Moderate traumatic brain injury causes acute dendritic and synaptic degeneration in the hippocampal dentate gyrus

Abstract

Hippocampal injury-associated learning and memory deficits are frequent hallmarks of brain trauma and are the most enduring and devastating consequences following traumatic brain injury (TBI). Several reports, including our recent paper, showed that TBI brought on by a moderate level of controlled cortical impact (CCI) induces immature newborn neuron death in the hippocampal dentate gyrus. In contrast, the majority of mature neurons are spared. Less research has been focused on these spared neurons, which may also be injured or compromised by TBI. Here we examined the dendrite morphologies, dendritic spines, and synaptic structures using a genetic approach in combination with immunohistochemistry and Golgi staining. We found that although most of the mature granular neurons were spared following TBI at a moderate level of impact, they exhibited dramatic dendritic beading and fragmentation, decreased number of dendritic branches, and a lower density of dendritic spines, particularly the mushroom-shaped mature spines. Further studies showed that the density of synapses in the molecular layer of the hippocampal dentate gyrus was significantly reduced. The electrophysiological activity of neurons was impaired as well. These results indicate that TBI not only induces cell death in immature granular neurons, it also causes significant dendritic and synaptic degeneration in pathohistology. TBI also impairs the function of the spared mature granular neurons in the hippocampal dentate gyrus. These observations point to a potential anatomic substrate to explain, in part, the development of posttraumatic memory deficits. They also indicate that dendritic damage in the hippocampal dentate gyrus may serve as a therapeutic target following TBI.

Figure 1. The anatomical structure of the hippocampus in POMC-Cre;Z/EG double transgenic mice.

There was no gross morphological change in the HDG between moderate TBI (a, a1) and sham control mice (b, b1). EGFP staining (green) showed that there were many more disconnected bulbs and swellings in the injured HDG (b2) compared with the sham (a2).

Figure 2. Moderate TBI causes dendrite degeneration in the HDG.

Golgi staining revealed the individual neurons including their processes and spines both in the control (a, c, d and g) and the moderate CCI injured hippocampus (b, e, f and h). The dendrites and spines of the spared neurons in the injured HDG were significantly damaged. Quantitative data showed that the number of dendrite beadings, the hallmark of an injured dendrite was increased dramatically in the HDG of TBI mice HDG as compared with sham control (i). (n = 5, ***, p<0.005).

Figure 3. Moderate TBI attenuate dendrite complexity in the HDG.

Neurolucida reconstruction of Golgi stained granule neurons in the HDG from sham mice (a) and moderate TBI mice (b). Both the dendrite total length and branches were decreased in spared neurons in HDG after TBI (c, d). There was no significant difference in average dendrite length between the 2 groups (e). Sholl analysis-derived distribution of granule neuron dendritic complexity and mean number of intersections of dendrite branches with consecutive 10 µm-spaced concentric spheres (f). A dramatic reduction of dendritic complexity was found in spared neurons in TBI mice compared with sham control. (n = 5, *, p<0.05, **, p<0.01).

Figure 4. Moderate TBI leads to spine degeneration in the HDG.

High power images of a single spine in control mice (a, a1–a4) and moderate TBI mice (b, b1–b4). Three types of spine, including mushroom (d), stubby (e) and fliopodia (f). Quantitative data showed that the density of the dendritic spine was significantly reduced in the spared neurons in the injured HDG (c), especially in mushroom and filopodia shaped spines (g). (n = 5, *, p<0.05, **, p<0.01).

Figure 5. Moderate TBI decreases the number of synapses in the injured area.

Synaptophysin staining (red) reveals the pre-synaptic puncta in the molecular layer of the HDG in control (a, c) and moderate TBI mice (b, d). Quantitative data showed a significant decrease of synapses in the HDG of injured mice (e). (n = 5, **, p<0.01).

Figure 6. Decreased neuronal excitability of granule cell after TBI.

(A), representative traces showing the voltage responses of granule cells to current pulses. The same amount of positive current evoked fewer spikes in post-traumatic cells. Meanwhile, post-traumatic cells exhibited smaller responses to negative currents. (B–E), summary data showing the post-traumatic changes of resting membrane potential (B), input resistance (C), and rheobase (D) in granule cells. The spike threshold showed no change after TBI (E). *, P<0.01; #, P<0.05.

Figure 7. Transient inhibition of excitatory synaptic inputs in granule cell after TBI.

(A), representative traces of mEPSCs recorded from control and post-traumatic cells, showing at lower scale (A1) and larger scale (A2), respectively. The traces in A2 were averaged from 150–260 events. (B), summary data showing the decrease of mEPSCs frequency, but not amplitude, rising time, and decay time constant. *, P<0.01.