Dear Colleagues,
I am enthusiastic about sending you the latest edition (10th) of neuroDEVELOPMENTS, a quarterly newsletter from the Lieber Institute for Brain Development dedicated to translating basic science about brain development into clinical relevance. In this issue, we review two recent papers related to expanding our understanding of excitatory and inhibitory neurons and their role in cortical development and psychiatric disease. As always, our editorial board provides their insights and comments for putting this latest work in a broader perspective.
I hope you find this issue of interest and informative. Past editions of neuroDEVELOPMENTS are available on our website at:
Please feel free to share this with colleagues and email us to subscribe so you do not miss future installments!!
Best wishes,
Karen Robinson
Director of Communications
Johns Hopkins Medical Campus
Human Excitation and Inhibition

Ron McKay, PhD, Chief Editor
Lieber Institute for Brain Development

Maltz Research Laboratories
The rise of mammals followed the global extinction of the dinosaurs 66 million years ago.  Since then, there has been an expansion in the size and complexity of the cerebral cortex in primates. Classic pulse-chase experiments using radioactive DNA precursors to trace the birthdates of excitatory neurons in the macaque brain first powerfully linked early developmental events to the functional architecture of the excitatory neurons in the primate cortex. Subsequent experiments in the mouse showed that inhibitory neurons were derived from a different region, the so-called ganglionic eminence of the ventral forebrain.

Most assumed that the organization in the mouse was also true in human brain. Recent work suggests our perspective must change. This issue of neuroDEVELOPMENTS reviews a new study based on experiments with human fetal cortex; the work suggests a large proportion of inhibitory neurons are derived from the dorsal neuroepithelium along with their excitatory neuron siblings. This study required access to primary human fetal brain tissue, a limited and regulated resource, but the rapid development of new stem cell technologies promise powerful access to the early stages of human cortical development. Remarkable progress using pluripotent stem cells to model psychiatric genetic risk in cortical development is illustrated by the second paper we discuss. In previous issues we have emphasized how new DNA sequencing tools provide high resolution maps of the neuronal lineages that construct the cortex. We now know genetic alteration specifically in these lineages causes highly penetrant psychiatric risk. Progress on these related tools raises the prospect of experimentally defining how this variation alters subsequent computations based on the interaction of excitatory and inhibitory neurons in cortical columns. In the commentary below and the insight from our board we hope to bring you a sense of this progress in our understanding of these risk mechanisms in the emerging vista of primate cortical development.

neuroDEVELOPMENTS Editorial Board
Fred 'Rusty' Gage, PhD
President, The Salk Institute for Biological Studies

Daniel Geschwind, MD, PhD
Professor, UCLA School of Medicine

Elizabeth Grove, PhD
Professor, University of Chicago
Jürgen Knoblich, PhD
Director, Institute of Molecular Biotechnology, Austrian Academy of Sciences 

Arnold Kriegstein, MD
Professor, UCSF

Pat Levitt, PhD
Professor, Keck School of Medicine of USC

Mu-Ming Poo, PhD
Director, Institute of Neuroscience, Chinese Academy of Sciences

John Rubenstein, MD, PhD
Professor of Psychiatry, UCSF

Nenad Sestan, MD, PhD
Professor, Yale University 

Flora Vaccarino, MD
Professor, Yale University 

Chris Walsh, MD, PhD
Chief, Division of Genetics & Genomics, Boston Children's Hospital


Venkata S. Mattay, MD
Managing Editor

Michele Solis, PhD
Science Writer 

Interneuron insights

Pity the inhibitory interneuron. Though contributing substantially to the brain’s cell inventory, the interneuron often stands in the shadows of its excitatory peers. Containing the usually hyperpolarizing chemical GABA (gamma-aminobutyric acid) and connecting with nearby neurons, inhibitory interneurons present a calm, provincial counterpart to the volatile, long-ranging excitatory neurons that connect far-flung brain regions. What’s more, excitatory neurons have distinguished themselves as a substrate of human brain evolution (as reviewed in neuroDEVELOPMENTs), whereas a special role for inhibitory neurons in the human brain remains to be discovered.

But now inhibitory interneurons in the human brain may be having a moment. Thanks to new techniques, two new studies track the proliferation and differentiation of these neurons from the earliest stages of human brain development, and suggest a link to disease. The first study from Tomas Nowakowski’s lab at the University of California San Francisco labels early progenitor cells in human embryonic cortex with a cellular barcode, and finds that these give rise to excitatory neurons as well as inhibitory interneurons — thus upending the textbook story of interneurons as born only elsewhere and migrating to the cortex (Delgado et al 2021). The second study from Paola Arlotta of Harvard University and colleagues engineers mutations related to autism spectrum disorder (ASD) into human induced pluripotent stem cells (iPSCs) and grows them into mini-brain organoids (Paulsen et al 2022). This reveals shifts in the brain’s composition of inhibitory and excitatory neurons, which likely reshapes brain circuit function. Together the studies reinforce the importance of understanding the details of brain development to get a focused understanding of what has gone awry in brain conditions like ASD, and holds up interneurons as a target worthy of study.


“The normal functions of the nervous system depend critically on the proper balance of excitation and inhibition of various neural circuits, making inhibitory neurons as important as excitatory neurons.” – Mu-ming Poo

“[I]t wouldn’t be surprising that the human cortex developed an interneuron repertoire different than mouse, and that humans have evolved different strategies for their generation.” – Flora Vaccarino

“Delgado et al published tantalizing data suggesting that human cortical progenitors can make neocortical interneurons, unlike in the mouse…I remain to be convinced that IN.3 are cortical interneurons, and not olfactory bulb interneurons.” – John Rubenstein

“One potential caveat is that the Delgado et al study was based on in vitro generated neurons. Such a surprising finding deserves to be validated based on in vivo observations…” – Arnold Kriegstein

Cortical kinship

The prevailing view on the origins of cortex holds that excitatory neurons arise from progenitors within cortex, while inhibitory interneurons are born elsewhere, in the ganglionic eminences, and then move tangentially into the cortex. The new paper from Nowakowski’s lab, however, shows that cortical progenitors are not exclusively devoted to making excitatory neurons. Through a tool called cellular barcoding, the team labeled cortical progenitor cells from human embryonic brain samples. This barcode was transferred to offspring cells, and so allowed identification of the lineages of differentiated cells.

After establishing these human cell cultures, first authors Ryan Delgado, Denise Allen, and Matthew Keefe let them grow for six weeks, then obtained the transcript profiles of over 1900 individual cells with single cell RNA-sequencing. This identified the cell types, which included the expected excitatory neurons and glia, but also unexpectedly, GABAergic interneurons. These cortically-derived interneurons did not differ obviously from those born the usual way, in the ganglionic eminences.

Examining the clonal kinships of cells arising from the same progenitor, they found that 51% of the clones contained a combination of excitatory neurons, inhibitory interneurons, and glia (Figure 1). To check that these interneurons were not just a fluke of growing up in a dish in vitro, the researchers also implanted barcoded cortical progenitors into postnatal mouse brain. There, these outsider progenitors still gave rise to inhibitory interneurons, which took on typical interneuron shapes. Overall, the results point to a new source for inhibitory interneurons, and cause a rethink of the potential of cortical progenitor cells.

Figure 1 Mixed kinships Bar graph shows average proportions of cell types derived from the same progenitor: inhibitory neurons (green), intermediate progenitor cells on an inhibitory neuron trajectory (DLX+ IPCs, blue), glia (orange), intermediate progenitor cells on an excitatory neuron trajectory (EOMES+ IPCs, purple) and excitatory neurons (red). From Delgado, Allen, Keefe et al., 2021.

Cellular convergences

Inhibitory interneurons are also a nexus for ASD risk, according to the study from Arlotta and colleagues. First authors Bruna Paulsen, Silvia Velasco, Amanda Kedaigle, and Martina Pigoni studied brain organoids derived from human iPSCs that had been engineered to carry loss-of-function mutations in one of three established risk genes for ASD: SUV420H1, ARID1B, or CHD8. These iPSCs developed into brain organoids, and were maintained for up to six months. At different time points, the researchers extracted and sequenced the RNA from more than 745,000 cells to obtain their transcriptome profiles and identify their cell type.

For all three mutations, disrupted differentiation was apparent, particularly for GABAergic interneurons and deep layer excitatory neurons. For example, SUV420H1 mutant organoids contained significantly more GABAergic neurons than control organoids did at one month, which suggests fast-tracked, premature interneuron development (Figure 2). Notably, the genetic background of each line mattered, too: different lines showed a different size effect on cell types, with some lines containing more than 50% of GABAergic interneurons and others only 5%. Also, the GABAergic phenotype persisted in some lines at later time points, but resolved in others. These shifts in cell composition had real ramifications for spontaneous neural activity in the SUV420H1 mutant organoids, with a decreased frequency of bursting activity compared to controls.

Though ARID1B and CHD8 organoids also showed premature GABAergic interneuron development and shifts in deep layer excitatory neuron differentiation, the mutations did not seem to act through the same molecular pathways. Though all three genes are chromatin modifiers, they seemed to act at different genomic regions, as revealed by ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) data. Finding that these risk genes act along distinct molecular pathways yet converge on a cell composition phenotype provides useful bookends for understanding the neurobiological basis of ASD.

“Organoids could provide a useful semi-in vivo system for studying the development of neural circuits, linking the transcriptomic and connectomic profiles of developing cortical neurons.” – Mu-ming Poo

“The phenotype for all three ASD genes converges on changes in the generation and maturation rates of neurons from deep layer projection neurons (PNs) and GABAergic lineages.” – Pat Levitt

Figure 2 Skewed cells Transcriptomic profiles from over 30,000 individual cells obtained from mini-brain organoids reveal multiple cell types (denoted by different colors). Bar graphs show the increased proportions of GABAergic interneurons (green) found in the organoids engineered to carry a mutation in SUV420H1, an ASD risk gene (“SUV”) compared to control organoids. Organoids with SUV420H1 mutations also had reduced proportions of deep layer projection neurons (pink) compared to control organoids. From Paulsen, Velasco, Kedaigle, Pigoni, et al. 2022.

Making sense of many

Genetic studies have found over 100 high confidence risk genes for ASD (Satterstrom et al 2020), and more are sure to follow. The methods used in these new studies will help researchers continue to do the hard work of making sense of these many risk genes in human brain tissue, and how they result in similar phenotypes that contribute to ASD and related neurodevelopmental disorders. The results also point to the importance of genetic background, and provide an in vitro venue in which to explore how even highly penetrant ASD risk genes result in varied phenotypes.

The findings also focus attention on interneuron development as a vulnerable process that is linked to disease risk. Other studies have suggested that interneuron development and function is also linked to schizophrenia. Understanding the details of interneuron birth, differentiation, migration, and integration into neural circuits may eventually suggest more precise therapeutic approaches targeted at the altered brain pathways.

“[T]he simultaneous generation of the two main neuronal subclasses and precise coordination between dorsal and ventral neurogenesis are crucial for proper formation of functional circuits in the human brain.” – Jürgen Knoblich

The full commentary from our Editorial Board on the two papers highlighted in this issue of neuroDEVELOPMENTS.
1. From Mu-ming Poo, PhD, Chinese Academy of Sciences:
The normal functions of the nervous system depend critically on the proper balance of excitation and inhibition of various neural circuits, making inhibitory neurons as important as excitatory neurons. Interestingly, the action of GABAergic neurons during early development is excitatory (i.e., producing membrane depolarization), due to GABA-induced outward rather than inward flow of chloride ions. As development proceeds, there is a switch of the relative expression levels of inward vs. outward chloride pumps (transporters), reducing cytoplasmic chloride concentration to a much lower level relative to the extracellular chloride concentration, thus the excitation-to-inhibition shift. The early excitatory action of GABA is by no means trivial – it generates large depolarizing waves across the brain and elevation of cytoplasmic calcium that triggers a myriad of developmental changes in gene expression. The exciting finding of Delgado et al 2021 that some cortical GABAergic neurons share common progenitors with glutamatergic neurons raises several questions: Do these GABAergic neurons differ in some subtle way in gene expression patterns and serve differential roles in physiological functions from those derived from the ganglionic eminences? Since previous studies using retroviral tagging of cortical progenitors showed that clonally-related neurons are electrically coupled early and become more synaptically connected and functionally-related later in development, is there distinct connectivity preference of cortical progenitor-derived GABAergic neurons with their clonally-related sister neurons? The timing of the appearance/arrival of two types of GABAergic neurons in the cortex may also result in differential contributions of the two populations of GABAergic neurons in the formation of mature local inhibitory circuits, including feedforward, feedback, and di-inhibitory connections with excitatory neurons.

The paper by Paulsen et al. 2022 nicely illustrates the power of organoids in studying cortical development. The abnormality in the composition of glutamatergic vs. GABAergic neurons due to ASD risk genes is also reflected in the physiological properties of the organoid, as shown by abnormal correlated spiking of neurons. There is now ample evidence that GABAergic activity is critical for cortical maturation, including the early excitatory action of GABA described above and the role of GABA in modulating the maturation of glutamatergic neuronal morphology and synapse formations. Notably, there is evidence linking birth-related stressful mechanisms, persistent excitatory GABA actions, perturbed network oscillations and autism (see Ben-Ari 2015). Organoids could provide a useful semi-in vivo system for studying the development of neural circuits, linking the transcriptomic and connectomic profiles of developing cortical neurons. In particular, it would allow us to address the link between genetic mutations and the pathology of brain disorders involving excitation-inhibition imbalance. Nevertheless, in order to understand the functional roles of GABAergic neurons, it is crucial to map out the temporal changes in the expression of various inward and outward chloride transporters in both glutamatergic and GABAergic neurons that underlie the excitation-to-inhibition shift of GABA action.

2. From Pat Levitt, PhD, University of Southern California:
Clinical heterogeneity, even in the context of highly penetrant single-gene mutations, is a well-known phenomenon. There is a long history of rodent models of psychiatric disorders using an “n of 1” strain approach to introduce a gene of choice into a single strain, typically C57BL6. This limits opportunities for systematically studying biological heterogeneity due to specific gene mutations. It thus has not been possible using this traditional approach to recapitulate the heterogeneity of traits, which is a major gap for developing intervention strategies to alter neurobiological and behavioral outcomes related to clinical phenotypes of psychiatric disorders. While behavioral disruption is not possible in brain organoids, the system can be applied to determine underlying factors that generate developing structural and physiological heterogeneity. This is the core message of Paulsen et al, using human brain organoids. The study reveals that individual iPS cell lines of origin, combined with the specific trait that is measured, results in broad variation. The study shows that the proteins encoded by three autism spectrum disorder (ASD) risk genes vary by genomic contexts, thus resulting in different heterozygous states. The phenotype for all three ASD genes converges on changes in the generation and maturation rates of neurons from deep layer projection neurons (PNs) and GABAergic lineages. Some of the rate changes are profound. The authors also report that disruption of one lineage may not be accompanied by changes to the other. While the authors note the technical challenges of comparing across organoids, and that within a single run there will be variation among organoids derived from a single iPS cell line, careful curation and sufficient sample power is bringing new insight for understanding the convergence of distinct molecular pathways on specific neurobiological processes that likely will lead to phenotypic heterogeneity.

The central findings of Delgado et al -- that some fraction of human dorsal pallial progenitors give rise to both glutamatergic and GABAergic neurons -- are in some sense not surprising. Though others have failed to show shared dorsal pallial origins, the authors note a handful of previous studies that are consistent with their findings, with Letinic et al 2001 being the most convincing, using slice cultures from human embryonic forebrain, lineage labeling and limited molecular characterization. Here, the report of Delgado et al adds substantially to a growing body of evidence that cerebral cortical progenitors are indeed heterogeneous, complex, and have extraordinary capacities to contribute to cellular diversity that is the hallmark of the cerebral cortex. Yet, perhaps more impressive is the report bringing to the field of neurodevelopment a new method, single-cell-RNA-sequencing-compatible tracer (STICR), which can be widely applied to determine lineage relations at a molecular level that has not been possible with other existing methods. Technology meets biological complexity and sorting out mechanisms will have an impact of our near-term understanding of psychiatric and neurodevelopmental disorder etiology.

3. From John Rubenstein, MD, PhD, University of California, San Francisco:
The search is on to identify evolutionary differences between the human brain and other mammals. Delgado et al published tantalizing data suggesting that human cortical progenitors can make neocortical interneurons, unlike in the mouse. They used a sophisticated method to perform lineage tracing of human cortical neural progenitors, and they obtained evidence of three types of GABAergic interneurons which they name IN.1, IN.2 and IN.3. The last group (IN.3) they conclude correspond to neocortical interneurons, generated by cortical progenitors, which are not found in the mouse. As it stands, I am not convinced that their evidence is definitive. First, their method involved dissociating and culturing human fetal cortex. In my experience, dissociating and culturing can generate cell fate artifacts. Furthermore, several groups have shown that mouse cortex generates olfactory bulb interneurons, and indeed their results support this based on their ­­IN.2 marker genes: TSHZ1, PBX3, MEIS2, CALB2, CDCA7L, SYNPR and ETV1. Delgado et al argue that the one class of their interneuron-like cells (IN.3) is similar to caudal ganglionic eminence (CGE)-derived cortical interneurons marked by the expression of NR2F1, NFIX, PROX1 and NR2F2, as well as, SOX6 and CXCR4. However, NR2F1 and NR2F2 are well-known to be expressed during olfactory bulb interneuron development; likewise, the Allen Developing Mouse Brain Atlas shows expression of NFIX, PROX1, SOX6 and CXCR4 in the developing olfactory bulb, so I remain to be convinced that IN.3 are cortical interneurons, and not olfactory bulb interneurons. For me, the most convincing evidence is that xenographed human PTPRZ1+ progenitors generate interneuron-like neurons that disperse and integrate in the cortex – previous work shows that rostral migratory stream-derived olfactory bulb interneurons do not have this property. However, I don’t know if the equivalent experiment, of transplanting purified mouse cortical progenitors, leads to the dispersion of some interneurons. Overall, this work should be followed up using additional experimental systems, and other primates, to further explore this important question.
The Paulsen et al study used the cortical organoid model of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency of three ASD risk nuclear proteins: SUV420H1, ARID1B and CHD8. In general, the mutants showed precocious generation of GABAergic neurons and deep layer projection neurons, although the implicated molecular mechanisms (differential RNA and protein expression) diverged for these cellular phenotypes. It is noteworthy that Mariani et al 2015 also found an expansion of GABAergic neurons in organoids of macrocephalic ASD individuals. These studies reinforce the utility of modeling neurodevelopmental disorders using organoids. Of course, there are caveats in assuming that these organoid phenotypes closely resemble in vivo human cortical phenotypes, but this approach is shedding insights into convergent phenotypes; these seem to be operating at the level of changing the timing and nature of cell fate decisions. Finally, it will be interesting to better understand the nature of the GABAergic neurons generated by these cortical organoids.

4. From Arnold Kriegstein, MD, University of California, San Francisco:
The discovery, made over 25 years ago, that a major and important class of cortical cells, namely the inhibitory interneurons, are not generated in the developing cortex at all, but in a different sub-cortical brain region was very surprising at the time. It is an odd way to build the cortex as the interneurons have to migrate relatively long distances from the ventral to the dorsal forebrain to reach their intended targets. It would be reasonable to ask: why has nature settled on this peculiar way to construct the cortex? One possible explanation is that the niche where progenitor cells reside provides instructional signals that help determine cell fate. Thus, the divergent programs that produce either excitatory or inhibitory neurons might need to be spatially segregated. However, this does not seem to be the case based on surprising new data presented by Delgado et al who show that clonal descendants derived from single cortical progenitor cells can consist of both excitatory projection neurons and inhibitory interneurons. All the more surprising, the transcriptomic identity of the cortically-derived inhibitory neurons matches that of interneurons generated at the sub-cortical site. This suggests that cortical interneurons can emerge with the same terminal identity despite arising from distinctly different lineages. This phenomenon is well-described in worms and flies where it is regulated by one or more specific terminal selector gene(s), and it would be very interesting to see if there are common terminal selector genes expressed in the distinct human inhibitory neuron cell lineages. One potential caveat is that the Delgado et al study was based on in vitro generated neurons. Such a surprising finding deserves to be validated based on in vivo observations, and the phenomenon of somatic mosaicism as outlined in a previous neuroDEVELOPMENTS report provides just such an opportunity to confirm in vivo that human cortical clones may contain both excitatory and inhibitory neurons.

The dual pallial and subpallial origin for certain classes of cortical interneuron may be an evolutionary adaptation to help generate the huge numbers of neurons required for the expanded human cerebral cortex. The need for producing large numbers of interneurons is made even more pressing in humans since the proportion of inhibitory interneurons relative to excitatory neurons has recently been shown to be much greater in human cortex compared to mouse. The regulation of interneuron production thus appears to be a feature under significant evolutionary pressure. It may therefore not be surprising that neurodevelopmental diseases might arise from altered regulation of the timing of inhibitory neuron production and even that three different mutations related to autism may converge on a similar phenotype related to interneuron production, as Paulsen and colleagues have shown.

5. From Flora Vaccarino, MD, Yale University:
The mouse has been the gold standard for setting up rules of brain development and patterning among mammals, yet, mice are extremely specialized organisms that have evolutionarily diverged from primate millions of years ago. So it wouldn’t be surprising that the human cortex developed an interneuron repertoire different than mouse, and that humans have evolved different strategies for their generation. Additionally, the human brain size and regional complexity present unique challenges to developmental processes, ie, increased distance cells must travel to reach target destination

Delgado et al performed a rigorous lineage analysis based upon lentivirally-transduced barcodes which can be analyzed together with single cell transcriptomes. Their lineage analyses identify three types of interneurons. The IN.1 express canonical markers for MGE-derived IN and indeed they are almost exclusively the progeny of MGE progenitor cells. In contrast, the IN.2 and IN.3 expressed canonical markers for the CGE-derived interneurons and were derived from cortical progenitors. The authors suggest that while IN.2, based on markers, are typical olfactory bulk interneurons, IN.3 are more similar to the subset of CGE-derived interneurons that populate the cortex. This is based upon ISH data showing differential localization in mouse OF and CTX of marker-expressing cells.

Their cortical progenitors generate two different types of interneurons, those that, based on markers, are olfactory bulb interneurons (and this curiously seems particularly prominent in PFC), and those that express markers typical of cortical interneurons originating from the GCE. These olfactory bulb and cortical interneurons are derived from cortical progenitors that are also concurrently generating excitatory neurons.

The mouse transplantation experiments quite convincingly demonstrate that these cortically derived interneurons display morphological features of mature interneurons. The experiment is designed to determine the developmental potential rather than actual fate, since human neuronal progenitors were labeled with the STICR viral library ex-vivo and then cultured or transplanted in the mouse cortex for 6 weeks. Nevertheless, many studies have shown that interneuron cell fate is irreversibly determined early in neurogenesis or even before neurogenesis starts, hence this strategy is in principle valid to assess the fate of human progenitors in vivo.

Although the Delgado et al study implies that the dorsally generated interneuron lineage generates interneurons that are similar to those generated in the CGE, it would be very important to follow up on potential differences between CGE-derived and cortically-derived human cortical interneurons.

6. From Jürgen Knoblich, PhD, Austrian Academy of Sciences:
This issue highlights the human-specific aspects of brain development with a particular focus on interneurons. Precise regulation of excitatory and inhibitory activity in the cortex is of crucial importance, and perturbations of this equilibrium are involved in multiple brain disorders. It is becoming increasingly evident that the regulation of this balance in the human brain involves developmental processes not seen in animal models, such as mice, which are the most common laboratory animal used to study brain development. Therefore, these processes cannot be investigated using the traditional animal model approach and we need innovative methodology to study them.

Delgado et al. describe an innovative lineage tracing approach to determine the origin of various subclasses of neurons. By dissecting human fetal brain tissue and labelling cells with viruses that express unique barcodes, they could trace back the origin of interneurons. Very surprisingly, they found that these neurons arise not only from the ventrally located ganglionic eminences, as they do in mice, but can also be generated by precursors located in the dorsal part of the developing cortex. The surprising conclusion of this work is that some cortical progenitors can switch between creating excitatory and inhibitory neurons, a hypothesis that had been very controversial before.

Organoids represent another approach to study human neurogenesis. Paulsen and colleagues use organoid models to address the role of three genes that convey susceptibility to autism spectrum disorder (ASD). Brain organoids (often called cerebral organoids) can be derived from healthy individuals or from patients carrying CrisPr-engineered DNA mutations responsible for ASD. The authors compare organoids derived from patients carrying mutations in KMT5B, ARID1B or CHD8. Using multiple cell lines per patient, they find that mutations in each of the genes affect different pathways, but they all perturb the synchronous generation of excitatory (glutamatergic) and inhibitory (GABA-ergic) neurons. Thus, the simultaneous generation of the two main neuronal subclasses and precise coordination between dorsal and ventral neurogenesis are crucial for proper formation of functional circuits in the human brain.

In fact, there are even more developmental processes that affect interneurons in humans that are not seen in mice: for example, the group of Arturo Alvarez-Buylla had previously identified the Arc, a stream of interneurons migrating into the human cortex even months after birth (Paredes et al., 2016). This late type of neurogenesis is not seen in mice and has strong implications for how we think about the development of human infants and their susceptibility to disease. The Arc neurons were identified using human fetal brain tissue. Although these post-mortem studies were absolutely crucial for their initial identification, they cannot be used to study the function of the Arc neurons in the developing brain and to characterize their role in neurodevelopmental disorders like epilepsy. Instead, recent work using organoids to model neurodevelopmental disease has shed some light on their origin and relevance. Analysis of Tuberous Sclerosis, a severe form of childhood epilepsy, in human 3D organoid culture identified an underlying mechanism that may involve the Arc, highlighting its relevance for human disease (Eichmueller et al., 2022). Tuberous sclerosis is accompanied by the formation of benign tumors and the study showed that in the organoid model, such tumors derive from cells that also give rise to the Arc. These experiments indicate that increased sensitivity to disease may be the price that we pay for the enormous complexity of our brain.

Increased complexity of the human brain is reflected not only by a larger number of interneuron subtypes, but also by a vastly increased complexity in the way they connect. A heroic effort using three-dimensional electron microscopy to reconstruct the connectome of a human brain (Loomba et al., 2022) has revealed that the increased complexity is largely due to an enormous increase in interneuron-to-interneuron connections and not the number of synapses onto excitatory neurons, as was expected before. This surprising finding highlights the need for experimental models to recapitulate human interneurons and their enormously complex connections. How are these interneuron-to-interneuron connections formed? What is their role in disease? And what is different in human and primate genomes to guide the formation of these types of connections? To answer these questions, we will need models recapitulating the pattern of synaptic connections formed in the human brain. Such models will allow us to study how complex interactions between interneurons change in patients suffering from brain diseases. Our recent findings on Tuberous sclerosis showcase how studying defects leading to human neurodevelopmental diseases can shed light on developmental processes that cannot be analyzed in laboratory animals. More and more sophisticated organoid models for the human brain offer great ways to test how disease mutations affect these unique processes in the human brain and have great potential for creating deep insights into the complexity of the human brain.
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