On the distribution of spinal premotor interneurons

The activity of flexor and extensor motor neurons is tightly regulated by a network of interneurons in the spinal cord. The introduction of rabies retrograde monosynaptic tracing has provided a powerful method to map interneurons directly connected to motor neurons so as to visualize premotor circuits. Previous strategies have used AAV for complementing rabies glycoprotein expression in motor neurons to obtain selectivity in transsynaptic transfer to identify premotor interneurons innervating specific motor neuron pools These studies revealed differences in the location of flexor and extensor premotor interneurons. Here, we report that by using a genetic approach to complement rabies glycoprotein expression in motor neurons, we did not observe any differences in the distribution of flexor and extensor premotor interneurons. In order to identify possible causes for these paradoxical findings, we discuss advantages and caveats of the experimental designs and suggest ways forward to resolve possible ambiguities. Furthermore, to obtain a complete picture of existing approaches and results we ask for contributions from the scientific community describing the use of additional mouse models, viral constructs, and complementation methods. The aim is to generate an open, comprehensive database to understand the specific organisation of premotor circuits

Organization and neurochemical properties of intersegmental interneurons in the lumbar enlargement of the adult rat

Intersegmental interneurons with relatively short axons perform an important role in the coordination of limb movement but surprisingly little is known about their organization and how they contribute to neuronal networks in the adult rat. We undertook a series of anatomical tract-tracing studies to label cell bodies and axons of intersegmental neurons in the lumbar cord and characterized their neurochemical properties by using immunocytochemistry. The b-subunit of cholera toxin was injected into L1 or L3 segments of seven rats in the vicinity of lateral or medial motor nuclei. In L5 lumbar segments, cells were found to be concentrated in contralateral lamina VIII, and in ipsilateral lamina VII and laminae V–VI following injections into the lateral and medial motor nuclei respectively. About 25% of labelled cells contained calbindin or calretinin or a combination of both. Calbindin positive cells were mainly distributed within the ipsilateral side of the L5 segment, especially within the ipsilateral dorsal horn whereas there was a concentration of calretinin cells in contralateral lamina VIII. A small population of cells around the central canal were cholinergic. We also examined axon terminals that projected from L1/3 to the L5 contralateral lateral motor nucleus. The majority of these axons were excitatory (75%) and made direct contacts with motoneurons. However, most inhibitory axons in L5 contained a mixture of GABA and glycine (20%) and about 22% of the total population of axons contained calbindin. In contrast, 19% of all intra-segmental axons in the L3 contralateral lateral motor nucleus were found to be purely glycinergic and 17% contained a mixture of GABA and glycine. This study shows that short range interneurons form extensive ipsi- and contralateral projections within the lumbar enlargement and that many of them contain calcium binding proteins. Those projecting contralaterally to motor nuclei are predominantly excitatory

Ascending and descending propriospinal pathways between lumbar and cervical segments in the rat: evidence for a substantial ascending excitatory pathway

Precise mechanisms are required to coordinate the locomotor activity of fore- and hind-limbs in quadrupeds and similar mechanisms persist to coordinate movement of arms and legs in humans. Propriospinal neurons (PSNs) are major components of the networks that coordinate these mechanisms. The b subunit of cholera toxin (CTb) was injected unilaterally into either L1 or L3 segments in order to label ascending and descending propriospinal pathways. Labelled cells were examined with light or confocal microscopy. Cells projecting to lumbar segments were evenly distributed, bilaterally throughout all cervical segments. However many more cells were labelled from L1 injections than L3 injections. Roughly 15% of cells in both sides of the C2 segment was found to be immunoreactive for calretinin and a small number (4%) was immunoreactive for calbindin. Axons projecting from L1 to cervical segments formed predominant ipsilateral projections to the cervical intermediate grey matter and ventral horn. Very large numbers of terminals were concentrated within the ventrolateral motor (VLM) nuclei of C7–8 segments but there was sparse innervation of the contralateral nucleus. The vast majority (85%) of these axon terminals in the ipsilateral VML was immunoreactive for the vesicular glutamate transporter 2 (VGLUT2) and the remaining 15% was immunoreactive for the vesicular GABA transporter (VGAT); many of these contained GABA and/or glycine. Inhibitory and excitatory terminals were also found in the contralateral VLM. Most of the terminals in the VLM made contacts with motoneurons. The major finding of this study is the existence of a substantial excitatory propriospinal pathway that projects specifically to the VLM. Motoneurons in the VLM supply muscles of the axilla therefore this pathway is likely to have a profound influence on the activity of the shoulder joint. This pathway may synchronise lumbar and cervical pattern generators and hence the coordination of locomotor activity in the fore- and hind limbs

Cholinergic terminals in the ventral horn of adult rat and cat: evidence that glutamate is a co-transmitter at putative interneuron synapses but not at central synapses of motoneurons

Until recently it was generally accepted that the only neurotransmitter to be released at central synapses of somatic motoneurons was acetylcholine. However, studies on young mice (P0-10) have provided pharmacological evidence indicating that glutamate may act as a co-transmitter with acetylcholine at synapses between motoneurons and Renshaw cells. We performed a series of anatomical experiments on axon collaterals obtained from intracellularly labelled motoneurons from an adult cat and labelled by retrograde transport in adult rats to determine if glutamate is co-localised with acetylcholine by these terminals. We could find no evidence for the presence of vesicular glutamate transporters in motoneuron axon terminals of either species. In addition, we were unable to establish any obvious relationship between motoneuron terminals and the GluR2 subunit of the AMPA receptor. However we did observe a population of cholinergic terminals in lamina VII which did not originate from motoneurons but were immunoreactive for the vesicular glutamate transporter 2 and formed appositions to GluR2 subunits. These were smaller than motoneuron terminals and, unlike them, formed no relationship with Renshaw cells. The evidence suggests that glutamate does not act as a co-transmitter with acetylcholine at central synapses of motoneurons in the adult cat and rat. However, glutamate is present in a population of cholinergic terminals which probably originate from interneurons where its action is via an AMPA recepto

Inhibitory inputs to four types of spinocerebellar tract neurons in the cat spinal cord

Spinocerebellar tract neurons are inhibited by various sources of input via pathways activated by descending tracts as well as peripheral afferents. Inhibition may be used to modulate transmission of excitatory information forwarded to the cerebellum. However it may also provide information on the degree of inhibition of motoneurons and on the operation of inhibitory premotor neurons. Our aim was to extend previous comparisons of morphological substrates of excitation of spinocerebellar neurons to inhibitory input. Contacts formed by inhibitory axon terminals were characterised as either GABAergic, glycinergic or both GABAergic/glycinergic by using antibodies against vesicular GABA transporter (VGAT), glutamic acid decarboxylase (GAD) and gephyrin. Quantitative analysis revealed the presence of much higher proportions of inhibitory contacts when compared with excitatory contacts on spinal border (SB) neurons. However similar proportions of inhibitory and excitatory contacts were associated with ventral spinocerebellar tract (VSCT) and dorsal spinocerebellar tract neurons located in Clarke´s column (ccDSCT) and the dorsal horn (dhDSCT). In all of the cells, the majority of inhibitory terminals were glycinergic. The density of contacts was higher on somata and proximal versus distal dendrites of SB and VSCT neurons but more evenly distributed in ccDSCT and dhDSCT neurons. Variations in the density and distribution of inhibitory contacts found in this study may reflect differences in information on inhibitory processes forwarded by subtypes of spinocerebellar tract neurons to the cerebellum

Neurotransmitter phenotypes of descending systems in the rat lumbar spinal cord

Descending systems from the brain exert a major influence over sensory and motor processes within the spinal cord. Although it is known that many descending systems have an excitatory effect on spinal neurons, there are still gaps in our knowledge regarding the transmitter phenotypes used by them. In this study we investigated transmitter phenotypes of axons in the corticospinal tract (CST); the rubrospinal tract (RST); the lateral component of the vestibulospinal tract (VST); and the reticulospinal tract (ReST). They were labelled anterogradely by stereotaxic injection of the b subunit of cholera toxin (CTb) in to the motor cortex, red nucleus, lateral vestibular nucleus and medial longitudinal fascicle to label CST, RST, VST and ReST axons respectively. Neurotransmitter content of labelled axons was investigated in lumbar segments by using immunofluroescence; antibodies against vesicular glutamate transporters (VGLUT1 and VGLUT2) were used to identify glutamatergic terminals and the vesicular GABA transporter (VGAT) was used to identify GABA- and glycinergic terminals. The results show that almost all CST (96%) axons contain VGLUT1 whereas almost all RST (97%) and VST (97%) axons contain VGLUT2. Although the majority of ReST axons contain VGLUT2 (59%), a sizable minority contain VGAT (20%) and most of these terminals can be subdivided into those that are GABAergic or those that are glycinergic because only limited evidence for co-localisation was found for the two transmitters. In addition, there is a population of ReST terminals that apparently does not contain markers for the transmitters tested and is not serotoninergic. We can conclude that the CST, RST and VST are 'pure' excitatory systems whereas the ReST consists of a heterogeneous population of excitatory and inhibitory axons. It is anticipated that this information will enable inputs to spinal networks to be defined with greater confidence

Networks of inhibitory and excitatory commissural interneurons mediating crossed reticulospinal actions

Axonal projections and neurotransmitters used by commissural interneurons mediating crossed actions of reticulospinal neurons were investigated in adult cats. Eighteen interneurons, located in or close to lamina VIM in midlumbar segments, that were monosynaptically excited by reticulospinal tract fibres and projected to contralateral motor nuclei were labelled by intracellular injection of tetra-methylrhodamine-dextran and Neurobiotin. The nine most completely labelled interneurons were analysed with combined confocal and light microscopy. None of the stem axons gave off ipsilateral axon collaterals. Seven cells had axon collaterals that arborized in the contralateral grey matter in the ventral horn of the same segments. Transmitters were identified by using antibodies raised against vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and the glycine transporter 2. The axons of two cells were immunoreactive for the glycine transporter 2 and hence were glycinergic. Three cells were immunoreactive for the vesicular glutamate transporter 2 and hence were glutamatergic. None of the axons displayed immunoreactivity for glutamic acid decarboxylase. Electron microscopy of two cells revealed direct synaptic connections with motoneurons and other neurons. Axonal swellings of one neuron formed synapses with profiles in motor nuclei whereas those of the other formed synapses with other structures, including cell bodies in lamina VII. The results show that this population of commissural interneurons includes both excitatory and inhibitory cells that may excite or inhibit contralateral motoneurons directly. They may also influence the activity of motoneurons indirectly by acting through interneurons located outside motor nuclei in the contralateral grey matter but are unlikely to have direct actions on interneurons in the ipsilateral grey matter

Spinal premotor interneurons controlling antagonistic muscles are spatially intermingled

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components