Gabriella Ugolini

Spinal and brain circuits to motoneurons of the bulbospongiosus muscle: retrograde transneuronal tracing with rabies virus.

Retrograde transneuronal tracing with rabies virus from the left bulbospongiosus muscle (BS) was used to identify the neural circuits underlying its peripheral and central activation. Rats were killed at 2, 3, 4, and 5 days post‐inoculation (p.i.). Rabies immunolabelling was combined with immunohistochemical detection of choline acetyltransferase and oxytocin. Virus uptake was restricted to ipsilateral BS motoneurons (2 days p.i.). The onset of transfer (3 days p.i.) visualized interneurons in the dorsal grey commissure (DGC), intermediate zone, and sacral parasympathetic nucleus (SPN), mainly in DGC at L5–S1, and revealed synaptic connections between BS and external urethral sphincter motoneurons. At 4 and 5 days p.i., higher‐order interneurons were labelled in other spinal areas and segments. Supraspinal labelling initially involved only Barringtons nucleus, nucleus reticularis magnocellularis, and paragigantocellularis lateralis (4 days p.i.). Later, labelling extended to other populations traditionally associated with control of sexual activity and micturition (periaqueductal grey, paraventricular nucleus, medial preoptic area, prefrontal cortex), but also indicated the intervention of somatic descending motor pathways (vestibulospinal and reticulospinal neurons, “hindlimb” regions of sensorimotor cortex and red nucleus) and cerebellar nuclei in multisynaptic innervation of the labelled motoneurons. Dual color immunofluorescence disclosed multisynaptic links between these motoneurons and thoracolumbar medial sympathetic (choline acetyltransferase‐immunoreactive) neurons. In contrast, preganglionic neurons in SPN and most oxytocinergic neurons in paraventricular hypothalamic nucleus remained unlabelled, suggesting that parasympathetic and somatic outflow to pelvic organs are probably controlled by separate interneuronal populations and that oxytocinergic spinal projections are more likely to influence sacral autonomic rather than somatic outflow. J. Comp. Neurol. 414:167–192, 1999.

Advances in viral transneuronal tracing.

Powerful transneuronal tracing technologies exploit the ability of some neurotropic viruses to travel across neuronal pathways and to function as self-amplifying markers. Two main classes of viral transneuronal tracers are available, derived from alpha-herpesviruses (Herpes Simplex virus type 1, Pseudorabies) and rabies virus. Depending on the virus type and strain, there are major differences with regard to host range, peripheral uptake, replication mechanisms, transport direction and specificity. While alpha-herpesviruses are the tracers of choice for studying autonomic innervation, rabies virus is the ideal tool for studying motor innervation, since its peripheral uptake occurs exclusively at motor endplates. Rabies virus is the only viral tracer that is entirely specific, as it moves exclusively across chemical synapses by strictly unidirectional (retrograde) transneuronal transfer without altering neuronal metabolism, allowing for the stepwise, time-dependent, identification of neuronal networks across an unlimited number of synapses. This review will highlight and contrast the different properties of these viral tracers, and summarize the methodological issues that are critical for the appropriate execution and interpretation of transneuronal tracing studies. Combinations of viral tracing with other methodologies will be evaluated. Emerging technologies, based on genetically modified herpes and rabies tracers, will be also discussed and put in perspective.

Horizontal eye movement networks in primates as revealed by retrograde transneuronal transfer of rabies virus: Differences in monosynaptic input to “slow” and “fast” abducens motoneurons

The sources of monosynaptic input to “fast” and “slow” abducens motoneurons (MNs) were revealed in primates by retrograde transneuronal tracing with rabies virus after injection either into the distal or central portions of the lateral rectus (LR) muscle, containing, respectively, “en grappe” endplates innervating slow muscle fibers or “en plaque” motor endplates innervating fast fibers. Rabies uptake involved exclusively motor endplates within the injected portion of the muscle. At 2.5 days after injections, remarkable differences of innervation of slow and fast MNs were demonstrated. Premotor connectivity of slow MNs, revealed here for the first time, involves mainly the supraoculomotor area, central mesencephalic reticular formation, and portions of medial vestibular and prepositus hypoglossi nuclei carrying eye position and smooth pursuit signals. Results suggest that slow MNs are involved exclusively in slow eye movements (vergence and possibly smooth pursuit), muscle length stabilization and gaze holding (fixation), and rule out their participation in fast eye movements (saccades, vestibulo‐ocular reflex). By contrast, all known monosynaptic pathways to LR MNs innervate fast MNs, showing their participation in the entire horizontal eye movements repertoire. Hitherto unknown monosynaptic connections were also revealed, such as those derived from the central mesencephalic reticular formation and vertical eye movements pathways (Y group, interstitial nucleus of Cajal, rostral interstitial nucleus of the medial longitudinal fasciculus). The different connectivity of fast and slow MNs parallel differences in properties of muscle fibers that they innervate, suggesting that muscle fibers properties, rather than being self‐determined, are the result of differences of their premotor innervation. J. Comp. Neurol. 498:762–785, 2006.

Modern Concepts of Brainstem Anatomy

Abstract: The extraocular muscles, unlike the skeletal muscles, contain non‐twitch muscle fibers. Recent experiments have located the non‐twitch motoneurons. They lie around the periphery of the oculomotor, trochlear and abducens nuclei, separate from the more usual twitch motoneurons that cluster within the boundaries of the classical motor nuclei. The premotor inputs to non‐twitch neurons were traced by the injection of rabies virus into the distal tip of the lateral rectus muscle. Retrogradely labeled cells were found in areas associated with the neural integrator, vergence and smooth pursuit premotor areas, but not the saccadic premotor burst neurons or the direct vestibulo‐ocular pathways. The rabies tracing emphasizes for the first time that the central mesencephalic reticular formation (cMRF) and the supraoculomotor area exert direct premotor control over the non‐twitch motoneurons. Because the two sets of motoneurons do not receive the same afferents, they must have different functions; these are not yet clarified. These results are not compatible with the concept of a single final common pathway from motoneurons to eye muscles.

Mapping the oculomotor system: the power of transneuronal labelling with rabies virus.

Neuronal networks underlying and related to horizontal eye movements were visualized by retrograde transneuronal tracing with rabies virus from the left medial rectus muscle in guinea pigs. Time‐sequenced labelling revealed distinct circuitries involved in particular oculomotor functions, i.e. vestibulo‐ocular reflex and saccade generation (brainstem circuitry), adaptive plasticity (cerebellar modules) and possibly motivation and navigation (limbic, hippocampal and cortical structures). Our results provide a first comprehensive road map of the oculomotor system that is unsurpassed by any previous tracing study. We report a number of unexpected findings that illustrate a much vaster and more complicated network for the control of the relatively simple horizontal eye movements than had been envisioned previously.

Rabies Virus as a Transneuronal Tracer of Neuronal Connections

Powerful transneuronal tracing technologies exploit the ability of some neurotropic viruses to travel across neuronal pathways and to function as self-amplifying markers. Rabies virus is the only viral tracer that is entirely specific, as it propagates exclusively between connected neurons by strictly unidirectional (retrograde) transneuronal transfer, allowing for the stepwise identification of neuronal connections of progressively higher order. Transneuronal tracing studies in primates and rodent models prior to the development of clinical disease have provided valuable information on rabies pathogenesis. We have shown that rabies virus propagation occurs at chemical synapses but not via gap junctions or cell-to-cell spread. Infected neurons remain viable, as they can express their neurotransmitters and cotransport other tracers. Axonal transport occurs at high speed, and all populations of the same synaptic order are infected simultaneously regardless of their neurotransmitters, synaptic strength, and distance, showing that rabies virus receptors are ubiquitously distributed within the CNS. Conversely, in the peripheral nervous system, rabies virus receptors are present only on motor endplates and motor axons, since uptake and transneuronal transmission to the CNS occur exclusively via the motor route, while sensory and autonomic endings are not infected. Infection of sensory and autonomic ganglia requires longer incubation times, as it reflects centrifugal propagation from the CNS to the periphery, via polysynaptic connections from sensory and autonomic neurons to the initially infected motoneurons. Virus is recovered from end organs only after the development of rabies because anterograde spread to end organs is likely mediated by passive diffusion, rather than active transport mechanisms.

Posterior parietal cortex areas MIP and LIPv receive eye position and velocity inputs via ascending preposito-thalamo-cortical pathways

Neuronal activity encoding eye position and gaze signals participates in updating the spatial representations found in the posterior parietal cortex and is necessary for spatial accuracy in goal‐directed movements. Using retrograde transneuronal transfer of rabies virus in combination with a conventional tracer, we studied direct and polysynaptic inputs to the ventral lateral intraparietal area (LIPv) and medial intraparietal area (MIP) in non‐human primates, to identify possible sources of eye position and gaze signals. We found that these areas receive disynaptic inputs from the brainstem horizontal eye position integrator network (nucleus prepositus hypoglossi, PH) via the central lateral and ventral lateral thalamic nuclei. Our findings provide the first demonstration that inputs from the horizontal eye position integrator reach cortical areas. We found important topographical differences between PH populations targeting MIP and LIPv that likely reflect transmission of different types of eye movement signals. LIPv receives projections from the ipsilateral rostral PH, which may transmit ipsilateral eye position signals. In addition to inputs from the rostral PH, MIP receives strong projections from the contralateral caudal PH, which may contribute to both eye position and velocity signals. Unlike the horizontal integrator, we found that the vertical eye position integrator network, the interstitial nucleus of Cajal, does not project to these posterior parietal areas, in keeping with findings that the thalamic nuclei targeting LIPv and MIP receive almost exclusively horizontal oculomotor signals.

Neuronal pathways for the propagation of herpes simplex virus type 1 from one retina to the other in a murine model.

Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. In order to determine the possible mechanisms leading to bilateral retinitis, we studied the pathways by which herpes simplex virus type 1 (HSV-1) is propagated from one retina to the other after intravitreal injection in mice. HSV-1 strain SC16 (90 p.f.u.) was injected into the vitreous body of the left eye of BALB/c mice. Animals were sacrificed 1, 2, 3, 4 and 5 days post-inoculation (p.i.). Histological sections were studied by immunochemical staining. Primary retinitis in the inoculated eye (beginning 1 day p.i.) was followed by contralateral retinitis (in the uninoculated eye) starting at 3 days p.i. Infected neurons of central visual pathway nuclei (lateral geniculate nuclei, suprachiasmatic nuclei and pretectal areas) were detected at 4 days p.i. Iris and ciliary body infection was minimal early on, but became extensive thereafter and was accompanied by the infection of connected sympathetic and parasympathetic pathways. The pattern of virus propagation over time suggests that the onset of contralateral retinitis was mediated by local (non-synaptic) transfer in the optic chiasm from infected to uninfected axons of the optic nerves. Later, retinopetal transneuronal propagation of the virus from visual pathways may have contributed to increase the severity of contralateral retinitis.

Transneuronal Tracing with Alpha-herpesviruses: A Review of the Methodology

Publisher Summary This chapter focuses on transneuronal tracing with alpha-herpesviruses. For a marker to be effective as a transneuronal tracer, transfer occurs specifically between connected neurons. Transneuronal labeling is easy to detect and involve all groups of neurons that are part of a given neuronal circuit. There are two classes of transneuronal markers: conventional tracers— namely, the wheat germ agglutinin-horseradish peroxidase conjugate and nontoxic fragments of tetanus toxin and some neurotropic viruses— namely, alpha-herpesviruses such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV), and rhabdoviruses. A unique feature of viruses is their ability to replicate in recipient neurons after transneuronal transfer, thus functioning as a self-amplifying marker. As a result, second- and third-order neurons show the same intensity of labeling as first-order neurons The success of transneuronal labeling with HSV-1 and PrV is dependent upon several experimental parameters, some related to the virus and others related to the host. The chapter reviews the methodological aspects regarding the use of HSV-1 and PrV as transneuronal tracers and hence, details the structure, tropism, and replication cycle of HSV-1 and PrV. There are several important parameters considered while designing viral transneuronal tracing studies: Host variables (species, strain, and age), viral strains (HSV and PrV), and virus dose that is measured in Plaque-forming units per milliliter (PFU/ml). Excellent visualization of HSV-1 and PrV is provided by standard immunohistochemical methods—that is, immunofluorescence and immunoperoxidase in frozen sections. Immunoperoxidase staining can also be carried out in paraffin sections. The interpretation of the kinetics of viral transfer is often complicated because viral infection causes degeneration of first-order neurons. This might include virus-induced neuronal degeneration and local transfer and asynchronous labeling of different groups of second-order neurons.

Density gradients of trans-synaptically labeled collicular neurons after injections of rabies virus in the lateral rectus muscle of the rhesus monkey.

We evaluated the two‐dimensional distribution of superior colliculus (SC) neurons visualized after retrograde transneuronal transport of rabies virus injected into the lateral rectus muscle of rhesus monkeys to test whether the density of projection neurons might play a role in the spatiotemporal transformation and vector decomposition. If this were the case, the number of horizontal eye movement‐related SC neurons should increase with their distance from the rostral pole of the SC and decrease with their distance from the representation of the horizontal meridian. Labeled neurons of the intermediate SC layers were counted inside a 1‐mm‐wide band that matched the horizontal meridian of the collicular motor map. Local areal densities were plotted against distance from the rostral SC pole. At 2.5 days after inoculation, there was no labeling in the SC. At 3 days, moderate labeling appeared on both sides, mostly in the intermediate layers. At 3.5 days, cell numbers substantially increased and the laminar distribution changed as cells appeared in the superficial SC layers. At 3 days, rostrocaudal density profiles were unimodal, with peaks at locations near 50 degrees (contralateral SC) and 25–30 degrees (ipsilateral SC) horizontal eccentricity. At 3.5 days, distributions were bimodal due to the appearance of a second high‐density region near the rostral pole of the SC. The distribution of SC neurons influencing the abducens nucleus, thus, was nonuniform. Caudal sites contained more neurons, but the experimentally observed density gradients were shallower than the theoretically predicted ones that would be necessary to fully account for the spatiotemporal transformation. Similarly, we studied the distributions of cell densities in the intermediate SC layers along an isoamplitude line (representing saccades of equal amplitudes but different directions). Consistent with theoretical estimates of the density gradients required for vector decomposition, we found that the concentrations of labeled cells were highest in the vicinity of the horizontal meridian but their decrease toward the periphery of the motor map was steeper than predicted. We conclude that SC cell density gradients cannot fully account for the spatiotemporal transformation and vector decomposition in the absence of an additional mechanism such as the previously demonstrated (Grantyn et al., [ 1997 ] Soc. Neurosci. Abstr. 23:1295; Moschovakis et al., [ 1998 ] J. Neurosci. 18:10219–10229) locus‐dependent weighting of the strength of efferent projections to the saccade generators. J. Comp. Neurol. 451:346–361, 2002.