Corticospinal and Reticulospinal Contacts on Cervical Commissural and Long Descending Propriospinal Neurons in the Adult Rat Spinal Cord; Evidence for Powerful Reticulospinal Connections

Abstract

Descending systems have a crucial role in the selection of motor output patterns by influencing the activity of interneuronal networks in the spinal cord. Commissural interneurons that project to the contralateral grey matter are key components of such networks as they coordinate left-right motor activity of fore and hind-limbs. The aim of this study was to determine if corticospinal (CST) and reticulospinal (RST) neurons make significant numbers of axonal contacts with cervical commissural interneurons. Two classes of commissural neurons were analysed: 1) local commissural interneurons (LCINs) in segments C4-5; 2) long descending propriospinal neurons (LDPNs) projecting from C4 to the rostral lumbar cord. Commissural interneurons were labelled with Fluorogold and CST and RST axons were labelled by injecting the b subunit of cholera toxin in the forelimb area of the primary somatosensory cortex or the medial longitudinal fasciculus respectively. The results show that LCINs and LDPNs receive few contacts from CST terminals but large numbers of contacts are formed by RST terminals. Use of vesicular glutamate and vesicular GABA transporters revealed that both types of cell received about 80% excitatory and 20% inhibitory RST contacts. Therefore the CST appears to have a minimal influence on LCINs and LDPNs but the RST has a powerful influence. This suggests that left-right activity in the rat spinal cord is not influenced directly via CST systems but is strongly controlled by the RST pathway. Many RST neurons have monosynaptic input from corticobulbar pathways therefore this pathway may provide an indirect route from the cortex to commissural systems. The cortico-reticulospinal-commissural system may also contribute to functional recovery following damage to the CST as it has the capacity to deliver information from the cortex to the spinal cord in the absence of direct CST input.

Fig 1. Injection sites for experiments with cervical local commissural interneurons for individual rats used in the study.

A: A1 Representative CTb injection site showing immunoperoxidase reaction in the cortex. A2-A4 are reconstructions of injection sites for 3 rats. Injections are focused on M1 with spread into M2 and S1. B: B1 is a representative fluorescence/dark field micrograph showing a FG injection site within a transverse section of the cervical cord (C4); B2-B4 are reconstructions of spinal injection sites for cortical experiments for the rats shown in A. C: C1 is a CTb injection site in the medial longitudinal fasciculus (MLF); C2-C4 are reconstructions of MLF injection sites for 3 rats. D: D1 is a representative fluorescence/dark field micrograph showing a FG injection site within a transverse section of the C5 Segment; D2-D4 are reconstructions of spinal injection sites for MLF experiments for rats shown in C. The darkest shading represents the core of the injection and lighter shading represents spread beyond the core. Brain and spinal templates are taken from the atlas of Paxinos & Watson, [38].

Fig 2. Injection sites for experiments with long descending propriospinal interneurons for individual rats used in the study.

A: A1 is a representative CTb injection site showing immunoperoxidase reaction in the cortex; A2-A4 are reconstructions of cortical injection sites for 3 rats. Injections are focused on M1 with spread into M2 and S1. B: B1 is a representative fluorescence/dark field micrograph showing a FG injection site within a transverse section of the lumbar spinal cord (L2); B2-B4 are reconstructions of spinal injection sites for cortical experiments for rats shown in A.C: C1 is a CTb injection site in the medial longitudinal fasciculus (MLF); C2-C4 are reconstructions of MLF injection sites for 3 rats. D: D1 is a representative fluorescence/dark field micrograph showing a FG injection site within a transverse section of the L2 segment; D2-D4 are reconstructions of spinal injection sites for MLF experiments for the rats shown in C. The darkest shading represents the core of the injection and lighter shading represents spread beyond the core. Brain and spinal templates are taken from the atlas of Paxinos & Watson [38].

Fig 3. Distribution of local commissural interneurons and long descending propriospinal interneurons in relation to corticospinal and reticulospinal terminations in the cervical spinal cord for each rat used in the study.

Cells are represented as green dots and axon terminals as small black dots on spinal outline diagrams of the C4/5 cervical segments. A1-4: Distribution of local commissural interneurons (LCINs) and corticospinal terminals (CST). A4 is a projected confocal image showing the distribution of LCINs (green) CST terminals (red) adjacent to the central canal. B1-4: Distribution of LCINs and reticulospinal (RST) terminals. B4 is a projected confocal image showing LCINs and RST terminals. C1-4: Distribution of long descending propriospinal neurons (LDPNs) and CST terminals. C4 is a projected confocal image showing LDPNs and CST terminals. D1-4: Distribution of LDPNs and RST terminals. D4 is a projected confocal image showing LDPNs and RST terminals. CC = central canal in confocal images. Scale bars = 50μm.

Fig 4. Confocal images of corticospinal and reticulospinal contacts on local commissural interneurons.

A: a projected confocal microscope image of a FG-labelled LCIN (green) and CTb-labelled CST terminals (red) taken from the medial region of lamina VII in segment C4. Scale bar = 20μm. Insets A1—A4 are single optical sections that correspond to the region demarcated in A showing a single CST contact on this cell (arrow). A1 immunoreactivity for FG; A2 immunoreactivity for CTb; A3 immunoreactivity for VGLUT1. A4 is a merged image of A1-3. Scale bar = 5 μm. B: a projected confocal microscope image of a FG-labelled cell (green) and CTb-labelled RST terminals (red) taken from the medial region of lamina VII in segment C5. Scale bar = 20μm. Insets B1—B4 are single optical sections that correspond to the region demarcated in B. B1 immunoreactivity for FG; B2 immunoreactivity for CTb; B3 immunoreactivity for VGLUT2. A4 is a merged image of B1-3: the arrows indicate VGLUT2 positive contacts. Scale bar = 5 μm. C: a projected confocal microscope image of a LCIN (green) and CTb-labelled RST terminals (red) in segment C5. Scale bar = 20μm. C1—C4 are single optical sections that correspond to the region demarcated in C. C1 immunoreactivity for FG; C2 immunoreactivity for CTb; C3 immunoreactivity for VGAT. A4 is a merged image of C1-3: the arrows indicate VGAT-positive contacts. Scale bar = 5 μm.

Fig 5. Confocal images of corticospinal and reticulospinal contacts on long descending propriospinal neurons.

A: a projected confocal microscope image of a FG-labelled LDPN (green) and CTb-labelled CST terminals (red) taken from the medial region of lamina VII (contralateral to the L1/L2 injection site; segment C5). Scale bar = 20μm. Insets A1—A4 are single optical sections that correspond to the region demarcated in A showing two CST contact on this cell (arrows). A1 immunoreactivity for FG; A2 immunoreactivity for CTb; A3 immunoreactivity for VGLUT1. A4 is a merged image of A1-3. Scale bar = 5 μm. B: a projected confocal microscope image of a FG-labelled cell (green) and CTb-labelled RST terminals (red) taken from the medial region of lamina VII (contralateral to lumbar injection; segment C5). Scale bar = 20μm. Insets B1—B4 are single optical sections that correspond to the region demarcated in B. B1 immunoreactivity for FG; B2 immunoreactivity for CTb; B3 immunoreactivity for VGLUT2. B4 is a merged image of B1-3: the arrows indicate VGLUT2-positive contacts. Scale bar = 5 μm. C: a projected confocal microscope image of a LDPN (green) and CTb-labelled RST terminals (red) in segment C5. Scale bar = 20μm. C1—C4 are single optical sections that correspond to the region demarcated in C. C1 immunoreactivity for FG; C2 immunoreactivity for CTb; C3 immunoreactivity for VGAT. C4 is a merged image of C1-3: the arrows indicate VGAT-positive contacts. Scale bar = 5 μm.

Fig 6. Contact densities on local commissural interneurons (LCINs) and long descending propriospinal interneurons (LDPNs) in the cervical spinal cord.

Each data point represents the contact density of an individual cell. Black dots represent somatic contacts and grey dots represent dendritic contacts. Units are in contacts/100 μm². CST = corticospinal tract; RST (VGLUT2) = contacts made by reticulospinal tract terminals immunoreactive for VGLUT2; RST (VGAT) = contacts made by reticulospinal tract terminals immunoreactive for VGAT. Note the difference in scale of Y axis of A and D showing paucity of CST contacts on both LCINs and LDPNs.

Fig 7. Contact densities of reticulospinal axons on ipsilateral and contralateral long descending propriospinal interneurons (LDPNs) in the cervical spinal cord.

A and B show total RST contacts and VGLUT2 contacts on somata and dendrites of ipsilateral and contralateral cells to the injection site. C and D show total RST contacts and VGAT contacts on somata and dendrites of ipsilateral and contralateral cells to the injection site. Units are in contacts/100 μm². VGLUT2^+CTb = contacts made by reticulospinal tract terminals immunoreactive for VGLUT2; VGAT^+CTb = contacts made by reticulospinal tract terminals immunoreactive for VGAT. I = Cells ipsilateral to L2 injection site; X = Cells contralateral to L2 injection site.