Formation of recurrent basal dendrites by relocation of existing apical dendrites
Analysis of explants paraformaldehyde-fixed after one week in culture (7 DIV) revealed that culture preparation alone was sufficient to drive recurrent basal dendrite formation. In 7 DIV explants, 20.26±3.66% of YFP-expressing cells had recurrent dendrites (n=7 explants, 136 cells). By contrast, only 2.78±2.78% of cells had recurrent basal dendrites in age-equivalent (14-day-old) intact control Thy1-YFP expressing mice (P=0.033, z-test).
To reveal the cellular processes underlying recurrent basal dendrite formation, 27 explants were serially imaged beginning at 6-7 DIV. Approximately 40 granule cells expressed YFP in each explant, giving a total of 1,030 cells. Forty-three of these cells were selected for analyses (see methods), representing 4.2% of the YFP-expressing cell population. Approximately half of these cultures were also treated with the excitotoxin kainic acid, but as this produced no significant increase in recurrent basal dendrite formation over culture preparation alone (Supplementary Table 1
), data from all cultures was pooled.
Eight of the 43 (18.6%) granule cells examined developed recurrent basal dendrites during the imaging period, revealing for the first time the cellular processes by which this pathology develops. Surprisingly, recurrent basal dendrites were not formed by growth of new dendritic processes from the basal pole of the cell (in fact, no examples of dendritic growth were observed). Rather, existing apical dendrites were observed to shift positions from the apical to the basal pole of the cell (), indicating – quite unexpectedly – that even though these cells are fully differentiated, dendritic origins are not stable relative to the cell body.
Figure 1 Serial confocal maximum projections of YFP-expressing granule cells showing conversion of apical dendrites into recurrent basal dendrites. (a-d) Images taken over one week show the formation of a recurrent basal dendrite at 10 DIV (white arrow). Blue (more ...)
Conversion of dendritic branches into primary dendrites
A second, entirely novel, form of neuronal plasticity observed was the conversion of dendritic branches into primary dendrites. Specifically, secondary dendritic branches moved onto the cell body, such that they now became, by definition, primary dendrites (). In the most extreme example observed, a secondary dendritic branch moved its origin to the cell body and existed as a primary dendrite for several days (). Following swelling and contraction of the cell body, this dendritic branch – now primary dendrite – moved back into position as a dendritic branch, but on a different dendrite. Branch-to-dendrite conversion was observed on 10 of 43 (23.3%) granule cells (two of these cells also developed recurrent basal dendrites).
Figure 2, a-c Serial confocal maximum projections of a YFP-expressing dentate granule cell exhibiting conversion of a dendritic branch to a primary dendrite. Images taken over six days exhibit the translocation of a dendritic branch (blue arrow) to the cell body at (more ...)
Somatic translocation contributes to dendritic dysmorphogenesis
Careful examination of serial images revealed that, as an apical dendrite shifted to the basal pole of the cell, the primary dendritic segment (the segment between the soma and the first branch point) of an adjacent apical dendrite frequently decreased in length (). To quantify this effect, changes in segment length were determined for the 16 cells exhibiting basal dendrite formation, branch point absorption or both. Eleven of these cells showed clear decreases in segment length, supporting the conclusion that somatic translocation accounts for the observed dendritic changes.
Based on these measurements, we were able to determine the total movement of the soma over the observation period, the average speed of movement and the maximum speed. The eleven cells exhibiting clear movement shifted their somata on average 11.04±1.47 μm towards the molecular layer, with the maximum movement observed being 20.45 μm. Average speed of movement for these cells was 2.20±0.24 μm/day, although somatic movement tended to occur in spurts, with a maximum speed observed of 9.6μm/day. It is unclear how dendritic changes developed in the five cells that did not show obvious somatic translocation (including the cell depicted in ). In some cases, the soma appeared to move first towards one dendrite, and then another, complicating interpretation; but the possibility that mechanisms apart from somatic translocation account for some plastic changes should not be discounted.
Granule cell dysmorphogenesis is correlated with cell layer dispersion
Evidence of somatic translocation in the present study raises the possibility that this phenomenon may contribute to granule cell layer dispersion. Dispersion of cell body layers is typical of organotypic hippocampal explants, as cultures thin and spread during the incubation period. To determine whether dendritic dysmorphogenesis was correlated with granule cell dispersion, dispersion in explants containing the most stable granule cells (n=10, Suppl. Fig.1
) was compared to explants containing cells that exhibited the most dramatic changes (n=11). The latter displayed significantly more dispersion relative to explants containing stable cells (percent change for explants with disrupted cells, 27.0±16.3%; stable cells -3.9±2.6%; P=0.007, Mann-Whitney RST).
Granule cell dysmorphogenesis does not reflect acute cellular degeneration
Cultures were maintained for at least three days after the last live-imaging session to ensure that the plastic changes described here did not reflect terminal degenerative changes. Serial-imaging data revealed a characteristic pattern of granule cell death. Dying cells first exhibited dendritic beading and a diminution of the YFP signal, followed by disintegration of the cell into a series of disconnected YFP-containing compartments (Suppl. Fig.2
). This process occurred rapidly, often leaving no evidence of the cell within 24 hours. Granule cells appearing to be in any stage of this process at the end of the experiment (3-5 days after the last imaging session) were excluded from the study.
Granule cells exhibiting plastic changes are fully differentiated
A subset of explant cultures was immunostained after 7 DIV to assess the developmental stage of YFP-expressing cells. Cultures were immunostained with calretinin, a marker of early postmitotic granule cells; NeuN, a postmitotic neuronal marker and calbindin, a marker of fully differentiated granule cells (for review of granule cell markers, see Zhao et al., 2008
). At 7 DIV, five of 138 YFP cells (3.62%) colocalized calretinin, 127 of 130 YFP cells (97.69%) colocalized NeuN and182 of 202 YFP cells (90.09%) colocalized calbindin (Suppl. Fig.3
). Morphologically, YFP-labeled cells appeared mature, exhibiting spine-coated dendrites projecting to the hippocampal fissure, somata located throughout the granule cell layer, and axons projecting into the hilus and stratum lucidum. Hilar basal dendrites were absent, as is typical for mature, but not immature, granule cells (Jones et al., 2003
; Shapiro and Ribak, 2005
Migration of granule cells and recurrent basal dendrite formation occur in vivo
The present study suggests that granule cell dispersion occurs following migration of the soma up a leading apical dendrite, which in turn leads to the formation of recurrent basal dendrites. If correct, granule cell dispersion and recurrent basal dendrite formation should occur in synchrony. To test this prediction, and to explore the cellular mechanisms by which recurrent basal dendrites form in vivo, we utilized the intrahippocampal kainic acid injection model of epilepsy (IHp-KA). In this model, kainic acid is injected unilaterally into the hippocampus, leading to status epilepticus, epileptogenesis and granule cell dispersion.
Measures of granule cell dispersion focused on cells located in the dentate molecular layer, as these cells presumably migrated the greatest distances to arrive at this location. The number of granule cells in the molecular layer was significantly increased one and two weeks after IHp-KA (; control, 15.6±1.4 ectopic cells/dentate; 1wk-IHpKA, 22.5±2.4; 2wk-IHpKA, 34.5±4.6; ANOVA on ranked data, p<0.001). The most parsimonious explanation for the appearance of these additional cells is that they migrated from the granule cell body layer to the molecular layer after IHp-KA. We next queried whether granule cells with recurrent basal dendrites were more common in the molecular layer after IHp-KA. The incidence of granule cells with recurrent basal dendrites () was increased one (p=0.025) and two (p=0.035) weeks after IHp-KA relative to controls (control, 0.75±0.29 ectopic cells with RBD's/dentate; 1wk-IHpKA, 3.25±0.85; 2wk-IHpKA, 3.25±1.07, ANOVA on ranked data, p<0.034). Interestingly, between one and two weeks, the number of cells with recurrent dendrites was stable, although whether this reflects different mechanisms of cell movement, partial recovery of neuronal structure (see ), neuronal loss, or other factors is not clear. Importantly, the data demonstrate a positive association between aberrant granule cell migration into the dentate molecular layer and formation of recurrent basal dendrites in the intact brain.
Figure 3 Granule cell dispersion induced by intrahippocampal injection of kainic acid. Confocal maximum projections illustrating the location of the granule cell layer (GCL), inner molecular layer (IML) and outer two-thirds of the molecular layer (MML+OML) in (more ...)