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.     

Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.     

Remote control of behavior through genetically targeted photostimulation of neurons

Optically gated ion channels were expressed in circumscribed groups of neurons in the Drosophila CNS so that broad illumination of flies evoked action potentials only in genetically designated target cells. Flies harboring the "phototriggers" in different sets of neurons responded to laser light with behaviors specific to the sites of phototrigger expression. Photostimulation of neurons in the giant fiber system elicited the characteristic escape behaviors of jumping, wing beating, and flight; photostimulation of dopaminergic neurons caused changes in locomotor activity and locomotor patterns. These responses reflected the direct optical activation of central neuronal targets rather than confounding visual input, as they persisted unabated in carriers of a mutation that eliminates phototransduction. Encodable phototriggers provide noninvasive control interfaces for studying the connectivity and dynamics of neural circuits, for assigning behavioral content to neurons and their activity patterns, and, potentially, for restoring information corrupted by injury or disease.     

Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons

Inducible and reversible silencing of selected neurons in vivo is critical to understanding the structure and dynamics of brain circuits. We have developed Molecules for Inactivation of Synaptic Transmission (MISTs) that can be genetically targeted to allow the reversible inactivation of neurotransmitter release. MISTs consist of modified presynaptic proteins that interfere with the synaptic vesicle cycle when crosslinked by small molecule "dimerizers." MISTs based on the vesicle proteins VAMP2/Synaptobrevin and Synaptophysin induced rapid ( approximately 10 min) and reversible block of synaptic transmission in cultured neurons and brain slices. In transgenic mice expressing MISTs selectively in Purkinje neurons, administration of dimerizer reduced learning and performance of the rotarod behavior. MISTs allow for specific, inducible, and reversible lesions in neuronal circuits and may provide treatment of disorders associated with neuronal hyperactivity.     

The transsynaptic regulation of the septal-hippocampal cholinergic neurons

There is not yet a complete understanding of the functional interactions among various septal nuclei which regulate hippocampal function. Nevertheless, much has been learned histologically and biochemically about the major connections of the distinct areas of the septal complex and the chemical character of some of these pathways. The cholinergic septal-hippocampal pathway serves as a well defined link between these two important structures of the limbic system. Acetylcholine turnover rates in the hippocampus have been shown to increase or decrease proportionally to the activity of the cholinergic neurons originating in the septum. Moreover, these turnover rates have been shown to be modulated by intraseptal injections of agonists or antagonists of various neurotransmitters or neuromodulators which are stored in various cell groups located in the septum. By coupling this biochemical approach with techniques to study the receptor organization, greater detail concerning the transmitter and cotransmitter interactions among the various neuromodulators can be obtained.     

Monosynaptic inhibitory postsynaptic potentials of cortical neurons

Monopolar intracortical stimulation of the auditory cortex was carried out in cats immobilized with D-tubocurarine. A macroelectrode (tip diameter 100 µ) or a microelectrode (tip diameter 10–15 µ) was used for stimulation. In both cases, besides excitatory responses, primary IPSPs with latent periods of 0.4–1.2 and 1.4–6.0 msec were recorded in cortical neurons close to the point of stimulation. The first group of IPSPs are considered to be generated in response to direct stimulation of bodies or axons of inhibitory cortical neurons, i.e., monosynaptically. The amplitude of these IPSPs varied in different neurons from 3 to 15 mV, and their duration from 4 to 150 msec. Additional later inhibitory responses were superposed on many of them. Of the IPSPs generated in auditory cortical neurons in response to stimulation of geniculocortical fibers 1.5% had a latency of 0.8–1.3 msec. They also are assumed to be monosynaptic. It is concluded that the duration of synaptic delay of IPSPs in cortical neurons and spinal motoneurons is the same, namely 0.3–0.4 msec. Axons of auditory cortical inhibitory neurons may be 1.5 mm long. The velocity of impulse conduction along these axons is 1.6–2.8 m/sec. The genesis of some special features of IPSPs of cortical neurons is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 7, No. 5, pp. 458–467, September–October, 1975.     

Local retinal circuits of melanopsin-containing ganglion cells identified by transsynaptic viral tracing

Intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) control important physiological processes, including the circadian rhythm, the pupillary reflex, and the suppression of locomotor behavior (reviewed in [1]). ipRGCs are also activated by classical photoreceptors, the rods and cones, through local retinal circuits [2, 3]. ipRGCs can be transsynaptically labeled through the pupillary-reflex circuit with the derivatives of the Bartha strain of the alphaherpesvirus pseudorabies virus(PRV) [4, 5] that express GFP [6-12]. Bartha-strain derivatives spread only in the retrograde direction [13]. There is evidence that infected cells function normally for a while during GFP expression [7]. Here we combine transsynaptic PRV labeling, two-photon laser microscopy, and electrophysiological techniques to trace the local circuit of different ipRGC subtypes in the mouse retina and record light-evoked activity from the transsynaptically labeled ganglion cells. First, we show that ipRGCs are connected by monostratified amacrine cells that provide strong inhibition from classical-photoreceptor-driven circuits. Second, we show evidence that dopaminergic interplexiform cells are synaptically connected to ipRGCs. The latter finding provides a circuitry link between light-dark adaptation and ipRGC function.     

Rapidly inducible, genetically targeted inactivation of neural and synaptic activity in vivo

Inducible and reversible perturbation of the activity of selected neurons in vivo is critical to understanding the dynamics of brain circuits. Several genetically encoded systems for rapid inducible neuronal silencing have been developed in the past few years offering an arsenal of tools for in vivo experiments. Some systems are based on ion-channels or pumps, others on G protein coupled receptors, and yet others on modified presynaptic proteins. Inducers range from light to small molecules to peptides. This diversity results in differences in the various parameters that may determine the applicability of each tool to a particular biological question. Although further development would be beneficial, the current silencing tool kit already provides the ability to make specific perturbations of circuit function in behaving animals.     

Selective photostimulation of genetically chARGed neurons.

To permit direct functional analyses of neural circuits, we have developed a method for stimulating groups of genetically designated neurons optically. Coexpression of the Drosophila photoreceptor genes encoding arrestin-2, rhodopsin (formed by liganding opsin with retinal), and the alpha subunit of the cognate heterotrimeric G protein--an explosive combination we term "chARGe"--sensitizes generalist vertebrate neurons to light. Illumination of a mixed population of neurons elicits action potentials selectively and cell-autonomously in its genetically chARGed members. In contrast to bath-applied photostimulants or caged neurotransmitters, which act indiscriminately throughout the illuminated volume, chARGe localizes the responsiveness to light. Distributed activity may thus be fed directly into a circumscribed population of neurons in intact tissue, irrespective of the spatial arrangement of its elements.     

Growth defects in the dorsal pallium after genetically targeted ablation of principal preplate neurons and neuroblasts: a morphometric analysis

The present study delineates the large-scale, organic responses of growth in the dorsal pallium to targeted genetic ablations of the principal PP (preplate) neurons of the neocortex. Ganciclovir treatment during prenatal development [from E11 (embryonic age 11) to E13] of mice selectively killed cells with shared S-phase vulnerability and targeted expression of a GPT [golli promoter transgene; GPT linked to HSV-TK (herpes simplex virus-thymidine kinase), τ-eGFP and lacZ reporters] localized in PP neurons and their intermediate progenitor neuroblasts. The volume, area and thickness of the pallium were measured in an E12–P4 (postnatal age 4) longitudinal study with comparisons between ablated (HSV-TK^+/0) and control (HSV-TK^0/0) littermates. The extent of ablations was also systematically varied, and the effect on physical growth was assessed in an E18 cross-sectional study. The morphological evidence obtained in the present study supports the conclusion that genetically targeted ablations delay the settlement of the principal PP neurons of the dorsal pallium. This leads to progressive and substantial reductions of growth, despite compensatory responses that rapidly replace the ablated cells. These growth defects originate from inductive cellular interactions in the proliferative matrix of the ventricular zone of the pallium, but are amplified by subsequent morphogenic and trophic cellular interactions. The defects persist during the course of prenatal and postnatal development to demonstrate a constrained dose–response relationship with the extent of specific killing of GPT neurons. The defects propagate simultaneously in both the horizontal and vertical cytoarchitectural dimensions of the developing pallium, an outcome that produces a localized shortfall of volume in the telencephalic vesicles.     

Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins

Optical imaging of electrical activity using voltage-sensitive dyes has been envisaged for many years as a powerful method to investigate multineuronal representation of information processing in brain tissue. This article describes the advent of novel genetically targeted voltage-sensitive fluorescent proteins. This new class of membrane voltage sensors overcomes previous limitations related to the nonselective staining of membranes associated with conventional voltage-sensitive dyes. Here, we discuss the methodology, applications, and potential advantages of this novel technique.     

Dipole characterization of single neurons from their extracellular action potentials

The spatial variation of the extracellular action potentials (EAP) of a single neuron contains information about the size and location of the dominant current source of its action potential generator, which is typically in the vicinity of the soma. Using this dependence in reverse in a three-component realistic probe + brain + source model, we solved the inverse problem of characterizing the equivalent current source of an isolated neuron from the EAP data sampled by an extracellular probe at multiple independent recording locations. We used a dipole for the model source because there is extensive evidence it accurately captures the spatial roll-off of the EAP amplitude, and because, as we show, dipole localization, beyond a minimum cell-probe distance, is a more accurate alternative to approaches based on monopole source models. Dipole characterization is separable into a linear dipole moment optimization where the dipole location is fixed, and a second, nonlinear, global optimization of the source location. We solved the linear optimization on a discrete grid via the lead fields of the probe, which can be calculated for any realistic probe + brain model by the finite element method. The global source location was optimized by means of Tikhonov regularization that jointly minimizes model error and dipole size. The particular strategy chosen reflects the fact that the dipole model is used in the near field, in contrast to the typical prior applications of dipole models to EKG and EEG source analysis. We applied dipole localization to data collected with stepped tetrodes whose detailed geometry was measured via scanning electron microscopy. The optimal dipole could account for 96% of the power in the spatial variation of the EAP amplitude. Among various model error contributions to the residual, we address especially the error in probe geometry, and the extent to which it biases estimates of dipole parameters. This dipole characterization method can be applied to any recording technique that has the capabilities of taking multiple independent measurements of the same single units.     

Reversible silencing of neuronal excitability in behaving mice by a genetically targeted, ivermectin-gated Cl- channel

Several genetic strategies for inhibiting neuronal function in mice have been described, but no system that directly suppresses membrane excitability and is triggered by a systemically administered drug, has been validated in awake behaving animals. We expressed unilaterally in mouse striatum a modified heteromeric ivermectin (IVM)-gated chloride channel from C. elegans (GluClalphabeta), systemically administered IVM, and then assessed amphetamine-induced rotational behavior. Rotation was observed as early as 4 hr after a single intraperitoneal IVM injection (10 mg/kg), reached maximal levels by 12 hr, and was almost fully reversed by 4 days. Multiple cycles of silencing and recovery could be performed in a single animal. In striatal slice preparations from GluClalphabeta-expressing animals, IVM rapidly suppressed spiking. The two-subunit GluCl/IVM system permits "intersectional" strategies designed to increase the cellular specificity of silencing in transgenic animals.     

Monosynaptic control of spinal motoneurons from different levels of the brain

In experiments on cats and monkeys it is established that reticulo-, rubro-, and corticomotoneuronal influences are characterized by a number of common features: 1) they are produced by fast conducting fibers of the descending tracts; 2) they do not attain the critical level needed for AP generation; and 3) they are caused by implication of synapses that are predominantly located on dendrites of the motoneurons. Results of experiments carried out on lampreys and rats indicate that reticulo-motoneuronal monosynaptic projections emerge already at the earliest stages of vertebrate evolution and retain their significance in mammals. The data of research on supraspinal influences during ontogenesis indicate early development of descending stem projections. This enables us to regard cerebro-motoneuronal monosynaptic connections as an important component of supraspinal control of motoneurons, a component whose functional role is in large measure determined by interaction with other synaptic inputs of the motoneuron.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 2, pp. 203–215, March–April, 1970.     

Electrophysiological and pharmacological properties of single spinal neurons isolated from adult bullfrogs

1. We developed an isolated spinal cell preparation from adult bullfrogs. 2. The average resting membrane potential was -60 mV, and an action potential was activated by positive current injection. 3. The cells retained their tetrodotoxin-sensitive Na+ channels and at least two kinetically different types of K+ channel. 4. Under K(+)-free conditions, responses to GABA were blocked by bicuculline while responses to glycine, taurine or beta-alanine were blocked by strychnine. 5. The potency of excitatory amino acids decreased in the order: kainic acid greater than glutamate greater than NMDA. 6. These studies demonstrated that the isolated cells are applicable for electrophysiological and pharmacological investigations.     

Untypical connectivity from olfactory sensory neurons expressing OR37 into higher brain centers visualized by genetic tracing

The OR37 subfamily of odorant receptors (ORs) exists exclusively in mammals. In contrast to ORs in general, they are highly conserved within and across species. These unique features raise the question, whether olfactory information gathered by the OR37 sensory cells is processed in specially designated brain areas. To elucidate the wiring of projection neurons from OR37 glomeruli into higher brain areas, tracing experiments were performed. The application of DiI onto the ventral area of the olfactory bulb, which harbors the OR37 glomeruli, led to the labeling of fibers not only in the typical olfactory cortical regions, but also in the medial amygdala and the hypothalamus. To visualize the projections from a defined OR37 glomerulus more precisely, transgenic mice were studied in which olfactory sensory neurons co-express the receptor subtype OR37C and the transsynaptic tracer wheat germ agglutinin (WGA). WGA became visible not only in the OR37C sensory neurons and the corresponding OR37C glomerulus, but also in cell somata located in the mitral/tufted cell layer adjacent to the OR37C glomerulus, indicating a transfer of WGA onto projection neurons. In the brain, WGA immunoreactivity was not detectable in typical olfactory cortical areas, but instead in distinct areas of the medial amygdala. Detailed mapping revealed that the WGA immunoreactivity was restricted to the posterior-dorsal subnucleus of the medial amygdala. In addition, WGA immunoreactivity was visible in some well-circumscribed areas of the hypothalamus. These results are indicative for a unique connectivity from OR37C sensory cells into higher brain centers.     

Interplay between hevin,SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

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