Renshaw cell mediated inhibition of Renshaw cells: patterns of excitation and inhibition from impulses in motor axon collaterals.
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Acetylcholine↗
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Postnatal maturation of gephyrin/glycine receptor clusters on developing Renshaw cells.
Adult mammalian Renshaw cells express large and complex postsynaptic gephyrin/glycine receptor clusters on their surface. Larger gephyrin clusters correlate with more "efficacious" inhibitory synapses, in terms of larger postsynaptic quantal size amplitudes, in part because they likely contain more postsynaptic receptors (Lim et al. [1999] J. Physiol. (Lond.) 516:505-512; Oleskevich et al. [1999] J. Neurophysiology 82:312-319). Here, we studied the postnatal development of the gephyrin/glycine receptor cluster size on Renshaw cells. Renshaw cells were identified by their calbindin immunoreactivity, location and morphology, and presence of cholinergic input. The populations of clusters over developing Renshaw cells immunoreactive to gephyrin or glycine receptor alpha1 subunits were comparable in number, size, and complexity and displayed a high degree of colocalization (>90%) at all ages. Quantitative morphologic analysis was performed on gephyrin-immunoreactive clusters. In neonatal animals, Renshaw cells expressed small punctate gephyrin-immunoreactive clusters (mean cluster size +/- SD = 0.19 +/- 0.19 microm(2)at 2 days; 0.22 +/- 0. 19 microm(2)at 5 days). By 10 and 15 days of age, Renshaw cells exhibited gephyrin-immunoreactive clusters that were larger and more complex (0.32 +/- 0.19 microm(2) at 10 days; 0.41 +/- 0.32 microm(2) at 15 days). Cluster growth reached a plateau in 25- and 60-day-old Renshaw cells (0.45 +/- 0.43 microm(2); 0.56 +/- 0.55 microm(2), respectively). By using electron microscopy, we confirmed that gephyrin-immunoreactive clusters were located at postsynaptic sites at both early and late postnatal ages on Renshaw cells. The potential significance of this gephyrin/glycine receptor cluster size maturation that sets Renshaw cells apart from other interneurons is discussed.
Animals↗
Peptidergic input to immunohistochemically-identified Renshaw cells.
Peptidergic influences on Renshaw cells were assessed in rat using gephyrin-immunoreactivity, as a Renshaw cell specific marker, in combination with substance P, calcitonin gene-related peptide- and nicotinic acetylcholine receptor-immunolabelling. An average of 3.9 substance P-, and 8.1 calcitonin gene-related peptide-, and 16.3 nicotinic acetylcholine receptor-immunoreactive close contacts or puncta were observed per Renshaw cell. Most appositions were somatic. These results provide neuroanatomical support for the peptidergic modulation of Renshaw cells.
Animals↗
Calbindin D28k expression in immunohistochemically identified Renshaw cells.
Double immunofluorescence was utilized to determine whether Renshaw cells contain calbindin D28k immunoreactivity. Renshaw cells were identified by their characteristic expression patterns of gephyrin immunoreactivity in sections of rat and cat lumbar spinal cord. In the rat, all neurons classified as Renshaw cells (n = 487) also contained calbindin D28k-immunoreactivity, and all calbindin D28k-immunoreactive cells located in the ventral-most region of lamina VII expressed the characteristic gephyrin labeling and morphology of Renshaw cells. In the cat, fewer than half of the Renshaw cells (47%; n = 128) were double-labeled. In both species, occasional calbindin D28k-immunoreactive Renshaw cells were identified within motor nuclei in lamina IX. The distinctive immunolabeling of Renshaw cells allowed us to estimate that there are about 250 Renshaw cells in each ventral horn of the fourth lumbar segment of rat spinal cord, and about 750 cells per ventral horn in the L6 segment of the cat. We conclude that the functional properties of Renshaw cells, including their ability to fire action potentials at high rates, likely require specific homeostatic mechanisms including strong intracellular calcium buffering, the precise mechanisms of which may vary between species.
Animals↗
Regulation of gephyrin cluster size and inhibitory synaptic currents on Renshaw cells by motor axon excitatory inputs.
Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for approximately 2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9-P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABA(A) receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABA(A) receptors.
Animals↗
Developmental changes in the expression of calbindin and potassium-channel subunits Kv3.1b and Kv3.2 in mouse Renshaw cells.
One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14.
Age Factors↗
Spontaneous respiratory activity of phrenic and intercostal Renshaw cells.
Activity of Renshaw cells evoked by electrical stimulation of either phrenic or internal intercostal axons was extracellularly recorded in anaesthetized spontaneously breathing cats. The response of all the studied units to antidromic invasion of the corresponding motoneurones was related to the respiratory cycle and some units displayed spontaneous respiratory activity. Recurrent IPSPs were recorded on phrenic and intercostal motoneurones.
Animals↗
Response of Renshaw cells to sinusoidal stretch of hindlimb extensor muscles.
1. Renshaw cells responding disynaptically to electrically induced group I volleys in the intact gastrocnemius-soleus (GS) nerve, were submitted to small-amplitude, high-frequency vibration applied longitudinally to the deefferented GS muscle in precollicular decerebrate cats. 2. Vibration of the GS muscle at 200/sec, 180 mu peak-to-peak amplitude for 80-100 msec produced a sudden increase in the discharge rate of Renshaw cells, which gradually decreased within 25-50 msec to reach a steady level higher than that recorded in the absence of vibration. 3. Excitation of Renshaw cells appeared at a threshold amplitude of vibration (at 200-250/sec) of 5-20 mu and increased to a maximum value for amplitudes of about 70-80 mu, i.e., when all the primary endings of the spindles from the GS muscle had been driven by the stimulus. Recruitment of the secondary endings of the muscle spindles, due to large amplitude muscle vibration, did not modify the response of the Renshaw cells to the mechanically induced group Ia volleys. 4. These findings were obtained with the GS muscle pulled at 8 mm of initial extension. A threshold response of Renshaw cells to vibration appeared at 4 mm of static stretch, while maximal responses occurred at 8 mm. No further increase and actually a slight decrease in the response appeared for initial extensions of the muscle of 10-12 mm. 5. For a given vibration amplitude, the response of the Renshaw cells increased with increasing frequencies of vibration to reach the maximum at frequencies of 150-250/sec. Bursts of Renshaw cell discharges synchronous to each stroke of vibrator occurred only for low frequencies of stimulation (less than 25/sec). 6. It is concluded that vibration of the GS muscle represents a very effective method in exciting the Renshaw cells and that this response depends upon selective stimulation of homonymous motoneurons monosynaptically excited by the orthodromic volleys originating from the primary endings of the corresponding muscle spindles.
Action Potentials↗
Primary afferent synapses on developing and adult Renshaw cells.
The mechanisms that diversify adult interneurons from a few pools of embryonic neurons are unknown. Renshaw cells, Ia inhibitory interneurons (IaINs), and possibly other types of mammalian spinal interneurons have common embryonic origins within the V1 group. However, in contrast to IaINs and other V1-derived interneurons, adult Renshaw cells receive motor axon synapses and lack proprioceptive inputs. Here, we investigated how this specific pattern of connectivity emerges during the development of Renshaw cells. Tract tracing and immunocytochemical markers [parvalbumin and vesicular glutamate transporter 1 (VGLUT1)] showed that most embryonic (embryonic day 18) Renshaw cells lack dorsal root inputs, but more than half received dorsal root synapses by postnatal day 0 (P0) and this input spread to all Renshaw cells by P10-P15. Electrophysiological recordings in neonates indicated that this input is functional and evokes Renshaw cell firing. VGLUT1-IR bouton density on Renshaw cells increased until P15 but thereafter decreased because of limited synapse proliferation coupled with the enlargement of Renshaw cell dendrites. In parallel, Renshaw cell postsynaptic densities apposed to VGLUT1-IR synapses became smaller in adult compared with P15. In contrast, vesicular acetylcholine transporter-IR motor axon synapses contact embryonic Renshaw cells and proliferate postnatally matching Renshaw cell growth. Like other V1 neurons, Renshaw cells are thus competent to receive sensory synapses. However, after P15, these sensory inputs appear deselected through arrested proliferation and synapse weakening. Thus, Renshaw cells shift from integrating sensory and motor inputs in neonates to predominantly motor inputs in adult. Similar synaptic weight shifts on interneurons may be involved in the maturation of motor reflexes and locomotor circuitry.
Afferent Pathways↗
Diisopropylfluorophosphate and tetanic stimulation fail to reverse mecamylamine antagonism of Renshaw cells.
Mecamylamine-induced antagonism of Renshaw cells was studied in the spinal cord of DIAL anesthetized adult male or spayed female mongrel cats. Renshaw cell unit responses to 1- or 2-Hz supramaximal antidromic stimulation of lumbar segment 7 ventral root were recorded by conventional means from glass micropipettes (2.7 M NaCl, tip diameter 1.0 to 1.6 micron, 2.0 to 4.0 M omega resistance). Data were analyzed by computer. Mecamylamine (1.0 mg/kg iv) antagonizes the response to 1- or 2-Hz stimulation. The onset occurs within 60 sec, only the first one or two spikes remain after 2 min, and this antagonism is observed 30 min after injection. Persistence of action of acetylcholine (ACh) induced by diisopropylfluorophosphate (DFP 2.0 to 2.5 mg/kg iv) increases the spike frequency of the Renshaw cell burst and reduces the variability in the number of spikes per discharge in response to 1- or 2-Hz antidromic stimulation. Excess ACh generated by 2 min of 20-Hz antidromic stimulation fails to reverse the mecamylamine-induced antagonism to 1- or 2-Hz antidromic stimulation. Also, DFP-induced persistence of action of ACh or 20-Hz (2 min) antidromic stimulation to induce excess ACh in the presence of DFP, fails to reverse the mecamylamine-induced antagonism of response to 1- or 2-Hz antidromic stimulation. It is concluded that the data agree with a mechanism of action of mecamylamine as a noncompetitive open channel blocker of the nicotinic receptor ion-channel complex which renders it nonresponsive to the agonist ACh and to the open channel blocker, DFP.
Acetylcholine↗
Higher-order non-linear phenomena in Renshaw cell responses to random motor axon stimulation.
Renshaw cell responses to random motor axon stimulation exhibit second-order non-linearities in that they depend on the occurrence of a preceding stimulus, although these non-linearities are not strong enough to significantly depress the coherence. However, higher-order non-linearities have not been checked for so far. This is carried out here. Lumbosacral Renshaw cells were recorded with micropipettes in anaesthetized cats. Their responses to random (pseudo-Poisson) stimulation of motor axons in peripheral nerves or ventral roots were quantified by calculating peristimulus-time histograms of various sorts, conventional and conditional. Conventional peristimulus-time histograms were computed with respect to all the stimuli in a train. Conditional peristimulus-time histograms were calculated with respect to "test" stimuli which were sorted out (by computer) from the original stimulus train when they were preceded by "conditional" stimuli at average intervals of delta 1 or delta 2 or both. These conditioned responses were compared with those to be expected from hypothetical linear superposition. Renshaw cell responses showed small third-order non-linearities to pairs of conditioning stimuli at small intervals (up to some tens of milliseconds before the test stimuli). These third-order effects were smaller than each of the second-order non-linearities elicited by any of the single-conditioning stimuli. Also, further higher-order non-linearities were apparent, but of little average significance. Hence, the non-linearities in Renshaw cell responses to random inputs are essentially of second-order and fairly small.
Animals↗
[Modulation of Renshaw cell activity during scratching].
The activity of the Renshaw cells was recorded during fictitious scratching in 7 decerebrated and 1 spinal curarized cats. The flexor and extensor Renshaw cells were identified according to the responses induced by orthodromic stimulation of muscle nerves. Before the beginning of rhythmical oscillations, the background activity of the flexor Renshaw cells was inhibited. When the rhythmical generation began, both flexor and and extensor cells generated short bursts of spikes at the end of the flexor phase and at the extensor phase of the cycle. The activity of flexor cells began and terminated earlier in the cycle than that of the extensor cells. The results show that the flexor Renshaw cells are active reciprocally with flexor motoneurons, while the extensor cells are coactivated with extensor motoneurons.
Animals↗
Enzyme histochemical profile of immunohistochemically identified Renshaw cells in rat lumbar spinal cord.
Activity levels of cytochrome oxidase, acid phosphatase, and NADPH diaphorase were examined in the perikarya of immunohistochemically identified Renshaw cells from sections of rat lumbar spinal cord. Renshaw cell profiles were identified on the basis of their characteristic anti-gephyrin-immunofluorescent labelling. Intrasomatic densities of enzyme histochemical reaction product were employed as indicators of relative mitochondrial activity (cytochrome oxidase), intracytoplasmic digestion (acid phosphatase), or putative nitrergic signalling (NAPDH-diaphorase). Approximately half of the Renshaw cell somata examined displayed moderate levels of cytochrome oxidase reaction product (142 of 262 Renshaw cells) or low levels of acid phosphatase activity (156 of 243 Renshaw cells). A majority (160 of 202 cells) of Renshaw cells contained low intrasomatic levels of NADPH-diaphorase activity but most of these cells were closely apposed by at least one NADPH-diaphorase reactive axonal varicosity. Our findings suggest that moderate levels of perikaryal oxidative metabolism and low levels of intracytoplasmic digestion are sufficient for, and support, the unique physiological capabilities of Renshaw cells. The presence of NADPH-diaphorase containing somatic close contacts indicate that nitric oxide may have at least a minor role in the regulation of Renshaw cell activity. These results are complementary and consistent with previous morphological and pharmacological demonstrations of Renshaw cell heterogeneity.
Acid Phosphatase↗
A pharmacological study of Renshaw cell inhibition.
1. In cats anaesthetized with pentobarbitone a pharmacological investigation was made of the inhibition of Renshaw cells by dorsal root afferent volleys and ventral root antidromic volleys, and of the inhibition of motoneurones by Renshaw cells. 2. The effects of strychnine, bicuculline and tetanus toxin indicate that both glycine and GABA operate as inhibitory transmitters released on Renshaw cells by dorsal root volleys. 3. The 'mutual' inhibition of Renshaw cells, and the recurrent inhibition of motoneurones by Renshaw cells, are suppressed by strychnine: Renshaw cells are thus glycinergic inhibitory neurones, a proposal consistant with recent evidence for strychnine-sensitive inhibition of Ia interneurones by Renshaw cells. 4. The 'pause' which follows high frequency synaptic excitation of Renshaw cells is insensitive to strychnine, bicuculline and tetanus toxin, and is considered unlikely to be the consequence of synaptic inhibition.
Action Potentials↗
The relative sensitivity of Renshaw cells to orthodromic group Ia volleys caused by static stretch and vibrations of extensor muscles.
1. Activity of Renshaw cells monosynaptically excited by ventral root stimulation and disynaptically excited by electric stimulation of the group Ia afferents in the gastrocnemius-soleus (GS) nerve, was recorded in precollicular decerebrate cats. The response of these units to prolonged vibration applied longitudinally to the deefferented GS muscle was then compared with that elicited by static stretch of the homonymous muscle, for comparable frequencies of discharge of the group Ia afferents. 2. Small-amplitude vibration of the GS muscle at 200/sec for one second produced a sudden increase in the discharge rate of Renshaw cells, which gradually decreased within the first 100 msec of vibration to reach steady albeit lower level than that obtained during the first part of vibration. The response of the Renshaw cells during the first 100 msec of vibration (phasic response) and that elicited during the last 500 msec of vibration (tonic response) were evaluated for different frequencies of sinusoidal stretch. The mean increase in the firing frequency per imp./sec in the Ia afferents was also calculated using the total one-second period. 3. The response of Renshaw cells to muscle vibration increased with the frequency of vibration and, over the value of 10/sec, appeared to be linearly related to the frequency of the input, at least up to the frequency of 150/sec. Since vibration was of sufficient amplitude to produce driving of all the primary endings of muscle spindles, the responses were expressed as mean increases in the discharge rate of Renshaw cells per average impulse/sec in the Ia afferents. The discharge of the Renshaw cell increased on the average by 2.90 and 1.08 imp./sec per each imp./sec in the Ia afferents during the phasic and the tonic component of the response respectively, while the response calculated during the whole period of vibration corresponded on the average to 1.45 imp./sec per each imp./sec in the Ia afferents. 4. The Renshaw cells tested above responded also with increasing frequencies of discharge to increasing levels of static extension of the GS muscle. In particular the discharge frequency of Renshaw cells was on the average linearly related to muscle extension, at least for values ranging from 0 to 8 mm. The mean increase in discharge rate as a function of the static extension corresponded on the average to 0.89 imp./sec/mm. Since the discharge rate of the primary endings of muscle spindles recorded from the deefferented GS muscle increased by 2.62 imp./sec/mm, it appears that the mean increase in the discharge rate of Renshaw cells as a function of static extension corresponded to 0.34 imp./sec per each imp./sec in the Ia afferents.
Action Potentials↗
Distribution of cholinergic contacts on Renshaw cells in the rat spinal cord: a light microscopic study.
1. Cholinergic terminals in the rat spinal cord were revealed by immunohistochemical detection of the vesicular acetycholine transporter (VAChT). In order to determine the relationships of these terminals to Renshaw cells, we used dual immunolabelling with antibodies against gephyrin or calbindin D28k to provide immunohistochemical identification of Renshaw cells in lamina VII of the ventral horn. 2. A total of 50 Renshaw cells were analysed quantitatively using a computer-aided reconstruction system to provide accurate localization of contact sites and determination of somatic and dendritic surface area. Dendrites could be traced for up to 413 microm from the soma in calbindin D28k-identified Renshaw cells and up to 184 microm in gephyrin-identified cells. 3. A total of 3330 cholinergic terminals were observed on 50 Renshaw cells, with a range of 21-138 terminal appositions per cell (mean 66.6 +/- 25.56 contacts per cell). The vast majority (83.5 %) of the terminals were apposed to dendrites rather than the soma. The overall density of cholinergic contacts increased from a little above 1 per 100 microm2 on the soma and initial 25 microm of proximal dendrites to 4-5 per 100 microm2 on the surface of dendritic segments located 50-250 microm from the soma. Single presynaptic fibres frequently formed multiple contacts with the soma and/or dendrites of individual Renshaw cells. 4. VAChT-immunoreactive terminals apposed to Renshaw cells varied in size from 0.6 to 6.9 microm in diameter (mean 2.26 +/- 0.94; n = 986) and were on average smaller than the cholinergic C-terminals apposed to motoneurones, but larger than VAChT-immunoreactive terminals contacting other ventral horn interneurones. 5. The high density and relatively large size of many cholinergic terminals on Renshaw cells presumably correlates with the strong synaptic connection between motoneurones and Renshaw cells. The fact that the majority of contacts are distributed over the dendrites makes the motoneurone axon collateral input susceptible to inhibition by the prominent glycinergic inhibitory synapses located on the soma and proximal dendrites. The relative positions and structural features of the excitatory cholinergic and inhibitory glycinergic synapses may explain why Renshaw cells, although capable of firing at very high frequency following motor axon stimulation, appear to fire at relatively low rates during locomotor activity.
Animals↗
The relative dependence of the activity of Renshaw cells on recurrent pathways during contraction of the triceps muscle.
Renshaw cell activity was recorded simultaneously with motoneuronal unit discharge during vibration and tetanic stimulation of triceps muscles in decerebrated cats. The experiments confirm that, in this preparation, the motoneurones are the main source of Renshaw cell firing during muscle stretch and vibration and when motoneuronal discharge was induced through the gamma loop. However they also show that a discharge of Renshaw cells, monosynaptically coupled with triceps motoneurones through their recurrent collaterals, could be elicited during contraction of the muscle at the time when the discharge of these motoneurones had been silenced. The recording of the stretch receptors and motoneuronal unit discharge during stretch, vibration, and ventral root stimulation gave evidence of the contribution of the withdrawal of excitation by primary endings to the occurrence of the silent period during tetanic contraction of the muscle. The measurements of the critical firing level in motoneuronal units responding reflexly to held stretch and vibration of the muscles, and silencing their discharge during muscle shortening, showed that these cells are amongst the lowest ranking in the pool For these reasons, these data suggest that Renshaw cell firing during vibration and tetanic contraction of the muscle cannot be attributed only to the alpha motoneurone excitation by the Ia fibres.
Animals↗
Evidence for separate morphological classes of Renshaw cells in the cat's spinal cord.
Although some functions of Renshaw cells are well defined, pharmacological evidence suggests that there may be more than one type of Renshaw cell involved in recurrent inhibition of motoneurons, or one type employing more than one inhibitory amino acid neurotransmitter. Identified Renshaw cells were intracellularly stained with horseradish peroxidase (HRP), revealing the existence of a distinctive group of fusiform neurons as well as the more common multipolar cells. The fusiform neurons may represent a subgroup of Renshaw cells having separate functions and/or synaptic mediators.
Animals↗