Interrelationships Between Spinal Sympathetic Preganglionic Neurons, Autonomic Systems and Electrical Synapses Formed by Connexin36-containing Gap Junctions.

Spinal sympathetic preganglionic neurons (SPNs) are among the many neuronal populations in the mammalian central nervous system (CNS) where there is evidence for electrical coupling between cell pairs linked by gap junctions composed of connexin36 (Cx36). Understanding the organization of this coupling in relation to autonomic functions of spinal sympathetic systems requires knowledge of how these junctions are deployed among SPNs. Here, we document the distribution of immunofluorescence detection of Cx36 among SPNs identified by immunolabelling of their various markers, including choline acetyltransferase, nitric oxide and peripherin in adult and developing mouse and rat. In adult animals, labelling of Cx36 was exclusively punctate and dense concentrations of Cx36-puncta were distributed along the entire length of the spinal thoracic intermediolateral cell column (IML). These puncta were also seen in association with SPN dendritic processes in the lateral funiculus, the intercalated and central autonomic areas and those within and extending medially from the IML. All labelling for Cx36 was absent in spinal cords of Cx36 knockout mice. High densities of Cx36-puncta were already evident among clusters of SPNs in the IML of mouse and rat at postnatal days 10-12. In Cx36BAC::eGFP mice, eGFP reporter was absent in SPNs, thus representing false negative detection, but was localized to some glutamatergic and GABAergic synaptic terminals. Some eGFP+ terminals were found contacting SPN dendrites. These results indicate widespread Cx36 expression in SPNs, further supporting evidence of electrical coupling between these cells, and suggest that SPNs are innervated by neurons that themselves may be electrically coupled.

Candidate Interneurons Mediating the Resetting of the Locomotor Rhythm by Extensor Group I Afferents in the Cat.

Sensory information arising from limb movements controls the spinal locomotor circuitry to adapt the motor pattern to demands of the environment. Stimulation of extensor group (gr) I afferents during fictive locomotion in decerebrate cats prolongs the ongoing extension, and terminates ongoing flexion with an initiation of the subsequent extension, i. e. "resetting to extension". Moreover, instead of the classical Ib non-reciprocal inhibition, stimulation of extensor gr I afferents produces a polysynaptic excitation in extensor motoneurons with latencies (∼3.5-4.0 ms) compatible with 3 interposed interneurons. We assume that some interneurons in this pathway actually belong to the rhythm-generating layer of the locomotor Central Pattern Generator (CPG), since their activity was correlated to a resetting of the rhythm. In the present work fictive locomotion was (mostly) induced by i.v. injection of nialamide followed by l-DOPA in paralyzed cats following decerebration and spinalization at C1 level. In some experiments, we extended previous observations during fictive locomotion on the emergence and locomotor state-dependence of polysynaptic excitatory postsynaptic potentials from extensor gr I afferents to ankle extensor motoneurons. However, the main focus was to record location and properties of interneurons (n = 62) that (i) were active during the extensor phase of fictive locomotion and (ii) received short-latency excitation (mono-, di- or polysynaptic) from extensor gr I afferents. We conclude that the interneurons recorded fulfill the characteristics to belong to the neuronal pathway activated by extensor gr I afferents during locomotion, and may contribute to the 'resetting to extension' as part of the locomotor CPG.

Connexin36 Expression in Primary Afferent Neurons in Relation to the Axon Reflex and Modality Coding of Somatic Sensation.

Electrical coupling mediated by connexin36-containing gap junctions that form electrical synapses is known to be prevalent in the central nervous system, but such coupling was long ago reported also to occur between cutaneous sensory fibers. Here, we provide evidence supporting the capability of primary afferent fibers to engage in electrical coupling. In transgenic mice with enhanced green fluorescent protein (eGFP) serving as a reporter for connexin36 expression, immunofluorescence labeling of eGFP was found in subpopulations of neurons in lumbar dorsal root and trigeminal sensory ganglia, and in fibers within peripheral nerves and tissues. Immunolabeling of connexin36 was robust in the sciatic nerve, weaker in sensory ganglia than in peripheral nerve, and absent in these tissues from Cx36 null mice. Connexin36 mRNA was detected in ganglia from wild-type mice, but not in those from Cx36 null mice. Labeling of eGFP was localized within a subpopulation of ganglion cells containing substance P and calcitonin gene-releasing peptide, and in peripheral fibers containing these peptides. Expression of eGFP was also found in various proportions of sensory ganglion neurons containing transient receptor potential (TRP) channels, including TRPV1 and TRPM8. Ganglion cells labeled for isolectin B4 and tyrosine hydroxylase displayed very little co-localization with eGFP. Our results suggest that previously observed electrical coupling between peripheral sensory fibers occurs via electrical synapses formed by Cx36-containing gap junctions, and that some degree of selectivity in the extent of electrical coupling may occur between fibers belonging to subpopulations of sensory neurons identified according to their sensory modality responsiveness.

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.

Ipsilateral actions from the feline red nucleus on hindlimb motoneurones.

The main aim of the study was to investigate whether neurones in the ipsilateral red nucleus (NR) affect hindlimb motoneurones. Intracellular records from motoneurones revealed that both EPSPs and IPSPs were evoked in them via ipsilaterally located premotor interneurones by stimulation of the ipsilateral NR in deeply anaesthetized cats in which only ipsilaterally descending tract fibres were left intact. When only contralaterally descending tract fibres were left intact, EPSPs mediated by excitatory commissural interneurones were evoked by NR stimuli alone while IPSPs mediated by inhibitory commissural interneurones required joint stimulation of the ipsilateral NR and of the medial longitudinal fascicle (MLF, i.e. reticulospinal tract fibres). Control experiments led to the conclusion that if any inadvertently coactivated axons of neurones from the contralateral NR contributed to these PSPs, their effect was minor. Another aim of the study was to investigate whether ipsilateral actions of NR neurones, pyramidal tract (PT) neurones and reticulospinal tract neurones descending in the MLF on hindlimb motoneurones are evoked via common spinal relay neurones. Mutual facilitation of these synaptic actions as well as of synaptic actions from the contralateral NR and contralateral PT neurones showed that they are to a great extent mediated via the same spinal neurones. A more effective activation of these neurones by not only ipsilateral corticospinal and reticulospinal but also rubrospinal tract neurones may thus contribute to the recovery of motor functions after injuries of the contralateral corticospinal tract neurones. No evidence was found for mediation of early PT actions via NR neurones.

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.

Uncrossed actions of feline corticospinal tract neurones on lumbar interneurones evoked via ipsilaterally descending pathways.

Effects of stimulation of ipsilateral pyramidal tract (PT) fibres were analysed in interneurones in midlumbar segments of the cat spinal cord in search of interneurones mediating disynaptic actions of uncrossed PT fibres on hindlimb motoneurones. The sample included 44 intermediate zone and ventral horn interneurones, most with monosynaptic input from group I and/or group II muscle afferents and likely to be premotor interneurones. Monosynaptic EPSPs evoked by stimulation of the ipsilateral PT were found in 12 of the 44 (27%) interneurones, while disynaptic or trisynaptic EPSPs were evoked in more than 75%. Both appeared at latencies that were either longer or within the same range as those of disynaptic EPSPs and IPSPs evoked by PT stimuli in motoneurones, making it unlikely that premotor interneurones in pathways from group I and/or II afferents relay the earliest actions of uncrossed PT fibres on motoneurones. These interneurones might nevertheless contribute to PT actions at longer latencies. Uncrossed PT actions on interneurones were to a great extent relayed via reticulospinal neurones with axons in the ipsilateral medial longitudinal fascicle (MLF), as indicated by occlusion and mutual facilitation of actions evoked by PT and MLF stimulation. However, PT actions were also relayed by other supraspinal or spinal neurones, as some remained after MLF lesions. Mutual facilitation and occlusion of actions evoked from the ipsilateral and contralateral PTs lead to the conclusion that the same midlumbar interneurones in pathways from group I or II muscle afferents may relay uncrossed and crossed PT actions.

Uncrossed actions of feline corticospinal tract neurones on hindlimb motoneurones evoked via ipsilaterally descending pathways.

Despite numerous investigations on the corticospinal system there is only scant information on neuronal networks mediating actions of corticospinal neurones on ipsilateral motoneurones. We have previously demonstrated double crossed pathways through which pyramidal tract neurones can influence ipsilateral motoneurones, via contralaterally descending reticulospinal neurones and spinal commissural interneurones. The aim of the present study was to examine the effects of stimulation of pyramidal tract (PT) fibres mediated via ipsilaterally descending pathways and to find out which neurones relay these effects. This was done by using intracellular recordings from 96 lumbar motoneurones in deeply anaesthetized cats. To eliminate actions of fibres descending on the side contralateral to the location of the motoneurones, the spinal cords were hemisected on this side at a low-thoracic level. Stimuli that selectively activated ipsilateral PT fibres evoked EPSPs and/or IPSPs in 34/47 motoneurones tested. These PSPs were evoked at latencies indicating that the most direct coupling between PT neurones and motoneurones in uncrossed pathways is disynaptic. Occlusion and spatial facilitation between actions evoked by stimulation of ipsilateral PT and of reticulospinal tract fibres in the ipsilateral medial longitudinal fascicle (MLF) indicated that PT actions are mediated by reticulospinal neurones with axons in the MLF. However, after transection of the MLF in the caudal medulla, stimulation of the ipsilateral PT continued to evoke EPSPs and IPSPs with characteristics similar to when the MLF was intact (in 15/49 motoneurones) suggesting the existence of parallel disynaptic pathways via other relay neurones.

Neuronal relays in double crossed pathways between feline motor cortex and ipsilateral hindlimb motoneurones.

Coupling between pyramidal tract (PT) neurones and ipsilateral hindlimb motoneurones was investigated by recording from commissural interneurones interposed between them. Near maximal stimulation of either the left or right PT induced short latency EPSPs in more than 80% of 20 commissural interneurones that were monosynaptically excited by reticulospinal tract fibres in the medial longitudinal fascicle (MLF). The EPSPs were evoked at latencies that were only 1-2 ms longer than those of EPSPs evoked from the MLF, compatible with a disynaptic coupling between PT fibres and these commissural interneurones. EPSPs evoked by PT stimulation were frequently associated with IPSPs which either followed or preceded the EPSPs. The latencies of the IPSPs (on average about 1 ms longer than latencies of the earliest EPSPs) indicated that they were mediated via single additional inhibitory interneurones. Records from a sample of nine commissural interneurones from a different population (with monosynaptic input from group I and/or II muscle afferents, and disynaptically excited from the MLF) suggest that actions of PT fibres on such interneurones are weaker because only four of them were excited by PT stimuli and at longer latencies. By demonstrating disynaptic coupling between PT neurones and commissural interneurones via reticulospinal fibres, the results provide a direct demonstration of trisynaptic coupling in the most direct pathways between PT neurones and ipsilateral motoneurones, and thereby strengthen the proposal that the double crossed pathways between PT neurones and ipsilateral motoneurones might be used to replace crossed actions of damaged PT neurones.

Same spinal interneurons mediate reflex actions of group Ib and group II afferents and crossed reticulospinal actions.

The aim of the study was to analyze interactions between neuronal networks mediating centrally initiated movements and reflex reactions evoked by peripheral afferents; specifically whether interneurons in pathways from group Ib afferents and from group II muscle afferents mediate actions of reticulospinal neurons on spinal motoneurons by contralaterally located commissural interneurons. To this end reticulospinal tract fibers were stimulated in the contralateral medial longitudinal fascicle (MLF) in chloralose-anesthetized cats in which the ipsilateral half of the spinal cord was transected rostral to the lumbosacral enlargement. In the majority of interneurons mediating reflex actions of group Ib and group II afferents, MLF stimuli evoked either excitatory or inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) or both EPSPs and IPSPs attributable to disynaptic actions by commissural interneurons. In addition, in some interneurons EPSPs were evoked at latencies compatible with monosynaptic actions of crossed axon collaterals of MLF fibers. Intracellular records from motoneurons demonstrated that both excitation and inhibition from group Ib and group II afferents are modulated by contralaterally descending reticulospinal neurons. The results lead to the conclusion that commissural interneurons activated by reticulospinal neurons affect motoneurons not only directly, but also by enhancing or weakening activation of premotor interneurons in pathways from group Ib and group II afferents. The results also show that both excitatory and inhibitory premotor interneurons are affected in this way and that commissural interneurons may assist in the selection of reflex actions of group Ib and group II afferents during centrally initiated movements.

Control of locomotor cycle durations.

In intact animals and humans, increases in locomotor speed are usually associated with decreases in step cycle duration. Most data indicate that the locomotor central pattern generator (CPG) shortens cycle duration mainly by shortening the durations of extensor rather than flexor phases of the step cycle. Here we report that in fictive locomotion elicited by electrical stimulation of the midbrain locomotor region (MLR) in the cat, spontaneous variations in cycle duration were due more to changes in flexor rather than extensor phase durations in 22 of 31 experiments. The locomotor CPG is therefore not inherently extensor- or flexor-biased. We coined the term "dominant" to designate the phase (flexion or extension) showing the larger variation. In a simple half-center oscillator model, experimental phase duration plots were fitted well by adjusting two parameters that corresponded to background drive ("bias") and sensitivity ("gain") of the oscillator's timing elements. By analogy we argue that variations in background drive to the neural timing elements of the CPG could produce larger variations in phase duration in the half-center receiving the lower background drive, i.e., background drive may determine which half-center is dominant. The fact that data from normal cats were also fitted well by the model indicates that sensory input and central drive combine to determine locomotor phase durations. We conclude that there is a considerable flexibility in the control of phase durations in MLR-induced fictive locomotion. We posit that this may be explained by changes in background excitation of neural timing elements in the locomotor CPG.