Xiaolong Jiang - US grants

Abstract Since Ramon y Cajal, neuroscientists have speculated that even the most complex brain functions might even- tually be understood at the level of neuronal cell types and their connections. More recently, while we have be- gun to understand the wiring principles of cortical microcircuit in rodents at the level of cell types, we are still in infancy in understanding the circuit organization of the primate neocortex at the level of cell types and their connections, slowing the progress toward a mechanistic understanding of complex cognitive capabilities char- acteristic of primates. For instance, the dorsolateral prefrontal cortex (DLPFC) of the primate brain is the most evolutionarily developed brain region that supports complex cognitive processes characteristic of primates, such as reasoning, planning, and abstract thinking. However, we know little about the constituent cell types comprising DLPFC circuit, how each cell type connects each other to form a functional circuit, and what circuit components specific to this circuit endow it with superb computational capabilities for complex cognitive pro- cesses. To fill in this knowledge gap, we scale up a cost-effective, interdisciplinary approach to macaque DLPFC, aiming at identifying all its consitiuent cell types and decipher their connectivity rules, with emphasis on highly diverse GABAergic interneurons. We propose to use multi-cell patch recordings, single-cell RNA sequencing (scRNA-seq), novel and rapid viral GABAergic labeling, and machine learning to achieve two main goals: 1) dissect macaque DLPFC microcircuit by generating a morphological taxonomy of cell types in DLPFC and mapping their connections; and 2) derive transcriptomic signatures of morphologically defined DLPFC neurons using Patch-seq method, a novel scRNA-seq protocol. We have demonstrated the feasibility and suc- cess of this approach in mouse neocortex, and our preliminary data indicate no technical issue in applying this approach to primates. Using multi-cell patch recordings, we will characterize electrophysiology and morphology of thousands of neurons and map connections between tens of thousands of cell pairs from DLPFC. Using Patch-seq method, we will combine patch recording with a novel/sensitive scRNA-seq method (Smart-seq2) to simultaneously obtain electrophysiology, morphology and transcriptome from single neurons, which can further substantiate cell type classification and identify novel molecular markers for each cell type. We will prioritize our effort on superficial layers of DLPFC, but eventually scale up our efforts to all layers if time permits. At the end, the project will uncover a high-resolution microcircuit blueprint of macaque DLPFC with each circuit com- ponent identified by specific genetic markers. Such a comprehensive dataset will provide the essential ground- work to design molecular tools for further functional dissection of the complex cognitive processes subserved by PFC. From a clinical perspective, having reference transcriptomes and connectivity patterns for different cell types in primate DLPFC will facilitate our understanding of the relationship between disease-associated genes, cell types, and circuit deficits in neuropsychiatric diseases, schizophrenia in particular.

Abstract Epilepsy affects over 2 million of people in the United States, causing significant morbidity with a high cost to society. While the behavioral and electrophysiological correlates of seizures in patients and animal models have been studied for over a century, the underlying circuit abnormalities are still being elucidated. Generalized spike-wave (SW) absence sei- zures are the most common seizure disorder in children and thought to be exclusively of genetic origin. While over 20 genes are discovered and studied in SW epilepsies, it is still unclear how each genetic lesion impairs normal circuit devel- opment and ultimately results in a seizure-prone cortical circuit. Since the SW seizure phenotype can be very similar de- spite disparate genetic etiologies, a stereotypical circuit deficit may exist which underlies the expression of this seizure pattern. More recently, as we have begun to understand the wiring principles of cortical microcircuits at the level of cell types, it has become possible to ask how disruption of these canonical networks may be responsible for initiating seizure activity and impairing cognitive functions in SW epilepsies, whether the pathogenic circuit changes overlap despite dis- parate molecular lesions, and how a seizure-prone circuit emerges from inherited molecular defects to favor seizure on- set at predictable developmental time-points. This information not only suggests novel and broadly-applicable therapeu- tic targets, but also leads to valuable insights into the functional roles of distinct cell types and specific connectivity principles in normal brain. To answer these important questions, we are taking advantage of three mouse models of ab- sence epilepsy, stargazer, tottering and Gabrg2 mutant mice, which harbor mutations in three unrelated genes but share the same SW phenotype, and propose a comprehensive microcircuit comparison among distinct genotypes at the level of cell types and their connections. We perform a large-scale circuit analysis across a whole column of the somatosen- sory cortex (S1) in three models along the seizure development, by leveraging a high-throughput multi-patching method (up to 12-patch) we recently developed. We will measure multiple neuronal features of distinct cell types within the S1 epileptic circuit, with an emphasis on connectivity and morphology of major groups of cortical GABAergic interneurons. In parallel, the same analysis will be performed on WT littermates as controls to reveal cell type-specific connectivity changes as a function of the genotype and developmental stage. These comprehensive, dynamic comparisons, based on large-scale circuit analyses with sensitive, state-of-the-art methods, will reveal the full extent of abnormal microcircuit structure and functions that are closely associated with seizure onset. Our preliminary data uncover several connectivity defects in these models. The most striking is that stereotypical connectivity and morphology of somatostatin-expressing Martinotti cells are severely disrupted, and this disruption appears to emerge only after seizure onset and is shared by models, suggesting a common circuit deficit underlying absence epilepsy. The potential causative circuit mechanisms will be further tested via network modeling and an in vivo chemogenetic assay. Identification of causative circuit deficits gen- eralized across genetically heterogeneous, yet highly stereotyped SW seizures will direct the field toward the develop- ment of innovative, broadly applicable circuit-based interventions for absence epilepsy and its related comorbidities.

Abstract Since Ramon y Cajal, neuroscientists have speculated that even the most complex brain functions might even- tually be understood at the level of neuronal cell types and their connections. More recently, while we have be- gun to understand cell types and their wiring principles in cortical circuits of rodents, we are still in infancy in understanding the circuit organization of the primate cortex at the level of cell types and their connections, slowing progress toward a circuit-level mechanistic understanding of complex cognitive capabilities of pri- mates. For instance, the human cortex houses two unique morphotypes, von Economo neurons (VENs) and fork cells, which are concentrated in the cortical regions that support complex social cognitive abilities and self- awareness. For a long time, the VENs and fork cells are believed to be unique to human and great apes, and thus are hypothesized to be the neural correlate of consciousness and human-like complex social behaviors. Despite their importance, their functions are deemed to be experimentally intractable given their exclusive re- striction to hominids. A recent study, however, provides compelling evidence that these unique morphotypes are also present in the anterior insula (AI) of macaque monkeys, providing an unprecedented opportunity for functional characterization of these novel neurons in the laboratory. Here, by partnering with a Chinese primate research laboratory to leverage the abundant macaque resources and lower cost of single-cell RNA- sequencing (scRNA-seq) in China, we propose to take this opportunity to dissect out the cortical circuit of mon- key AI and characterize these novel cell types in terms of electrophysiology, morphology, transcriptome, and connectivity. By taking advantage of a novel set of cost-effective, high-throughput approaches, including large- scale droplet-based scRNA-seq, Patch-seq, and multi-cell patch recordings, we aim to identify and character- ize all the cell types that comprise monkey AI with molecular, spatial and functional annotations. Particularly, this comprehensive interrogation of macaque cortical circuit will lead to a detailed, functional characterization of VENs and FCs for the first time. Importantly, by complementing the strength and unique resources of two collaborating labs in USA and in China, we expect to accomplish this otherwise infeasible, costly primate re- search at this scale within a reasonable budget and time period, providing unprecedented knowledge and re- source for the field to understand the emergence of human-like social intelligence and related neuropsychiatric disorders. Particularly, identifying the specific marker genes for those novel cell types will promote the field to develop genetically targeted tools for studying human-like social cognitive abilities in the context of behaviors. With all information and tools available, our understanding of human intelligence, previously perceived as ex- perimentally intractable and largely speculative, will finally gain solid ground and are ready to take off in the near future.