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Axonal Plasticity of Age-Defined Dentate Granule Cells in a Rat Model of Mesial Temporal Lobe Epilepsy.
Althaus AL, Zhang H, Parent JM. Neurobiol Dis 2016;86:187–196. [PubMed]
Dentate granule cell (DGC) mossy fiber sprouting (MFS) in mesial temporal lobe epilepsy (mTLE) is thought to underlie the creation of aberrant circuitry which promotes the generation or spread of spontaneous seizure activity. Understanding the extent to which populations of DGCs participate in this circuitry could help determine how it develops and potentially identify therapeutic targets for regulating aberrant network activity. In this study, we investigated how DGC birthdate influences participation in MFS and other aspects of axonal plasticity using the rat pilocarpine-induced status epilepticus (SE) model of mTLE. We injected a retrovirus (RV) carrying a synaptophysin-yellow fluorescent protein (syp-YFP) fusion construct to birthdate DGCs and brightly label their axon terminals, and compared DGCs born during the neonatal period with those generated in adulthood. We found that both neonatal and adult-born DGC populations participate, to a similar extent, in SE-induced MFS within the dentate gyrus inner molecular layer (IML). SE did not alter hilar MF bouton density compared to sham-treated controls, but adult-born DGC bouton density was greater in the IML than in the hilus after SE. Interestingly, we also observed MF axonal reorganization in area CA2 in epileptic rats, and these changes arose from DGCs generated both neonatally and in adulthood. These data indicate that both neonatal and adult-generated DGCs contribute to axonal reorganization in the rat pilocarpine mTLE model, and indicate a more complex relationship between DGC age and participation in seizure-related plasticity than was previously thought.
Since its initial discovery in animals (1) and its description in human patients with temporal lobe epilepsy (2), hippocampal granule cell mossy fiber sprouting has received more attention and generated more controversy than almost every other anatomic change of the epileptic brain. Like the mythologic Hydra, hippocampal granule cells have proved to be challenging neurons to subdue. Their unforeseen regenerative capabilities have raised a host of new, exciting questions and inspired the development of new technologies to pin them down.
Mossy fiber sprouting, defined as the growth of granule cell axons into their own dendritic field in the inner molecular layer, creates de novo recurrent excitatory circuits. These new circuits provide an appealing explanation for the epileptic brain's increased excitability. Recent findings, however, call into question the functional significance of the phenomenon: mossy fiber sprouting is not required for epileptogenesis, and sprouting can be blocked without reducing seizure occurrence (3). Nonetheless, given the robust nature of mossy fiber sprouting in animals and patients with temporal lobe epilepsy, it is hard to imagine sprouting does not impact brain function—perhaps with a greater role in cognitive disruption or other comorbidities than seizures, per se.
The study by Althaus and colleagues addresses one of many unresolved questions about mossy fiber sprouting. Specifically, they seek to determine the roles of newborn and mature granule cells in the phenomena. The dentate gyrus generates new neurons throughout life in mammals. These new neurons go through a transient developmental stage in the weeks after they are generated, during which they exhibit enhanced excitability and plasticity relative to more mature (>6 weeks) granule cells (4). The role played by granule cells of various ages, therefore, is an important new question in epilepsy.
Early work implicated newborn granule cells—generated after pilocarpine-induced status epilepticus—in mossy fiber sprouting (5). This finding was intellectually appealing given the heightened plastic capabilities of the new cells. Irradiation treatment to ablate newborn cells, however, failed to significantly reduce mossy fiber sprouting (6), implicating cells born before the insult. Later studies confirmed that both cells born up to 4 weeks before the insult (juvenile granule cells) and cells born after the insult (newborn granule cells) contribute to mossy fiber sprouting (7). Intriguingly, however, newborn cells don't contribute to sprouting until they are more than 1 month old. This accounts for the negative finding with irradiation (6), since animals in this study were collected before the youngest cells sprouted.
For the present study, Althaus and colleagues examined granule cells that were mature at the time of the insult to determine whether they contribute to mossy fiber sprouting. They infected granule cell progenitors with retrovirus containing a synaptophysin–yellow fluorescent protein (YFP) fusion protein driven by the synapsin-1 promoter. This approach allowed the investigators to fate-map different-age cohorts of granule cells, while the synaptophysin-YFP fusion protein revealed the finest details of their synaptic terminals—a notable advance over prior work using cytoplasmic green fluorescent protein (GFP) (7). Retrovirus was injected into the dentate on either postnatal day 7 or 60, while pilocarpine status epilepticus was induced on postnatal day 56, producing YFP-labeled granule cells born either 7 weeks before (mature), or 4 days after (newborn) status epilepticus, respectively. Subsequent anatomic studies clearly revealed that both populations contribute to mossy fiber sprouting, extending the age range of mossy fiber plasticity to mature, 7-week-old cells. Notably, however, 7-week-old cells still border the age range during which granule cells exhibit heightened plasticity (4). In future studies, it will be important to examine even older cells to determine whether they reach an age when sprouting is no longer possible. Such studies would be particularly relevant for human epilepsy, where decades-old granule cells exist alongside newborn cells.
Additionally, the investigators found that mature and newborn granule cells can extend axons beyond their traditional CA3 pyramidal cell targets to innervate CA2 pyramidal cells. Granule cell input to CA2 was only recently identified in normal animals, and the revelation by Althaus and colleagues that this pathway is strengthened during epileptogenesis draws important attention to a new means for excitatory information to traverse the hippocampus in epilepsy.
Beyond the current work, the technique developed by Althaus and colleagues holds the potential to resolve a number of unanswered questions. One in particular concerns the impact of antineurogenic treatments on sprouting: specifically, while blocking neurogenesis with methylazoxymethanol acetate (MAM) proved effective at reducing sprouting (in a study by Liu and colleagues ), recent studies using transgenic thymidine–kinase and diphtheria toxin–mediated cell-ablation strategies found no impact on eliminating juvenile and newborn granule cells on mossy fiber sprouting (9, 10). Future studies will be needed to resolve these discrepancies. Indeed, granule cells may pose a labor reminiscent of slaying the Lernaean hydra; their ability to change their structure and regenerate creates similar challenges. Ablation studies can be complicated by the brain's response to ablation. Since both adult and neonatally generated granule cells can contribute to mossy fiber sprouting, loss of one age cohort might encourage the remaining cohort to sprout more robustly, resulting in no net loss of terminals in the inner molecular layer. Consistent with this idea, some evidence suggests that homeostatic mechanisms may act to maintain a set level of mossy fiber sprouting (3). The robust axonal labeling approach developed by Althaus may reveal such an effect in future work.
The nature of the stimuli that drive mossy fiber sprouting may also shed new light on the role of different-age granule cells in epilepsy. A number of likely candidates for mediating sprouting have been explored—such as mossy cell loss and seizure activity—but a final, common mediator remains elusive. Indeed, there may be multiple factors that mediate mossy fiber sprouting. In this latter case, different-age cohorts of granule cells may be more or less responsive to different mediators, leading to variable contributions depending on model or disease state.
In summary, the work by Althaus and colleagues elegantly demonstrates that both newborn and mature granule cells contribute to mossy fiber sprouting. The direct observational approach minimally perturbs a very dynamic system. Application of this approach to other models of epilepsy and different-age cohorts of granule cells in the future will help determine if the findings are generalizable, and whether even older granule cells are capable of mossy fiber sprouting.
Editor's Note: Authors have a Conflict of Interest disclosure which is posted under the Supplemental Materials link.