the average filopodial motility per cell were made using ANOVA. Immunocytochemical detection of GluR1 and GluR2 Cultures transfected with GFP and GluR1 or GluR2 were processed immunochemically to confirm expression of AMPA receptor transgenes. Briefly cultures were fixed by the addition of 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Cells were rinsed in PBS for a further 30 min and incubated overnight at 4 °C with primary antibodies against GluR1 (rabbit polyclonal Chemicon Temecula CA USA; 1:200 in 1% normal goat serum (NGS) with 0.1% Triton X-100) or GluR2 (rabbit polyclonal ChemiconA; 1:500 in 1% NGS with 0.1% Triton X-100). Cells were rinsed with PBS and incubated for 1 h at room temperature in a rhodamine–tetramethyl rhodamine iso-thiocyanate–conjugated secondary antibody (goat anti-rabbit 1:200; Chemicon) in PBS containing 1% NGS. Cells were rinsed with PBS and coverslipped and immunoreactivity for GluR1 and GluR2 was detected by fluorescence microscopy and analyzed as described above. To estimate the degree of transgene expression mean fluorescence intensities were quantified for the cell body and dendrites of transfected cells and compared with non-transfected cells within the same coverslip as described previously (Robert et al. 2002). For each group the fluorescence intensities of five cells were analyzed and compared statistically using Student’s t-test (unpaired two-tailed).

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
PSD95