A disynaptic basal ganglia connection to the inferior olive: potential for basal ganglia influence on cerebellar learning.

Recent studies have shown that the cerebellum and the basal ganglia are interconnected at subcortical levels. However, a subcortical basal ganglia connection to the inferior olive (IO), being the source of the olivocerebellar climbing fiber system, is not known. We have used classical tracing with CTb, retrograde transneuronal infection with wildtype rabies virus, conditional tracing with genetically modified rabies virus, and examination of material made available by the Allen Brain Institute, to study potential basal ganglia connections to the inferior olive in rats and mice. We show in both species that parvalbumin-positive, and therefore GABAergic, neurons in the entopeduncular nucleus, representing the rodent equivalent of the internal part of the globus pallidus, innervate a group of cells that surrounds the fasciculus retroflexus and that are collectively known as the area parafascicularis prerubralis. As these neurons supply a direct excitatory input to large parts of the inferior olivary complex, we propose that the entopeduncular nucleus, as a main output station of the basal ganglia, provides an inhibitory influence on olivary excitability. As such, this connection may influence olivary involvement in cerebellar learning and/or could be involved in transmission of reward properties that have recently been established for olivocerebellar signaling.

An open cortico-basal ganglia loop allows limbic control over motor output via the nigrothalamic pathway.

Cortico-basal ganglia-thalamocortical loops are largely conceived as parallel circuits that process limbic, associative, and sensorimotor information separately. Whether and how these functionally distinct loops interact remains unclear. Combining genetic and viral approaches, we systemically mapped the limbic and motor cortico-basal ganglia-thalamocortical loops in rodents. Despite largely closed loops within each functional domain, we discovered a unidirectional influence of the limbic over the motor loop via ventral striatum-substantia nigra (SNr)-motor thalamus circuitry. Slice electrophysiology verifies that the projection from ventral striatum functionally inhibits nigro-thalamic SNr neurons. In vivo optogenetic stimulation of ventral or dorsolateral striatum to SNr pathway modulates activity in medial prefrontal cortex (mPFC) and motor cortex (M1), respectively. However, whereas the dorsolateral striatum-SNr pathway exerts little impact on mPFC, activation of the ventral striatum-SNr pathway effectively alters M1 activity. These results demonstrate an open cortico-basal ganglia loop whereby limbic information could modulate motor output through ventral striatum control of M1.

Anatomical and physiological foundations of cerebello-hippocampal interaction.

Multiple lines of evidence suggest that functionally intact cerebello-hippocampal interactions are required for appropriate spatial processing. However, how the cerebellum anatomically and physiologically engages with the hippocampus to sustain such communication remains unknown. Using rabies virus as a retrograde transneuronal tracer in mice, we reveal that the dorsal hippocampus receives input from topographically restricted and disparate regions of the cerebellum. By simultaneously recording local field potential from both the dorsal hippocampus and anatomically connected cerebellar regions, we additionally suggest that the two structures interact, in a behaviorally dynamic manner, through subregion-specific synchronization of neuronal oscillations in the 6-12 Hz frequency range. Together, these results reveal a novel neural network macro-architecture through which we can understand how a brain region classically associated with motor control, the cerebellum, may influence hippocampal neuronal activity and related functions, such as spatial navigation.

Multizonal Cerebellar Influence Over Sensorimotor Areas of the Rat Cerebral Cortex.

The cerebral cortex requires cerebellar input for optimizing sensorimotor processing. However, how the sensorimotor cortex uses cerebellar information is far from understood. One critical and unanswered question is how cerebellar functional entities (zones or modules) are connected to distinct parts of the sensorimotor cortices. Here, we utilized retrograde transneuronal infection of rabies virus (RABV) to study the organization of connections from the cerebellar cortex to M1, M2, and S1 of the rat cerebral cortex. RABV was co-injected with cholera toxin ß-subunit (CTb) into each of these cortical regions and a survival time of 66-70 h allowed for third-order retrograde RABV infection of Purkinje cells. CTb served to identify the injection site. RABV+ Purkinje cells throughout cerebellar zones were identified by reference to the cerebellar zebrin pattern. All injections, including those into S1, resulted in multiple, zonally arranged, strips of RABV+ Purkinje cells. M1 injections were characterized by input from Purkinje cells in the vermal X-zone, medial paravermis (C1- and Cx-zones), and lateral hemisphere (D2-zone); M2 receives input from D2- and C3-zones; connections to S1 originate from X-, Cx-, C3-, and D2-zones. We hypothesize that individual domains of the sensorimotor cortex require information from a specific combination of cerebellar modules.

Changes in innervation of lumbar motoneurons and organization of premotor network following training of transected adult rats.

Rats with complete spinal cord transection (SCT) can recover hindlimb locomotor function under strategies combining exercise training and 5-HT agonist treatment. This recovery is expected to result from structural and functional re-organization within the spinal cord below the lesion. To begin to understand the nature of this reorganization, we examined synaptic changes to identified gastrocnemius (GS) or tibialis anterior (TA) motoneurons (MNs) in SCT rats after a schedule of early exercise training and delayed 5-HT agonist treatment. In addition, we analyzed changes in distribution and number of lumbar interneurons (INs) presynaptic to GS MNs using retrograde transneuronal transport of rabies virus. In SCT-untrained rats, we found few changes in the density and size of inhibitory and excitatory inputs impinging on cell bodies of TA and GS MNs compared to intact rats, whereas there was a marked trend for a reduction in the number of premotor INs connected to GS MNs. In contrast, after training of SCT rats, a significant increase of the density of GABAergic and glycinergic axon terminals was observed on both GS and TA motoneuronal cell bodies, as well as of presynaptic P-boutons on VGLUT1 afferents. Despite these changes in innervation the number of premotor INs connected to GS MNs was similar to control values although some new connections to MNs were observed. These results suggest that adaptation of gait patterns in SCT-trained rats was accompanied by changes in the innervation of lumbar MNs while the distribution of the spinal premotor circuitry was relatively preserved.

Disynaptic Subthalamic Input to the Posterior Cerebellum in Rat.

In the last decade, the interplay between basal ganglia and cerebellar functions has been increasingly advocated to explain their joint operation in both normal and pathological conditions. Yet, insight into the neuroanatomical basis of this interplay between both subcortical structures remains sparse and is mainly derived from work in primates. Here, in rodents, we have studied the existence of a potential disynaptic connection between the subthalamic nucleus (STN) and the cerebellar cortex as has been demonstrated earlier for the primate. A mixture of unmodified rabies virus (RABV: CVS 11) and cholera toxin B-subunit (CTb) was injected at places in the posterior cerebellar cortex of nine rats. The survival time was chosen to allow for disynaptic retrograde transneuronal infection of RABV. We examined the STN for neurons infected with RABV in all nine cases and related the results with the location of the RABV/CTb injection site, which ranged from the vermis of lobule VII, to the paravermis and hemispheres of the paramedian lobule and crus 2a. We found that cases with injection sites in the vermis of lobule VII showed prominent RABV labeling in the STN. In contrast, almost no subthalamic labeling was noted in cases with paravermal or hemispheral injection sites. We show circumstantial evidence that not only the pontine nuclei but also the pedunculotegmental nucleus may act as the intermediary in the connection from STN to cerebellar cortex. This finding implies that in the rat the STN links disynaptically to the vermal part of lobule VII of the cerebellar cortex, without any major involvement of the cerebellar areas that are linked to sensorimotor functions. As vermal lobule VII recently has been shown to process disynaptic input from the retrosplenial and orbitofrontal cortices, we hypothesize that in the rat the subthalamic input to cerebellar function might be used to influence more prominently non-motor functions of the cerebellum than motor functions. This latter aspect seems to contradict the primate results and could point to a more elaborate interaction between basal ganglia and cerebellum in more demanding motor tasks.

Caveats in Transneuronal Tracing with Unmodified Rabies Virus: An Evaluation of Aberrant Results Using a Nearly Perfect Tracing Technique.

Apart from the genetically engineered, modified, strains of rabies virus (RABV), unmodified 'fixed' virus strains of RABV, such as the 'French' subtype of CVS11, are used to examine synaptically connected networks in the brain. This technique has been shown to have all the prerequisite characteristics for ideal tracing as it does not metabolically affect infected neurons within the time span of the experiment, it is transferred transneuronally in one direction only and to all types of neurons presynaptic to the infected neuron, number of transneuronal steps can be precisely controlled by survival time and it is easily detectable with a sensitive technique. Here, using the 'French' CVS 11 subtype of RABV in Wistar rats, we show that some of these characteristics may not be as perfect as previously indicated. Using injection of RABV in hind limb muscles, we show that RABV-infected spinal motoneurons may already show lysis 1 or 2 days after infection. Using longer survival times we were able to establish that Purkinje cells may succumb approximately 3 days after infection. In addition, some neurons seem to resist infection, as we noted that the number of RABV-infected inferior olivary neurons did not progress in the same rate as other infected neurons. Furthermore, in our hands, we noted that infection of Purkinje cells did not result in expected transneuronal labeling of cell types that are presynaptic to Purkinje cells such as molecular layer interneurons and granule cells. However, these cell types were readily infected when RABV was injected directly in the cerebellar cortex. Conversely, neurons in the cerebellar nuclei that project to the inferior olive did not take up RABV when this was injected in the inferior olive, whereas these cells could be infected with RABV via a transneuronal route. These results suggest that viral entry from the extracellular space depends on other factors or mechanisms than those used for retrograde transneuronal transfer. We conclude that transneuronal tracing with RABV may result in unexpected results, as not all properties of RABV seem to be ubiquitously valid.

Early postnatal maturation in vestibulospinal pathways involved in neck and forelimb motor control.

To assess the organization and functional development of vestibulospinal inputs to cervical motoneurons (MNs), we have used electrophysiology (ventral root and electromyographic [EMG] recording), calcium imaging, trans-synaptic rabies virus (RV) and conventional retrograde tracing and immunohistochemistry in the neonatal mouse. By stimulating the VIIIth nerve electrically while recording synaptically mediated calcium responses in MNs, we characterized the inputs from the three vestibulospinal tracts, the separate ipsilateral and contralateral medial vestibulospinal tracts (iMVST/cMVST) and the lateral vestibulospinal tract (LVST), to MNs in the medial and lateral motor columns (MMC and LMC) of cervical segments. We found that ipsilateral inputs from the iMVST and LVST were differentially distributed to the MMC and LMC in the different segments, and that all contralateral inputs to MMC and LMC MNs in each segment derive from the cMVST. Using trans-synaptic RV retrograde tracing as well as pharmacological manipulation of VIIIth nerve-elicited synaptic responses, we found that a substantial proportion of inputs to both neck and forelimb extensor MNs was mediated monosynaptically, but that polysynaptic inputs were also significant. By recording EMG responses evoked by natural stimulation of the vestibular apparatus, we found that vestibular-mediated motor output to the neck and forelimb musculature became more robust during the first 10 postnatal days, concurrently with a decrease in the latency of MN discharge evoked by VIIIth nerve electrical stimulation. Together, these results provide insight into the complexity of vestibulospinal connectivity in the cervical spinal cord and a cogent demonstration of the functional maturation that vestibulospinal connections undergo postnatally. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1061-1077, 2016.

Differential organization of cortical inputs to striatal projection neurons of the matrix compartment in rats.

In prior studies, we described the differential organization of corticostriatal and thalamostriatal inputs to the spines of direct pathway (dSPNs) and indirect pathway striatal projection neurons (iSPNs) of the matrix compartment. In the present electron microscopic (EM) analysis, we have refined understanding of the relative amounts of cortical axospinous vs. axodendritic input to the two types of SPNs. Of note, we found that individual dSPNs receive about twice as many axospinous synaptic terminals from IT-type (intratelencephalically projecting) cortical neurons as they do from PT-type (pyramidal tract projecting) cortical neurons. We also found that PT-type axospinous synaptic terminals were about 1.5 times as common on individual iSPNs as IT-type axospinous synaptic terminals. Overall, a higher percentage of IT-type terminals contacted dSPN than iSPN spines, while a higher percentage of PT-type terminals contacted iSPN than dSPN spines. Notably, IT-type axospinous synaptic terminals were significantly larger on iSPN spines than on dSPN spines. By contrast to axospinous input, the axodendritic PT-type input to dSPNs was more substantial than that to iSPNs, and the axodendritic IT-type input appeared to be meager and comparable for both SPN types. The prominent axodendritic PT-type input to dSPNs may accentuate their PT-type responsiveness, and the large size of axospinous IT-type terminals on iSPNs may accentuate their IT-type responsiveness. Using transneuronal labeling with rabies virus to selectively label the cortical neurons with direct input to the dSPNs projecting to the substantia nigra pars reticulata, we found that the input predominantly arose from neurons in the upper layers of motor cortices, in which IT-type perikarya predominate. The differential cortical input to SPNs is likely to play key roles in motor control and motor learning.

Segmental organization of vestibulospinal inputs to spinal interneurons mediating crossed activation of thoracolumbar motoneurons in the neonatal mouse.

Vestibulospinal pathways activate contralateral motoneurons (MNs) in the thoracolumbar spinal cord of the neonatal mouse exclusively via axons descending ipsilaterally from the vestibular nuclei via the lateral vestibulospinal tract (LVST; Kasumacic et al., 2010). Here we investigate how transmission from the LVST to contralateral MNs is mediated by descending commissural interneurons (dCINs) in different spinal segments. We test the polysynaptic nature of this crossed projection by assessing LVST-mediated ventral root (VR) response latencies, manipulating synaptic responses pharmacologically, and tracing the pathway transynaptically from hindlimb extensor muscles using rabies virus (RV). Longer response latencies in contralateral than ipsilateral VRs, near-complete abolition of LVST-mediated calcium responses in contralateral MNs by mephenesin, and the absence of transsynaptic RV labeling of contralateral LVST neurons within a monosynaptic time window all indicate an overwhelmingly polysynaptic pathway from the LVST to contralateral MNs. Optical recording of synaptically mediated calcium responses identifies LVST-responsive ipsilateral dCINs that exhibit segmental differences in proportion and dorsoventral distribution. In contrast to thoracic and lower lumbar segments, in which most dCINs are LVST responsive, upper lumbar segments stand out because they contain a much smaller and more ventrally restricted subpopulation of LVST-responsive dCINs. A large proportion of these upper lumbar LVST-responsive dCINs project to contralateral L5, which contains many of the hindlimb extensor MNs activated by the LVST. A selective channeling of LVST inputs through segmentally and dorsoventrally restricted subsets of dCINs provides a mechanism for targeting vestibulospinal signals differentially to contralateral trunk and hindlimb MNs in the mammalian spinal cord.

Reorganization of supramammillary-hippocampal pathways in the rat pilocarpine model of temporal lobe epilepsy: evidence for axon terminal sprouting.

In mesial temporal lobe epilepsy (MTLE), spontaneous seizures likely originate from a multi-structural epileptogenic zone, including several regions of the limbic system connected to the hippocampal formation. In this study, we investigate the structural connectivity between the supramammillary nucleus (SuM) and the dentate gyrus (DG) in the model of MTLE induced by pilocarpine in the rat. This hypothalamic nucleus, which provides major extracortical projections to the hippocampal formation, plays a key role in the regulation of several hippocampus-dependent activities, including theta rhythms, memory function and emotional behavior, such as stress and anxiety, functions that are known to be altered in MTLE. Our findings demonstrate a marked reorganization of DG afferents originating from the SuM in pilocarpine-treated rats. This reorganization, which starts during the latent period, is massive when animals become epileptic and continue to evolve during epilepsy. It is characterized by an aberrant distribution and an increased number of axon terminals from neurons of both lateral and medial regions of the SuM, invading the entire inner molecular layer of the DG. This reorganization, which reflects an axon terminal sprouting from SuM neurons, could contribute to trigger spontaneous seizures within an altered hippocampal intrinsic circuitry.

Socioeconomic factors and mortality in diastolic heart failure.

BACKGROUND: Mortality in patients with heart failure with preserved ejection fraction (HFPEF) has remained stable over recent decades. Few studies have explored prognostic characteristics specifically in HFPEF, and none of them has assessed the potential impact of socioeconomic factors. We aimed to evaluate the impact of socioeconomic factors on all-cause and cardiovascular mortality in HFPEF patients. MATERIALS AND METHODS: We used data from the French ODIN cohort. All patients with heart failure and a left ventricular ejection fraction (LVEF) > 45%, included in ODIN between July 2007 and July 2010, were eligible here. Socioeconomic, demographic, clinical, biological and therapeutic data were collected at inclusion. The endpoints were all-cause and cardiovascular mortality between inclusion and 30 September 2011. The impact of patient socioeconomic characteristics on mortality was assessed using Cox regression models. RESULTS: Of 575 HFPEF patients considered, 58·6% were male; their mean age was 71·1 ± 13·5 years, and their mean LVEF was 58·1 ± 8·5%. After adjustment for confounders, living alone and limitations on activities of daily living were associated with all-cause mortality [HR = 1·77, 95%CI(1·11-2·81) and 2·61(1·35-5·03), respectively] and cardiovascular mortality [2·26 (1·24-4·10) and 3·16 (1·33-7·54), respectively]. Having a professional occupation was associated with a lower cardiovascular mortality only [0·37(0·15-0·94)]. CONCLUSIONS: Poor social conditions impair survival in patients with HFPEF. These findings may shed new light on how best to detect HFPEF patients with high health-care needs.

Organization of cerebral projections to identified cerebellar zones in the posterior cerebellum of the rat.

The cerebrocerebellar connection makes use of two of the largest fiber tracts in the mammalian brain, i.e., the cerebral and medial cerebellar peduncles. Neuroanatomical approaches aimed to elucidate the organization of this important connection have been hindered by its multisynaptic nature, the complex organization of its components, and the dependency of conventional tracers on precisely placed injections. To overcome these problems, we used rabies virus (RV) as a retrograde transneuronal tracer. RV was injected simultaneously with cholera toxin ß subunit (CTb) into selected areas of the cerebellar cortex of 18 male Wistar rats. A survival time of 48-50 h resulted in first- and second-order labeling of RV in combination with first-order labeling of CTb. The distribution of CTb-labeled neurons in the inferior olive established the zonal identity of the injection site. In this way, it was possible to examine the cortical distribution of neurons from which disynaptic cerebrocerebellar projections to specific cerebellar loci originate. The results show that this distribution covaries with the identity of the injected cerebellar lobule. More subtle changes were present when different zones of the same lobule were injected. The C1 zone of lobule VIII receives a more prominent projection from the somatosensory cortex compared with the C2/D zones. The laterally positioned D zones receive information from more rostral regions of the cerebral cortex. The vermis of lobule VII receives a prominent input from the retrosplenial and orbitofrontal cortices. Different injection sites also result in differences in laterality of the connections.

Characterization of last-order premotor interneurons by transneuronal tracing with rabies virus in the neonatal mouse spinal cord.

We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V-VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles.

Chapter 1--importance of chloride homeostasis in the operation of rhythmic motor networks.

GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. The postsynaptic action depends on the intracellular concentration of chloride ions ([Cl(-)](i)), which is regulated by proteins in the plasma membrane: the K(+)-Cl(-) cotransporter KCC2 and the Na(+)-K(+)-Cl(-) cotransporter NKCC1, which extrude and intrude Cl(-) ions, respectively. A high [Cl(-)](i) leads to a depolarizing (excitatory) action of GABA and glycine, as observed in mature dorsal root ganglion neurons and in motoneurons both early during development and in several pathological conditions, such as following spinal cord injury. Here, we review some recent data regarding chloride homeostasis in the spinal cord and its contribution to network operation involved in locomotion.

The added value of rabies virus as a retrograde tracer when combined with dual anterograde tract-tracing.

Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of connected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibodies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further characterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution.

Changes to interneuron-driven striatal microcircuits in a rat model of Parkinson's disease.

Striatal interneurons play key roles in basal ganglia function and related disorders by modulating the activity of striatal projection neurons. Here we have injected rabies virus (RV) into either the rat substantia nigra pars reticulata or the globus pallidus and took advantage of the trans-synaptic spread of RV to unequivocally identify the interneurons connected to striatonigral- or striatopallidal-projecting neurons, respectively. Large numbers of RV-infected parvalbumin (PV+/RV+) and cholinergic (ChAT+/RV+) interneurons were detected in control conditions, and they showed marked changes following intranigral 6-hydroxydopamine injection. The number of ChAT+/RV+ interneurons innervating striatopallidal neurons increased concomitant with a reduction in the number of PV+/RV+ interneurons, while the two interneuron populations connected to striatonigral neurons were clearly reduced. These data provide the first evidence of synaptic reorganization between striatal interneurons and projection neurons, notably a switch of cholinergic innervation onto striatopallidal neurons, which could contribute to imbalanced striatal outflow in parkinsonian state.

Prenatal activation of 5-HT2A receptor induces expression of 5-HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice.

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

Multiple cerebellar zones are involved in the control of individual muscles: a retrograde transneuronal tracing study with rabies virus in the rat.

To identify cerebellar regions that are involved in the control of limb muscles, rabies virus was injected into the tibialis anterior (TA), the gastrocnemius (GC) or, for comparison, into the flexor digitorum (FD) muscles of the rat. Progression of retrograde transneuronal infection at supraspinal levels was assessed after variable time spans and was divided into three groups. Initially, infected neurons were observed in the reticular formation, lateral vestibular nucleus, red nucleus and motor cortex (group 1). Group 2 was characterized by labelling within the cerebellar nuclei as well as of two vermal strips of Purkinje cells (PCs). Double-labelling with zebrin enabled identification of these strips as the lateral part of the A1- and B-zone. For TA both zones were ipsilateral, whereas for GC the A1 strip predominated contralaterally. Group 3 infections showed additional labelling of multiple, in part bilateral, identifiable strips of PCs in vermis, paravermis and hemisphere. FD injections resulted in less robust labelling of vermal strips and more pronounced labelling within paravermal and hemispheral zonal regions. Only sporadic labelling in corresponding regions of the inferior olive and no labelling of cortical interneurons or granule cells was observed. Prolonged infection was seen to result in degeneration of PCs and possibly of motoneurons. We conclude that vermal, paravermal as well as hemispheral zones of the cerebellar cortex converge upon motoneurons that innervate a particular muscle. In addition, individual zones may control motorpools of different muscles and thus contribute to muscle synergies.

High-resolution neuroanatomical tract-tracing for the analysis of striatal microcircuits.

Although currently available retrograde tracers are useful tools for identifying striatal projection neurons, transported tracers often remained restricted within the neuronal somata and the thickest, main dendrites. Indeed, thin dendrites located far away from the cell soma as well as post-synaptic elements such as dendritic spines cannot be labeled unless performing intracellular injections. In this regard, the subsequent use of anterograde tracers for the labeling of striatal afferents often failed to unequivocally elucidate whether a given afferent makes true contacts with striatal projections neurons. Here we show that such a technical constraint can now be circumvented by retrograde tracing using rabies virus (RV). Immunofluorescence detection with a monoclonal antibody directed against the viral phosphoprotein resulted in a consistent Golgi-like labeling of striatal projection neurons, allowing clear visualization of small-size elements such as thin dendrites as well as dendritic spines. The combination of this retrograde tracing together with dual anterograde tracing of cortical and thalamic afferents has proven to be a useful tool for ascertaining striatal microcircuits. Indeed, by taking advantage of the trans-synaptic spread of RV, different subpopulations of local-circuit neurons modulating striatal efferent neurons can also be identified. At the striatal level, structures displaying labeling were visualized under the confocal laser-scanning microscope at high resolution. Once acquired, confocal stacks of images were firstly deconvoluted and then processed through 3D-volume rendering in order to unequivocally identify true contacts between pre-synaptic elements (axon terminals from cortical or thalamic sources) and post-synaptic elements (projection neurons and/or interneurons labeled with RV).