Sprouted Mossy Fiber Connections of Adult-Born Granule Cells: Detonate or Fizzle?

Short-Term Depression of Sprouted Mossy Fiber Synapses from Adult-Born Granule Cells

Hendricks WD, Chen Y, Bensen AL, Westbrook GL, and Schnell E. J Neurosci 2017;37:5722–5735.

Epileptic seizures potently modulate hippocampal adult neurogenesis, and adult-born dentate granule cells contribute to the pathologic retrograde sprouting of mossy fiber axons, both hallmarks of temporal lobe epilepsy. The characteristics of these sprouted synapses, however, have been largely unexplored, and the specific contribution of adult-born granule cells to functional mossy fiber sprouting is unknown, primarily due to technical barriers in isolating sprouted mossy fiber synapses for analysis. Here, we used DcxCreER^T2 transgenic mice to permanently pulse-label age-defined cohorts of granule cells born either before or after pilocarpine-induced status epilepticus (SE). Using optogenetics, we demonstrate that adult-born granule cells born before SE form functional recurrent monosynaptic excitatory connections with other granule cells. Surprisingly, however, although healthy mossy fiber synapses in CA3 are well characterized “detonator” synapses that potently drive postsynaptic cell firing through their profound frequency-dependent facilitation, sprouted mossy fiber synapses from adult-born cells exhibited profound frequency-dependent depression, despite possessing some of the morphological hallmarks of mossy fiber terminals. Mature granule cells also contributed to functional mossy fiber sprouting, but exhibited less synaptic depression. Interestingly, granule cells born shortly after SE did not form functional excitatory synapses, despite robust sprouting. Our results suggest that, although sprouted mossy fibers form recurrent excitatory circuits with some of the morphological characteristics of typical mossy fiber terminals, the functional characteristics of sprouted synapses would limit the contribution of adult-born granule cells to hippocampal hyperexcitability in the epileptic hippocampus.

Commentary

The continual generation of selective neuron populations in the adult brain remains one of the least well-understood types of experience-dependent neuroplasticity in mammals, including humans. After seizures and other brain insults, the process of adult neurogenesis is accelerated in the hippocampal subgranular zone giving rise to a transient surge in the number of newly born dentate granule cells (DGCs), and these cells have been hypothesized to participate in the process of epileptogenesis (1). Newly born DGC progenitors migrate to target regions, differentiate into mature DGCs, and integrate within existing neural circuitry, eventually playing important roles in memory and cognition (2–7). Sprouting of DGC axons into the molecular layer of the dentate gyrus (i.e., mossy fiber sprouting; MFS), and the consequent development of new recurrent synaptic excitability within the dentate gyrus, is a well-documented and intriguing component of the synaptic reorganization that occurs during development of temporal lobe epilepsy (TLE), although the contribution of this synaptic reorganization to epilepsy remains a controversial issue. Because normal mossy fiber synaptic connections with CA3 pyramidal cells potently drive postsynaptic action potentials, MFS and synaptic reorganization among DGCs remains an attractive mechanism to explain specific components of the process of epileptogenesis. Adult-born DGCs contribute to MFS (8), but the function of neural circuits formed by these neurons is not adequately characterized and whether these neurons contribute to epileptogenesis or seizures has been difficult to ascertain. As a result, the contribution of adult-born neurons to mature brain function in healthy and disease states remains poorly understood. To help close this knowledge gap, Hendricks and colleagues took advantage of relatively recent technological advances that allow selective labeling and optogenetic stimulation of adult-born neurons to assess synaptic characteristics of mossy fiber synapses with CA3 pyramidal neurons and also to determine, for the first time, the contribution of adult-born DGCs to functional synaptic reorganization in the dentate gyrus after development of TLE.

After carefully documenting the birth dates of DGCs in a transgenic mouse that selectively expresses a fluorescent marker (EYFP) and the blue light-sensitive channelrhodopsin in adult born DGCs, groups of mice were used in which the EYFP/channelrhodopsin construct was expressed in DGCs born neonatally, those generated just prior to induction of status epilepticus (SE), or just after SE. Two months after SE induction, at a time when mice develop MFS and spontaneous seizures, electrophysiological recordings were made from unlabeled, presumably mature DGCs. Using blue light to selectively stimulate channelrhodopsin-expressing adult-born DGCs in vitro, the authors compared the synaptic physiology of mossy fiber connections arising from adult-born DGCs that were fully mature (neonatal) with those of neurons born just prior to and just after the epileptogenic insult. Similar to a previous report (8), adult-born DGCs of all three post-mitotic ages contributed robustly to MFS. Consistent with the anatomic results, optogenetic activation of DGCs born prior to SE induction resulted in excitatory synaptic responses in mature DGCs, confirming their contribution to the new recurrent circuitry in the dentate gyrus that correlates with the establishment of TLE. Compared with “detonator” mossy fiber synapses with CA3 pyramidal cells in healthy mice, adult-born DGC to mature DGC synapses that formed after SE expressed different release properties, having a higher initial release probability but decreased ability to maintain synaptic release with repetitive activation. These new synapses may therefore be more likely to initially excite their targets, but may be less likely to sustain this excitation over time. It was recently reported that ablation of DGCs born shortly before SE reduced seizure frequency, but increased seizure duration (9). If there is a relationship between MFS and seizures, then new synapses formed by adult-born neurons might therefore participate mainly in seizure initiation, due to their increased probability of synaptic release, rather than in seizure maintenance.

The finding that ablation of neural progenitors prior to SE induction reduces epilepsy severity is somewhat frustrating from a translational perspective. Conversely, modification of disease progression by preventing neurogenesis after an epileptogenic injury might be more amenable to clinical translation. Perhaps Hendricks and colleagues’ most intriguing finding was that activation of DGCs born just after SE failed to elicit synaptic responses in other DGCs, even though MFS arising from these cells was prevalent. Absence of evidence is not evidence of absence and, as the authors suggest, the new connections may simply not be mature enough at 2 months to contribute to the synaptically reorganized circuit or they may remain functionally silent indefinitely. Longer term studies could resolve this issue. From a technical standpoint, optogenetically-evoked glutamate release from isolated synaptic terminals can require experimentally increased terminal excitability to be effective (10), and synaptic terminals can be isolated from their somata during slice preparation. Enhancing mossy fiber terminal excitability with, for example, 4-aminopiridine, could unmask responses at the immature synapses. In addition, activation of adult-born DGCs in healthy mice yields synaptic inhibition of other DGCs (6), and DGCs could release the inhibitory neurotransmitter, GABA at immature synapses. Some inhibitory interneurons might express the channelrhodopsin construct in the doublecortin transgenic mouse used in this study, so GABA receptors were blocked, and thus plausible inhibitory responses at immature mossy fiber synapses would be undetected. It is therefore probably premature to conclude that the post-SE surge in adult neurogenesis contributes nothing to the development of recurrent excitation in the dentate gyrus during epileptogenesis; the temporal and physiological characteristics of this circuit require further assessment before reaching that conclusion. That stated, seizures or SE may indeed change essential characteristics of mossy fiber connections. This metaplasticity could also impact cognitive comorbidities long-term, because adult-born DGCs contribute importantly to memory and cognition in healthy animals (7).

Mounting evidence highlights the important contribution of adult-born DGCs to MFS and now, to functional synaptic reorganization in the dentate gyrus that is associated with the development of TLE. The finding that MFS is not always and obviously coupled to functional changes highlights the necessity of rigorous and multidisciplinary approaches to identify changes in the brain that occur during epileptogenesis. Whether synaptic reorganization attributed to DGCs born near the time of the initiating insult (or, for that matter, any DGC) is necessary or sufficient to support spontaneous seizures remains an unanswered question, especially because mature, neonatally-born DGCs also participate. Likewise, the integration of these neurons into hippocampal circuitry seems likely to impact cognitive comorbidities associated with seizure disorders. Hendricks et al. have demonstrated that neonatally- and adult-born DGCs contribute to the functional synaptic reorganization of the dentate gyrus during epileptogenesis. Understanding the mechanisms underlying this abnormal integration and the true contribution of this synaptic reorganization to TLE will be vital to preventing acquired epileptogenesis.