Abstract
Kang YJ, Lee SH, Boychuk JA, Butler CR, Juras JA, Cloyd RA, Smith BN. J Neurosci. 2022;42(37):7077-7093. doi:10.1523/JNEUROSCI.2263-21.2022. PMID: 36002261; PMCID: PMC9480876. Post-traumatic epilepsy (PTE) and behavioral comorbidities frequently develop after traumatic brain injury (TBI). Aberrant neurogenesis of dentate granule cells (DGCs) after TBI may contribute to the synaptic reorganization that occurs in PTE, but how neurogenesis at different times relative to the injury contributes to feedback inhibition and recurrent excitation in the dentate gyrus is unknown. Thus, we examined whether DGCs born at different postnatal ages differentially participate in feedback inhibition and recurrent excitation in the dentate gyrus using the controlled cortical impact (CCI) model of TBI. Both sexes of transgenic mice expressing channelrhodopsin2 (ChR2) in postnatally born DGCs were used for optogenetic activation of three DGC cohorts: postnatally early born DGCs, or those born just before or after CCI. We performed whole-cell patch-clamp recordings from ChR2-negative, mature DGCs and parvalbumin-expressing basket cells (PVBCs) in hippocampal slices to determine whether optogenetic activation of postnatally born DGCs increases feedback inhibition and/or recurrent excitation in mice 8-10 weeks after CCI and whether PVBCs are targets of ChR2-positive DGCs. In the dentate gyrus ipsilateral to CCI, activation of ChR2-expressing DGCs born before CCI produced increased feedback inhibition in ChR2-negative DGCs and increased excitation in PVBCs compared with those from sham controls. This upregulated feedback inhibition was less prominent in DGCs born early in life or after CCI. Surprisingly, ChR2-positive DGC activation rarely evoked recurrent excitation in mature DGCs from any cohort. These results support that DGC birth date-related increased feedback inhibition in of DGCs may contribute to altered excitability after TBI. Significance Statement: Dentate granule cells (DGCs) control excitability of the dentate gyrus through synaptic interactions with inhibitory GABAergic interneurons. Persistent changes in DGC synaptic connectivity develop after traumatic brain injury, contributing to hyperexcitability in post-traumatic epilepsy (PTE). However, the impact of DGC neurogenesis on synaptic reorganization, especially on inhibitory circuits, after brain injury is not adequately described. Here, upregulation of feedback inhibition in mature DGCs from male and female mice was associated with increased excitation of parvalbumin-expressing basket cells by postnatally born DGCs, providing novel insights into underlying mechanisms of altered excitability after brain injury. A better understanding of these inhibitory circuit changes can help formulate hypotheses for development of novel, evidence-based treatments for post-traumatic epilepsy by targeting birth date-specific subsets of DGCs.
Commentary
Aberrant integration of adult-generated hippocampal dentate granule cells is hypothesized to contribute to the development of temporal lobe epilepsy. Speaking broadly, the hypothesis postulates that granule cells born in the weeks before and after an epileptogenic brain injury are uniquely vulnerable to disruption due to their immature state. The immature neurons develop abnormal connectivity and properties that facilitate epileptogenesis. The hypothesis has been extensively examined in rodent status epilepticus models of epilepsy, providing strong evidence in support of key tenets. 1 Specifically, granule cells born shortly before and after status epilepticus contribute to mossy fiber sprouting and develop aberrant basal dendrites, creating recurrent excitatory circuits among granule cells. They migrate to ectopic locations and exhibit altered physiological properties that can promote network hyperexcitability. Finally, and perhaps most notably, several groups have demonstrated that selective ablation and silencing of newborn granule cells reduces seizure incidence in epileptic rodents. 2 –5
An important consideration, though, is that rodent status epilepticus models may be limited in their translatability to human epilepsy. It is important, then, to evaluate the extent to which aberrant granule cell neurogenesis might contribute to other models of epilepsy, and if so, how the age of the new cells is influential. As post-traumatic epilepsy (PTE) is a common form of acquired epilepsy in people, Kang and colleagues have begun to address these gaps in knowledge through their examination of adult neurogenesis in the controlled cortical impact (CCI) model of PTE. 6 Specifically, utilizing Gli1-CreERT2, ChR2/eYFP (channelrhodopsin/enhanced yellow fluorescent protein) bitransgenic mice providing for tamoxifen-dependent control of cre-recombinase activity in neural progenitor cells, they selectively expressed a ChR2/eYFP fusion protein in 3 age-specific cohorts of granule cells: (1) postnatally generated cells that were mature at the time of the insult, (2) immature cells labeled just before CCI, and (3) newborn cells labeled just after CCI. Animals were examined 8 to 10 weeks after CCI, a time point at which spontaneous seizures are common in the model.
Expression of the light-activated excitatory channelrhodopsin ChR2 in the different age-cohorts of granule cells allowed the investigators to selectively excite these cells while recording from neighboring ChR2-negative granule cells. The dentate gyrus circuitry supports robust feedback inhibition, whereby granule cell firing either directly or indirectly excites GABAergic inhibitory interneurons, which in turn inhibit granule cells. ChR2 activation of different age-cohorts of granule cells, therefore, allowed the investigators to assess the strength of the recurrent inhibitory pathway. Interestingly, the investigators found that activation of granule cells born just before CCI produced more inhibitory input to adjacent granule cells than activation of older or younger cohorts. Both the number and amplitude of evoked inhibitory postsynaptic currents were increased. Parvalbumin-expressing basket cells are one of the key interneuron types providing feedback inhibition to granule cells, so the investigators next queried whether granule cell input to these cells was increased after CCI. ChR2 activation of granule cells born just before CCI produced larger amplitude evoked excitatory postsynaptic currents among recorded basket cells relative to controls, supporting the conclusion that the granule cell >> basket cell >> granule cell feedback inhibitory circuit is enhanced after CCI. In contrast to findings showing enhanced feedback inhibition, there was no evidence that any of the 3 cohorts supported enhanced feedback excitation of granule cells. Specifically, optogenetic activation of the different cohorts of granule cells did not increase the number of evoked excitatory postsynaptic currents among ChR2-negative granule cells. Adult-generated granule cells in status epilepticus models exhibit significant mossy fiber sprouting which contributes to recurrent excitation. By contrast, sprouting is more modest in the CCI model and, as findings by Kang and colleagues indicate, the contribution of new cells to a recurrent excitatory pathway was minimal. These findings suggest an overall antiepileptogenic role—increased feedback inhibition—for granule cells born shortly before CCI injury, although increased feedback inhibition might also enhance granule cell synchrony, which could be proepileptogenic.
The work by Kang and colleagues highlights several new avenues for research. While not examined in their study, only a subset of animals in the CCI model develop epilepsy, providing an opportunity to query whether new cells show different integration patterns in epileptic versus nonepileptic animals. Interventional studies directly manipulating newborn granule cell numbers or activity with simultaneous seizure monitoring are also needed to definitively assess the role of these neurons in epilepsy. To date, studies designed to directly silence or ablate newborn granule cells in different epilepsy models have found either no effect of treatment or have observed seizure reductions, supporting a net harmful role for the new cells. 1 On the other hand, adult-generated granule cells in healthy animals appear to play a specialized role by activating GABAergic neurons to inhibit older granule cells. 7 Indeed, having a large population of healthy newborn granule cells provides protection against seizures. 8 Taken together, these prior findings suggest that adult-generated granule cells normally act to restrain seizure activity, but in the setting of epilepsy, they can be replaced by proconvulsant granule cells. The new work by Kang and colleagues paints a more complicated picture and highlights the need for further research in epilepsy models with better pathogenic validity.
So what accounts for the differing behaviors of adult-generated granule cells? Firstly, the nature of granule cells themselves is likely critical. Although granule cell excitation of glutamatergic CA3 pyramidal cells is a canonical part of the hippocampal trisynaptic circuit, and de novo formation of recurrent excitatory granule cell >> granule cell contacts is a hallmark of temporal lobe epilepsy, normal granule cells actually form more synapses with interneurons. 9 Newborn granule cells must strike the right balance between excitatory and inhibitory targets as they integrate to maintain circuit stability. Secondly, granule cell integration is regulated by numerous factors that are disrupted by epileptogenic brain injuries, including network activity levels, input from local interneurons, inflammation, and vascular signals. 10 Disruption of these factors varies widely among epilepsy models. Systemic chemoconvulsant models of status epilepticus, for example, produce bilateral injury and extensive hippocampal and extrahippocampal cell loss, whereas CCI produces unilateral injury with a relatively preserved contralateral hippocampus. Therefore, immature and newborn granule cells find themselves in very different environments among epilepsy models. A final factor to consider, highlighted by the work by Kang and colleagues, is the significant impact of granule cell age. The dentate is clearly not a homogeneous cell population. Granule cells of different ages exhibit distinct functional properties and appear to mediate the formation of distinct neuronal circuits. Seemingly conflicting roles of granule cells, therefore, may result from experiments examining different populations of granule cells within the same dentate gyrus.
Evidence for potentially protective effects of adult-generated cells raises some interesting questions for human epilepsy. Notably, whether adult neurogenesis occurs in humans is still controversial, with studies for and against appearing in the literature on a regular basis. 11,12 An absence of adult neurogenesis in humans would preclude these cells from promoting epilepsy. But if new cells in rodents are sometimes protective, then would a lack of adult neurogenesis in humans mean that our brains are not capable of this potentially adaptive response? How might this difference impact the development of epilepsy across species? If neurogenesis is restricted to development in humans, might impaired developmental neurogenesis produce a lifelong susceptibility to epilepsy? Although these intriguing questions lack answers, a thorough understanding of newborn granule cell integration in multiple epilepsy models is certain to yield novel insights into how the brain regulates (or fails to regulate) excitability and may provide useful clues for epilepsy prevention.
