Corticobulbar projections change according to motor ... · after a lesion of the CST, spinal cord...
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Corticobulbar projections change according to motor pathology in macaque monkeys Fregosi M., Contestabile A., Hamadjida, A., Badoud, S., Borgognon, S., Cottet, J., Rouiller E.M. University of Fribourg, Departement of Medecine, Neuroscience The corticobulbar projection, together with the corticospinal tract (CST), act in parallel with projections from the brainstem (such as the reticulospinal tract) to ensure direct or indirect control of movement on motoneurons in the spinal cord. In monkeys little is known about the projections coming from the motor cortex on the brainstem as well as on their influence. Previous studies suggested a role of the reticulospinal tract in the control of reaching movement and in the recovery after a lesion of the CST, spinal cord or cerebral cortex. The aim of the present study was to anatomically analyze the corticobulbar projections coming from different motor cortical areas (premotor cortex (PM), primary motor cortex (M1) and supplementary motor cortex area (SMA)) on the reticular formation of the brainstem, possibly influencing the reticulospinal neurons. We analysed the projections in intact monkeys (n=7, tracer injections in either PM, M1 or SMA) and in monkeys subjected to different pathologies: M1 cortical lesion (PM injection (n=4)); spinal cord injury (M1 injection, (n=5)) or Parkinson's disease (PD; tracer injection in either PM (n=2) or M1 (n=2)). The anterograde tracer biotinylated dextran amine (BDA) was injected unilaterally in either PM, SMA or M1 in twenty macaque monkeys (Macaca fascicularis). The corticobulbar projections labeled anterogradelly by BDA were then analyzed in 12 consecutive histological sections (50 µm thick), 250 micrometers apart. Axons and terminals, including boutons en passant, were then plotted using the software Neurolucida. An adjacent series of 12 sections was stained with Creysil violet revealing Nissl bodies. On these sections we delineated the brainstem nuclei. The Neurolucida software is connected to a light microscope (Olympus BX40). We used the objective 4x to trace the contours of the sections and the Pyramidal tract, the 10x to trace the axons and finally the 20x to plot the boutons en passant and terminaux. For the series stained with Nissl we used the 1.25x objective to delineate the nuclei and to acquire pictures. Both series of sections (BDA and Nissl staining) were overlapped in order to match the zone of terminals and the nuclei delineated with Nissl staining. In the group of the intact monkeys (n=7), 3 were injected in PM, 3 in M1 and one in SMA; monkeys subjected to cortical lesion of M1 hand area (n=4) were injected in PM; animals subjected to MPTP lesion mimicking PD (n=4) were injected in either PM (n=2) or M1 (n=2); finally monkeys subjected to unilateral cervical hemisection at C7/C8 (n=5; SCI) were injected in M1. Statistics were calculated on the basis of the number of boutons in each nucleus and were derived from the Paired t-test /Wilcoxon; * p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001 (bilateral comparison). METHODS INTRODUCTION RESULTS The greater number of corticobulbar projections was found in the main nuclei of the Pontomedullary reticular formation (PMRF), namely in PnO, PnC, Gi, IRt and LRt. Intact animals injected in either PM or SMA showed denser corticobulbar projections as compared to M1 (Fig. 3A + 3B). For Mk-R13 (PM) on both contralateral and ipsilateral sides the largest percentage of terminals was found in the Gi nucleus. The same was true for Mk-CH (PM); however the three most rostral sections were unavailable for this animal. In contrast, Mk-R12 (PMd) showed a comparable percentage of axonal boutons in PnO+PnC and Gi on both the ipsilateral and contralateral sides (Fig. 2A). Animals injected in M1 showed a large percentage of projections to the ipsilateral Gi and contralateral IRt and LRt. For Mk-93-80 the projections to IRt and LRt was mostly contralateral. Few axonal boutons were found in PnO+PnC (Fig. 2B). Mk-M93-81 (SMA) showed the largest percentage of axonal boutons on both sides in both the Gi nucleus and the PnO+PnC nuclei (Fig. 2A). In M1 lesioned monkeys the corticobulbar projections from PM decreased in density (Fig. 3A and 3C), with a majority of boutons in PnO+PnC and Gi nuclei (Fig. 2C). In animals with Parkinson's disease (PD) there was a strong decrease of corticobulbar projections from PM (Fig. 3A), whereas projections from M1 showed a slighter decrease as compared to intact animals (Fig. 3B). In general a comparable number of projections to PnO+PnC and Gi nuclei was observed, even in M1 injected monkeys (Fig. 2D). In SCI animals injected in M1, we found an increase of corticobulbar projections, mainly contralateral, in monkeys subjected to anti-Nogo A antibody treatment (Fig. 3B and 3D). The majority of corticobulbar projections were observed in Gi nucleus and in IRt + LRt nuclei (Fig. 2E). Intact monkeys: The corticobulbar projections originating from PM and SMA are denser then from M1 (Fig. 3C and D). Moreover, the corticobulbar projection from PM and SMA tend to be more prominent on the ipsilateral PMRF than contralaterally; this is the other way around for the corticobulbar projection from M1. M1 lesion and PD: For both pathologies, there was a decrease of corticobulbar projections (as compared to intact monkeys), from PM after M1 lesion and from both PM and M1 in case of PD (Fig. 3C and D). Spinal cord injury (SCI): As compared to intact monkeys, no change in the two control SCI monkeys; in contrast, 2 out of 3 anti-Nogo A antibody treated monkeys showed an increase of the corticobulbar projections from M1, with contralateral predominance as well (Fig. 3D). CONCLUSIONS Supported by: Swiss National Science Fundation (SNF), Grants to E.M.R. No.31-61857.00, 310000-110005, 31003A-132465, 310030B-149643; The National Center of Competence in Research (NCCR) on «Neural Plasticity and Repair»; Novartis Foundation; The Swiss Primate Competence Center for Research (SPCCR): http://www.unifr.ch/spccr/home/ Figure 2: Percentage distribution of boutons across brainstem nuclei. Percentage of boutons en passant or terminaux ipsilateral (blue) and contralateral (orange) calculated on the total number of boutons found in the whole brainstem ipsilaterally or contralaterally to the BDA injection site. In each graph, the sum of all orange bars is 100%. The sum of all blue bars is 100%. The blue frame shows data obtained from animals injected in PM, the pink frame shows data obtained from monkeys injected in SMA and finally the green frame shows the data obtained from animals injected in M1. Injection sites of BDA tracer into one area of the motor cortex (PM) Micrographs of BDA anterograde labelling Micrographs of Nissl staining 50µm 50µm 1mm Figure 3: Histograms showing the total numbers of boutons en passant and terminaux in the whole brainstem (Tot), in its caudal half (C; from section 7 to section 12) and in its rostral half (R; from section 1 to section 6). Black bars are for ipsilateral projections and white bars for contralateral ones. Panel A) Histogram representing the row data (absolute numbers of boutons) for projections arising either from SMA (pink background) or from PM (blue background) in intact animals, in monkeys subjected to M1 hand lesion and in PD animals. Panel B) Histogram showing the row data for animals injected in M1 (green background) in intact animals, in monkeys subjected to cervical SCI and in PD animals. Panel C) The same data as in A but normalized. Panel D) The same data as in B but normalized. Normalization was performed with reference to the number of BDA labelled corticospinal axons observed above the Decussatio pyramidum. Mk-CG 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC Mk-CP 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC * Mk-AC 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC Mk-AP 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC ** Mk-AG 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC * E Cervical SCI Anti-Nogo A antibody treatment Control Ab M1 injection 100 % Mk-Robby 0 20 40 60 80 Undefined areas Others Raphe IRt+LRt Gi PnO + PnC * Undefined areas Others Raphe IRt+LRt Gi PnO + PnC Mk-Bill 0 20 40 60 80 100 % Mk-Mono 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC * Mk-Vassia 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC * * Anti-Nogo A treatment C M1 lesion Untreated PM injection Patholgies D 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC Mk-LL 0 20 40 60 80 100 % Undefined areas Others Raphe IRt+LRt Gi PnO + PnC Mk-MY Undefined areas Others Raphe IRt+LRt Gi PnO + PnC 0 20 40 60 80 100 % Mk-LY Undefined areas Others Raphe IRt+LRt Gi PnO + PnC 0 20 40 60 80 100 % Mk-MI M1 PD lesion M1 injection PM injection Figure 1: brainstem drawings of coronal sections of Mk-R13 arranged from rostral (section 1) to caudal (section 12). All nuclei are delineated with a different color (see list of abbreviations). Axons located ipsilateral to the BDA injection are marked in blue whereas those located contralaterally are marked in bordeaux. Boutons en passant and terminaux ipsilateral to the BDA injection are marked as green circles whereas the contralateral terminals are marked as blue squares. Mk-R13 (BDA injected in PM) 10 8 4 6 12 n n n n n n n n n n nnn n n n n nn n n n n n nnn nnn n nn n n n n n nnn n n n n nnn nn n n n n n n nnn nnnn n n n n n n n nnn nn n n n n n n n nn n n n n n nnnn n n n n n n nnn n n n n nn nnn nn n n n n n n n n n n n n n n n n n n n n n n nn n nn n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n nn n n n n n n n n n n n n n n n n n nn n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nnn n n n n n n n n n n n n n n nn nn n n n n n n n n n n n n n n n n n nn n nn n n n n n n n n n n n n nn n nn nn n n nnn nn n nn n nn nn nn n n nnn n n nnnn nnnn n nn n n n n n n nn n n n n n n n n nn nnn nn nn nn nnn n n n n n n nnn n nnn n n n n n n n n n nn n n nn nn n nn n n n n n n n n n n n n n nn n n n n n n nnn n nn n n n nn n n nnn nn nn n n n n n n n n n n n n n n nn n nnn n nn n n n n nn n n nnn n n n n nnn nnn nn n n nn n n n n n n n n n n n n n nn n n n n n n n n n nn n n n n n n nn n n n n n n n n nn n n n n nn n nn nn n n nn n nn n n nn n nn nn n n n n nn n n nn n n nn n n nnn n n n n n n n n n n n n nn n n n n n n nn n n n nn n nn nn n n n n nn nn n n n n n n n n nn n nn n nn nn n nnnn n n n nn nn n n n n n n n n n n n n n n nn n n nn n nn n n n n nn n n n n n n n n n n n n n n nn n n n n n n n n n n n n n nn n n n n n n nnn n n n n nn n n n n n n n n n n n n n n n nn n n n n n n n n nn n n n n n nn n n n n n n n n n n n n n n nn n n n n nn nn n n n nn n nn n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n nn n n n n nn nn n nn nn n n n n n n n n nn n n n n n n nn n n nn n n n n n n n n n n nn n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n nn n n n nn nn n n n n n n n n n n n n n n nn nn nnn n n n n n n n n nn n n n n n n n n nn n n n n n n n n n n n n nn n n n n n n n n n nn n n nn nn n n nn n n n n n n n n n n n n n n n n n n n n n nn n n n n n n nn n n n n n n n n n n nn n n n n n n n nn n nn n n n nn n n n n n n n n nnn n n n n n n n n nn n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n nn n n n nn n n n n n n nn n n n n n n nn n n n n nn n n n n n n n nn n n n n n n n n n n n n n n n n n n n n nn nnn n n n nn n nn n n n n n n nn n n n n n n n n n n n n nn nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n nn n n n nn n n n n n nn n n n n n n n n n n n n n n n n nn n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n nn n n n n n n n n n n n n n n n nnn n n n n n n nn n n n n n n n n n nn n n nn n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n nn nn n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn nn n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n nn n n n nn n n n n n n n nn n n n n n n n n n n n n n nn n n nn n n nn n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n nn n n n n nnn n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n nn n n n n n n n n nn n n n n n n n n n n nn n n n n n n n n n n n n n n n n n nn n n n n n nn n n n n n n n n n nn n n nn nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n nn n n n n n n n nn n n n n n n n nn n n n n n n n nn n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n nn n n n n n n n n n n n n n n n n n n n n n nnn n n n nn n n n n n n n n n n n n SOC Py SOC Pr5 6N Raphe PnC mcp VC ml Gi Py ml Gi IO IRt IRt Gi ml Py Raphe IO IRt Gi IO Py ml Raphe LRt 12N Py IO IR Sol LRt 12N 2 Stem axons Boutons en passant and terminaux 11 9 7 5 3 1 n n nn n n nn n n n n nn n n n n n nn nnn nnn n n nn nn nn n n n n n n n n n n n nn n n n nn n nn n n n n n nn nn n n n nn n n n nn n nn nnn n n n n n n nn n n nn n n nn nn n nn n n n n nn n n n nnn nnnn n n nn nn n nnn n n n nn n nnn n n n n n n n nnn n n n n n nnn n n nn n n n n n n nn nn n n nn n nn n n nn nn nn n n n nn n n n n n n n n n n n n n n n n n n n nn n n n n n nnn n nn n n n n nn n n n n nn n n n n n nnn n nn nn n nn nn n nn n nn n n n n n n nn nn nnn n n n nnn n n n nn n n n n n n n n n nn n n n n nn n n n n n nn nnn n n n n nnnn n nn n n n n n nnnn nnn n n nn n nnnnn nn n nn n n n n n nnn n n nn nn n n n nnn n n n n n n n n n nn n n nn n n n n n n n n nnn n n nn nn n n nnn n n n n n nnn nn n n n nn n n n n n n n n n n n n nnn n nn nnn n n n n n n 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nnn n n n n nn n nn nn n n n nn n nnn n n n n n n n nn nn n n n nn n n n n n n n n n n n nn n n nn n n n n n n nn nn n n nn n nnnn n nn n n n nn n n n n n n n n nn nn n n n n n n n n n n n n n n n n n n nn n n n n n n nn n n n n n n n n n nn n n n n n n n n n n n nn n n nn n n n n n n n n n nnn n n n n n n nnn n n n n n nnn n n n n n nn n n n nn n nn n nn n nnn nn n nn n nnn n n nn n n n n nn n n n n n n nn n n n nnn n nnn nn n n n n n n n n n n n n n n n n n n n nn n n n n nnnn n n nn nnn n n n nnnn nn nn n n n n nnn n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nnn n n nn n n n n n n n n n nn n n n n n n nn n n n n nn n n n n n n n n n n n n n n n n n nn nn n n n n n n n nn n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n nn n n n n nn n n n n n n n n n n n n n n n n n n n n n n nn nn n n n nn n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n nn n n n n n n n n n n n n n n n n nn n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn nn n n n n n n nnn n n n n n n n n n n n n n n n nn n n n n n n n n n n nn n n n n n n n n n n n n nn nnn n n n n n n n nn n n n n nnn n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n nn n n nn n n n n n n n n n n n n n nn n n nn n n n n n n n n n n n nn n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n nn n n n n n n n n n n n n nn n n n n n n n n n n nn n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn nn n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n nnn nn n n n n n n n n n n n n n n n nn nn n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nnnn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n nn n nn nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n nn n n nn n n n nn n n n n n nn n n n n n n nn n n n n nn n n n n nn n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n n nn n n n n n nn n n n n n n n n n n n n n nn n n n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n nn n n n n n n n n n n n n n n n n n nnn n n n n n n n nn nn n n n n n n nn n n n n n n n n n n n n n n n n n n n n n n n n nn n n n n n n nn n n n n n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n nn n n n n n n n n n n n n n n n n nn n n n n n n nn n n n n n n n n n n n nn n nn n n n nnnn nn n n n n n nnnn n n nn n n n n nn n nn n n n n n n nnn n nnn n n n n nn n n n nnn n nn nn n n nn nn n n n nn n n n n n n n n n n n n n n n n nnn n n n n nn n nnn n n n n nnnnn n n n n nn nn n n n n n nn n n n n n n nnn nn n n nnnnn n n n n nnn n n n n n n n nnn nn n n n n n n n n n nn n n n nn n n n n n n n n nnn nn n nn nnn n n n n n n n n n n n n n n n n n n nn n n n n n nn n n nn n n Stem axons Pn Py mcp Mo5 PnO Raphe RtTg ml LC VC Py ml Sp5 7N 8n Gi CN Pr5 6N PnC Py ml Mo5 Raphe Sp5 IRt Gi VC Sp5 IRt Gi VC VC Sp5 Gi Cu Ecu n n IRt Sol Boutons en passant and terminaux 0 1000 2000 3000 Mk-LY Tot C R Mk-MI Tot C R Tot C R Tot C R Mk-Z182 Mk-M310 Tot C R Mk-M93-80 Tot C R Tot C R Mk-CG Mk-CP Tot C R Mk-AC Tot C R Mk-AP Tot C R Mk-AG Normalized number of boutons * *** * * * *** * * * ** ** * * * * 0 1000 2000 3000 4000 Absolute number of boutons Intact SCI Anti Nogo A Ab PD Control Ab Tot C R Tot C R Mk-Z182 Mk-M310 Tot C R Mk-M93-80 Tot C R Tot C R Mk-CG Mk-CP Tot C R Mk-AC Tot C R Mk-AP Tot C R Mk-AG Mk-LY Tot C R Mk-MI Tot C R * *** * * * *** * * * ** ** * * * * B M1 injection D Normalized number of boutons 0 1000 2000 3000 Mk-R13 Mk-R12 Mk-CH Tot C R Tot C R Tot C R Tot C R Mk-MO Tot C R Mk-VA Tot C R Mk-RO Tot C R Mk-BI Tot C R Mk-M93-81 Tot C R Mk-LL Tot C R Mk-MY *** * ** *** *** * *** * * * * ** Intact Absolute number of boutons 0 1000 2000 3000 4000 Tot C R Mk-LL Tot C R Mk-MY Mk-R13 Mk-R12 Mk-CH Tot C R Tot C R Tot C R Tot C R Mk-MO Tot C R Mk-VA Tot C R Mk-RO Tot C R Mk-BI Tot C R Mk-M93-81 Intact M1 lesion PD Anti-Nogo A Ab Untreated *** * ** *** *** * *** * * * * ** A PM injection SMA injection C Abbreviations Medial leminiscus Trigeminal motor nucleus Ponto Medullary Reticular Formation Pontine nuclei Pontine reticular nucleus caudalis Pontine reticular nucleus oralis Prepositus nucleus Principal sensory trigeminal nucleus Pyramidal tract Raphe nuclei Reticulo tegmental nucleus of Pons Solitary nucleus Superior olivary complex Spinal sensory trigeminal nucleus Vestibular complex ml Mo5 PMRF Pn PnC PnO Pr Pr5 Py Raphe RtTg Sol SOC Sp5 VC 6N 7N 5n 7n 8n 9n 12N CN Cu Ecu Gi IO LC mcp Abducens nucleus Facial nucleus Trigeminal nerve Facial nerve Vestibulocochlear nerve Glossopharyngeal nerve Hypoglossal nucleus Cochlear nucleus Cuneate nucleus External cuneate nucleus Gigantocellular reticular nucleus Inferior olive Intermediate reticular nucleus Locus coeruleus Lateral reticular nucleus Middle cerebellar peduncle Rt L Rt I
Transcript of Corticobulbar projections change according to motor ... · after a lesion of the CST, spinal cord...
Corticobulbar projections change according to motor pathology in macaque monkeys
Fregosi M., Contestabile A., Hamadjida, A., Badoud, S., Borgognon, S., Cottet, J., Rouiller E.M.University of Fribourg, Departement of Medecine, Neuroscience
The corticobulbar projection, together with the corticospinal tract (CST), act in parallel with projections from the brainstem (such as the reticulospinal tract) to ensure direct or indirect control of movement on motoneurons in the spinal cord. In monkeys little is known about the projections coming from the motor cortex on the brainstem as well as on their influence. Previous studies suggested a role of the reticulospinal tract in the control of reaching movement and in the recovery after a lesion of the CST, spinal cord or cerebral cortex.The aim of the present study was to anatomically analyze the corticobulbar projections coming from different motor cortical areas (premotor cortex (PM), primary motor cortex (M1) and supplementary motor cortex area (SMA)) on the reticular formation of the brainstem, possibly influencing the reticulospinal neurons. We analysed the projections in intact monkeys (n=7, tracer injections in either PM, M1 or SMA) and in monkeys subjected to different pathologies: M1 cortical lesion (PM injection (n=4)); spinal cord injury (M1 injection, (n=5)) or Parkinson's disease (PD; tracer injection in either PM (n=2) or M1 (n=2)).
The anterograde tracer biotinylated dextran amine (BDA) was injected unilaterally in either PM, SMA or M1 in twenty macaque monkeys (Macaca fascicularis). The corticobulbar projections labeled anterogradelly by BDA were then analyzed in 12 consecutive histological sections (50 µm thick), 250 micrometers apart. Axons and terminals, including boutons en passant, were then plotted using the software Neurolucida.An adjacent series of 12 sections was stained with Creysil violet revealing Nissl bodies. On these sections we delineated the brainstem nuclei. The Neurolucida software is connected to a light microscope (Olympus BX40). We used the objective 4x to trace the contours of the sections and the Pyramidal tract, the 10x to trace the axons and finally the 20x to plot the boutons en passant and terminaux. For the series stained with Nissl we used the 1.25x objective to delineate the nuclei and to acquire pictures. Both series of sections (BDA and Nissl staining) were overlapped in order to match the zone of terminals and the nuclei delineated with Nissl staining. In the group of the intact monkeys (n=7), 3 were injected in PM, 3 in M1 and one in SMA; monkeys subjected to cortical lesion of M1 hand area (n=4) were injected in PM; animals subjected to MPTP lesion mimicking PD (n=4) were injected in either PM (n=2) or M1 (n=2); finally monkeys subjected to unilateral cervical hemisection at C7/C8 (n=5; SCI) were injected in M1.Statistics were calculated on the basis of the number of boutons in each nucleus and were derived from the Paired t-test /Wilcoxon; * p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001 (bilateral comparison).
METHODSINTRODUCTION
RESULTS
The greater number of corticobulbar projections was found in the main nuclei of the Pontomedullary reticular formation (PMRF), namely in PnO, PnC, Gi, IRt and LRt. Intact animals injected in either PM or SMA showed denser corticobulbar projections as compared to M1 (Fig. 3A + 3B). For Mk-R13 (PM) on both contralateral and ipsilateral sides the largest percentage of terminals was found in the Gi nucleus. The same was true for Mk-CH (PM); however the three most rostral sections were unavailable for this animal. In contrast, Mk-R12 (PMd) showed a comparable percentage of axonal boutons in PnO+PnC and Gi on both the ipsilateral and contralateral sides (Fig. 2A). Animals injected in M1 showed a large percentage of projections to the ipsilateral Gi and contralateral IRt and LRt. For Mk-93-80 the projections to IRt and LRt was mostly contralateral. Few axonal boutons were found in PnO+PnC (Fig. 2B). Mk-M93-81 (SMA) showed the largest percentage of axonal boutons on both sides in both the Gi nucleus and the PnO+PnC nuclei (Fig. 2A). In M1 lesioned monkeys the corticobulbar projections from PM decreased in density (Fig. 3A and 3C), with a majority of boutons in PnO+PnC and Gi nuclei (Fig. 2C). In animals with Parkinson's disease (PD) there was a strong decrease of corticobulbar projections from PM (Fig. 3A), whereas projections from M1 showed a slighter decrease as compared to intact animals (Fig. 3B). In general a comparable number of projections to PnO+PnC and Gi nuclei was observed, even in M1 injected monkeys (Fig. 2D). In SCI animals injected in M1, we found an increase of corticobulbar projections, mainly contralateral, in monkeys subjected to anti-Nogo A antibody treatment (Fig. 3B and 3D). The majority of corticobulbar projections were observed in Gi nucleus and in IRt + LRt nuclei (Fig. 2E).
Intact monkeys: The corticobulbar projections originating from PM and SMA are denser then from M1 (Fig. 3C and D). Moreover, the corticobulbar projection from PM and SMA tend to be more prominent on the ipsilateral PMRF than contralaterally; this is the other way around for the corticobulbar projection from M1.M1 lesion and PD: For both pathologies, there was a decrease of corticobulbar projections (as compared to intact monkeys), from PM after M1 lesion and from both PM and M1 in case of PD (Fig. 3C and D). Spinal cord injury (SCI): As compared to intact monkeys, no change in the two control SCI monkeys; in contrast, 2 out of 3 anti-Nogo A antibody treated monkeys showed an increase of the corticobulbar projections from M1, with contralateral predominance as well (Fig. 3D).
CONCLUSIONS
Supported by: Swiss National Science Fundation (SNF), Grants to E.M.R. No.31-61857.00, 310000-110005, 31003A-132465, 310030B-149643; The National Center of Competence in Research (NCCR) on «Neural Plasticity and Repair»; Novartis Foundation; The Swiss Primate Competence Center for Research (SPCCR): http://www.unifr.ch/spccr/home/
Figure 2: Percentage distribution of boutons across brainstem nuclei. Percentage of boutons en passant or terminaux ipsilateral (blue) and contralateral (orange) calculated on the total number of boutons found in the whole brainstem ipsilaterally or contralaterally to the BDA injection site. In each graph, the sum of all orange bars is 100%. The sum of all blue bars is 100%. The blue frame shows data obtained from animals injected in PM, the pink frame shows data obtained from monkeys injected in SMA and finally the green frame shows the data obtained from animals injected in M1.
Injection sites of BDA tracer into one area of the
motor cortex (PM)
Micrographs of BDA anterograde labelling
Micrographs of Nissl staining
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Figure 3: Histograms showing the total numbers of boutons en passant and terminaux in the whole brainstem (Tot), in its caudal half (C; from section 7 to section 12) and in its rostral half (R; from section 1 to section 6). Black bars are for ipsilateral projections and white bars for contralateral ones.Panel A) Histogram representing the row data (absolute numbers of boutons) for projections arising either from SMA (pink background) or from PM (blue background) in intact animals, in monkeys subjected to M1 hand lesion and in PD animals. Panel B) Histogram showing the row data for animals injected in M1 (green background) in intact animals, in monkeys subjected to cervical SCI and in PD animals. Panel C) The same data as in A but normalized. Panel D) The same data as in B but normalized. Normalization was performed with reference to the number of BDA labelled corticospinal axons observed above the Decussatio pyramidum.
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Figure 1: brainstem drawings of coronal sections of Mk-R13 arranged from rostral (section 1) to caudal (section 12). All nuclei are delineated with a different color (see list of abbreviations). Axons located ipsilateral to the BDA injection are marked in blue whereas those located contralaterally are marked in bordeaux. Boutons en passant and terminaux ipsilateral to the BDA injection are marked as green circles whereas the contralateral terminals are marked as blue squares.
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Stem axonsPn
Py
mcp
Mo5 PnORaphe
RtTg ml
LCVC
Py
ml
Sp5
7N8n
GiCN
Pr56NPnC
Py
ml
Mo5 Raphe
Sp5
IRtGi
VC
Sp5
IRtGi
VC VC
Sp5
Gi
Cu
Ecu
n
nn
IRt
Sol
Boutons en passant and terminaux
0
1000
2000
3000
Mk-LY
Tot C R
Mk-MI
Tot C RTot C RTot C R
Mk-Z182 Mk-M310
Tot C R
Mk-M93-80
Tot C RTot C R
Mk-CG Mk-CP
Tot C R
Mk-AC
Tot C R
Mk-AP
Tot C R
Mk-AG
Norm
aliz
ed n
um
ber
of bouto
ns
*
***
*
**
***
**
*
**
**
** *
*
0
1000
2000
3000
4000
Abso
lute
num
ber
of bouto
ns
Intact SCI
Anti Nogo A Ab
PD
Control Ab
Tot C RTot C R
Mk-Z182 Mk-M310
Tot C R
Mk-M93-80
Tot C RTot C R
Mk-CG Mk-CP
Tot C R
Mk-AC
Tot C R
Mk-AP
Tot C R
Mk-AG Mk-LY
Tot C R
Mk-MI
Tot C R
*
***
* **
***
**
*
**
**
** *
*
B M1 injection
D
Norm
aliz
ed n
um
ber
of bouto
ns
0
1000
2000
3000
Mk-R13 Mk-R12 Mk-CH
Tot C R Tot C R Tot C RTot C R
Mk-MO
Tot C R
Mk-VA
Tot C R
Mk-RO
Tot C R
Mk-BI
Tot C R
Mk-M93-81
Tot C R
Mk-LL
Tot C R
Mk-MY
***
*
*****
****
****
*
*
*
**
Intact
Abso
lute
num
ber
of bouto
ns
0
1000
2000
3000
4000
Tot C R
Mk-LL
Tot C R
Mk-MYMk-R13 Mk-R12 Mk-CH
Tot C R Tot C R Tot C RTot C R
Mk-MO
Tot C R
Mk-VA
Tot C R
Mk-RO
Tot C R
Mk-BI
Tot C R
Mk-M93-81
Intact M1 lesion PD
Anti-Nogo A Ab Untreated
***
*
*****
****
****
**
*
**
A PM injectionSMA injection
C
Abbreviations
Medial leminiscusTrigeminal motor nucleus
Ponto Medullary Reticular FormationPontine nuclei
Pontine reticular nucleus caudalisPontine reticular nucleus oralis
Prepositus nucleusPrincipal sensory trigeminal nucleus
Pyramidal tractRaphe nuclei
Reticulo tegmental nucleus of PonsSolitary nucleus
Superior olivary complexSpinal sensory trigeminal nucleus
Vestibular complex
mlMo5
PMRFPn
PnCPnOPr
Pr5Py
RapheRtTgSol
SOCSp5VC
6N7N5n7n8n9n
12NCNCuEcuGiIO
LC
mcp
Abducens nucleusFacial nucleus
Trigeminal nerveFacial nerve
Vestibulocochlear nerveGlossopharyngeal nerve
Hypoglossal nucleusCochlear nucleusCuneate nucleus
External cuneate nucleusGigantocellular reticular nucleus
Inferior oliveIntermediate reticular nucleus
Locus coeruleusLateral reticular nucleus
Middle cerebellar peduncleRtL
RtI
