{'fraction_non_alphanumeric': 0.03208918452820892, 'fraction_numerical': 0.01604459226410446, 'mean_word_length': 5.022440652818991, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Pyramidal cell regulation of interneuron survival sculpts cortical networks

Complex neuronal circuitries such as those present in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers for these cells is essential for brain function and behaviour, our understanding of this fundamental process is very fragmentary. Here we show that interneuron cell survival in mice depends on the activity of pyramidal cells during a critical window of postnatal development, in which excitatory synaptic input to individual interneurons predicts their outcome. Pyramidal cells regulate interneuron survival through the negative modulation of PTEN signalling, which effectively drives interneuron cell death during this period. Taken together, our findings indicate that activity-dependent mechanisms dynamically adjust the number of inhibitory cells in nascent local cortical circuits, ultimately establishing the appropriate proportions of excitatory and inhibitory neurons in the cerebral cortex.


Complex neuronal circuitries such as those present in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers for these cells is essential for brain function
In the adult neocortex, approximately one in six neurons are inhibitory gamma-aminobutyric acid-containing (GABAergic) interneurons 1,2 , and this ratio is relatively stable across cortical regions and species regardless of total neuronal numbers 3-6 . The cellular balance between excitation and inhibition is critical for brain function and is likely disrupted in a number of neuropsychiatric conditions 7-9 . However, the mechanisms regulating the establishment of appropriate numbers of excitatory and inhibitory neurons in the cerebral cortex remain largely unknown.
Programmed cell death, also known as apoptosis, is an essential mechanism that sculpts the central and peripheral nervous systems during development [10][11][12] . The death of developing neurons is mediated by an evolutionarily conserved signalling pathway that involves the proapoptotic Bcl2 family members Bax and Bak 13 . Previous studies have shown that both cortical pyramidal cells and GABAergic interneurons undergo extensive cell death during postnatal development 14,15 , which suggests that apoptosis may contribute to the establishment of balanced networks of excitatory and inhibitory neurons in the cerebral cortex. However, the temporal relationship and interdependency of the programmed cell death periods for both populations of neurons have not been explored in detail.

Concatenated waves of neuronal death
To determine the developmental sequence that establishes the final ratio of excitatory and inhibitory neurons in the cerebral cortex, we estimated the absolute numbers and relative We observed that the total number of excitatory neurons in the neocortex decreases (~12%) between postnatal day (P) 2 and P5, and then remains stable into adulthood (Fig. 1a, b, e). The reduction in excitatory neurons affects all layers of the neocortex and not only subplate cells , which are known to undergo programmed cell death during this period 19 . By contrast, we found that the number of interneurons is steady until P5, drops extensively between P5 and P10 (~30%), and remains constant into adulthood (Fig. 1c-e).
Interneuron cell loss follows the normal maturation sequence of MGE/POA interneurons 20 , with deep layer interneurons adjusting their numbers ahead of superficial layer interneurons (Fig.   1f). These results revealed that consecutive waves of programmed cell death adjust the final ratio of excitatory and inhibitory neurons in the developing cerebral cortex.

Interneuron activity predicts cell death
Our results indicated that the adjustment of interneuron numbers is preceded by a wave of pyramidal cell death, which suggest that these two processes might be directly linked. Since previous work has shown that neuronal activity and apoptosis rates are inversely correlated in the developing brain 21-23 , we hypothesised that pyramidal cells may impact interneuron survival by increasing the activity of the cells they connect to. We began testing this idea by monitoring the activity of MGE/POA interneurons in the superficial layers of the barrel cortex (S1BF) during the period of interneuron cell death. To this end, we generated mice expressing the fluorescent reporter tdTomato and the genetically encoded calcium sensor GCaMP6s in MGE/POA interneurons (Nkx2-1-Cre;RCL tdT/GCaMP6s ) 24 and performed long-term Ca 2+ imaging in the same interneurons from layer 2/3 S1BF of awake, head-restrained pups (Fig. 2a). To select the most appropriate time for these experiments, we estimated interneuron cell death in S1BF during postnatal development and found comparable dynamics to the rest of the neocortex (Extended Data Fig. 2). For layer 2/3, we observed the most prominent decrease in the number of MGE/POA interneurons between P7 and P8 (Fig. 2b).
We first established our ability to identify surviving interneurons at both times. As expected, we observed that the majority of tdTomato+ interneurons in a region of interest (ROI) were present in the same location the following day (Fig. 2c). However, we also observed that a fraction of tdTomato+ interneurons disappeared between P7 and P8 (Fig. 2c). Since MGE/POA interneurons have ceased migration at the end of the first postnatal week 25 , these observations are consistent with the idea that the cells disappearing between P7 and P8 have undergone apoptosis.
We next wondered whether neural activity at P7 in interneurons that die by P8 was different from that of cells that lived past P8. Analysis of calcium event rates (events/min) at P7 indicated that interneurons that died at P8 exhibited significantly fewer calcium events than neurons that lived past P8 (Fig. 2d, e). We next analysed whether P7 event rates could discriminate between neurons that die at P8 and neurons that live beyond this day. Receiver-operating characteristic (ROC) analysis revealed that event rate at P7 performs significantly better than chance in discriminating between these two populations (Fig. 2f). These results suggested that interneurons with relatively low levels of activity immediately before the period of interneuron cell death have an increased probability to undergo apoptosis 26,27 .

Pyramidal cells regulate interneuron death
The previous experiments led us to hypothesise that interneurons receiving abundant or particularly strong inputs during the period of interneuron cell death would have increased chances to survive. Since PV+ and SST+ interneurons receive most of their inputs from local pyramidal cells during the first postnatal week 28 , we reasoned that modifying the activity of cortical excitatory neurons during the period of interneuron cell death would influence interneuron survival. To test this idea, we transiently modified the activity of pyramidal cells using a chemogenetic approach based on Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that induce neuronal activation or inhibition 29 . We injected the primary somatosensory cortex (S1) of P0 Nex Cre/+ (pyramidal cell-specific) mice with the adenoassociated virus (AAV) encoding mutant G protein-coupled receptors that induce neuronal activation (hM3Dq) or inhibition (hM4Di) following administration of the pharmacologically inert molecule clozapine-N-oxide (CNO) (Fig. 3a). We then injected pups with CNO twice daily between P5 and P8, and examined the distribution of interneurons at P21 (Fig. 3a). We found that increasing the activity of pyramidal cells during the period of interneuron cell death prevents this process and leads to a significant increase in the density of PV+ and SST+ interneurons at P21 compared to control mice . This effect was not due to activitydependent changes in the expression of PV or SST or in the density of pyramidal cells (Extended Data Fig. 3a, b). We also found that dampening the activity of pyramidal cells decreases the density of PV+ and SST+ interneurons at P21 compared to controls, which indicates that interneuron cell death can be bi-directionally modulated by modifying the activity of pyramidal cells (Fig. 3b-d). In both types of experiments, changes in the density of interneurons were homogenously distributed across layers containing PV+ and SST+ interneurons (Extended Data Fig. 3c, d). CNO administration did not cause a redistribution of interneurons from neocortical areas adjacent to the injection site (Extended Data Fig. 3e, f).
Instead, we observed a prominent increase in the density of cleaved caspase-3 cells following inhibition of the activity of pyramidal cells during the normal period of interneuron cell death (Extended Data Fig. 4a-c). Importantly, control experiments revealed that CNO did not modify the density of PV or SST interneurons in pups not infected with AAV-expressing DREADDs (Extended Data Fig. 4d, e). Similarly, CNO administration between P10 and P13 in hM3Dqand hM4Di-injected mice revealed no significant changes in the density of PV+ and SST+ interneurons at P21 (Extended Data Fig. 5). Altogether, these experiments demonstrated that pyramidal cells activity is an essential regulator of interneuron survival during the normal period of interneuron cell death.

Interneurons match pyramidal cell numbers
The previous experiments suggest that pyramidal cells 'rescue' appropriate numbers of cortical interneurons from programmed cell death through an activity-dependent mechanism. Based on this idea, we reasoned that modifying the number of pyramidal cells prior to the period of interneuron cell death should also influence the number of surviving interneurons. To test this hypothesis, we generated conditional mice in which pyramidal cells specifically lack Bak and Bax, whose combined function is critical for apoptosis 30 . As expected, we observed that the number of excitatory neurons in the cerebral cortex of Nex Cre/+ ;Bak -/-;Bax fl/fl mutant mice does not decline between P2 and P21 (Extended Data Fig. 6). Consequently, Nex Cre/+ ;Bak -/-;Bax fl/fl mutant mice have approximately 12% more pyramidal cells than control animals ( Fig. 1b and Extended Data Fig. 6).
We next quantified PV+ and SST+ interneurons in S1 of control and Nex Cre/+ ;Bak -/-;Bax fl/fl mutant mice at P21. We found that the density of both PV+ and SST+ interneurons increases by roughly 30% in Nex Cre/+ ;Bak -/-;Bax fl/fl mutant mice compared to controls (Fig. 4a, c), which suggests that interneuron cell death is suppressed when pyramidal cell death is prevented. This increase is homogenously distributed across layers containing PV+ and SST+ interneurons (Extended Data Fig. 7a), and was also observed in other neocortical areas (Extended Data Fig. 7c,d). To evaluate whether the increase in the number of PV+ and SST+ interneurons represents the entire population of cells that should have normally died through programmed cell death, we generated conditional mice lacking Bax and Bak in MGE/POA interneurons. We found that the density of PV+ and SST+ interneurons also increases by approximately 30% in Nkx2-1-Cre;Bak -/-;Bax fl/fl mutant mice compared to controls (Fig. 4b, c). Indeed, fold changes for PV+ and SST+ interneurons were identical for Nex Cre/+ ;Bak -/-;Bax fl/fl and Nkx2-1-Cre;Bak -/-;Bax fl/fl mutant mice (Extended Data Fig. 7b). These results revealed that preventing pyramidal cell death is sufficient to abolish programmed cell death in MGE/POA interneurons, which reinforces the idea that excitatory input from pyramidal cells onto interneurons during early postnatal development is critical for establishing the appropriate ratio of excitatory and inhibitory cells in the cerebral cortex.

PTEN regulates interneuron cell death
We next investigated the molecular mechanisms through which pyramidal cell activity prevents programmed cell death in cortical interneurons. In the developing nervous system, the serinethreonine kinase Akt is a critical mediator of neuronal survival 31,32 that is antagonised by the activity of the phosphatase and tensin homolog PTEN 33,34 . Consistent with this notion, we observed that the relative levels of activated Akt (P-Akt/Akt ratio) transiently increase in the neocortex during the period of interneuron cell death (Fig. 5a). Interestingly, PTEN levels are very heterogeneous among MGE/POA interneurons during the same period (Fig. 5b). PTEN levels are transiently elevated in sparse interneurons in deep and superficial layers of S1 and this increase is concurrent with the peak of interneuron cell death in these layers (Fig. 5c, d and Extended Data Fig. 8a, b). These observations led us to hypothesise that high PTEN levels during this period may drive interneurons towards cell death, and that pyramidal cells might influence this process by regulating PTEN in interneurons.
To test this hypothesis, we generated mice in which we conditionally deleted Pten from postmitotic MGE interneurons 35,36 . We observed that Lhx6-Cre;Pten fl/fl mutant mice have abnormally large jaws and reduced body weight compared to their littermates by P16, probably due to the embryonic expression of Lhx6 in the first branchial arch 37 , which prevented their analysis at later developmental stages. We nevertheless found that Pten conditional mutants have a significant increase in the density of PV and SST interneurons in S1 compared to control mice (Fig. 5e,f,g and Extended Data Fig. 8c,d), without changing their relative distribution across layers (Extended Data Fig. 8e). Since Lhx6-Cre drives recombination in endothelial cells in addition to MGE interneurons 35 , we examined whether a change in the organisation of neocortical blood vessels might contribute to increased survival of interneurons in conditional Pten mutants. We found that the density of blood vessels increases in conditional Pten mutants compared to controls (Extended Data Fig. 8c, d). However, this change does not affect the density of pyramidal cells (Extended Data Fig. 8c, d), which rules out an indirect effect of blood vessels on interneuron survival through an increase in pyramidal cell density. To discard a direct effect of blood vessels on interneuron survival, we carried out a second series of experiments using acute pharmacological inhibition of PTEN. We injected the PTEN inhibitor bpV(pic) systemically at P7 and P8 in wild type mice and analysed blood vessel density in S1 at P10 (Extended Data Fig. 9b,c). Mice injected with the PTEN inhibitor did not exhibit increased blood vessel coverage (Extended Data Fig. 9b,c). In contrast, transient PTEN inhibition during the period of interneuron cell death increased the density of MGE interneurons compared to control mice (Extended Data Fig. 9a,d,e). Mice injected with the PTEN inhibitor outside the normal window of interneuron programmed cell death showed similar densities of PV and SST interneurons than controls (Extended Data Fig. 9f-h). These results revealed that PTEN is most likely required cell-autonomously for interneuron apoptosis during the normal period of interneuron cell death.
Finally, we examined whether pyramidal cell activity influences the survival of interneurons by non-cell autonomously regulating the expression of PTEN levels in these cells during the period of interneuron cell death. To this end, we carried out DREADDs experiments similar to those that led to an increased number of cortical interneurons following transient activation of pyramidal cells between P5 and P8 (Fig. 3), but here we analysed PTEN levels in cortical interneurons at P8 (Fig. 5h). We found that PTEN levels are significantly decreased in GABAergic interneurons following the activation of pyramidal cells (Fig. 5i, j). These results strongly suggested that pyramidal cells influence the normal process of interneuron programmed cell death through the activity-dependent inhibition of PTEN, which tips the balance between survival and apoptotic signalling pathways in developing interneurons.

Discussion
Our results suggest that interneuron programmed cell death has evolved as a mechanism responsible for adjusting the final ratio of excitatory and inhibitory neurons in the cerebral cortex, a critical milestone in the assembly of cortical circuits 38 . Although synaptic mechanisms are known to stabilise excitatory-inhibitory ratios in cortical circuits [39][40][41] , this effectively requires that the relative proportions of pyramidal cells and interneurons are within certain parameters [42][43][44] . Considering the disproportionate expansion of neocortical areas during human evolution 45,46 , it is tempting to speculate that the dependency of interneuron survival on pyramidal cells provides an evolutionary advantage for the preservation of appropriate ratios of excitatory and inhibitory cells during the rapid increase of pyramidal cell numbers in the primate lineage.
Our work indicates that interneuron cell death is non-cell autonomously regulated by pyramidal cells, which seem to be able to 'rescue' connected interneurons from their intrinsically determined cell death 14 by inhibiting the activity of PTEN during a critical window in postnatal development. It is worth noting that a sizable proportion of individuals with autism spectrum disorders (ASD) and macrocephaly carry deleterious mutations in the PTEN gene 47,48 .
Our observations indicate that loss of PTEN function is sufficient to disrupt interneuron programmed cell death, which may in turn alter the cellular balance of excitation and inhibition in the cerebral cortex. This mechanism may contribute to deregulation of cortical information processing and social dysfunction in ASD patients carrying PTEN mutations.
The apoptosis rate of pyramidal cells varies among functionally different neocortical areas and even across layers within the same cortical area 49 . This suggests that the proposed mechanism may sculpt the heterogeneous patterns of interneuron distributions that exist across the cerebral cortex 50 . Consequently, the regulation of interneuron programmed cell death by pyramidal cells likely contributes to the cytoarchitectonical specialisation of cortical areas. 1 Beaulieu, C. Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res. 609, 284-292 (1993).       following AAV injection at P0. b, c, Coronal sections through S1BF from P21 Nex Cre/+ mice injected with hM3Dq-mCherry or hM4Di-mCherry viruses followed by vehicle or CNO treatment. d, Quantification of the density of PV and SST cells at P21. Two-tailed Student's unpaired t-test, for hM3DQ, ***p = 0.0002, **p = 0.003; for hM4DI, **p = 0.006 [PV], **p = 0.004 [SST]; for hM3DQ, n = 7 and 9 animals for -CNO PV and SST, respectively; 6 and 7 animals for +CNO PV and SST, respectively; for hM4DI, n = 7 animals for -CNO and 5 animals for +CNO for both PV and SST. Data is shown as mean ± SEM. Scale bars, 500 µm (a) and 100 µm (b). µm.

Some interneurons have much higher PTEN levels (arrowhead) than most (open arrowheads).
c, Coronal sections through layer 2/3 S1BF from Nkx2-1-Cre;RCL tdTomato mice at P5, P7, P8 and   Stereology. The total number of pyramidal neurons and MGE interneurons in the cerebral cortex were estimated using the optical dissector method 53 ,

Animals
where ΣQis the total number of cells counted, t the mean section thickness, h the height of the optical dissector (17 µm for pyramidal neuron stereology, 18 µm for MGE stereology), adjusting for the guard zones (1 µm) above and below the dissector, asf stands for the area sampling fraction and ssf stands for the section sampling fraction (frequency of sampling  In vivo imaging. P6 animals were anesthetized with 2% isoflurane and held in a nose-clamp. Isoflurane concentration during surgery was maintained between 1-2% and the body temperature was maintained at 37 o C by a heating pad. The scalp was cleaned with Betadine and cut open to expose the skull covering the dorsal neocortex. The periosteal tissue, surrounding the skull, was gently scraped with a scalpel. The skull was cleaned with Betadine and Ringer's solution. A circular custom-made metal head-post (Luigs and Neumann) was attached over the left-hemisphere with cyanoacrylate glue (Henkel). A thin protective layer of glue was applied over the skull. The glue was allowed to dry for 10 minutes. Dental cement (Paladur) was used to reinforce the attachment of the head-post to the skull. The animal was injected with Buprenorphine (2 µl/g of a 50 μg/ml solution) and returned to its home cage.
At P7, the animal was anesthetized and head-restrained in a custom-made head holder. A 3mm craniotomy was opened over the posterior-lateral neocortex. This craniotomy encompassed the primary somatosensory cortex (S1). Care was taken not to damage the dura mater. A circular coverslip (3 mm diameter, Harvard Apparatus) was placed over the craniotomy and its edges were sealed with cyano-acrylate glue and reinforced with dental cement. Following surgery, Dexamethasone (5 µl/g of a 38 μg/ml solution) was injected subcutaneously. The animal was allowed to recover for at least 2 hours in its home cage, following which we commenced imaging at P7.
Imaging sessions lasted for 40-60 min and we imaged the same field of view for consecutive days in 3 mice. TdTomato and GCaMP6s 54 were excited using a Ti-Sapphire laser (Coherent Chameleon) tuned to λ = 930 nm. The emitted photons were collected by two GaAsP detectors through a 20x objective (Olympus, 1.0 NA). The field of view (FOV) measured 385 x 385 μm (512 x 512 pixels). The scan speed was set to 30 Hz and image sequences were obtained in sweeps of 1 minute (1800 images/channel/minute). The average excitation power was between 40 and 50 mW, and this was kept constant over all imaging days.
To correct for motion artefacts, image registration was carried out using custom written spatial cross-correlation methods on the tdTomato channel. Briefly, on every 1 min sweep, a part of the tdTomato image sequence, where the animal was not moving, was chosen and 20 frames were averaged to give a non-moving reference image. Every frame of the tdTomato image sequence was spatially cross-correlated to this reference image and offset along the Xand Y-axes to match the cross-correlation peak. The offsets obtained for each tdTomato frame was applied to the corresponding GCaMP6s frame. Calcium events were detected by setting a threshold of 3% change in fluorescence from baseline.

Receiver-operating characteristic (ROC) curves.
To identify whether the calcium event rate (events/min) at P7 could act as a binary classifier in distinguishing cells that will live and cells that die at P8, we plotted the ROC curve 55 by varying the discrimination threshold (which, in this case, is the P7 event rate) and calculated the area under the curve (AUC). To test for statistical significance, the cell labels were randomly shuffled 5000 times. On each shuffle, we calculated the ROC curve and the corresponding AUC. We then compared our observed AUC to the distribution of shuffled AUCs. The p-value is the fraction of shuffled AUCs ≥ observed AUC.
Injections were targeted for the somatosensory cortex with an injection rate of 10nl/s.

Drugs.
For DREADDs experiments, Clozapine-N-Oxide (CNO, Tocris) was dissolved in 5% dimethyl sulfoxide (Sigma) and then diluted with 0.9% saline to either 1 mg/ml or 5 mg/ml. probed with rabbit anti-P-Akt (Ser473, Cell Signalling, 1:1000) overnight at 4°C, followed by an HRP-conjugated donkey anti-rabbit antibody (Thermo Fisher, 1:10,000). The blots were developed using ECL femto Western blotting detection reagents and following read-out, they were stripped (Thermo Fisher). After confirming stripping efficiency, an HRP-conjugated mouse anti-Akt antibody (Cell Signalling, 1:1000) was added overnight at 4°C. The blots were developed using ECL western blotting detection reagents the signals were registered and following stripping, an HRP-conjugated rabbit anti-Actin (Sigma, 1:20,000) was added for 1 h at room temperature. Pico ECL Western blotting reagent was used to detect Actin levels.
Signals were read on a Li-COR Odyssey Imaging Band intensities were analysed using ImageStudioLite.
Image acquisition. Images used for analysis were obtained from an ApoTome (Zeiss), epifluorescence microscope (Leica), or SP8 confocal microscope (Leica). ApoTome images were taken using the ApoTome function on the Zen2 software. Images obtained with the confocal and epifluorescence microscope were taken using the LAS AF software.
Cell counting. Cortical layers were identified based on their distinct histological characteristics. Layer 1 was identified as a sparsely populated cell layer. The border between layers 2/3 and 4 was distinguished by the higher nuclei density of layer 4. Layer 5 was identified as the layer basal to layer 4 and above layer 6, which contains less densely packed nuclei. Cell density, within cortical layers, was quantified either manually or using custom routines written in Matlab (MathWorks). For manual quantification, all analyses were conducted blind and cells were counted in a rectangular area, 551.5 µm wide at the pia surface within the somatosensory cortex, auditory cortex or motor cortex. Cells were counted without using pseudocolour in Fiji.
Automatic quantification was carried out by image segmentation using morphological operations.
To identify PTEN staining intensity in tdTomato+ or GABA+ interneurons, self-designed Cell Profiler 58 pipelines were used. Briefly, tdTomato+ or GABA+ interneurons were identified as primary objects using the global Otsu thresholding method and any objects outside the preset diameter range (25-100 pixels) were excluded. PTEN intensity was measured under this cell mask.
Blood vessel analysis. The fraction of the total area covered by blood vessels and the average vessel diameter were quantified using "Vessel Analysis", an ImageJ plugin All experiments were replicated at least in two different litters.
Data availability. All data and/or analyses generated during the current study are available from the corresponding author upon reasonable request.
Code availability. For automatic quantification, the code was written in Matlab (Mathworks) and is available from the corresponding author on reasonable request.

Stereology
The total number of pyramidal neurons and MGE interneurons in the cerebral cortex were estimated using the optical dissector method 55 , where ΣQis the total number of cells counted, t the mean section thickness, h the height of the optical dissector (17 µm for pyramidal neuron stereology, 18 µm for MGE stereology), adjusting for the guard zones (1 µm) above and below the dissector, asf stands for the area sampling fraction and ssf stands for the section sampling fraction (frequency of sampling).
An ApoTome (Zeiss) equipped with a motorised stage and colour camera was connected to a computer with the Stereo Investigator software (MBF Biosciences). The boundaries of the neocortex were first defined with a 2.5x objective (Zeiss). At P7, the animal was anesthetized and head-restrained in a custom-made head holder. A 3 mm craniotomy was opened over the posterior-lateral neocortex. This craniotomy encompassed the primary somatosensory cortex (S1). Care was taken not to damage the dura mater. A circular coverslip (3 mm diameter, Harvard Apparatus) was placed over the craniotomy and its edges were sealed with cyano-acrylate glue and reinforced with dental cement. Following surgery, Dexamethasone (5 µl/g of a 38 μg/ml solution) was injected subcutaneously. The animal was allowed to recover for at least 2 hours in its home cage, following which we commenced imaging at P7. To correct for motion artefacts, image registration was carried out using custom written spatial cross-correlation methods on the tdTomato channel. Briefly, on every 1 min sweep, a part of the tdTomato image sequence, where the animal was not moving, was chosen and 20 frames were averaged to give a non-moving reference image. Every frame of the tdTomato image sequence was spatially cross-correlated to this reference image and offset along the Xand Y-axes to match the cross-correlation peak. The offsets obtained for each tdTomato frame was applied to the corresponding GCaMP6s frame. Calcium events were detected by setting a threshold of 3% change in fluorescence from baseline.

Receiver-operating characteristic (ROC) curves.
To identify whether the calcium event rate (events/min) at P7 could act as a binary classifier in distinguishing cells that will live and cells that die at P8, we plotted the ROC curve 57 by varying the discrimination threshold (which, in this case, is the P7 event rate) and calculated the area under the curve (AUC). To test for statistical significance, the cell labels were randomly shuffled 5000 times. On each shuffle, we calculated the ROC curve and the corresponding AUC. We then compared our observed AUC to the distribution of shuffled AUCs. The p-value is the fraction of shuffled AUCs ≥ observed AUC.
P0 mice were anesthetized with isoflurane and were mounted in a stereotaxic frame. Pups were injected with 600 nl of virus diluted in PBS and coloured with 0.5% Fast Green (Sigma).
Injections were targeted for the somatosensory cortex with an injection rate of 10nl/s.
Samples were denatured in Laemmli Sample Buffer 59 and run on 10% SDS-PAGE gels.
Separated proteins were electrophoretically transferred onto PVDF membranes. Membranes were blocked with 5% BSA in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Tween20) for 1 h and probed with rabbit anti-P-Akt (Ser473, Cell Signalling, 1:1000) overnight at 4°C, followed by an HRP-conjugated donkey anti-rabbit antibody (Thermo Fisher, 1:10,000). The blots were developed using ECL femto Western blotting detection reagents and following read-out, they were stripped (Thermo Fisher). After confirming stripping efficiency, an HRP-conjugated mouse anti-Akt antibody (Cell Signalling, 1:1000) was added overnight at 4°C. The blots were developed using ECL western blotting detection reagents the signals were registered and following stripping, an HRP-conjugated rabbit anti-Actin (Sigma, 1:20,000) was added for 1 h at room temperature. Pico ECL Western blotting reagent was used to detect Actin levels. Signals were read on a Li-COR Odyssey Imaging Band intensities were analysed using ImageStudioLite.
Image acquisition. Images used for analysis were obtained from an ApoTome (Zeiss), epifluorescence microscope (Leica), or SP8 confocal microscope (Leica). ApoTome images were taken using the ApoTome function on the Zen2 software. Images obtained with the confocal and epifluorescence microscope were taken using the LAS AF software.
Cell counting. Cortical layers were identified based on their distinct histological characteristics. Layer 1 was identified as a sparsely populated cell layer. The border between layers 2/3 and 4 was distinguished by the higher nuclei density of layer 4. Layer 5 was identified as the layer basal to layer 4 and above layer 6, which contains less densely packed nuclei. Cell density, within cortical layers, was quantified either manually or using custom routines written in Matlab (MathWorks). For manual quantification, cells were counted in a rectangular area, 551.5 µm wide at the pia surface within the somatosensory cortex, auditory cortex or motor cortex. Cells were counted without using pseudocolour in Fiji. Automatic quantification was carried out by image segmentation using morphological operations.
To identify PTEN staining intensity in tdTomato+ or GABA+ interneurons, self-designed Cell Profiler 60 pipelines were used. Briefly, tdTomato+ or GABA+ interneurons were identified as primary objects using the global Otsu thresholding method and any objects outside the pre-set diameter range (25-100 pixels) were excluded. PTEN intensity was measured under this cell mask.
Blood vessel analysis. The fraction of the total area covered by blood vessels and the average vessel diameter were quantified using "Vessel Analysis", an ImageJ plugin Data availability. Datasets generated and analysed during the current study are available in the online version of the paper.