The human cerebral cortex houses 1000 times more neurons than that of the cerebral cortex of a mouse, but the possible differences in synaptic circuits between these species are still poorly understood. We used three-dimensional electron microscopy of mouse, macaque, and human cortical samples to study their cell type composition and synaptic circuit architecture. The 2.5-fold increase in interneurons in humans compared with mice was compensated by a change in axonal connection probabilities and therefore did not yield a commensurate increase in inhibitory-versus-excitatory synaptic input balance on human pyramidal cells. Rather, increased inhibition created an expanded interneuron-to-interneuron network, driven by an expansion of interneuron-targeting interneuron types and an increase in their synaptic selectivity for interneuron innervation. These constitute key neuronal network alterations in the human cortex. The difference between human and mouse Over the past few decades, the mouse has become a model organism for brain research. Because of the close evolutionary similarity of ion channels, synaptic receptors, and other key molecular constituents of the brain to that of humans, corresponding similarity has been assumed for cortical neuronal circuits. However, comparative synaptic-resolution connectomic studies are required to determine the degree to which circuit structure has evolved between species. Using three-dimensional electron microscopy, Loomba et al . compared mouse and human/macaque cortex synaptic connectivity. Although human cells are much larger compared with mouse neurons and are more numerous, on average, they do not receive more synapses. And, even though there are three times more interneurons in the human cortex than in the mouse, the excitation-to-inhibition ratio is similar between the species. —PRS Three-dimensional electron microscopy of mouse, macaque, and human brain samples elucidates cell type composition and synaptic circuit architecture. INTRODUCTION The analysis of the human brain is a central goal of neuroscience, but for methodological reasons, research has focused on model organisms, the mouse in particular. Because substantial homology was found at the level of ion channels, transcriptional programs, and basic neuronal types, a strong similarity of neuronal circuits across species has also been assumed. However, a rigorous test of the configuration of local neuronal circuitry in mouse versus human—in particular, in the gray matter of the cerebral cortex—is missing. The about 1000-fold increase in number of neurons is the most obvious evolutionary change of neuronal network properties from mouse to human. Whether the structure of the local cortical circuitry has changed as well is, however, unclear. Recent data from transcriptomic analyses has indicated an increase in the proportion of inhibitory interneurons from mouse to human. But what the effect of such a change is on the circuit configurations found in the human cerebral cortex is not known. This is, however, of particular interest also to the study of neuropsychiatric disorders because in these, the alteration of inhibitory-to-excitatory synaptic balance has been identified as one possible mechanistic underpinning. RATIONALE We used recent methodological improvements in connectomics to acquire data from one macaque and two human individuals, using biopsies of the temporal, parietal, and frontal cortex. Human tissue was obtained from neurosurgical interventions related to tumor removal, in which access path tissue was harvested that was not primarily affected by the underlying disease. A key concern in the analysis of human patient tissue has been the relation to epilepsy surgery, when the underlying disease has required often year-long treatment with pharmaceuticals, plausibly altering synaptic connectivity. Therefore, the analysis of nonepileptic surgery tissue seemed of particular importance. We also included data from one macaque individual, who was not known to have any brain-related pathology. RESULTS We acquired three-dimensional electron microscopy data from temporal and frontal cortex of human and temporal and parietal cortex of macaque. From these, we obtained connectomic reconstructions and compared these with five connectomes from mouse cortex. On the basis of these data, we were able to determine the effect of the about 2.5-fold expansion of the interneuron pool in macaque and human cortex compared with that of mouse. Contrary to expectation, the inhibitory-to-excitatory synaptic balance on pyramidal neurons in macaque and human cortex was not substantially altered. Rather, the interneuron pool was selectively expanded for bipolar-type interneurons, which prefer the innervation of other interneurons, and which further increased their preference for interneuron innervation from mouse to human. These changes were each multifold, yielding in effect an about 10-fold expanded interneuron-to-interneuron network in the human cortex that is only sparsely present in mouse. The total amount of synaptic input to pyramidal neurons, however, did not change according to the threefold thickening of the cortex; rather, a modest increase from about 12,000 synaptic inputs in mouse to about 15,000 in human was found. CONCLUSION The principal cells of the cerebral cortex, pyramidal neurons, maintain almost constant inhibitory-to-excitatory input balance and total synaptic input across 100 million years of evolutionary divergence, which is particularly noteworthy with the concomitant 1000-fold expansion of the neuronal network size and the 2.5-fold increase of inhibitory interneurons from mouse to human. Rather, the key network change from mouse to human is an expansion of almost an order of magnitude of an interneuron-to-interneuron network that is virtually absent in mouse but constitutes a substantial part of the human cortical network. Whether this new network is primarily created through the expansion of existing neuronal types, or is related to the creation of new interneuron subtypes, requires further study. The discovery of this network component in human cortex encourages detailed analysis of its function in health and disease. Connectomic screening across mammalian species: Comparison of five mouse, two macaque, and two human connectomic datasets from the cerebral cortex. ( A ) Automated reconstructions of all neurons with their cell bodies in the volume shown, using random colors. The analyzed connectomes comprised a total of ~1.6 million synapses. Arrows indicate evolutionary divergence: the last common ancestor between human and mouse, approximately 100 million years ago, and the last common ancestor between human and macaque, about 20 million years ago. ( B ) Illustration of the about 10-fold expansion of the interneuron-to-interneuron network from mouse to human.