To investigate the relationship of spine changes in learning to patterns of neural activity associated with the learning event we used the GFP-GluR1cfos transgenic mouse. The mouseexpresses a tetracycline response element linked GFP-GluR1 fusion protein in an activity-dependent and dox dependent manner through regulation by a second c-fos-tTA transgene. Discrete windows for mapping brain activity can be opened by keeping mice on dox for several weeks prior to experiments, and then removing dox from the diet prior to experimental manipulations ().
to contextual control training alone (CT), contextual fear conditioning (FC) and unpaired shock (UP) protocols results in strong GFP-GluR1 expression above home cage (HC) controls. HC control levels were low, as previously described, but FC resulted in a three-fold increase in the percentage of CA1 neurons expressing GFP-GluR1 () (Matsuo et al., 2008
). This was paralleled by an ~80% increase in GFP-GluR1 signal within the dendritic layers of CA1 (). Co-staining hippocampal sections with GFP and DiI allowed us to identify active neurons and compare dendrites from these and from inactive neurons. We could thereby relate the structure of dendrites to the pattern of neural activity during learning (). Mice kept on dox from weaning through maturity were grouped into HC, CT, FC or UP experimental protocols at maturity (), and removed from dox for four days prior to training. The HC experimental group remained within their homecage throughout the experiment. In a separate group of mice we confirmed that our FC protocol resulted in a strong contextual freezing and that our UP protocol did not ().
Fear conditioning results in a specific decrease in spine density on active versus inactive neurons
Twenty-four hours after training animals were sacrificed and brain sections processed for imaging of dendritic spines. Our experimental protocols significantly affected total spine density (active and inactive neurons combined) with total spine density lower in the experimental groups that received footshocks (F(3, 249) =7.1918, p=0.0001) (HC vs FC, p<0.05; HC vs UP, p<0.001; CT vs UP, p<0.01) ().
In order to determine the relationship of neural activity at the time of training to spine density changes, we dissociated spine changes on dendrites from active (GFP+) and inactive (GFP−) neurons. Neural activity was associated with lower spine density on active compared to inactive neurons (F(1,429)=10.292, p=0.00144). This is presented as percent spine density in active neurons relative of inactive neurons within each group (). The decrease in spine density was found specifically on active relative to inactive neurons of FC animals (**p<0.01). This change was not seen in HC mice or mice exposed to CT or UP (, , ).
Statistical Analysis of Total Spine Density on Active and Inactive Neurons
Recent findings show that synaptic inputs cluster on active neurons as mechanism of circuit remodeling associated with learning (Takahashi et al., 2012). We therefore investigated whether spine clustering was seen in our experiment and whether this was selective for active neurons. If this occurred on the active cells of our fear conditioned group, we hypothesized that clustered inputs would be identified as a discrete population of dendrite segments enriched in spines, occurring amidst an overall increase in pruned segments.
We did not see clustering but instead found that the FC resulted in a generalized increase in pruned segments in the active circuit without any evidence of a clustered spine population. As opposed to the FC group, the HC, CT and UP groups had similar and overlapping spine density distributions for both active and inactive cells (). These data do not suggest that spine changes are anatomically restricted to particular dendritic regions, at least at the 30 μm level of resolution.
We further classified spines into mushroom, branched, stubby, and thin morphologies, since distinctive roles for each of these subtypes are implicated in various plasticity paradigms (). For example, studies have implicated mushroom spines in memory (Bourne and Harris, 2007
; Matsuo et al., 2008
) and changes in branched spines have been identified in LTP (Desmond and Levy, 1986
; Trommald et al., 1996
). To assess whether spine elimination is restricted to a specific spine morphology, we analyzed the changes occurring in each morphological type ().
Morphological Analysis of Spine Density Changes
Our experimental protocols significantly affected mushroom spines (, , F(3,429)=9.9801, p<0.001). CT significantly increased mushroom spines relative to their levels in in the HC group (p<0.05). Relative to the CT group, though, mushroom spines were fewer in the groups that received footshock (FC vs CT, p<0.05; UP vs CT, p<0.001). Between the groups that received a footshock, mushroom spines were lower in the UP compared to the FC group (p<0.01) ().
Statistical Analysis of Spine Morphologies on Active and Inactive Neurons
Our protocols also led to changes in mushroom spines when analyzed by their relative density on active and inactive neurons. There were fewer mushroom spines on active compared to inactive neurons of the FC group (p<0.05), but this activity-related change was not seen in HC mice or mice exposed to UP or CT training (, ). Thus, while both groups that received a foot-shock showed reductions in mushroom-type spines compared to CT, in the UP group this was a generalized decrease across neurons while in the FC group the change was specific to the circuit active during learning. This activity-specific decease in mushroom spines accounted for ~27% of the total spine decrease on active neurons with FC, and was hence representative of activity-specific elimination within the total spine population, but did not account for all of the spine loss. We further examined other spine morphologies for their contribution to activity-specific decreases with learning.
Our protocols significantly affected branched spines (F(3,429)=2.6562, p=0.04803), with more branched spines on the neurons of the UP group compared to mice subject to CT (p<0.05) and FC (p<0.05). Neural activity was associated with lower branched spine density on active compared to inactive neurons (F(1,429)=4.9105. p=0.02722.) The decrease in branched spine density was specifically found on active but not on inactive neurons of FC and UP animals (p<0.001 and p<0.05, respectively), and it was not seen in HC mice or mice exposed to CT (, ).
Experimental protocols led to small but significant changes in stubby and thin spines with fewer stubby spines on neurons of mice in the UP group compared to the HC (p<0.05) and CT groups (p<0.05). Within experimental groups, we did not find activity-specific changes for stubby spine morphology (, ). FC resulted in a decrease in the thin spines relative to HC (p<0.05). There were fewer thin spines on active compared to inactive neurons of the FC group (p<0.05), but this activity-related change was not seen in HC mice or mice exposed to UP or CT training (, ).