A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons

Neurotropic viruses that conditionally infect or replicate in molecularly defined neuronal subpopulations, and then spread transsynaptically, are powerful tools for mapping neural pathways. Genetically targetable retrograde transsynaptic tracer viruses are available to map the inputs to specific neuronal subpopulations, but an analogous tool for mapping synaptic outputs is not yet available. Here we describe a Cre recombinase-dependent, anterograde transneuronal tracer, based on the H129 strain of herpes simplex virus (HSV). Application of this virus to transgenic or knockin mice expressing Cre in peripheral neurons of the olfactory epithelium or the retina reveals widespread, polysynaptic labeling of higher-order neurons in the olfactory and visual systems, respectively. Polysynaptic pathways were also labeled from cerebellar Purkinje cells. In each system, the pattern of labeling was consistent with classical circuit-tracing studies, restricted to neurons, and anterograde specific. These data provide proof-of-principle for a conditional, nondiluting anterograde transsynaptic tracer for mapping synaptic outputs from genetically marked neuronal subpopulations.     

Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.     

Similarity of visual selectivity among clonally related neurons in visual cortex

Neurons in rodent visual cortex are organized in a salt-and-pepper fashion for orientation selectivity, but it is still unknown how this functional architecture develops. A recent study reported that the progeny of single cortical progenitor cells are preferentially connected in the postnatal cortex. If these neurons acquire similar selectivity through their connections, a salt-and-pepper organization may be generated, because neurons derived from different progenitors are intermingled in rodents. Here we investigated whether clonally related cells have similar preferred orientation by using a transgenic mouse, which labels all the progeny of single cortical progenitor cells. We found that preferred orientations of clonally related cells are similar to each other, suggesting that cell lineage is involved in the development of response selectivity of neurons in the cortex. However, not all clonally related cells share response selectivity, suggesting that cell lineage is not the only determinant of response selectivity.     

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.     

The structure of the nervous system of the nematode Caenorhabditis elegans

The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.     

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)¹. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site^2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)^4,5 . This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3''-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites^6-11.     

A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.     

Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer

This report describes a method of marking nerve cells which is approximately 100 times more sensitive than those previously available. The method depends upon intracellular injection of a new, highly fluorescent dye, Lucifer Yellow CH, which can be viewed both in living tissue and after fixation and embedding. The intense fluorescence of the dye makes injected neurons visible in cleared wholemounts, where the complex three-dimensional structure of neurons is readily apparent.Three new observations have been made with Lucifer Yellow. First, many of the invertebrate neurons studied possess an extensive and complex array of fine processes not visible with other techniques. Second, dye spreads rapidly within an injected cell. Third, dye frequently spreads from the injected cell directly to certain other cells. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily, but not exclusively, between cells known to be electrically coupled.Dye-coupling in the turtle retina revealed striking and distinctive patterns of connections. Type I horizontal cells appear to be multiply connected to each other in an extensive net. Type II horizontal cells are often connected to each other in a hexagonal array. Individual type I and type II cells, widely separated, are frequently dye-coupled; in one case, they were connected by a dyefilled axon.Dye-coupling, readily observed because of the low molecular weight and the intense fluorescence of the new dye, may serve as a general method of tracing certain functional connections by morphological means, and of studying the transfer of small molecules between cells. Preliminary results suggest that systems of dye-coupled cells are substantially more common than was previously believed.     

C-terminal fragment of tetanus toxin: its use in neuronal network analysis and its potential as non-viral vector

The atoxic C-terminal fragment of tetanus neurotoxin or TTC fragment presents similar retrograde and transsynaptic properties to that of holotoxin. Detection of this fragment is easier when it is associated with a fluorescent marker or with beta-galactosidase activity by genetic fusion or chemical conjugation. Thus, these tracers have been used to study and analyse the synaptic connections of a neural network. In this article, we shortly review the various methods used with this aim including: injection of the fusion protein, adenovirus in vivo expression and transgenesis. Since neural activity is essential for neuronal TTC binding and internalization, the functionality of connections can be also evaluated. Moreover, modifications of the retrograde transport can be detected by using this fragment. Thus, TTC fragment is an excellent tracer to analyse the connectivity and functionality of a neural network. The TTC fragment was also soon proposed as potential therapeutic vector to transport and to deliver a biological activity or gene in a neural network. With this aim, the efficiency of a translocation domain to induce the cytosolic release of the associated activity has been evaluated. The use of the TTC fragment to target specifically a neurotrophic factor to neurons and thus avoid secondary effects has been tested with interesting results.     

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G¹. This chimeric G specifically infects cells expressing the TVA receptor¹. The gene encoding TVA can been delivered by various methods^2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)² onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.We recently modified this technique to use VSV as a transsynaptic tracer⁹. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.⁴ and Beier et al.⁹ permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).     

Cell type specificity of local cortical connections

The function of the cerebral cortex is dependent on the precise organization of the circuits formed by its component neurons. The connections between neurons are not random, but are specific at multiple levels of organization. For example, each cortical area connects to only a selected subset of other areas and within any given area the axonal and dendritic arbors of individual neurons arborize in precise, layer-specific patterns (for review see Felleman & Van Essen, 1991; Callaway, 1998) . In each layer there are dendrites from multiple cell types including cells with somata both within and outside that layer. Anatomical studies have shown that axons arborizing in a particular cortical layer can connect selectively onto dendrites of some cell types in the layer, while avoiding the dendrites of other cell types (e.g. Freund & Gulyas, 1991; Hornung & Celio, 1992; Staiger et al., 1996). These cell type specific connections are, however, difficult to elucidate with anatomical methods, so the frequency of such specificity has remained elusive. Recent experimental methods combining intracellular recording of single neurons with focal neuronal stimulation by uncaging glutamate with light (“photostimulation”) have made the analysis of cell type specific cortical connections more tractable. These studies show that cell type specificity of connections is prevalent in cortex. Here I review photostimulation-based studies investigating the laminar sources of cortical input to distinct cell types in the visual and somatosensory cortices of rats and the primary visual cortex of monkeys.     

Parallel motor pathways from thoracic interneurons of the ventral giant interneuron system of the cockroach, Periplaneta americana

The data described here complete the principal components of the cockroach wind-mediated escape circuit from cercal afferents to leg motor neurons. It was previously known that the cercal afferents excite ventral giant interneurons which then conduct information on wind stimuli to thoracic ganglia. The ventral giant interneurons connect to a large population of interneurons in the thoracic ganglia which, in turn, are capable of exciting motor neurons that control leg movements. Thoracic interneurons that receive constant short latency inputs from ventral giant interneurons have been referred to as type A thoracic interneurons (TIAs). In this paper, we demonstrate that the motor response of TIAs occurs in adjacent ganglia as well as in the ganglion of origin for the TIA. We then describe the pathway from TIAs to motor neurons in both ganglia. Our observations reveal complex interactions between thoracic interneurons and leg motor neurons. Two parallel pathways exist. TIAs excite leg motor neurons directly and via local interneurons. Latency and amplitude of post-synaptic potentials (PSPs) in motor neurons and local interneurons either in the ganglion of origin or in adjacent ganglia are all similar. However, the sign of the responses recorded in local interneurons (LI) and motor neurons varies according to the TIA subpopulation based on the location of their cell bodies. One group, the dorsal posterior group, (DPGs) has dorsal cell bodies, whereas the other group, the ventral median cells, (VMC) has ventral cell bodies. All DPG interneurons either excited postsynaptic cells or failed to show any connection at all. In contrast, all VMC interneurons either inhibited postsynaptic cells or failed to show any connection. It appears that the TIAs utilize directional wind information from the ventral giant interneurons to make a decision on the optimal direction of escape. The output connections, which project not only to cells within the ganglion of origin but also to adjacent ganglia and perhaps beyond, could allow this decision to be made throughout the thoracic ganglia as a single unit. However, nothing in these connections indicates a mechanism for making appropriate coordinated leg movements. Because each pair of legs plays a unique role in the turn, this coordination should be controlled by circuits dedicated to each leg. We suggest that this is accomplished by local interneurons between TIAs and leg motor neurons.     

The vertebrate retina: a model for neuronal polarization in vivo

The vertebrate retina develops rapidly from a proliferative neuroepithelium into a highly ordered laminated structure, with five distinct neuronal cell types. Like all neurons, these cells need to polarize in appropriate orientations order integrate their neuritic connections efficiently into functional networks. Its relative simplicity, amenability to in vivo imaging and experimental manipulation, as well as the opportunity to study varied cell types within a single tissue, make the retina a powerful model to uncover how neurons polarize in vivo. Here we review the progress that has been made thus far in understanding how the different retinal neurons transform from neuroepithelial cells into mature neurons, and how the orientation of polarization may be specified by a combination of pre-established intrinsic cellular polarity set up within neuroepithelial cells, and extrinsic cues acting upon these differentiating neurons.     

Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins

Background

The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections.

Results

We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity.

Conclusions

Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.

     

Dye-coupling visualizes networks of large-field motion-sensitive neurons in the fly

In the fly, visually guided course control is accomplished by a set of 60 large-field motion-sensitive neurons in each brain hemisphere. These neurons have been shown to receive retinotopic motion information from local motion detectors on their dendrites. In addition, recent experiments revealed extensive coupling between the large-field neurons through electrical synapses. These two processes together give rise to their broad and elaborate receptive fields significantly surpassing the extent of their dendritic fields. Here, we demonstrate that the electrical connections between different large-field neurons can be visualized using Neurobiotin dye injection into a single one of them. When combined with a fluorescent dye which does not cross electrical synapses, the injected cell can be identified unambiguously. The Neurobiotin staining corroborates the electrical coupling postulated amongst the cells of the vertical system (VS-cells) and between cells of the horizontal system (HS-cells and CH-cells). In addition, connections between some cells are revealed that have so far not been considered as electrically coupled.     

Neural development features: spatio-temporal development of the Caenorhabditis elegans neuronal network

The nematode Caenorhabditis elegans, with information on neural connectivity, three-dimensional position and cell linage, provides a unique system for understanding the development of neural networks. Although C. elegans has been widely studied in the past, we present the first statistical study from a developmental perspective, with findings that raise interesting suggestions on the establishment of long-distance connections and network hubs. Here, we analyze the neuro-development for temporal and spatial features, using birth times of neurons and their three-dimensional positions. Comparisons of growth in C. elegans with random spatial network growth highlight two findings relevant to neural network development. First, most neurons which are linked by long-distance connections are born around the same time and early on, suggesting the possibility of early contact or interaction between connected neurons during development. Second, early-born neurons are more highly connected (tendency to form hubs) than later-born neurons. This indicates that the longer time frame available to them might underlie high connectivity. Both outcomes are not observed for random connection formation. The study finds that around one-third of electrically coupled long-range connections are late forming, raising the question of what mechanisms are involved in ensuring their accuracy, particularly in light of the extremely invariant connectivity observed in C. elegans. In conclusion, the sequence of neural network development highlights the possibility of early contact or interaction in securing long-distance and high-degree connectivity.     

Whole-brain patterns of the presynaptic inputs and axonal projections of BDNF neurons in the paraventricular nucleus

Brain-derived neurotrophic factor (BDNF) plays a crucial role in human obesity. Yet, the neural circuitry supporting the BDNF-mediated control of energy homeostasis remains largely undefined. To map key regions that might provide inputs to or receive inputs from the paraventricular nucleus (PVN) BDNF neurons, a key type of cells in regulating feeding and thermogenesis, we used rabies virus-based transsynaptic labeling and adeno-associated virus based anterograde tracing techniques to reveal their whole-brain distributions. We found that dozens of brain regions provide dense inputs to or receive dense inputs from PVN BDNF neurons, including several known weight control regions and several novel regions that might be functionally important for the BDNF-mediated regulation of energy homeostasis.Interestingly, several regions show very dense reciprocal connections with PVN BDNF neurons, including the lateral septum, the preoptic area, the ventromedial hypothalamic nucleus, the paraventricular thalamic nucleus, the zona incerta, the lateral parabrachial nucleus, the subiculum, the raphe magnus nucleus, and the raphe pallidus nucleus. These strong anatomical connections might be indicative of important functional connections. Therefore, we provide an outline of potential neural circuitry mediated by PVN BDNF neurons, which might be helpful to resolve the complex obesity network.     

Feedback loops link odor and pheromone signaling with reproduction

Pheromones can have profound effects on reproductive physiology and behavior in mammals. To investigate the neural circuits underlying these effects, we used a genetic transneuronal tracer to identify neurons that synapse with GnRH (LHRH) neurons, the key regulators of reproduction. We then asked whether the connected neurons are presynaptic or postsynaptic to GnRH neurons and analyzed their responses to chemosensory cues. Surprisingly, these experiments indicate that GnRH neurons receive pheromone signals from both odor and pheromone relays in the brain and may also receive common odor signals. Moreover, feedback loops are evident whereby GnRH neurons could influence both odor and pheromone processing. Remarkably, approximately 800 GnRH neurons communicate with approximately 50,000 neurons in 53 functionally diverse brain areas, with some connections exhibiting sexual dimorphism. These studies reveal a complex interplay between reproduction and other functions in which GnRH neurons appear to integrate information from multiple sources and modulate a variety of brain functions.     

Cellular mechanisms governing synapse formation: lessons from identified neurons in culture

The accessibility of embryonic and adult neurons within invertebrate nervous systems has made them excellent subjects for neurobiological study. The ability to readily identify individual neurons, together with their great capacity for regeneration, has been especially beneficial to investigations of synapse formation and the specificity of neuronal connectivity. Many invertebrate neurons survive for long periods following isolation into primary cell culture. In addition, they readily extend new neuritic arbors and form electrical and chemical connections at sites of contact. Thus, cell culture approaches have allowed neuroscientists greater access to, and resolution of, events underlying neurite outgrowth and synaptogenesis. Studies of identified neuromuscular synapses ofHelisoma have determined a number of signaling mechanisms involved in transsynaptic communication at sites of neuron-target contact. At these sites, both anterograde and retrograde signals regulate the transformation of growth cones into functional presynaptic terminals. We have found that specific muscle targets induce both global and local changes in neurotransmitter secretion and intracellular calcium handling. Here we review recent studies of culturedHelisoma synapses and discuss the mechanisms thought to govern chemical synapse formation in these identified neurons and those of other invertebrate species.