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.

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) into the motor cortex, red nucleus, lateral vestibular nucleus and medial longitudinal fascicle (MLF) to label CST, RST, VST and ReST axons respectively. Neurotransmitter content of labelled axons was investigated in lumbar segments by using immunoflurescence; 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 contains 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.

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, glutamic acid decarboxylase 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.

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.

Cholinergic terminals in the ventral horn of adult rat and cat: evidence that glutamate is a cotransmitter 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 cotransmitter with acetylcholine at synapses between motoneurons and Renshaw cells. We performed a series of anatomical experiments on axon collaterals obtained from intracellularly labeled motoneurons from an adult cat and labeled by retrograde transport in adult rats to determine if glutamate is co-localized 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 R2 subunit of the AMPA receptor (GluR2). 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 cotransmitter 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 receptor.

Commissural interneurons with input from group I and II muscle afferents in feline lumbar segments: neurotransmitters, projections and target cells.

The aim of this study was to analyse neurotransmitter content, projection areas and target cells of commissural interneurons with input from group I and/or II muscle afferents in lumbar segments in the cat. Axonal projections of 15 intracellularly labelled commissural interneurons were reconstructed. Ten interneurons (nine located in laminae VI-VII, one in lamina VIII) were glutamatergic; only one interneuron (located in lamina VIII) was glycinergic. Contralateral terminal projections were found both in motor nuclei and within laminae VI-VIII. In order to identify target cells of commissural interneurons, effects of stimulation of contralateral group I and II muscle afferents were investigated on interneurons within these laminae. Three tests were used: intracellular records from individual interneurons, modulation of probability of activation of extracellularly recorded interneurons and modulation of their actions on motoneurons using disynaptic PSPs evoked in motoneurons as a measure. All these tests revealed much more frequent and/or stronger excitatory actions of contralateral afferents. The results indicate that commissural interneurons with input from contralateral group I and II afferents target premotor interneurons in disynaptic pathways from ipsilateral group I and II afferents and that excitatory disynaptic actions of contralateral afferents on these interneurons are mediated primarily by intermediate zone commissural interneurons. A second group of commissural interneurons activated by reticulospinal neurons, previously described, frequently had similar, but occasionally opposing, actions to the cells described here, thus indicating that these two subpopulations may act on the same premotor interneurons and either mutually enhance or counteract each other's actions.

Excitatory and inhibitory intermediate zone interneurons in pathways from feline group I and II afferents: differences in axonal projections and input.

The aim of the present study was to compare properties of excitatory and inhibitory spinal intermediate zone interneurons in pathways from group I and II muscle afferents in the cat. Interneurons were labelled intracellularly and their transmitter phenotypes were defined by using immunocytochemistry. In total 14 glutamatergic, 22 glycinergic and 2 GABAergic/glycinergic interneurons were retrieved. All interneurons were located in laminae V-VII of the L3-L7 segments. No consistent differences were found in the location, the soma sizes or the extent of the dendritic trees of excitatory and inhibitory interneurons. However, major differences were found in their axonal projections; excitatory interneurons projected either ipsilaterally, bilaterally or contralaterally, while inhibitory interneurons projected exclusively ipsilaterally. Terminal projections of glycinergic and glutamatergic cells were found within motor nuclei as well as other regions of the grey matter which include the intermediate region, laminae VII and VIII. Cells containing GABA/glycine had more restricted projections, principally within the intermediate zone where they formed appositions with glutamatergic axon terminals and unidentified cells and therefore are likely to be involved in presynaptic as well as postsynaptic inhibition. The majority of excitatory and inhibitory interneurons were found to be coexcited by group I and II afferents (monosynaptically) and by reticulospinal neurons (mono- or disynaptically) and to integrate information from several muscles. Taken together the morphological and electrophysiological data show that individual excitatory and inhibitory intermediate zone interneurons may operate in a highly differentiated way and thereby contribute to a variety of motor synergies.

Premotor interneurones contributing to actions of feline pyramidal tract neurones on ipsilateral hindlimb motoneurones.

The aim of the study was to analyse the potential contribution of excitatory and inhibitory premotor interneurones in reflex pathways from muscle afferents to actions of pyramidal tract (PT) neurones on ipsilateral hindlimb motoneurones. Disynaptic EPSPs and IPSPs evoked in motoneurones in deeply anaesthetized cats by group Ia, Ib and II muscle afferents were found to be facilitated by stimulation of the ipsilateral, as well as of contralateral, PT. The ipsilateral actions were evoked by either uncrossed or double-crossed pathways. The results show that interneurones mediating reflex actions of muscle afferents may be activated strongly enough by PT stimulation to contribute to movements initiated by ipsilateral PT neurones and that PT actions relayed by them might be enhanced by muscle stretches and/or contractions. However, in some motoneurones disynaptic IPSPs and EPSPs evoked from group Ib or II afferents were depressed by PT stimulation. In order to analyse the basis of this depression, the transmitter content in terminals of 11 intracellularly labelled interneurones excited by PT stimulation was defined immunohistochemically and their axonal projections were reconstructed. The interneurones included 9 glycinergic and 2 glutamatergic neurones. All but one of these neurones were mono- or disynaptically excited by group I and/or II afferents. Several projected to motor nuclei and formed contacts with motoneurones. However, all had terminal projections to areas outside the motor nuclei. Therefore both inhibitory and excitatory interneurones could modulate responses of other premotor interneurones in parallel with direct actions on motoneurones.

On coupling and decoupling of spinal interneuronal networks.

This review addresses the question of interrelations between spinal interneuronal networks. On the basis of electrophysiological, pharmacological, morphological and immunohistochemical analysis of interneurones mediating various reflex actions from muscle receptors and of reticulospinal neurones a considerable degree of interweaving between networks of these neurones has been established. The coupling has been found to occur at the level of several sites of these networks but the review focuses on two of these sites. The first is between dorsal horn interneurones with group II input and their target ipsilaterally and contralaterally projecting intermediate zone and commissural interneurones. The second is between commissural interneurones with input from reticulospinal neurones and their target interneurones. Several ways of both strengthening and weakening of coupling between various interneuronal networks are also briefly reviewed.

New growth elicited in adult leech mechanosensory neurones by peripheral axon damage.

1. New growth in cutaneous mechanosensory neurones elicited by axotomy or axon crush was studied using intracellular injection of horseradish peroxidase at different times after the lesion, ranging from a few days to over a year. 2. Cutting or crushing major, large-calibre axon branches of mechanosensory neurones elicits sprouting of new processes, either centrally within the ganglion neuropile or at the site of the lesion in the peripheral nerve. In contrast, cutting or crushing fine-calibre axon branches supplying accessory parts of the receptive field does not elicit sprouting of the main arbor or main axon branches. 3. Different modalities of mechanosensory neurone respond differently to lesions of their axons. Cutting the axons of high-threshold units responding to noxious stimulation of the skin elicits sprouting of additional processes from the axon hillock region within the central nervous system (CNS), whereas cutting or crushing the axons of low-threshold cells responding to light touch of the skin elicits sprouting at the site of the lesion only, and not within the CNS. 4. In addition to the new growth directed into the peripheral nerve, damaged nociceptive neurones also form new processes that wrap the somata of particular cells within the ganglion. 5. Sprouted processes of axotomized neurones are retained for long periods after the lesion (up to 425 days). 6. The electrical properties of touch and nociceptive cells were studied between 1 and 60 days after axotomy, by intracellular recording from the centrally located cell bodies. The amplitude, width and maximum dV/dt of the action potential and after-hyperpolarization, as well as the resting potential and input resistance, did not change significantly after axotomy, despite the considerable process sprouting known to occur during this time.

Fine structure of synapses associated with characterized postsynaptic dorsal column neurons in the cat.

Fourteen dorsal horn neurons with axons projecting through the dorsal columns were identified either by electrophysiological methods (and subsequently injected with horseradish peroxidase) or by retrograde labelling with horseradish peroxidase in cats. All neurons were contacted by small (less than 2 micron) boutons containing spherical or elongated agranular vesicles. One neuron with its soma located in lamina III received additional contacts from central elements of glomerular complexes. Neurons with somata located more ventrally (deep lamina IV and V) were also postsynaptic to large (greater than 2 microns) electron lucent profiles which formed multiple synapses with the labelled cells. Some boutons presynaptic to postsynaptic dorsal column neurons were themselves postsynaptic to profiles containing pleiomorphic agranular vesicles at axoaxonic synapses. They also occasionally participated in triadic complexes. It is concluded that the synaptic arrangements formed by boutons in association with postsynaptic dorsal column neurons differ significantly from those associated with spinocervical neurons. Such differences might provide the anatomical substrate for the observed receptive field characteristics of these neurons.

Light and electron microscopy of contacts between primary afferent fibres and neurones with axons ascending the dorsal columns of the feline spinal cord.

In addition to primary afferent fibres, the dorsal columns of the cat spinal cord contain ascending second-order axons which project to the dorsal column nuclei. The aim of the present study was to obtain morphological evidence that certain primary afferent axons form monosynaptic contacts with cells of origin of this postsynaptic dorsal column pathway. In ten adult cats, neurones with axons ascending the dorsal columns were retrogradely labelled with horseradish peroxidase using a pellet implantation method in the thoracic dorsal columns. In the lumbosacral regions of the same animals, primary afferent fibres were labelled intra-axonally with ionophoretic application of horseradish peroxidase. Tissue containing labelled axons was prepared for light and combined light and electron microscopy. Ultrastructural examination demonstrated that slowly adapting (Type I), hair follicle, Pacinian corpuscle and group Ia muscle spindle afferents formed monosynaptic contacts with labelled cells and light microscopical analysis suggested that they also received monosynaptic input from rapidly adapting (Krause) afferents. This evidence suggests that sensory information from large-diameter cutaneous and muscle spindle afferent fibres is conveyed disynaptically via the postsynaptic dorsal column pathway to the dorsal column nuclei. Some of the input to this pathway is probably modified in the spinal cord as the majority of primary afferent boutons forming monosynaptic contacts were postsynaptic to other axon terminals. The postsynaptic dorsal column system appears to constitute a major somatosensory pathway in the cat.

Fine structure of primary afferent axon terminals of slowly adapting cutaneous receptors in the cat.

Three slowly adapting type I and two slowly adapting type II afferent fibres from the lumbosacral cord of the cat were intra-axonally labelled with horseradish peroxidase and processed for light and electron microscopy. Terminals from both types of afferent exhibited similar ultrastructural features in that both formed contacts with one to five post-synaptic profiles, including dendritic shafts and spine heads, some of which contained vesicles. The stained axons were themselves post-synaptic in axo-axonic synapses. Maximum diameters of slowly adapting boutons and the dendritic shafts on which they terminated were measured. The present results indicate that there is considerable overlap in the morphological characteristics studied for all large myelinated cutaneous afferent boutons. It is not possible therefore to distinguish between these on ultrastructural grounds alone.

Fine structure of primary afferent axon terminals projecting from rapidly adapting mechanoreceptors of the toe and foot pads of the cat.

Two Pacinian corpuscle afferents and two rapidly adapting afferents from Krause corpuscles were intra-axonally labelled with horseradish peroxidase in the lumbosacral enlargement of the cat's spinal cord. Tissue was prepared for combined light and electron microscopical analysis. Boutons from both classes of afferent had similar ultrastructural appearances. They both formed from one to three synaptic junctions with dendritic shafts and spines and received axo-axonic synapses. In addition, both categories of bouton were seen to be presynaptic to structures interpreted as vesicle-containing dendrites. It is concluded that both types of afferent fibre are subject to presynaptic control and that they synapse with dorsal horn neurones which are possibly interneurones involved in primary afferent depolarization and post-synaptic dorsal column neurones.

Ultrastructure of muscle spindle afferent terminations in lamina VI of the cat spinal cord.

Two group Ia muscle spindle afferents were impaled in the lumbosacral enlargement of the cat's spinal cord and intra-axonally labeled with horseradish peroxidase. Terminations in lamina VI were examined with the electron microscope. Boutons formed synaptic associations with somata and large dendritic shafts in the lamina VI neuropile and received axo-axonic contacts. It is concluded that group Ia terminals synapse with the proximal regions of lamina VI neurons and are under strong presynaptic control.

Ultrastructure of hair follicle afferent fibre terminations in the spinal cord of the cat.

In acute electrophysiological experiments on anaesthetized cats, single identified hair follicle afferent fibres were injected with horseradish peroxidase (HRP). The HRP was injected from an intra-axonal microelectrode in the lumbosacral spinal cord. One to six hours after injection the animals were perfused and the tissue prepared for light and electron microscopy (EM). Axon collateral arborizations containing HRP reaction product were identified in thick sections under the light microscope and the same tissue then cut on the ultramicrotome for EM study. The terminal branches of the collaterals kept their myelin sheaths until they were 0.45-1.0 micron in diameter, just before they formed synaptic boutons. Synaptic boutons (1.0-4.0 microns in diameter) were usually of the en passant variety and made contact with dendrites. The contacts were asymmetrical (Type I) and contained round, clear synaptic vesicles of 35-60 nm diameter. Both the non-myelinated portion of the terminal axon and the synaptic boutons received axo-axonic contacts. These axo-axonic boutons contained clear (agranular) vesicles irregular in profile.