We first characterized the developmental cell death of cortical interneurons by measuring the expression of the apoptotic marker, cleaved caspase-3, in GAD67-GFP mice9
(). The number of cleaved caspase-3-labeled neocortical GAD67-GFP neurons increased from postnatal days 1 to 5 (P1 to P5), reached a maximum around P7, and declined towards zero by approximately P15 (; Analysis of Variance (ANOVA), F = 84.0 and P < 0.0001). The majority (75%) of cleaved caspase-3-positive cells were observed between P7 and P11 (), approximately 11 to 18 days after the cells were produced in the embryonic ventral forebrain10
. The temporal profile of cleaved caspase-3 expression in GAD67-GFP cells was similar to that observed across the total cellular population of the neocortex (Figure S2
), which may preserve the relative sizes of different cellular populations11
. Because the GAD67-GFP knock-in reduces brain gamma-aminobutyric acid (GABA) content by approximately 20 to 40%9
, we examined whether this in turn affected cell death in GAD67-GFP mice. Across the entire cellular population of the neocortex, neither the temporal profile nor the extent of apoptosis was significantly different between GAD67-GFP mice and wild type mice (Figure S3
Bax-dependent programmed cell death eliminates 40% of developing interneurons from the postnatal mouse neocortex
We next measured the GAD67-GFP population size during postnatal life and adulthood (). The number of GAD67-GFP neurons reached a maximum around P5 (mean, 1.65 ± 0.03 × 106
cells), and then declined by approximately 40% during the period of interneuron cell death (), reaching a stable size of 1.01 ± 0.02 × 106
cells by P120 (mean; ANOVA, F = 32.1 and P < 0.0001). The developmental cell death of cortical interneurons depended on Bax function: at P7, when GAD67-GFP cell death reached a maximum in wild type mice (), GAD67-GFP cell death was nearly absent in Bax−/−
(; Student’s t-test, P = 0.0034). Between P5 and P120, the cortical GAD67-GFP population did not decline in Bax
mutants (; ANOVA, F = 2.28, P = 0.18), and, at P120, the cortical interneuron population was 33% smaller in wild type GAD67-GFP mice than in Bax−/−
;GAD67-GFP littermates (1.02 ± 0.04 × 106
cells versus 1.52 ± 0.08 × 106
cells, respectively; Student’s t-test, P = 0.0041). In wild type and Bax
mutant mice, similar proportions of GAD67-GFP neurons were labeled by parvalbumin, somatostatin, neuropeptide Y, and calretinin (Figure S4
), indicating that Bax
-dependent cell death occurred uniformly across neurochemically defined interneuron subtypes. These findings indicate that Bax
-dependent programmed cell death eliminates roughly 40% of neocortical interneurons during postnatal life.
After characterizing neocortical interneuron cell death in vivo, we examined whether neocortical interneurons undergo a similar pattern of cell death in vitro. We placed interneuron precursors from the embryonic day 13.5 (E13.5) GAD67-GFP medial ganglionic eminence (MGE) onto P0 to P2 neocortical feeder layers13
(), and quantified the expression of cleaved caspase-3 expression at various timepoints (). GAD67-GFP neurons underwent cell death in vitro, with cleaved caspase-3 expression reaching a maximum at 13 days (; ANOVA, F = 9.12 and P < 0.0001). Approximately 66% of cell death occurred between 11 and 15 days in vitro (DIV), around which time the GAD67-GFP cell number declined by approximately 30% (; ANOVA, F = 4.53 and P = 0.0012). As previously mentioned, in vivo, the majority of interneuron cell death occurred between P7 and 11, when the developing cells were likewise between 11 and 18 days old (). Interneuron cell death thus manifests in vitro, with a temporal pattern resembling that observed in vivo.
In vitro, and following heterochronic transplantation, interneuron precursors undergo programmed cell death during a period defined by their intrinsic cellular age
We next transplanted embryonic interneuron precursors into the postnatal neocortex during the period of endogenous interneuron cell death14, 15
. We postulated that, if the timing of interneuron cell death reflects the maturation of interneurons into a trophic signal-dependent state, then transplanted interneuron precursors would undergo developmental cell death asynchronously from endogenous interneurons. We transplanted 5 × 105
cells from the MGE of E13.5 to E14.5 beta-actin:GFP mice16
into P3 wild type recipients (Figure S5
) and then quantified cleaved caspase-3 expression at various timepoints after transplantation. Given that mouse gestation ends around E19, the transplanted interneuron precursors were approximately 6 to 10 days younger than their endogenous counterparts10
. As previously described14, 15, 17
, transplanted MGE cells dispersed in the recipient cortex, developed the morphological features of GABAergic interneurons (Figure S5
), and formed synaptic contacts with recipient neurons (Figure S6
). We did not observe Ki-67 labeling of the transplanted interneuron precursors, indicating that the cells did not proliferate in the recipient (Figure S7
). Cleaved caspase-3 expression increased 200% in the transplanted population between 7 and 15 DAT, reached a maximum at 15 DAT, then declined to undetectable levels by 45 DAT (; ANOVA, F = 17.79 and P < 0.0001). By contrast, in endogenous cells of the recipient neocortex, cleaved caspase-3 expression reached a relative maximum at 7 DAT, then declined approximately 80% between 7 and 15 DAT (Figure S8
; ANOVA, F = 401.20 and P < 0.0001). The addition of transplanted cells did not affect endogenous cell death, as cleaved caspase-3 expression was similar between hemispheres that received transplanted cells and hemispheres that received media vehicle injections (Figure S8
; Student’s t-test, P = 0.76 (7 DAT), P = 0.83 (15 DAT), P = 0.89 (25 DAT), P = 0.67 (45 DAT)). Transplanted interneuron cell death thus reached a maximum around 15 DAT, when the transplanted cells reached a cellular age equivalent to that of endogenous interneurons during the peak of normal developmental cell death ( and ). Taken together with the in vitro data (), these findings suggest that interneuron cell death is timed by the intrinsic maturational state of the developing cells.
We also used heterochronic transplantation to introduce varying numbers of embryonic interneuron precursors into the neocortex. We expected that, if interneuron cell death is determined by intercellular competition for extrinsically derived signals, then the amount of interneuron cell death should increase with larger transplant sizes. Surprisingly, however, across initial transplant sizes that varied 200-fold (5 × 103, 5 × 104, 5 × 105, and 106 cells), similar fractions of the transplanted cells survived in the recipient neocortical hemisphere at 60 DAT (20.8 ± 2.4%, 22.2 ± 1.4%, 17.8 ± 0.6% and 15.3 ± 0.3%, respectively; ; ANOVA, F = 0.34 and P = 0.12). When 106 or 2 × 106 cells were transplanted, similar numbers of cells survived (1.65 ± 0.18 × 105 cells versus 1.53 ± 0.01 × 105 cells, respectively; Student’s t-test, P = 0.58), suggesting that the neocortical hemisphere has a limited capacity for approximately 1.6 × 105 additional interneurons. However, when the initial transplant size was far smaller than this theoretical limit, transplanted cell death still occurred, and it occurred at a constant rate. This finding indicates that interneuron cell death is not governed by competition for limited trophic signals derived from other cell types.
Transplanted interneuron cell death is not governed by competition for survival signals derived from other cell types in the recipient neocortex
To further examine whether soluble neurotrophic signals regulate interneuron cell death, we studied the survival of mutant interneurons lacking the neurotrophin receptor, TrkB. We transplanted interneuron precursors from TrkB−/−
into P2 wild type recipients and examined the survival of the cells at 60 DAT. Surprisingly, the survival of transplanted TrkB−/−
interneurons was similar to that of transplanted wild type cells (; 2.32 ± 0.32 × 104
wild type cells versus 2.20 ± 0.20 × 104
cells; Student’s t-test, P = 0.75), indicating that the cell death of transplanted interneurons is not governed by neurotrophin signaling through TrkB. This finding is consistent with other reports suggesting that the death of developing CNS neurons is regulated by mechanisms other than neurotrophin signaling6, 19
To confirm that transplanted interneuron cell death occurred through Bax-dependent apoptosis, we examined the survival of transplanted Bax−/−
, and compared their survival to that of transplanted wild type and Bax+/−
and cells. We pooled counts of wild type and Bax+/−
interneurons because endogenous interneuron cell death was not disrupted in P20 Bax+/−
GAD67-GFP mutants (8.88 ± 0.03 × 105
wild type cells versus 9.63 ± 0.04 × 105
cells; Student’s t-test, P = 0.20). At 60 DAT into P2 recipients, transplanted Bax
null interneurons survived in greater numbers than transplanted Bax
heterozygous and wild-type interneurons (; 4.31 ± 0.21 × 104
and wild type cells versus 9.11 ± 1.63 × 104
wild type cells; Student’s t-test, P = 0.03), indicating that the death of transplanted interneurons, like that of endogenous interneurons, occurs at least partially through a Bax-dependent mechanism.
While our transplantation experiments strongly suggested that interneuron cell death is not determined through competition for extrinsic survival signals, it was possible that the transplanted cells competed with endogenous cells, and the survival of the transplanted interneurons occurred at the expense of endogenous interneuron survival. To examine this possibility, we transplanted 106
beta-actin:DsRed MGE cells20
to one neocortical hemisphere of P2 to P3 GAD67-GFP recipients, and then compared the number of endogenous interneurons between the recipient and contralateral control hemispheres (). As expected (), we observed an average of approximately 1.7 × 105
transplanted interneurons in the recipient cortical hemisphere at 60 DAT (; mean 1.69 ± 0.41 × 105
cells). In the recipient and control hemispheres, we observed equal numbers of endogenous interneurons (; mean endogenous cell count, recipient hemisphere = 4.81 ± 0.12 × 105
; mean endogenous cell count, control hemisphere = 5.04 ± 0.15 × 105
; Student’s t-test, P = 0.28), consistent with the findings presented in Figure S8
, which indicated that transplantation did not affect cleaved caspase-3 expression in endogenous cells. The neocortex is thus able to support approximately 35% additional interneurons, with no effect on the endogenous interneuron population size. This suggests that developmental cell death does not tune the number of developing interneurons towards a cellular limit, as would occur if interneuron number is determined by the availability of limited, extrinsically derived survival signals.
Given that transplantation increases the number of interneurons in the neocortex, it offers a strategy for studying the relationship between interneuron number and cortical inhibition. To explore this relationship, we transplanted varying numbers of interneuron precursors into P2 to P3 recipients, and then performed in vitro patch-clamp recordings on endogenous neocortical pyramidal neurons at 30 to 40 DAT. We recorded the amplitudes and frequencies of spontaneous inhibitory post-synaptic currents (sIPSCs; ) and then performed post-hoc quantification of transplanted interneuron cell densities. Consistent with previous findings15, 17
, transplanted interneurons increased the frequency of sIPSCs onto endogenous pyramidal neurons (; controls, 18.4 ± 3.4 Hz; transplant recipients, 31.7 ± 3.9 Hz; Wilcoxon rank-sum test, P = 0.02). The amplitudes of inhibitory events, however, were not significantly increased by transplantation (; controls, 37.3 ± 1.9 pA; transplant recipients, 42.4 ± 2.5 pA; Wilcoxon rank-sum test, P = 0.22). Remarkably, inhibitory event frequencies did not increase with transplanted interneuron density (linear regression analysis, slope = 0.0003 and r2
= 0.0003; ). Thus, the extent of cortical inhibition is more likely determined by mechanisms that adjust synaptic strength and number, rather than mechanisms that govern interneuron population size. These findings indicate that transplantation can add a limited amount of new inhibition to the neocortex, and this limit is reached with transplanted cell numbers much smaller than that which the neocortex can support.
Interneuron population size is not a primary determinant of the level of functional cortical inhibition
In summary, our findings suggest that interneuron cell death is regulated by intrinsically defined mechanisms. When interneuron precursors were cultured in vitro or heterochronically transplanted, they died when they reached a cellular age equivalent to that of endogenous interneurons during the peak of endogenous interneuron cell death ( and ). This suggests that interneuron cell death is timed by the expression of a maturational program intrinsic to interneurons, rather than the developmental state of the cortex itself. Likewise, the extent of interneuron cell death appears to be intrinsically defined: across a range of transplant sizes, a constant fraction of the transplanted interneurons died in the recipient cortex, even when the transplant size was significantly below the number of interneurons the cortex could support (). As such, interneuron cell death is unlikely to follow from intercellular competition for limiting survival signals derived from other cell types.
We propose two mechanisms that may govern the developmental cell death of cortical interneurons (Figure S1
). In the first, which we refer to as ‘cell-autonomous,’ interneuron cell death is intrinsically determined within each embryonic interneuron precursor. In this scenario, interneuron precursors would be individually destined to die in a manner independent from their interactions with other cell types. For example, the production of interneurons could occur with a certain rate of error21
such that a fraction of defective interneuron precursors cannot survive past a certain cellular age. Similarly, a fixed fraction of interneuron precursors may be cell-autonomously programmed to die during a specific stage of their development. Alternatively, in a ‘population-autonomous’ mechanism, developing interneurons may require and compete for limiting survival signals produced by other isochronic interneurons. These neurotrophic signals, which may be obtained via cell-cell contact, synaptic transmission, or neurotrophin signaling independent of TrkB, would be present in a quantity that scales to the number of isochronic developing interneurons. Either a cell-autonomous or population-autonomous mechanism could account for why (1) cell death occurred at a constant rate across broad range of interneuron transplant sizes, and, (2) the survival of endogenous interneurons was not affected by the transplantation of additional interneurons.
Interneurons play a critical role in cortical physiology, and their dysfunction has been implicated in neurological disorders such as epilepsy, schizophrenia, and Alzheimer’s disease22-24
. The detailed examination of interneuron cell death is thus expected to yield new insights into cortical development, the pathophysiology of brain disorders, and the therapeutic application of neuronal transplantation.