Postsynaptic versus presynaptic inhibition in antagonistic stretch reflexes.

Motoneurons of the cat gastrocnemius-soleus muscle were studied intracellularly with conventional glass micropipettes. Each of these motoneurons was made to fire repetitively by stretch of its own muscle (gastrocnemius-soleus), and by current injected through the impaling microelectrode. By comparing the amount of inhibitory influence from antagonistic stretch of posterior biceps on the repetitive firing in these two different situations, an estimate could be obtained of the relative contribution of postsynaptic inhibition in this type of antagonistic stretch reflex. Even when the experimental conditions were such as to favor presynaptic inhibition, only strong postsynaptic inhibitory effects were seen; presynaptic inhibition was not found.

Substance p: localization in the central nervous system and in some primary sensory neurons.

Antibodies to substance P with a high titer have been produced and used in immunohistochemical studies on the peripheral and central nervous system of the rat and the cat. Evidence was obtained for the localization of substance P in a certain population of primary sensory neurons, probably small nerve cells with unmyelinated processes. Substance P or a peptide similar to it was also observed in cell bodies in the medial habenula and in probable nerve terminals in many brain areas. The results give morphological support for a transmitter (or modulator) role of substance P in the nervous system.

A quantitative light microscopic study of the dendrites of cat spinal alpha-motoneurons after intracellular staining with horseradish peroxidase.

The cell bodies and dendrites of cat spinal alpha-motoneurons were studied after intracellular staining with horseradish peroxidase. The mean diameter of the soma was positively correlated to both the mean diameter and the combined diameter of the first-order dendrites, but not to the number of first-order dendrites. On the average, 11.2 dendrites originated from the soma. The dendritic trees were more extensive than has been described previously. The mean value for the combined length of a whole dendrite was 4.7 mm, while the mean values for the total surface area and volume of a dendrite were 33.0 x 10(3) micron (2) and 27.2 x 10(3) micron (3), respectively. The diameter of the first-order dendrite was positively correlated to the combined length of the entire dendrite, the number of dendritic branching points, and the number of dendritic end branches. The diameter of the first-order dendrite was also directly proportional to the volume and the surface area of the entire dendrite. About 75% of the dendritic surface area and 55% of the dendritic volume was located more than 300 micron away from the soma. The dendrites constituted about 97% of the surface area and about 75% of the volume of the entire motoneuron (excluding the axon). The dendritic tapering was moderate. On the average, the distal decrease in dendritic diameters caused a reduction in the combined dendritic parameter (sigma d 3/2) by 1.5% and 15% at 500 micron and 800 micron distance, respectively, from the soma.

Light microscopic observations on cat Renshaw cells after intracellular staining with horseradish peroxidase. I. The axonal systems.

Five intracellularly HRP-stained Renshaw cells were subjected to light microscopic analysis of the trajectories, branching patterns, and projections of the axonal systems. The cell bodies were located ventrally in lamina VII. In three neurons the axon originated from the cell body and in the remaining two cells from a dendrite. After a 600-870-microns distance the axons entered the ventral funiculus, where all of them continued rostrally. Two axons also gave off a caudal branch in the funiculus. The diameters of the main axons varied between 2.1 and 10.0 microns. The main axons gave off one to four first-order collaterals before entering the ventral funiculus and up to three collaterals could be seen to originate from the same node of Ranvier. In the ventral funiculus up to five first-order collaterals could be traced from the same main axon. The axon collateral trees were often very extensive and daughter branches up to the 22nd order were observed. The distance between two successive branching points varied between 4 and 410 microns. A large number of boutonlike swellings were found along (59%) or at the ends of the collateral branches. At the most, 1,278 swellings originated from a single axon collateral tree. Most of the swellings were located in lamina IX, but they also appeared ventrally and dorsolaterally in lamina VII.

Light microscopic observations on cat Renshaw cells after intracellular staining with horseradish peroxidase. II. The cell bodies and dendrites.

The cell bodies and dendritic trees of five lumbosacral Renshaw cells of adult cats were studied in the light microscope (LM) after intracellular injection with horseradish peroxidase (HRP). The cell bodies were all located in the ventral part of lamina VII. The dendrites extended up to 0.7 mm from the cell body into the neighbouring parts of laminae VIII and IX as well as into more dorsal parts of lamina VII. The dendritic branching was sparse and about half the dendrites were unbranched. The mean diameter of the cell body was positively correlated to both the combined and mean diameters of the first-order dendrites. Between four and eight dendrites originated from the cell bodies. The number of dendritic end-branches, the combined dendritic length, the mean dendritic length from the cell body to the termination of the end branches, the distance from the cell body to the termination of the most remote end-branch, the dendritic surface area, and the dendritic volume all correlated positively with the diameter of the parent first-order dendrite. The dendritic tapering was somewhat more pronounced in the Renshaw cells than previously observed in alpha- and gamma-motoneurons. The present data are discussed in relation to previous morphological observations on Renshaw cells and alpha- and gamma-motoneurons.

A morphological study of the axons and recurrent axon collaterals of cat sciatic alpha-motoneurons after intracellular staining with horseradish peroxidase.

Utilizing the centrifugal neuronal transport of intracellularly injected horseradish peroxidase (HRP), we have performed a light microscopic (LM) investigation of the intramedullary parts of the axons and axon collaterals of sciatic alpha-motoneurons in the adult cat. The intramedullary parts of the alpha-motor axons had comparatively short internodes (down to 75 microns) and were thinner than reported in earlier studies on the ventral root. Positive correlations were obtained when relating nodal diameters (2.8-7.8 micron) or the mean diameters of the motor axons in the white matter (4.4-9.0 micron) to the diameters of the initial axonal segments (2.3-4.9 micron). Eighty percent of the motor axons gave off one to five collaterals. There was no correlation between the numbers of collaterals and the lengths of the parent motor axons in the gray matter. The branching patterns of the axon collaterals showed considerable variation and the number of end branches from a single collateral ranged between 1 and 39. The rostro-caudal distribution of the collateral end branches was arranged symmetrically within a narrow space (+/- 300 micron) around the origins of the first order collaterals. Outbulgings of the motor axon collaterals, interpreted as synaptic terminals, were found along (59%) or at the ends (41%) of the collateral branches, and were located 200-700 micron away from the origin of the first order collateral. No characteristic LM feature of the outbulgings was distinguished.

Electron microscopic studies of serially sectioned cat spinal alpha-motoneurons. II. A method for the description of architecture and synaptology of the cell body and proximal dendritic segments.

The paper presents a method for ultrastructural analysis and description of neuronal architecture and synaptology of cat spinal alpha-motoneurons from complete series of consecutive ultrathin sections through the cell body and proximal parts of the dendrites. The method implies that sections are selected for analysis only at certain constant intervals in the series. The occurrence of boutons of different morphological types on the neuronal surface was expressed by their percentage covering of the neuronal membrane. The neuronal surface was divided into a number of compartments and the synaptic covering was calculated separately for each compartment. An interval of 6 micrometer between the sections was used for these calculations, and the obtained values for synaptic covering were found not to differ significantly from those obtained in controls at 3 micrometer intervals. The number and location of individual large boutons (C- and M-types) were studied at 3 micrometer section intervals, and the escape of boutons connected to this procedure was estimated from control observations at 1 micrometer intervals. It is concluded that detailed information on neuronal synaptology can be obtained with this method, which will be used in three subsequent studies on functionally identified and intracellularly stained cat alpha-motoneurons.

Electron microscopic studies of serially sectioned cat spinal alpha-motoneurons. I. Effects of microelectrode impalement and intracellular staining with the fluorescent dye "Procion Yellow".

Cat spinal alpha-motoneurons were studied in the light and electron microscope after intracellular recording and staining with the fluorescent dye Procion Yellow. Generally, the ultrastructural preservation of the stained neurons improved when the amount of dye delivered was decreased, and when the duration of the microelectrode impalement of the neuron as well as the time between the intracellular staining and the tissue fixation was kept as short as possible. Utilizing the optimal experimental procedure finally arrived at, about one-third of the stained neurons could be used for further quantitative morphometric analysis. With respect to synaptology and gross architecture these cells appeared to differ from control motoneurons mainly with regard to a focal disarrangement of the cell body periphery, probably a result of the microelectrode injury, and a certain degree of damage to some large boutons.

Electron microscopic studies of serially sectioned cat spinal alpha-motoneurons. III. Motoneurons innervating fast-twitch (type FR) units of the gastrocnemius muscle.

Two intracellularly stained cat gastrocnemius alpha-motoneurons of the FR-type (Burke et al., '73) were studied ultrastructurally. The architecture and synaptology of the cell body and proximal parts of the dendrites were analyzed from a long series of consecutive sections, according to a method presented in a preceding paper (Conradi et al., '79a). Several of the dendrites had a base diameter exceeding 10 micrometer. The proportion of the surface covered by boutons was 40-50% for the cell body and about 80% for the proximal dendrites. In both regions, about 20% of the boutons were of the S-type and 70% of the F-type. The large C-boutons were clustered around the dendritic roots and were also present on the axon hillock. M-boutons of dorsal root origin were located on the proximal parts of the majority of the dendrites.

Changes in size and dendritic arborization patterns of adult cat spinal alpha-motoneurons following permanent axotomy.

This study was performed to analyse quantitatively the changes in dimensions and dendritic branching patterns of adult cat spinal alpha-motoneurons following permanent axotomy, i.e., in a situation in which the transected motoraxons are prevented from reinnervating their peripheral target muscle. After transection and ligation of the medial gastrocnemius nerve of adult cats, homonymous alpha-motoneurons were intracellularly labelled with horseradish peroxidase and subjected to quantitative light microscopic analyses. The cell bodies and proximal dendrites were studied at 3, 6, and 12 weeks after the axotomy. An initial increase in cell body size at 3 weeks was followed by a gradual return towards normal values. The mean diameter of the stem dendrites was decreased at all time periods studied, and the combined diameter of the stem dendrites was reduced at 12 weeks after the axotomy. Entire dendritic trees were reconstructed at 12 weeks postoperatively, and the regression equations describing the correlations between dendritic stem diameter, on one hand, and the size of the entire dendrite, on the other, were used to calculate the total dendritic length, volume, and membrane area of whole axotomized motoneurons. The dendritic branching patterns were also analysed. In comparison with normal medial gastrocnemius alpha-motoneurons, the dendritic membrane area and volume of the axotomized cells had decreased by 36% and 29%, respectively, at 12 weeks after the axotomy. This reduction in dendritic size was due to a loss of preterminal and terminal dendritic segments. Abnormal dendritic elongations were observed in 2 of 16 completely reconstructed dendrites.

Restorative effects of reinnervation on the size and dendritic arborization patterns of axotomized cat spinal alpha-motoneurons.

In a preceding paper [Brännström, et al. (1992) J. Comp. Neurol. 318:439-451] a marked reduction in dendritic size was observed in cat spinal motoneurons following permanent axotomy. The aim of the present study was to analyse the possible restorative effects of peripheral reinnervation on the size and dendritic branching patterns of cat spinal motoneurons which had been deprived of neuromuscular contact for an extended period of time. In adult cats the medial gastrocnemius (MG) nerve was transected and ligated. After 6 weeks the nerve was allowed to reinnervate its muscle through a nerve graft. With approximately 6 weeks needed for muscle reinnervation [Foehring, et al. (1986) J. Neurophysiol. 55:947-965], the MG motoneurons were devoid of neuromuscular contact for altogether about 12 weeks. Two years later reinnervated MG alpha-motoneurons were intracellularly labelled with horseradish peroxidase to allow quantitative analyses of the cell bodies and dendritic trees. Comparisons were made with previous data from normal and permanently axotomized MG motoneurons. The reinnervated motoneurons exhibited positive correlations between dendritic stem diameter, on one hand, and combined length, volume, membrane area, and number of end branches of the whole dendrite, on the other. By using the regression equations for these correlations, the total dendritic size of whole reinnervated motoneurons could be estimated. Such calculations showed that in comparison with the reduction in dendritic size found at 12 weeks after permanent axotomy (Brännström et al., see above), peripheral reinnervation caused the dendritic volume and membrane area to return to normal values. However, the values for combined dendritic length and number of dendritic end branches were still reduced by more than 25% as compared to the normal situation. The results indicate that following reinnervation of the target muscle, the axotomized motoneurons did not recover their original number of dendritic branches. The normalization of dendritic membrane area and volume was instead accomplished by two other mechanisms, namely an increase in dendritic diameters and an increased number of dendrites per neuron.

Electron microscopic studies of serially sectioned cat spinal alpha-motoneurons. IV. Motoneurons innervating slow-twitch (type S) units of the soleus muscle.

Two intracellularly stained cat alpha-motoneurons of the soleus-S type (Burke et al., '74) were studied ultrastructurally. The architecture and synaptology of the cell body and proximal parts of the dendrites were analyzed from a long series of consecutive sections (Conradi et al., '79). Only few of the dendrites had a base diameter exceeding 10 micrometer. The proportion of the membrane surface of the cell body and dendrites covered by boutons was 40-45% and 50-80%, respectively. Out of this, 15-20% was constituted by S-boutons and 70-75% by F-boutons in both regions. In contrast to the situation in the gastrocnemius FR-neurons (Kellerth et al., '79) the large boutons of the C-type showed no clustering around the dendritic roots and were absent on the axon hillock. In addition, the M-boutons of dorsal root origin were more sparse on the proximal parts on the dendrites of the soleus-S neurons.

BDNF abolishes the survival effect of NT-3 in axotomized Clarke neurons of adult rats.

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) have previously been shown to support survival and axonal regeneration in various types of neurons. Also, synergistic neuroprotective effects of these neurotrophins have been reported in descending rubrospinal neurons after cervical spinal cord injury (Novikova et al., [2000] Eur. J. Neurosci. 12:776-780). The present study investigates the effects of intrathecally delivered NT-3 and BDNF on the survival and atrophy of ascending spinocerebellar neurons of Clarke nucleus (CN) after cervical spinal cord injury in adult rats. At 8 weeks after cervical spinal cord hemisection, 40% of the axotomized CN neurons had been lost, and the remaining cells exhibited marked atrophy. Microglial activity was significantly increased in CN of the operated side. Intrathecal infusion of NT-3 for 8 weeks postoperatively resulted in 91% cell survival and a reduction in cell atrophy, but did not reduce microglial activity. In spite of the fact that the CN neurons expressed both TrkC and TrkB receptors, only NT-3 had a neuroprotective effect, whereas BDNF was ineffective. Furthermore, when a combination of BDNF and NT-3 was administered, the neuroprotective effect of NT-3 was lost. The present results indicate a therapeutic potential for NT-3 in the treatment of spinal cord injury, but also demonstrate that in certain neuronal populations the neuroprotection obtained by a combination of neurotrophic factors may be less than that of a single neurotrophin.

Brain-derived neurotrophic factor promotes axonal regeneration and long-term survival of adult rat spinal motoneurons in vivo.

This study shows that in adult rat spinal motoneurons brain-derived neurotrophic factor exerts a neuroprotective effect which extends several weeks beyond the duration of treatment. In addition, brain-derived neurotrophic factor strongly enhances regeneration of avulsed motor axons across the border between the central and peripheral nervous systems. Treatment with brain-derived neurotrophic factor is known to rescue adult rat spinal motoneurons from retrograde cell death induced by ventral root avulsion. The present experiments were designed to test whether this survival effect remains over an extended period of time following cessation of treatment and, also, whether brain-derived neurotrophic factor promotes regeneration of avulsed motor axons. After avulsion of a spinal ventral root, four weeks of treatment with brain-derived neurotrophic factor (10 microg/day) or vehicle was initiated. By using different retrograde tracers to obtain pre- and postoperative labelling of avulsed and regenerating motoneurons, respectively, the number of surviving motoneurons as well as the extent of motor axonal regeneration could be analysed. The expression of nitric oxide synthase in the lesioned motoneurons was also studied. In the vehicle-treated rats, only 10% of the avulsed motoneurons remained at 12 weeks postoperatively, 20-40% of which displayed nitric oxide synthase activity. Treatment with brain-derived neurotrophic factor during the initial four postoperative weeks resulted in 45% motoneuron survival and a complete blockage of nitric oxide synthase expression at 12 weeks postoperatively. Brain-derived neurotrophic factor also induced abundant regeneration of the avulsed motor axons, which formed extensive fibre bundles along the surface of the spinal cord and adjacent ventral roots. The long-term effect by brain-derived neurotrophic factor seemed to be even stronger on motor axonal regeneration than on motoneuron survival. The present results indicate a therapeutic potential for brain-derived neurotrophic factor in the early treatment of traumatic injuries to spinal nerves and roots.

Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.

Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates.

The permanence of retrograde neuronal labeling by the fluorescent tracers Fast Blue, Fluoro-Gold, Mini-Ruby, Fluoro-Ruby and Fluoro-Emerald was investigated in adult rat spinal motorneurons at 1, 4, 12 and 24 weeks after tracer application to a transected muscle nerve. After 1 week, the largest number of retrogradely labeled motoneurons was found with Mini-Ruby, Fluoro-Gold and Fluoro-Ruby, while Fluoro-Emerald yielded a smaller number of labeled cells. With increasing survival time, all of these tracers exhibited a marked decrease in the number of labeled neurons. Fast Blue also produced very efficient staining after 1 week and, in addition, the number of Fast Blue-labeled cells remained constant over the entire time period studied. Also in embryonic spinal cord tissue exposed to Fast Blue. the label persisted for at least 6 months after transplantation into adult spinal cord. Double-labeling experiments combining Fast Blue with Fluoro-Gold, Mini-Ruby, Fluoro-Ruby or Fluoro-Emerald showed that all these substances were non-toxic and that the time-related decrease in the number of neurons labeled by the latter tracers was due to degradation or leakage of the dyes. Thus, Fast Blue would be the tracer of choice for motoneuronal labeling in long-term experiments, whereas the usage of the other tracers should be restricted to experiments of limited duration.

A quantitative morphological study of HRP-labelled cat alpha-motoneurones supplying different hindlimb muscles.

Cat alpha-motoneurones supplying the quadriceps (Q), posterior biceps (PB), gastrocnemius (G), soleus (SOL) and short intrinsic plantar foot (SP) muscles were studied after retrograde or intracellular labelling with HRP. The average soma sizes were rather similar for the different pools, the SOL cells being the smallest. The median number of first-order dendrites ranged from 10 (PB) to 12 (SOL). The median diameters of the first-order dendrites ranged from 6 (SOL) to 8.5 (PB, G) micrometer. The dendritic projection patterns were rather similar for the different motoneurone groups, except for a prominent dorsomedial projection of SP dendrites. A considerable fraction of the dendrites extended into the white matter. The diameter of the first-order dendrite correlated positively to the number of end branches as well as to the combined length, surface area and volume of the whole dendrite. These relations appeared to be independent of motoneurone group and dendritic orientation. The combined diameter of the first-order dendrites, which reflects the total dendritic size of a motoneurone, exhibited median values between 82 micrometers (SOL) and 112 micrometers (Q). With respect to the relative scaling of soma and dendrites, motoneurones with large somas tended to have proportionally larger dendritic trees. The distribution of dendritic diameters, number of branches, dendritic surface area and volume, and the combined dendritic parameter (epsilon d3/2) at various distances from the soma were quite similar for the different motoneurone groups.

Does alpha-motoneurone size correlate with motor unit type in cat triceps surae?

The cell bodies and first-order dendrites of alpha-motoneurones supplying different functional types of muscle units in the cat gastrocnemius (type FF, FR and S units) and soleus (type SOL-S units) muscles, were studied after intracellular injection of horseradish peroxidase. The SOL-S neurones had smaller values for cell body diameter in comparison with both the FF and FR neurones. The SOL-S neurones also had significantly thinner first-order dendrites than the FF, FR and S neurones. In the gastrocnemius pool the S neurones had smaller values for dendritic diameters than the FF and FR cells. The values for combined diameter of the first-order dendrites indicated that the dendritic trees of the FF and FR neurones are, on the average, larger than those of the S and SOL-S neurones. Furthermore, the relationship between the combined dendritic diameter and the mean soma diameter, indicated that a difference in relative scaling of soma and dendrites exists between the FF and FR neurones on the one hand and the S and SOL-S neurones on the other. Similar results were obtained also when relating the combined dendritic parameter sigma d3/2 to the soma surface area. Although a certain statistical relation seems to exist between motoneurone size and motoneurone type, it should be emphasized, however, that the range of values for each parameter studied overlapped considerably between the different types of motoneurones.

Physiological adjustments in a reflex pathway following partial loss of target neurons.

This investigation was undertaken to study plasticity in a reflex pathway following partial elimination of target neurons. Adult cats were subjected to unilateral avulsion of the L7 spinal ventral root, which induces retrograde cell death among the motoneurons of the L7 segment. At 1, 3, 6 and 12 weeks after the lesion, the monosynaptic reflexes were recorded in the L6 and S1 ventral roots during stimulation of the L6, L7 and S1 dorsal roots. Since the group Ia muscle spindle afferents passing through these dorsal roots were deprived of their target motoneurons in the L7 segment, compensatory reflex changes were searched for in the remaining monosynaptic contacts with the intact target motoneurons of the L6 and S1 segments. The results indicate that a partial loss of target motoneurons triggers changes leading to increased monosynaptic reflexes of the remaining intact target motoneurons. On average, the reflexes had more than doubled their size at 12 weeks postoperatively. Possible mechanisms for this reflex potentiation are discussed.

A physiological study of the monosynaptic reflex responses of cat spinal alpha-motoneurons after partial lumbosacral deafferentation.

In adult cats the whole S1 and rostral half of the L7 dorsal roots were cut on the left side of the spinal cord to produce a partial monosynaptic deafferentation of the ipsilateral alpha-motoneurons. Three, 6 or 12 weeks later, monosynaptic reflexes (MSRs) were recorded from the L6, L7 and S1 ventral roots or from various peripheral nerves during stimulation of the L6 and remaining parts of the L7 dorsal roots. Also, monosynaptic excitatory postsynaptic potentials (EPSPs) were recorded intracellularly in different types of medial gastrocnemius alpha-motoneurons of the L7 segment during stimulation of various hind limb muscle nerves. The right side with an identical acute deafferentation served as control. On the chronically lesioned side the MSRs were increased in size, also during post-tetanic potentiation. The monosynaptic EPSPs had increased amplitudes in all motoneuron types, but the relation in EPSP size between different motoneuron types as well as between different synergistic inputs remained largely unchanged. EPSP rise times were not changed, and aberrant monosynaptic connections from non-synergist muscles were not observed. It is concluded that the extent of reactive reflex changes may be related to both the number of vacant synaptic sites and the degree of functional synergism between the eliminated and remaining monosynaptic pathways. Possible underlying mechanisms are discussed.