SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1.

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

Neuronal SAM68 differentially regulates alternative last exon splicing and ensures proper synapse development and function.

Alternative splicing in the 3'UTR of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key splicing regulator that controls the diversity of 3'UTR isoforms through alternative last exon (ALE) selection. However, the tissue/cell type-specific mechanisms underlying the splicing control at the 3' end and its functional significance remain unclear. Here, we show that SAM68 regulates ALE splicing in a dose-dependent manner and the neuronal splicing is differentially regulated depending on the characteristics of the target transcript. Specifically, we found that SAM68 regulates interleukin-1 receptor-associated protein splicing through the interaction with U1 small nuclear ribonucleoprotein. In contrast, the ALE splicing of protocadherin-15 (Pcdh15), a gene implicated in several neuropsychiatric disorders, is independent of U1 small nuclear ribonucleoprotein but modulated by the calcium/calmodulin-dependent protein kinase signaling pathway. We found that the aberrant ALE selection of Pcdh15 led to a conversion from a membrane-bound to a soluble isoform and consequently disrupted its localization into excitatory and inhibitory synapses. Notably, the neuronal expression of the soluble form of PCDH15 preferentially affected the number of inhibitory synapses. Moreover, the soluble form of PCDH15 interacted physically with α-neurexins and further disrupted neuroligin-2-induced inhibitory synapses in artificial synapse formation assays. Our findings provide novel insights into the role of neuron-specific alternative 3'UTR isoform selections in synapse development.

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Maternal immunoglobulin G affects brain development of mouse offspring.

Maternal immunoglobulin (Ig)G is present in breast milk and has been shown to contribute to the development of the immune system in infants. In contrast, maternal IgG has no known effect on early childhood brain development. We found maternal IgG immunoreactivity in microglia, which are resident macrophages of the central nervous system of the pup brain, peaking at postnatal one week. Strong IgG immunoreactivity was observed in microglia in the corpus callosum and cerebellar white matter. IgG stimulation of primary cultured microglia activated the type I interferon feedback loop by Syk. Analysis of neonatal Fc receptor knockout (FcRn KO) mice that could not take up IgG from their mothers revealed abnormalities in the proliferation and/or survival of microglia, oligodendrocytes, and some types of interneurons. Moreover, FcRn KO mice also exhibited abnormalities in social behavior and lower locomotor activity in their home cages. Thus, changes in the mother-derived IgG levels affect brain development in offsprings.

Neuron-derived neurotrophic factor protects against dexamethasone-induced skeletal muscle atrophy.

Skeletal muscle atrophy caused by various conditions including aging, nerve damage, and steroid administration, is a serious health problem worldwide. We recently reported that neuron-derived neurotrophic factor (NDNF) functions as a muscle-derived secreted factor, also known as myokine, which exerts protective actions on endothelial cell and cardiomyocyte function. Here, we investigated whether NDNF regulates skeletal muscle atrophy induced by steroid administration and sciatic denervation. NDNF-knockout (KO) mice and age-matched wild-type (WT) mice were subjected to continuous dexamethasone (DEX) treatment or sciatic denervation. NDNF-KO mice exhibited decreased gastrocnemius muscle weight and reduced cross sectional area of myocyte fiber after DEX treatment or sciatic denervation compared with WT mice. Administration of an adenoviral vector expressing NDNF (Ad-NDNF) or recombinant NDNF protein to gastrocnemius muscle of WT mice increased gastrocnemius muscle weight after DEX treatment. NDNF-KO mice showed increased expression of ubiquitin E3-ligases, including atrogin-1 and MuRF-1, in gastrocnemius muscle after DEX treatment, whereas Ad-NDNF reduced expression of atrogin-1 and MuRF-1 in gastrocnemius muscle of WT mice after DEX treatment. Pretreatment of cultured C2C12 myocytes with NDNF protein reversed reduced myotube diameter and increased expression of atrogin-1 and MuRF-1 after DEX stimulation. Treatment of C2C12 myocytes increased Akt phosphorylation. Pretreatment of C2C12 myotubes with the PI3-kinase/Akt inhibitor reversed NDNF-induced increase in myotube fiber diameter after DEX treatment. In conclusion, our findings indicated that NDNF prevents skeletal muscle atrophy in vivo and in vitro through reduction of ubiquitin E3-ligases expression, suggesting that NDNF could be a novel therapeutic target of muscle atrophy.

Distinct Expression of SLM2 Underlies Splicing-Dependent Trans-Synaptic Signaling of Neurexin Across GABAergic Neuron Subtypes.

The mammalian brain contains multiple types of neuronal cells with complex assemblies and distinct structural and functional properties encoded by divergent gene programs. There is increasing evidence that alternative splicing (AS) plays fundamental roles in transcriptomic diversity and specifying synaptic properties of each neuronal cell type. However, the mechanisms underlying AS regulation and whether it controls synapse formation across GABAergic interneurons have not been fully elucidated. Here we show the differential expression levels of Sam68-like molecule 2 (SLM2), a major splicing regulator of neurexin (NRX), in GABAergic neuronal subtypes and its contribution to GABAergic synapse specification. Cortical SLM2 is strongly expressed not only in excitatory neurons but also in a subpopulation of GABAergic interneurons, especially in VIP-positive neurons that are originated from late-born caudal ganglionic eminence (GE)- derived cells. Using artificial synapse formation assay, we found that GE containing cortices form a strong synapse with LRRTM2, a trans-synaptic receptor of the alternatively spliced segment 4 (AS4)(-) of NRX. SLM2 knock-down reduced the NRX AS4(-) isoform expression and hence weaken LRRTM2-induced synapse formation. The addition of NRX AS4(-) was sufficient to rescue the synaptic formation by LRRTM2 in SLM2 knock-down neurons. Thus, our findings suggest a novel function of SLM2 in modifying network formation of a specific population of GABAergic interneurons and contribute to a better understanding of the roles AS plays in regulating synapse specificity and neuronal molecular diversity.

Neuroligin-induced presynaptic differentiation through SLM2-mediated splicing modifications of neurexin in cerebellar cultures.

Neurexins (NRXs) and neuroligins (NLs) play important roles in synapse specification. The alternatively spliced segment 4 (AS4) of NRX genes (Nrxn) is a critical element in selective trans-synaptic interactions. However, the role of splicing of NRXs and NLs in synapse specification is not fully understood. To investigate the exact role of splice-dependent NRX-NL interaction in the specification of glutamatergic and gamma-aminobutyric acid (GABA)-ergic synapses in the cerebellum, we evaluated the synaptogenic receptor activity of NL1/2/3 isoforms in a neuron-fibroblast co-culture system, in which the Nrxn AS4 segments are manipulated using SLM2, a selective and dominant regulator of AS4 splicing. We show that ectopic SLM2 expression (SLM2 E/E) causes marked skipping of exon 20 of AS4 in cerebellar neuron culture. Whereas NLs can induce VAMP2+ presynaptic contacts from mainly glutamatergic neurons in both uninfected (control) and SLM2 E/E co-cultures, they induce VGAT+ GABAergic contacts in the control culture, but not properly in the SLM2 E/E culture. Furthermore, Nrxn3 is responsible for the NL-induced assembly of GABAergic synapses in co-culture. Importantly, lentivirus-based expression of Nrxn3 containing exon 20 restores the reduced NL-induced GABAergic contacts in the SLM2 E/E co-culture. Therefore, our findings may provide further insights into NRX-NL mediated synapse specification.

Activity-dependent repression of Cbln1 expression: mechanism for developmental and homeostatic regulation of synapses in the cerebellum.

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is released from cerebellar granule cells and plays a crucial role in forming and maintaining excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells not only during development but also in the adult cerebellum. Although neuronal activity is known to cause morphological changes at synapses, how Cbln1 signaling is affected by neuronal activity remains unclear. Here, we show that chronic stimulation of neuronal activity by elevating extracellular K(+) levels or by adding kainate decreased the expression of cbln1 mRNA within several hours in mature granule cells in a manner dependent on L-type voltage-dependent Ca(2+) channels and calcineurin. Chronic activity also reduced Cbln1 protein levels within a few days, during which time the number of excitatory synapses on Purkinje cell dendrites was reduced; this activity-induced reduction of synapses was prevented by the addition of exogenous Cbln1 to the culture medium. Therefore, the activity-dependent downregulation of cbln1 may serve as a new presynaptic mechanism by which PF-Purkinje cell synapses adapt to chronically elevated activity, thereby maintaining homeostasis. In addition, the expression of cbln1 mRNA was prevented when immature granule cells were maintained in high-K(+) medium. Since immature granule cells are chronically depolarized before migrating to the internal granule layer, this depolarization-dependent regulation of cbln1 mRNA expression may also serve as a developmental switch to facilitate PF synapse formation in mature granule cells in the internal granule layer.

Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins.

Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system.

Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells in vitro and in vivo.

Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

Cbln1 binds to specific postsynaptic sites at parallel fiber-Purkinje cell synapses in the cerebellum.

Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis.

A Perspective on the Role of Dynamic Alternative RNA Splicing in the Development, Specification, and Function of Axon Initial Segment.

Alternative splicing is a powerful mechanism for molecular and functional diversification. In neurons, alternative splicing extensively controls various developmental steps as well as the plasticity and remodeling of neuronal activity in the adult brain. The axon initial segment (AIS) is the specialized compartment of proximal axons that initiates action potential (AP). At the AIS, the ion channels and cell adhesion molecules (CAMs) required for AP initiation are densely clustered via the scaffolding and cytoskeletal proteins. Notably, recent studies have elucidated that multiple AIS proteins are controlled by extensive alternative splicing in developing and adult brains. Here, we argue the potential role of dynamic regulation of alternative splicing in the development, specification, and functions of the AIS. In particular, we propose the novel concept that alternative splicing potentially modulates the structural and functional plasticity at the AIS.

SAM68-Specific Splicing Is Required for Proper Selection of Alternative 3' UTR Isoforms in the Nervous System.

Neuronal alternative splicing is a core mechanism for functional diversification. We previously found that STAR family proteins (SAM68, SLM1, SLM2) regulate spatiotemporal alternative splicing in the nervous system. However, the whole aspect of alternative splicing programs by STARs remains unclear. Here, we performed a transcriptomic analysis using SAM68 knockout and SAM68/SLM1 double-knockout midbrains. We revealed different alternative splicing activity between SAM68 and SLM1; SAM68 preferentially targets alternative 3' UTR exons. SAM68 knockout causes a long-to-short isoform switch of a number of neuronal targets through the alteration in alternative last exon (ALE) selection or alternative polyadenylation. The altered ALE usage of a novel target, interleukin 1 receptor accessory protein (Il1rap), results in remarkable conversion from a membrane-bound type to a secreted type in Sam68KO brains. Proper ALE selection is necessary for IL1RAP neuronal function. Thus the SAM68-specific splicing program provides a mechanism for neuronal selection of alternative 3' UTR isoforms.

Impaired motor functions in mice lacking the RNA-binding protein Hzf.

Local protein synthesis in dendrites plays an important role in some aspects of neuronal development and synaptic plasticity. Neuronal RNA-binding proteins regulate the transport and/or translation of the localized mRNAs. Previously, we reported that hematopoietic zinc finger (Hzf) is one of the neuronal RNA-binding proteins that regulate these processes. The Hzf protein is highly expressed in neuronal cells including hippocampal pyramidal neurons and cerebellar Purkinje cells, and plays essential roles in the dendritic mRNA localization and translation. In the present study we demonstrated that mice lacking Hzf (Hzf(-/-) mice) exhibited severe impairments of motor coordination and cerebellum-dependent motor learning. These findings raise the possibility that the post-transcriptional regulation by Hzf may contribute to some aspects of synaptic plasticity and motor learning in the cerebellum.

Spatio-temporal and dynamic regulation of neurofascin alternative splicing in mouse cerebellar neurons.

Alternative splicing is crucial for molecular diversification, which greatly contributes to the complexity and specificity of neural functions in the central nervous system (CNS). Neurofascin (NF) is a polymorphic cell surface protein that has a number of splicing isoforms. As the alternative splicing of the neurofascin gene (Nfasc) is developmentally regulated, NF isoforms have distinct functions in immature and mature brains. However, the molecular mechanisms underlying the alternative splicing of Nfasc in neurons are not yet understood. Here, we demonstrate that, alongside developmental regulation, Nfasc alternative splicing is spatially controlled in the mouse brain. We then identified distinct Nfasc splicing patterns at the cell-type level in the cerebellum, with Nfasc186 being expressed in Purkinje cells and absent from granule cells (GCs). Furthermore, we show that high K+-induced depolarization triggers a shift in splicing from Nfasc140 to Nfasc186 in cerebellar GCs. Finally, we identified a neural RNA-binding protein, Rbfox, as a key player in neural NF isoform selection, specifically controlling splicing at exons 26-29. Together, our results show that Nfasc alternative splicing is spatio-temporally and dynamically regulated in cerebellar neurons. Our findings provide profound insight into the mechanisms underlying the functional diversity of neuronal cell-adhesive proteins in the mammalian CNS.

Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases.

Distinct Defects in Synaptic Differentiation of Neocortical Neurons in Response to Prenatal Valproate Exposure.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders characterized by impairments in social interactions and stereotyped behaviors. Valproic acid (VPA) is frequently used to treat epilepsy and bipolar disorders. When taken during pregnancy, VPA increases the risk of the unborn child to develop an ASD. In rodents, in utero VPA exposure can precipitate behavioral phenotypes related to ASD in the offspring. Therefore, such rodent models may allow for identification of synaptic pathophysiology underlying ASD risk. Here, we systematically probed alterations in synaptic proteins that might contribute to autism-related behavior in the offspring of in utero VPA-exposed mice. Moreover, we tested whether direct VPA exposure of cultured neocortical neurons may recapitulate the molecular alterations seen in vivo. VPA-exposed neurons in culture exhibit a significant increase in the number of glutamatergic synapses accompanied by a significant decrease in the number of GABAergic synapses. This shift in excitatory/inhibitory balance results in substantially increased spontaneous activity in neuronal networks arising from VPA-exposed neurons. Pharmacological experiments demonstrate that the alterations in GABAergic and glutamatergic synaptic proteins and structures are largely caused by inhibition of histone deacetylases. Therefore, our study highlights an epigenetic mechanism underlying the synaptic pathophysiology in this ASD model.

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways.

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.

Neuronal cell type-specific alternative splicing is regulated by the KH domain protein SLM1.

The unique functional properties and molecular identity of neuronal cell populations rely on cell type-specific gene expression programs. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Neuronal cells exhibit particularly extensive alternative splicing regulation. We report a highly selective expression of the KH domain-containing splicing regulators SLM1 and SLM2 in the mouse brain. Conditional ablation of SLM1 resulted in a severe defect in the neuronal isoform content of the polymorphic synaptic receptors neurexin-1, -2, and -3. Thus, cell type-specific expression of SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo.