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Background : The spinal cord's central pattern generators (CPGs) have been explained by the symmetrical half-center hypothesis, the bursts generator, computational models, and more recently by connectome circuits. Asymmetrical models, at odds with the half-center paradigm, are composed of extensor and flexor CPG modules. Other models include not only flexor and extensor motoneurons but also motoneuron pools controlling biarticular muscles. It is unknown whether a preferred model can explain some particularities that fictive scratching (FS) in the cat presents. The first aim of this study was to investigate FS patterns considering the aiming and the rhythmic periods, and second, to examine the effects of serotonin (5HT) on and segmental inputs to FS. Methods : The experiments were carried out first in brain cortex-ablated cats (BCAC), then spinalized (SC), and for the midcollicular (MCC) preparation. Subjects were immobilized and the peripheral nerves were used to elicit the Monosynaptic reflex (MR), to modify the scratching patterns and for electroneurogram recordings. Results : In BCAC, FS was produced by pinna stimulation and, in some cases, by serotonin. The scratching aiming phase (AP) initiates with the activation of either flexor or extensor motoneurons. Serotonin application during the AP produced simultaneous extensor and flexor bursts. Furthermore, WAY 100635 (5HT1A antagonist) produced a brief burst in the tibialis anterior (TA) nerve, followed by a reduction in its electroneurogram (ENG), while the soleus ENG remained silent. In SC, rhythmic phase (RP) activity was recorded in the soleus motoneurons. Serotonin or WAY produced FS bouts. The electrical stimulation of Ia afferent fibers produced heteronymous MRes waxing and waning during the scratch cycle. In MCC, FS began with flexor activity. Electrical stimulation of either deep peroneus (DP) or superficial peroneus (SP) nerves increased the duration of the TA electroneurogram. Medial gastrocnemius (MG) stretching or MG nerve electrical stimulation produced a reduction in the TA electroneurogram and an initial MG extensor burst. MRes waxed and waned during the scratch cycle. Conclusion : Descending pathways and segmental afferent fibers, as well as 5-HT and WAY, can change the FS pattern. To our understanding, the half-center hypothesis is the most suitable for explaining the AP in MCC., (Copyright © 2020 Aguilar Garcia, Dueñas-Jiménez, Castillo, Osuna-Carrasco, De La Torre Valdovinos, Castañeda-Arellano, López-Ruiz, Toro-Castillo, Treviño, Mendizabal-Ruiz and Duenas-Jimenez.)

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

We studied the serotonin effect on monosynaptic reflex potentials (MSR) of spinal motorneurons in the decerebrated rats in control and after intraperitoneal administration of serotonin precursor – 5-Hydroxytryptophan (5-HTP). MSR of motorneurons in the lumbar spinal cord were registered using electrical stimulation of dorsal root of the 5th lumbar section. During stimulation physiological saline or 5-hydroxytryptophan was injected intraperitoneally. In comparison with average amplitude of the control MSR there were registered significant increase in amplitudes of the MSR (169% and +172%, P <0,001) in animals with injection 5-HTP. These data suggest that serotonin release after 5-HTP administration leads to activation of motorneurons in the lumbar spinal cord. The mechanism of this activation may be related to the weakening of the inhibitory control of interneurons in the transmission pathways of the excitatory influences from muscle afferent to motorneurons and to the postural (antigravity) reflex reactions which necessary for the initiation of locomotion.

Background: The spinal cord is a prime site of action for analgesia. Here we characterize the effects of established analgesics on segmental spinal reflexes. The aim of the study was to look for the pattern of action or signature of analgesic effects on these reflexes., Methods: We used a spinal cord in vitro preparation of neonate mice to record ventral root responses to dorsal root stimulation. Pregabalin, clonidine, morphine and duloxetine and an experimental sigma-1 receptor antagonist (S1RA) were applied to the preparation in a cumulative concentration protocol. Drug effects on the wind-up produced by repetitive stimulation of C-fibres and on responses to single A- and C-fibre intensity stimuli were analysed., Results: All compounds produced a concentration-dependent inhibition of total spikes elicited by repetitive stimulation. Concentrations producing ∼50% reduction in this parameter were (in μM) clonidine (0.01), morphine (0.1), pregabalin (1), duloxetine (10) and S1RA (30). At these concentrations clonidine, pregabalin and S1RA had significant effects on the wind-up index and little depressant effects on responses to single stimuli. Morphine and duloxetine did not depress wind-up index and showed large effects on responses to single stimuli. None of the compounds had strong effects on the amplitude of the non-nociceptive monosynaptic reflex., Conclusions: morphine and duloxetine had general depressant effects on spinal reflexes, whereas the effects of clonidine, pregabalin and S1RA appeared to be restricted to signals originated by strong repetitive activation of C-fibres. Results are discussed in the context of reported behavioural effects of the compounds studied., (© 2014 European Pain Federation - EFIC®)

Activity-based interventions such as locomotor training or passive cycling have a positive influence on the spinal circuitry and recovery following a spinal cord injury (SCI). The use of quipazine in combination with exercise training has demonstrated a greater functional recovery than has exercise training alone. However, the influence of exercise or training on the responsiveness of the spinal cord to quipazine has not been examined following a chronic spinal transection. The purpose of this study was to characterize the flexor and extensor monosynaptic reflex (MSR) response pre- and post-quipazine in chronic complete spinally transected rats that either underwent daily passive cycling for 3 months or did not receive passive cycling. Following a chronic spinal transection, the extensor MSR demonstrated a hyperreflexive response (fivefold increase) to afferent stimuli, and did not respond to quipazine injection. With daily passive cycling, the extensor MSR hyperexcitability was attenuated, and the MSR amplitude increased 72% following quipazine injection (p<0.004), which was comparable to the extensor MSR response (94%) in the control group. For both chronic spinal transection groups, the flexor MSR amplitudes were not altered following quipazine injection, whereas in the control group the flexor MSR amplitude increased 86% in response to quipazine (p<0.004). These results demonstrate that passive cycling attenuates the hyperreflexive response of the extensor MSR following a chronic SCI, and restores the MSR response to quipazine.

The purpose of this study was to determine if quipazine, a serotonergic agonist, differentially modulates flexor and extensor motor output. This was achieved by examining the monosynaptic reflex (MSR) of the tibial (extensor) and peroneal (flexor) nerves, by determining the basic and rhythmic properties of extensor and flexor motoneurons, and by recording extracellular Ia field potentials of the tibial and peroneal nerves in the in vivo adult decerebrate rat in both spinal intact and acute spinalized preparations. In the spinal intact preparation, the tibial and peroneal MSR amplitude significantly increased compared with baseline in response to quipazine, with no difference between nerves (P < 0.05). In the spinalized preparation, the MSR was significantly increased in both the tibial and peroneal nerves with the latter increasing more than the former (5.7 vs. 3.6 times; P < 0.05). Intracellular motoneuron experiments demonstrated that rheobase decreased, while input resistance, afterhyperpolarization amplitude, and the firing rate at a given current injection increased in motoneurons following quipazine administration with no differences between extensor and flexor motoneurons. Both the tibial and peroneal nerve extracellular Ia field potentials increased with the peroneal demonstrating a significantly greater increase (7 vs. 38%; P < 0.05) following quipazine. It is concluded that in the spinal intact preparation quipazine does not have a differential effect on flexor or extensor motor output. However, in the acute spinalized preparation, quipazine preferentially affects the flexor MSR compared with the extensor MSR, likely due to the removal of a descending tonic inhibition on flexor Ia afferents.

Peripheral nerve transection, even with optimal repair, can result in an extensive disruption of central connectivity, which can lead to long-lasting impairments in motor and sensory function. We hypothesised that removal of spinal cord chondroitin sulphate proteoglycans (CSPGs) would promote plasticity in the spinal cord, allowing compensation for inaccurate peripheral reinnervation. In adult rats, the median and radial nerves were cut and repaired, either correctly (median to median and radial to radial), or incorrectly (median to radial and vice versa). This produced two levels of inaccuracy of peripheral reinnervation. Whole nerve recordings from a third brachial plexus nerve, the ulnar, were made during median or radial nerve stimulation. Low and high threshold reflexes were characterised in uninjured animals and a clear difference in the pattern of ulnar response to flexor (median) or extensor (radial) stimulation was established. This included the phenomenon of wind-up, where repetitive median nerve stimulation at supramaximal C-fibre threshold leads to a progressive increase in the number of spikes recorded. To achieve digestion of CSPGs a lentiviral vector expressing ChABC was delivered to the spinal cord via intraspinal injection. Following ChABC treatment, we found several indicators of reorganisation of central connections. Firstly, we found that the amplitude of a low threshold, polysynaptic reflex could be increased after nerve injury, only following treatment with ChABC. Secondly, wind-up of motor responses in the ulnar nerve to supramaximal stimulation of afferents in the median nerve, which collapses after nerve injury (to ~25% of uninjured value), could be restored by ChABC after correct repair (to ~90% of uninjured value). Thirdly, wind-up in ulnar motor axons to stimulation of radial nerve afferents, which is minimal in the uninjured state, becomes significantly stronger after nerve injury and ChABC treatment (a 10 fold increase). We propose that application of a plasticity-promoting treatment to the spinal cord allows the amplification of adaptive changes in response to inaccurate wiring in the periphery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Muscle sensory axons induce the development of specialized intrafusal muscle fibers in muscle spindles during development, but the role that the intrafusal fibers may play in the development of the central projections of these Ia sensory axons is unclear. In the present study, we assessed the influence of intrafusal fibers in muscle spindles on the formation of monosynaptic connections between Ia (muscle spindle) sensory axons and motoneurons (MNs) using two transgenic strains of mice. Deletion of the ErbB2 receptor from developing myotubes disrupts the formation of intrafusal muscle fibers and causes a nearly complete absence of functional synaptic connections between Ia axons and MNs. Monosynaptic connectivity can be fully restored by postnatal administration of neurotrophin-3 (NT-3), and the synaptic connections in NT-3-treated mice are as specific as in wild-type mice. Deletion of the Egr3 transcription factor also impairs the development of intrafusal muscle fibers and disrupts synaptic connectivity between Ia axons and MNs. Postnatal injections of NT-3 restore the normal strengths and specificity of Ia-motoneuronal connections in these mice as well. Severe deficits in intrafusal fiber development, therefore, do not disrupt the establishment of normal, selective patterns of connections between Ia axons and MNs, although these connections require the presence of NT-3, normally supplied by intrafusal fibers, to be functional.

Glutamate is a putative neurotransmitter at Ia-alpha motoneuron synapse in the spinal cord and mediate the action via N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. Since NMDA receptors are not involved in M. tamulus Pocock (MBT) venom-induced depression of spinal monosynaptic reflex (MSR), the present study was undertaken to evaluate the role of AMPA receptors in mediating the depression of MSR by MBT venom. The experiments were performed on isolated hemisected spinal cord from 4-6 day old rats. Stimulation of a dorsal root with supramaximal voltage evoked MSR and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. Superfusion of MBT venom (0.3 microg/ml) depressed the spinal reflexes in a time-dependent manner. The maximum depression of MSR(approximately 66%) was seen at 10 min and it was 25 min for PSR (approximately 75%). The time to produce 50% depression of MSR and PSR was 6.7+/- 1.5 and 10.8 +/- 2.6 min, respectively. Pretreatment of the cords with 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 0.1 microM), an AMPA receptor antagonist, blocked the venom-induced depression of MSR but not PSR. The results indicate that venom-induced depression of MSR is mediated via AMPA receptors.

Aims: The present study was undertaken to evaluate the role of nitric oxide (NO) in Mesobuthus tamulus (MBT) venom-induced depression of spinal reflexes., Main Methods: Experiments were performed on isolated hemisected spinal cords from 4 to 6day old rats. Stimulation of a dorsal root with supramaximal strength evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root., Key Findings: Superfusion of MBT venom (0.3μg/ml) depressed the spinal reflexes in a time-dependent manner and the maximum depression was seen at 10min (MSR by 63%; PSR by 79%). The time to produce 50% depression (T-50) of MSR and PSR was 7.7±1.3 and 5.7±0.5min, respectively. Pretreatment with bicuculline (1μM; GABA(A) receptor antagonist) or strychnine (1μM; glycine(A) receptor antagonist) did not block the venom-induced depression of spinal reflexes. However, Nω-nitro-L-arginine methyl ester (L-NAME, 100 or 300μM; NO synthase inhibitor) or hemoglobin (Hb, 100μM; NO scavenger) antagonized the venom-induced depression of MSR. Further, soluble guanylyl cylase inhibitors (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ; 1μM or methylene blue, 100μM) also antagonized the venom-induced depression of MSR but not PSR. Nitrite concentration (indicator of NO activity) of the cords exposed to venom (0.3μg/ml) was not different from the control group., Significance: The results indicate that venom-induced depression of MSR is mediated via NO-guanylyl cyclase pathway without involving GABAergic or glycinergic system., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mesobuthus tamulus (MBT) venom is shown to depress the spinal reflexes through a mechanism unrelated to the NMDA receptors. 5-Hydroxytryptamine (5-HT) is another excitatory transmitter in the spinal cord therefore, the present study was undertaken to examine the involvement of 5-HT in the venom-induced depression of reflexes. The experiments were performed on isolated hemisected spinal cords from 4 to 6-day-old rats. Stimulation of a dorsal root with supramaximal strength evoked monosynaptic reflex (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. MBT venom (0.3 microg/ml) depressed the spinal reflexes in a time-dependent manner and the maximal depression was seen at 10 min. The time to produce 50% depression (T-50) of MSR and PSR was 8.1+/-1.41 and 6.8+/-0.5 min, respectively. Pretreatment with pindolol (1 microM; 5-HT(1A/1B) receptor antagonist) blocked the reflexes up to 15 min. On the other hand, ketanserin (10 microM; 5-HT(2A/2C) receptor antagonist) or ondansetron (0.1 microM; 5-HT(3) receptor antagonist) blocked the venom-induced depression of MSR and PSR during entire exposure time (30 min). The 5-HT concentration of the cords exposed to venom (1.6+/-0.04 microg/g tissue) was significantly greater than the control group (0.98+/-0.08 microg/g tissue). The results indicate that venom-induced depression of spinal reflexes is mediated via 5-HTergic transmission involving 5-HT(1A/1B), 5-HT(2A/2C) and 5-HT(3) receptors., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

There is growing evidence that activation of high affinity extrasynaptic GABA(A) receptors in the brain, cerebellum and spinal cord substantia gelatinosa results in a tonic inhibition controlling postsynaptic excitability. The aim of the present study was to determine if GABA(A) receptors mediating tonic inhibition participate in the modulation of monosynaptic reflex (MSR) in the vertebrate spinal cord. Using an in vitro turtle lumbar spinal cord preparation, we show that conditioning stimulation of a dorsal root depressed the test monosynaptic reflex (MSR) at long condition-test intervals. This long duration inhibition is similar to the one seen in mammalian spinal cord and it is dependent on GABA(A) as it was completely blocked by 20 microm picrotoxin (PTX) or bicuculline (BIC) or 1 microm gabazine, simultaneously depressing the dorsal root potential (DRP) without MSR facilitation. Interestingly 100 microm picrotoxin or BIC potentiated the MSR, depressed the DRP, and produced a long lasting motoneurone after-discharge. Furosemide, a selective antagonist of extrasynaptic GABA(A) receptors, affects receptor subtypes with alpha(4/6) subunits, and in a similar way to higher concentrations of PTX or BIC, also potentiated the MSR but did not affect the DRP, suggesting the presence of alpha(4/6) GABA(A) receptors at motoneurones. Our results suggest that (1) the turtle spinal cord has a GABA(A) mediated long duration inhibition similar to presynaptic inhibition observed in mammals, (2) GABA(A) receptors located at the motoneurones and primary afferents might produce tonic inhibition of monosynaptic reflex, and (3) GABA(A) receptors modulate motoneurone excitability reducing the probability of spurious and inappropriate activation.

The objective of the present investigation is to study the involvement of nitric oxide (NO) in 3-nitropropionic acid (3-NPA)-induced depression of spinal reflexes. Experiments were conducted on preparations of hemisected spinal cord isolated from 4 to 8 day old rats. Stimulation of a dorsal root evoked reflex potentials (monosynaptic, MSR; polysynaptic, PSR) in the corresponding segmental ventral root. Superfusion of 3-NPA (3.4 mM) depressed the spinal reflexes in a time-dependent manner and the reflexes were abolished after 35 min. The time required to produce 50% depression of the reflexes (T-50) was 17.8+/-5.3 min for MSR and 17.5+/-2.1 min for PSR. L-NAME (Nomega-nitro-L-arginine methyl ester; 100 microM), a nitric oxide synthase inhibitor, antagonized the 3-NPA (3.4 mM)-induced depression of reflexes and increased the T-50 values (34 and 30 min for MSR and PSR, respectively) significantly (P<0.05). In addition, hemoglobin (Hb, 100 microM), a NO scavenger, blocked the 3-NPA-induced depression of reflexes significantly (P<0.05). T-50 values in Hb pretreated cords were 57 and 45 min for MSR and PSR, respectively which were greater than the cords pretreated with L-NAME. The nitrite (NO(2)(-)) content of the 3-NPA exposed cords was 84 microM/g of tissue which was significantly greater than the control (13 microM/g; P<0.05). Pretreatment of cords with L-NAME or Hb antagonized the 3-NPA-induced increase in NO(2)(-). The results indicate that NO produced by 3-NPA is involved in the 3-NPA-induced depression of spinal reflexes.

Introduction: Neuropeptides are known to modulate female receptivity. However, even though receptivity is a spinal reflex, the role of neuropeptides within the spinal cord remains to be elucidated., Aim: The aims were to (i) investigate neuropeptides in the lumbosacral region; and (ii) determine how neuropeptides modulate glutamate release from stretch Ia fibers, touch sensation Abeta fibers and Adelta/C pain fibers., Main Outcome Measures: Neuropeptide modulation of the lumbosacral dorsal-root ventral-root reflex in vitro., Methods: Spinal cords were removed from Sprague-Dawley rats in compliance with UK Home Office guidelines. Hemisected cords were superfused with aCSF and the dorsal root (L4-S1) was stimulated to evoke glutamate release. A biphasic reflex response was evoked from the opposite ventral root consisting of a monosynaptic (Ia fibers) and polysynaptic (Abeta, Adelta/C fibers) component., Results: The micro opioid receptor (MOR) agonist DAMGO inhibited the monosynaptic (EC(50) 0.02 +/- 0.02 nM) and polysynaptic area (EC(50) 125 +/- 167 nM) but not polysynaptic amplitude. Oxytocin and corticotrophin releasing factor (CRF) inhibited the monosynaptic amplitude (EC(50), 1.4 +/- 1.0 nM and EC(50) 4.3 +/- 3.5 nM, respectively), polysynaptic amplitude (EC(50) 18.2 +/- 28.0 nM and EC(50), 9.5 +/- 13.3 nM, respectively), and area (EC(50) 11.6 +/- 13.0 nM and EC(50), 2.8 +/- 3.3 nM, respectively); effects that were abolished by oxytocin and CRF(1) antagonists, L-368899 and 8w. Melanocortin agonists solely inhibited the monosynaptic component, which were blocked by the MC(3/4) receptor antagonist SHU9119., Conclusion: These data suggest endogenous neuropeptides are released within the lumbosacral spinal cord. Melanocortin agonists, oxytocin, CRF, and DAMGO via MC(4), oxytocin, CRF(1), and MOR inhibit glutamate release but with differing effects on afferent fiber subtypes. Melanocortins, oxytocin, CRF, and DAMGO have the ability to modulate orgasm whereas oxytocin, CRF and DAMGO can increase pain threshold. Oxytocin and CRF may dampen touch sensation.

Receptor subtypes involved in the 5-hydroxytryptamine (5-HT)-induced depression of synaptic transmission in neonatal rat spinal cords in vitro were evaluated in the absence or presence of Mg(2+) in the medium. Stimulation of a dorsal root evoked monosynaptic reflex potential (MSP) and polysynaptic reflex potential (PSP) in the segmental ventral root in Mg(2+)-free medium where the voltage-dependent blockade of NMDA receptors is absent. The 5-HT (0.3-50 microM) in the Mg(2+)-free medium depressed the MSP and PSP in a concentration-dependent manner. At 30 microM of 5-HT, the depression was 57% and 95% for MSP and PSP, respectively, and no further depression was seen at 50 microM. The 5-HT-induced depression of the reflexes in the Mg(2+)-free medium was blocked by ondansetron (5-HT(3) receptor antagonist), but not by spiperone (5-HT(2A/2C) antagonist). In the Mg(2+)-free medium, phenylbiguanide (5-HT(3) agonist) also depressed the MSP and PSP in a concentration-dependent manner and was blocked by ondansetron. Addition of Mg(2+) (1.3 mM) to the medium abolished the PSP and decreased the MSP by 30%. In the presence of Mg(2+), 5-HT (1-50 microM) also depressed the MSP in a concentration-dependent manner. At 10 microM of 5-HT, there was approximately 20% depression and at 50 microM the depression was 100%. The 5-HT-induced depression of MSP in the Mg(2+)-containing medium was antagonized by spiperone (p < 0.05, two-way ANOVA), but not by ondansetron. The results indicate that the 5-HT-induced depression of MSP involves 5-HT(3) receptors in the Mg(2+)-free medium and 5-HT(2A/2C) in the presence of Mg(2+) when NMDA receptors are in the closed state.

Effect of 3-nitropropionic acid (3-NPA) and ischemia (glucose- and O(2)-free solution) on synaptic transmission in hemisected spinal cord from 4 to 8 day old rats was examined in vitro. Stimulation of a dorsal root (L3-5 segments) evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. Superfusion of 3-NPA (0.17-3.4 mM) depressed the reflexes in a concentration- and time-dependent manner. At 3.4 mM of 3-NPA, the reflexes were abolished by 35 min. Time required to produce 50% depression (T-50) was around 170, 80, 40 and 17 min for MSR and 110, 70, 25 and 16 min for PSR at 0.17, 0.51, 1.7 and 3.4mM of 3-NPA, respectively. Ischemia also produced a time-dependent depression of reflexes and abolished them by 35 min and the T-50 values were around 18 min. Presence of creatine phosphate (10mM) in the superfusing medium did not alter the time course of 3-NPA-induced depression of reflexes but prolonged the ischemia-induced depression. dl-2-amino-5-phosphonovaleric acid (NMDA receptor antagonist; 10 microM) failed to block the 3-NPA (3.4 mM)-induced depression of reflexes, but blocked the ischemia-induced depression. The results indicate that 3-NPA-induced depression of spinal reflexes does not involve NMDA receptors and is different from ischemia-induced depression.

Labyrinthine nerve-evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in second-order vestibular neurons (2 degrees VN) sum with disynaptic inhibitory postsynaptic potentials (IPSPs) that originate from the thickest afferent fibers of the same nerve branch and are mediated by neurons in the ipsilateral vestibular nucleus. Pharmacological properties of the inhibition and the interaction with the afferent excitation were studied by recording monosynaptic responses of phasic and tonic 2 degrees VN in an isolated frog brain after electrical stimulation of individual semicircular canal nerves. Specific transmitter antagonists revealed glycine and GABA(A) receptor-mediated IPSPs with a disynaptic onset only in phasic but not in tonic 2 degrees VN. Compared with GABAergic IPSPs, glycinergic responses in phasic 2 degrees VN have larger amplitudes and a longer duration and reduce early and late components of the afferent nerve-evoked subthreshold activation and spike discharge. The difference in profile of the disynaptic glycinergic and GABAergic inhibition is compatible with the larger number of glycinergic as opposed to GABAergic terminal-like structures on 2 degrees VN. The increase in monosynaptic excitation after a block of the disynaptic inhibition in phasic 2 degrees VN is in part mediated by a N-methyl-d-aspartate receptor-activated component. Although inhibitory inputs were superimposed on monosynaptic EPSPs in tonic 2 degrees VN as well, the much longer latency of these IPSPs excludes a control by short-latency inhibitory feed-forward side-loops as observed in phasic 2 degrees VN. The differential synaptic organization of the inhibitory control of labyrinthine afferent signals in phasic and tonic 2 degrees VN is consistent with the different intrinsic signal processing modes of the two neuronal types and suggests a co-adaptation of intrinsic membrane properties and emerging network properties.

The purpose of the present study was to investigate the 5-HT(2C) receptor-mediated effects on the spinal monosynaptic mass reflex activities and also its functional interactions with 5-HT(1A) receptors in anesthetized, acutely spinalized mammalian adult spinal cord in vivo. Intravenous administration of (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (0.1 mg/kg), an agonist of 5-HT(2A/2C) receptors, significantly increased the excitability of spinal motoneurons as reflected by an increase in the spinal monosynaptic mass reflex amplitude to 150-200% of the control. 5-HT(2A/2C) receptor-induced motoneuron excitability was slow, persistent and long-lasting for more than 2h that was significantly inhibited by 5-HT(2C) receptor specific antagonist SB 242084 administered 10 min prior to DOI. Simultaneous administration of DOI (0.1 mg/kg, i.v.) along with (+/-)-8-hydroxy dipropylaminotetraline hydrobromide (8-OH-DPAT) (0.1 mg/kg, i.v.) completely inhibited DOI-induced spinal monosynaptic mass reflex facilitation. In another separate study, administration of 8-OH-DPAT (0.1 mg/kg, i.v.) at the maximum response of DOI also inhibited the motoneuron's excitability; however, the inhibition lasted only for a period of 40-60 min after administration of 8-OH-DPAT, after which the spinal monosynaptic mass reflex amplitude reached its maximum level. These findings suggest that the 5-HT(2C) receptor is primarily involved in the mediation of the long-lasting excitability of spinal motoneurons and possibly interacts with its functional counterpart, 5-HT(1A) receptors in the mammalian adult spinal cord.

Introduction: Already low blood concentrations of ethanol acutely impair motor control and coordination. In vitro experiments have given evidence that spinal effects of ethanol contribute to this by reducing spinal excitability and enhancing presynaptic inhibition of Ia fibers. In this study, we investigated the influence of 0.7 g per kilogram of bodyweight ethanol on motoneuronal excitability and presynaptic inhibition in humans., Methods: The study was performed in 10 volunteers. Spinal excitability was measured by the maximal H-reflex of the soleus muscle normalized to the maximal muscular response (Hmax/Mmax). Presynaptic inhibition was measured by changes in heteronymous Ia-facilitation of the soleus H-reflex, which is achieved by stimulation of the femoral nerve. A decrease in facilitation can be ascribed to an increase in presynaptic inhibition. Changes of these parameters under the influence of 0.7 g per kilogram of bodyweight ethanol were assessed in comparison to control measurements before ethanol application., Results: Both parameters, Hmax/Mmax and Heteronymous facilitation, were significantly reduced under the influence of ethanol (Wilcoxon signed-rank test with Bonferroni correction for each, p<0.01)., Discussion: The increase in presynaptic inhibition by ethanol is probably caused by an increase in GABAA receptor-mediated Cl-conductance, which has been shown in spinal cord cultures. The role of presynaptic inhibition in movement is assumed to be there to control the afferent input of muscle spindles and tendon organs as a mechanism of specific input-selection. This study demonstrated that ethanol reduces spinal excitability and increases GABAergic presynaptic inhibition on Ia afferent fibers in humans.

The effects of 7- and 21-day haloperidol treatment on the spinal serotonergic system were examined in vivo in acutely spinalized adult rats. Intravenous administration of a selective 5-HT(2A/2C) receptor agonist, (+/-)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride (0.1 mg/kg) significantly increased the excitability of spinal motoneurones as reflected by increased monosynaptic mass reflex amplitude. This was significantly reduced in rats treated with haloperidol (1 mg/kg/day, i.p.) for 7 and 21 days. Administration of a 5-HT(1A/7) receptor agonist, (+/-)-8-Hydroxy dipropylaminotetraline hydrobromide (0.1 mg/kg, i.v.) significantly inhibited the monosynaptic mass reflex. This inhibition was greatly prolonged in haloperidol treated animals. These results demonstrate that the effects of haloperidol on the activation and desensitization of 5-HT(1A) and 5-HT(2A/2C) receptors respectively, may be mediated via intracellular mechanisms shared by these receptors with dopamine D(2) receptors in the mammalian spinal cord. The above serotonergic mechanisms may be partly responsible for haloperidol-induced extrapyramidal motor dysfunction.

We studied changes in the spinal segmental reflex and serotonergic (5-HT) responses in rats after spinal cord injury (SCI) produced by the weight-dropping method at the T8 level. The spinal monosynaptic reflex amplitude (MSR) was recorded from the L5 ventral root following stimulation of the ipsilateral L5 dorsal root. The 5-HT precursor l-5-hydroxytryptophan (L-5-HTP) depressed MSR in the spinal cord injured rats but not in normal rats. We investigated whether the SCI-specific depression of MSR by L-5-HTP was attributable to postsynaptic supersensitivity of 5-HT receptors or presynaptic loss of the 5-HT uptake system. Sumatriptan, a selective 5-HT(1B/1D) receptor agonist that is not taken up by 5-HT transporters, depressed the MSR similarly in both SCI and normal rats, suggesting that SCI resulted in the loss of 5-HT terminals and not postsynaptic supersensitivity of 5-HT receptors.

The neuronal pathways involved in the muscle relaxant effect of tizanidine were examined by measurement of spinal reflexes in rats. Tizanidine (i.v. and intra-4th ventricular injection) decreased the mono- and disynaptic (the fastest polysynaptic) reflexes (MSR and DSR, respectively) in non-spinalized rats. Depletion of central noradrenaline by 6-hydroxydopamine abolished the depressant effect of tizanidine on the MSR almost completely and attenuated the effect on the DSR. Co-depletion of serotonin by 5,6-dihydroxytryptamine and noradrenaline resulted in more prominent attenuation of tizanidine-induced inhibition of the DSR. Supraspinal receptors were then studied using yohimbine- and some imidazoline-receptor ligands containing an imidazoline moiety. Idazoxan (I1, I2, I3, and alpha2), efaroxan (I1, I3, and alpha2), and RX821002 (I3 and alpha2), but not yohimbine, an alpha2-adrenergic receptor antagonist with no affinity for I receptors, antagonized the inhibitory effects of tizanidine. Thus, supraspinal I receptors (most likely I3) and descending monoaminergic influences are necessary for tizanidine-induced inhibition of spinal segmental reflexes.

The effects of nonsteroidal antiinflammatory drugs, acetylsalicylate and metamizol, on spinal monosynaptic reflexes were investigated in spinalized and normal rats. Adult rats (n=36) weighing 150-200 g were anesthetized with ketamine and artificially ventilated. Half of rats were spinalized at C1 level. A laminectomy was performed in the lumbosacral region. Following electrical stimulation of the sciatic nerve by single pulses, reflex potentials were recorded from the ipsilateral L5 ventral root. Acetylsalicylate was administered orally (100 mg/kg for both spinalized and normal rats). Metamizol was administered intramuscularly (15 mg/kg for both spinalized and normal rats). These drug administrations significantly decreased the amplitude of reflex response in all groups (p < 0.05). These data verify that observed inhibition by acetylsalicylicate and metamizol may be at the level of spinal cord. Also we suggested that the cyclooxygenase products of arachidonic acid may play an important role in regulating the reflex potential.

Descending monoaminergic systems modulate spinal cord function, yet spinal dopaminergic actions are poorly understood. Using the in vitro lumbar cord, we studied the effects of dopamine and D2-like receptor ligands on spinal reflexes in wild-type (WT) and D3-receptor knock-out mice (D3KO). Low dopamine levels (1 microM) decreased the monosynaptic "stretch" reflex (MSR) amplitude in WT animals and increased it in D3KO animals. Higher dopamine concentrations (10-100 microM) decreased MSR amplitudes in both groups, but always more strongly in WT. Like low dopamine, the D3 receptor agonists pergolide and PD 128907 reduced MSR amplitude in WT but not D3KO mice. Conversely, D3 receptor antagonists (GR 103691 and nafadotride) increased the MSR in WT but not in D3KO mice. In comparison, D2-preferring agonists bromocriptine and quinpirole depressed the MSR in both groups. Low dopamine (1-5 microM) also depressed longer-latency (presumably polysynaptic) reflexes in WT but facilitated responses in D3KO mice. Additionally, in some experiments (e.g., during 10 microM dopamine or pergolide in WT), polysynaptic reflexes were facilitated in parallel to MSR depression, demonstrating differential modulatory control of these reflex circuits. Thus, low dopamine activates D3 receptors to limit reflex excitability. Moreover, in D3 ligand-insensitive mice, excitatory actions are unmasked, functionally converting the modulatory action of dopamine from depression to facilitation. Restless legs syndrome (RLS) is a CNS disorder involving abnormal limb sensations. Because RLS symptoms peak at night when dopamine levels are lowest, are relieved by D3 agonists, and likely involve increased reflex excitability, the D3KO mouse putatively explains how impaired D3 activity could contribute to this sleep disorder.

The adenosine A(1) receptor is involved in spinal cord antinociception. As its role at supraspinal sites is not well known, we studied the systemic effects of its agonist N-cyclopentyl-adenosine (CPA) in single motor units from adult-spinalized, intact and sham-spinalized rats. CPA was not effective after spinalization, but it was very effective in intact animals (ID50: 92+/-1.3 microg/kg, noxious pinch) and over 10-fold more potent in sham-spinalized animals (ID50 of 8.3+/-1 microg/kg). Wind-up was also inhibited by CPA. We also studied the effect of CPA in the immature spinal cord preparation, where CPA dose-dependently inhibited responses to low (IC50s: 9+/-0.7 and 7.7+/-1.3 nM) and high intensity stimulation (IC50s: 4.9+/-0.5 and 12.1+/-2 nM). We conclude that the integrity of the spinal cord is crucial for the antinociceptive activity of systemic CPA in adult rats but not in immature rats, not yet influenced by a completely developed supraspinal control.

Background: Spinal actions of halogenated ethers are widely recognized, whereas spinal actions of intravenous anaesthetics like propofol are less clear. The aim of this study was to compare the spinal effects of propofol and sevoflurane., Methods: We used an isolated spinal cord in vitro preparation from rat pups and superfused the anaesthetics at known concentrations. Responses of motoneurones to single and repetitive C-fibre intensity stimulation (trains of 20 stimuli at 1 Hz) of a lumbar dorsal root were recorded from the corresponding ventral root via a suction electrode., Results: Stimulation trains produced a wind-up of action potentials in motoneurones. Both propofol and sevoflurane produced a significant concentration-dependent depression of the evoked wind-up, although at clinically relevant concentrations sevoflurane exhibited a larger intrinsic efficacy. Applied at anaesthetic concentrations, sevoflurane 250 micro M abolished action potentials whereas propofol 1 micro M only produced a reduction close to 50%. At these concentrations, sevoflurane produced a large depressant effect on the monosynaptic reflex whereas propofol was ineffective., Conclusions: Sevoflurane produces large inhibitory effects on nociceptive and non-nociceptive reflexes which are likely to contribute to immobility during surgery. Compared with sevoflurane, propofol appears to have much weaker effects on spinal reflexes such as those recorded in an isolated preparation.

Although analgesic action of xenon has been reported, little is known about the effect of xenon at the spinal cord, which plays a crucial role in nociceptive transmission. We studied the effect of xenon on nociceptive reflex (the slow ventral root potential) and the monosynaptic reflex in neonatal rat spinal cord in vitro in comparison with nitrous oxide. Xenon (30%) and nitrous oxide (30%) were applied for 17 min through superfusing artificial cerebrospinal fluid. Xenon and nitrous oxide significantly reduced the amplitude of nociceptive reflex by approximately 70% and approximately 25%, respectively. Xenon and nitrous oxide also significantly reduced the amplitude of the monosynaptic reflex by approximately 35% and approximately 15%, respectively. These results indicate that xenon suppressed the synaptic transmission at the spinal cord, especially those of the slow ventral root potential, which reflect nociceptive transmission.

The novel antiepileptic drug gabapentin was designed as a structural analog of gamma-aminobutyric acid (GABA). However, its mechanism of action remains unclear. In the present study, we investigated the effect of gabapentin on spinal reflexes in anesthetized rats. The mono- and polysynaptic reflex potentials were recorded from the ipsilateral L5 ventral root after stimulation of the L5 dorsal root. The dorsal root reflex potential, an index of presynaptic inhibition, was recorded from the ipsilateral L4 dorsal root. In non-spinalized (intact) and spinalized rats, intravenously administered gabapentin reduced the mono- and polysynaptic reflex potentials in a dose-dependent manner. These inhibitory effects of gabapentin were not suppressed by the GABA(A) antagonist picrotoxin. Moreover, gabapentin also decreased spinal reflexes in spinalized rats depleted of spinal GABA with semicarbazide, an inhibitor of the GABA-synthesizing enzyme. The dorsal root reflex potentials were not affected by gabapentin. These results suggest that endogenous GABA does not mediate the inhibitory effects of gabapentin on spinal reflexes.

In the spinal cord, various subtypes of serotonin (5-hydroxytryptamine; 5-HT) receptors are involved in the modulation of motor output. Although the excitatory role of 5-HT(2) receptors is known, the receptor subtypes mediating the inhibitory effect of 5-HT on monosynaptic reflex transmission remain unclear. In this study, segmental spinal reflexes were recorded to examine the receptor subtypes underlying 5-HT-mediated inhibition of monosynaptic reflex transmission in spinalized rats. Under conditions of monoamine oxidase blockade with clorgyline, the 5-HT precursor L-5-hydroxytryptophan depressed the monosynaptic reflex. 3-Hydroxybenzylhydrazine dihydrochloride (NSD-1015), a centrally active decarboxylase inhibitor, abolished this inhibition, confirming that the depression of the monosynaptic reflex by L-5-hydroxytryptophan was due to 5-HT. In the presence of GR127935 or isamoltane, which show high affinity for 5-HT(1B) receptors, L-5-hydroxytryptophan did not suppress the monosynaptic reflex, whereas 5-HT(1A), 5-HT(1D), 5-HT(2) and 5-HT(7) receptor antagonists did not alter the inhibitory effect of L-5-hydroxytryptophan. These results suggest that serotonergic depression of monosynaptic reflex transmission is mediated by 5-HT(1B) receptors.

The involvement of frequency-dependent depression (FDD) of synaptic transmission for the depressant action of the Ptychodiscus brevis toxin (PbTx) was investigated in neonatal rat spinal cord in vitro. The stimulation of a dorsal root by train of pulses (five stimuli) at different frequencies evoked potentials in the ventral root (monosynaptic reflex, MSR). Amplitude of the fifth response as percent of first response at 0.1, 0.2, 0.5, 1.0 and 2.0 Hz were 90, 80, 75, 70 and 50%, respectively. In Mg2+-free medium, PbTx depressed the MSR and also enhanced the FDD in a concentration-dependent manner. Further, the PbTx-induced depression can well be correlated with the enhancement of FDD (r=0.98). In the presence of Mg2+ (1.3 mM), the FDD was greater than that in the absence of Mg2+. But in the presence of Mg2+ PbTx did not alter FDD, even though there was 25% depression at 28 microM (significantly lesser than in Mg2+-free medium). The results indicate that the Mg2+-sensitive component of PbTx-induced depression of MSR is mediated via the neuronal systems involving FDD.

The effects of acetylsalicylate and metamizol on spinal monosynaptic reflexes were tested in spinal rats. Adult rats were anesthetized with ketamine, artificially ventilated, and spinalized at the C1 level. A laminectomy was performed in the lumbosacral region. Following electrical stimulation of the sciatic nerve by single pulses, the reflex potentials were recorded from the ipsilateral L5 ventral root. Acetylsalicylate was administered orally via nasogastric tube and metamizol intramuscularly. Acetylsalicylate (50 and 100 mg/kg) and metamizol (15 mg/kg) significantly decreased the amplitude of the reflex response (p < 0.05). But the 10-mg/kg metamizol dose did not significantly decrease the amplitude of the reflex response. The cyclooxygenase products of arachidonic acid may play an important role in regulating the reflex potential., (Copyright 2003 S. Karger AG, Basel)

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex. These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.

In the 1st part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex (PFC) were evoked by electrical stimulation of layer I. Recordings with intracellular sharp, microelectrodes showed a concentration-dependent inhibition of the EPSP by adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), when given alone depressed the EPSP and in addition antagonized the effect of ADP-beta-S. Exclusion of the N-methyl-D-aspartate (NMDA) component of the EPSP by D(.)-amino-5-phosphonopentanoic acid (AP-5) abolished the ADP-beta-S-induced depression. The pressure-application of both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused reproducible depolarizations. ADP-beta-S inhibited the effect of NMDA, but did not alter that of AMPA. PPADS was also under these conditions antagonistic with ADP-beta-S. In the 2nd part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ADP-beta-S caused a concentration-dependent inhibition of the responses to NMDA. PPADS alone did not alter the NMDA-currents but again antagonized the action of ADP-beta-S; 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) also abolished the NMDA effect. The ADP-beta-S-induced inhibition persisted in the presence of tetrodotoxin (TTX) or guanosine 5'-O-(3-thiodiphosphate) (GDP-beta-S) applied to the external medium and the pipette solution, respectively. The 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) moderately decreased the ADP-beta-S effect. The inhibitory function of ADP-beta-S on EPSPs and the interaction with PPADS was observed also in layer V pyramidal neurons of the parietal somatosensory cortex. In conclusion, metabotropic P2Y(1) receptors appear to exert a new modulatory influence on fast excitatory amino acid transmission in the cerebral cortex.

The present study aimed at determining the role of the serotonin(1A) (5-HT(1A)) receptor subtype on the modulation of the mono-synaptic reflex (MSR) elicited by dorsal root stimulation and recorded from ventral roots in the hemisected spinal cord obtained from rat pups. Serotonin and 5-carboxamidotryptamine (5-CT) depressed both the MSR and the cumulative depolarisation (CD) produced by repetitive dorsal root stimulation, whereas the specific 5-HT(1A) receptor agonist (R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) applied at 1 microM showed a selective depressant effect on the MSR. Superfusion of the 5-HT(1A) receptor antagonist (+)-N-tert-butyl-3(4(2-methoxyphenyl)-piperazin-1-yl)-2-phenylprpanamide ((+)WAY 100135) produced a complete blockade of 8-OH-DPAT effects. In addition (+)WAY 100135 blocked partially the effects of 5-HT and 5-CT on the MSR but not the effects of these compounds on the CD. The results are consistent with an intervention of 5-HT(1A) receptors in the modulation of non-nociceptive reflexes but do not support a prominent role for this receptor in the modulation of nociceptive reflexes., (Copyright 2002 Elsevier Science Ireland Ltd.)

Our earlier studies demonstrated that in the hippocampus, cannabinoids suppress inhibitory synaptic transmission via CB(1) cannabinoid receptors, whereas a novel cannabinoid-sensitive receptor modulates excitatory synapses (Katona, I. et al., Journal of Neuroscience 19 (1999) 4544; Hájos, N. et al., European Journal of Neuroscience 12 (2000) 3239; Hájos, N. et al., Neuroscience 106 (2001) 1). The novel receptor does not correspond to CB(2), since this receptor type is not expressed in the brain (Munro, S. et al., Nature 365 (1993) 61). Recent binding experiments revealed that the synthetic cannabinoid WIN 55,212-2 binds with lower affinity to brain membranes of CB(1) receptor-knockout mice indicating that pharmacological differences exist between these two types of cannabinoid receptors in the hippocampus (Breivogel et al., Molecular Pharmacology 60 (2001) 155). To analyze this difference in detail, we first determined the EC(50) values of WIN 55,212-2 for excitatory and inhibitory transmission in rat hippocampal slices using whole-cell patch-clamp recordings. The estimated EC(50) value for inhibitory postsynaptic currents (IPSC) evoked by electrical stimulation in CA1 pyramidal cells was 0.24 microM, whereas for excitatory postsynaptic currents (EPSC) it was 2.01 microM, respectively. The cannabinoid antagonist, AM251, blocked the WIN 55,212-2-induced inhibition of evoked IPSCs, but not of EPSCs, providing evidence for its selectivity for CB(1). We then tested the hypothesis of whether the cannabinoid effect on hippocampal excitatory neurotransmission is mediated via receptors with an affinity for vanilloid ligands. Co-application of the vanilloid receptor antagonist capsazepine (10 microM) with cannabinoids (WIN55,212-2 or CP55,940) prevented the reduction of EPSCs, but not of IPSCs. The amplitude of evoked EPSCs was also suppressed by superfusion of the vanilloid receptor agonist capsaicin (10 microM), an effect which could also be antagonized by capsazepine. In contrast, capsaicin did not change the amplitude of evoked IPSCs. These results demonstrate that WIN 55,212-2 is an order of magnitude more potent in reducing GABAergic currents via CB(1) than in inhibiting glutamatergic transmission via the new CB receptor. The sensitivity of the new CB receptor (and EPSCs) to vanilloid ligands, but not to the cannabinoid antagonist AM251, represents another pharmacological tool to distinguish the two receptors, since CB(1) (and its effect on IPSCs) is not modulated by vanilloids, but is antagonized by AM251.

1. The identity of the serotonin (5-HT) receptors modulating the transmission of segmental C-fibre mediated signals was studied using an in vitro preparation of the hemisected spinal cord from rat pups. 2. Responses to trains of stimuli delivered to a lumbar dorsal root were recorded from the corresponding ventral root. The resulting cumulative depolarization (CD) mediated by unmyelinated fibres was quantified in terms of integrated area. The amplitude of the mono-synaptic reflex was also measured. Serotonergic agents were superfused at known concentrations and their effects on the reflexes evaluated. 3. 5-HT had depressant effects on the CD (EC(50) 34 microM). The rank order of potency of agonists for the depression of the CD was 5-carboxamidotryptamine (5-CT)>alpha-methylserotonin (alpha-met-5-HT) approximately 5-HT>42-methylserotonin (2-met-5-HT)approximately 8-OH-DPAT. 4. All the agonists including 2-met-5-HT and 8-OH-DPAT had strong depressant effects on the mono-synaptic reflex with the following order of potency: 5-CT>48-OH-DPAT>4alpha-met-5-HT approximate5-HTapproximate2-met-5-HT. 5. The inhibitory effects of 5-HT, alpha-met-5-HT and 5-CT were attenuated by the non-specific 5-HT antagonist methiothepin (1 microM) and by the 5-HT(1A/1B) antagonist SDZ 21009 (100 nM) but not by the selective 5-HT(1A) antagonist WAY 100135 (1 microM). 6. Other antagonists known to block 5-HT(2), 5-HT(6) and/or 5-HT(7) receptors (ketanserin, RO 04-6790, ritanserin and clozapine) did not change the effect of the agonists. 7. The data suggest an important contribution of 5-HT(1B) receptors to the inhibition of spinal C-fibre mediated nociceptive reflexes but no experimental support was found for the intervention of 5-HT(2), 5-HT(6) or 5-HT(7) receptors in this in vitro model.

The role of muscle ischemia and fatigue in modulating the monosynaptic reflex was investigated in decerebrate and spinalized rats. Field potentials and fast motoneuron single units in the lateral gastrocnemious (LG) motor pool were evoked by dorsal root stimulation. Muscle ischemia was induced by occluding the LG vascular supply and muscle fatigue by prolonged tetanic electrical stimulation of the LG motor nerve. Under muscle ischemia the monosynaptic reflex was facilitated since the size of the early and late waves of the field potential and the excitability of the motoneuron units increased. This effect was abolished after L3-L6 dorsal rhizotomy, but it was unaffected after L3-L6 ventral rhizotomy. By contrast, the monosynaptic reflex was inhibited by muscle fatiguing stimulation, and this effect did not fully depend on the integrity of the dorsal root. However, when ischemia was combined with repetitive tetanic muscle stimulation the inhibitory effect of fatigue was significantly enhanced. Both the ischemia and fatigue effects were abolished by capsaicin injected into the LG muscle at a dose that blocked a large number of group III and IV muscle afferents. We concluded that muscle ischemia and fatigue activate different groups of muscle afferents that are both sensitive to capsaicin, but enter the spinal cord through different roots. They are responsible for opposite effects, when given separately: facilitation during ischemia and inhibition during fatigue; however, in combination, ischemia enhances the responsiveness of the afferent fibres to fatigue.

The role of NMDA receptors in the regulation of spinal motor output was studied in rats. Muscle tension of the hind limbs of decerebrate animals and spinal reflex potentials in anesthetized animals were recorded. Intrathecal injection as well as systemic or intra-4th ventricular injection of (+)-5-methyl-10-11-dihydro-5H-dibenzo[a,d]cyclohepta-5-10-imine maleate (MK-801) reduced muscle tension. Systemic MK-801 did not alter monosynaptic reflexes either in intact or spinal rats, but attenuated polysynaptic reflexes in spinal rats. Thus spinal NMDA receptors participate in spinal motor output in the presence of some specific factors such as descending facilitation and preceding segmental depolarization, which remove the Mg2+ blocking.

(R)-8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) depressed the monosynaptic reflex. This effect was not antagonized by 5-HT(1A) receptor antagonists. We examined whether 5-HT(1D) and 5-HT(7) receptors are involved in (R)-8-OH-DPAT-induced inhibition of the monosynaptic reflex in spinalized rats. Pretreatment with methiothepin and mesulergine, but not clozapine, inhibited (R)-8-OH-DPAT-induced monosynaptic reflex depression. Pretreatment with 2a-(4-phenyl-1,2,3,6-tetrahydropyridal)butyl)-2a,3,4,5-tetrahydrobenzo[c,d]indol-2(1H)-one (DR4004) and (R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrolidine (SB-269970), new selective 5-HT(7) receptors antagonists, and N-[methoxy-3-(4-methyl-l-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide (GR127935), a selective 5-HT(1D) receptor antagonist, had no effect on (R)-8-OH-DPAT-induced depression. These results suggested that 5-HT(7) and 5-HT(1D) receptors are not involved in (R)-8-OH-DPAT-induced monosynaptic reflex depression.

The effect produced by the nitric oxide donor SIN-1 on monosynaptic reflexes was examined. Experiments were performed on anesthetized, paralyzed and spinalized cats. Lumbar monosynaptic reflexes were produced by stimulation of Ia afferents. I.v. application of SIN-1 (500 microg/kg) produced a mean marked potentiation of 704% of pre-drug control (100%) in the amplitude of monosynaptic reflexes. In addition, in other experiments a concentration-dependent effect on the amplitude of monosynaptic reflexes was observed after microinjections of SIN-1 into the ventral horn (1 microl; 10(-12) - 10(-3) M), with a mean facilitatory effect of 355%. In both cases, the potentiation was reversible 45 min after i.v. or local application of SIN-1. These results provide the first evidence that monosynaptic reflexes can be potentiated by nitric oxide.

Mice were used to record the spinal reflex potentials and to examine the effects of some drugs upon them. In anesthetized mice, laminectomy was performed in the lumbo-sacral region, and monosynaptic reflex potential (MSR) and polysynaptic reflex potential were recorded from the L5 ventral root after stimulation of the L5 dorsal root. Thyrotropin-releasing hormone (TRH) and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI) produced transient and long-lasting increases in the MSR amplitude, respectively. Tolperisone hydrochloride and baclofen produced transient and long-lasting MSR depressions, respectively. These results show that mice can be used to record spinal reflex potentials, and that it may be possible to study the spinal cord function of mutant and knockout mice using this method.

Despite the importance of GABAergic input to cardiac vagal neurons the electrophysiological properties and possible origins of this innervation have not yet been studied. Individual cardiac vagal neurons were identified by a retrograde fluorescent tracer and were studied in an in vitro slice preparation using patch-clamp electrophysiology. Cardiac vagal neurons received spontaneous GABAergic inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA(A) receptor antagonist bicuculline. The spontaneous presynaptic GABAergic input to cardiac vagal neurons in the nucleus ambiguus occurred at a significantly lower frequency than that recorded in cardiac vagal neurons in the dorsal motor nucleus of the vagus. To identify a possible source of the GABAergic innervation to cardiac vagal neurons the nucleus tractus solitarius was electrically stimulated. GABAergic synaptic currents in cardiac vagal neurons, in both the dorsal motor nucleus of the vagus (DMNX) and the nucleus ambiguus (NA), were consistently evoked upon stimulation of the nucleus tractus solitarius and these responses were also blocked by bicuculline.

We examined the effects of (R)- and (S)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) on the monosynaptic spinal reflex in rats. In intact rats, (R)-8-OH-DPAT (10 microg/kg, i.v.) enhanced the amplitude of the monosynaptic reflex, whereas at 100 microg/kg, it reduced the amplitude. (S)-8-OH-DPAT enhanced the monosynaptic reflex dose-dependently. In spinalized rats, (R)-8-OH-DPAT produced dose-dependent inhibition, but the (S)-enantiomer did not affect the monosynaptic reflex. Pretreatment with spiroxatrine or 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190) inhibited (R)-8-OH-DPAT-induced monosynaptic reflex enhancement in intact rats, as did 5-hydroxytryptamine (5-HT) depletion. Ketanserin reduced the effect of (R)-8-OH-DPAT. These pretreatment regimens had no effect on the monosynaptic reflex depression produced by the (R)-enantiomer in intact and spinalized rats. Pretreatment with prazosin inhibited (S)-8-OH-DPAT-induced monosynaptic reflex enhancement in intact rats, as did noradrenaline and 5-HT depletion. These results suggest that supraspinal 5-HT1A receptors and the descending serotonergic system are involved in the stimulatory effect of (R)-8-OH-DPAT on the monosynaptic reflex, while both the descending serotonergic and noradrenergic systems, the latter acting via alpha1-adrenoceptors, are involved in the effect of the (S)-enantiomer on this reflex.

1. The temporal interaction of evoked synaptic excitation and GABAA-mediated inhibition was examined in CA1 pyramidal cells. Single and paired intracellular recordings were carried out in pyramidal cell dendrites and somata, and interneurons of the guinea-pig hippocampal slice. Current-clamp, sharp electrode and whole-cell voltage-clamp recordings were made. 2. Kinetics of dendritic and somatic inhibitory responses were similar. Notably, kinetics of dendritic unitary IPSPs were as fast as kinetics of somatic unitary IPSPs. 3. GABAA-mediated influences were present throughout the orthodromic pyramidal cell EPSP/EPSC. Comparison of the kinetics of pharmacologically isolated monosynaptic IPSPs, IPSCs and inhibitory conductances (g GABAA), showed fastest kinetics for g GABAA. Close temporal overlap was observed between monosynaptic g GABAA and the rising phase of the evoked EPSP/EPSC. The onset of g GABAA coincided with or preceded onset of the EPSP/EPSC. 4. Onsets of feedforward IPSPs coincided with the rising phase of the pyramidal cell EPSP in > 80 % of paired recordings. Fastest feedforward inhibitory responses exerted near complete overlap with evoked excitation. 5. Onsets of recurrent IPSPs did not occur during the rising phase of the evoked EPSP, but > 3.0 ms after the peak of the pyramidal cell EPSP. 6. Orthodromically evoked interneuron spikes were observed at stimulation intensities that were below the threshold for eliciting EPSPs in concomitantly recorded pyramidal cells. The activation of feedforward inhibitory responses by weakest excitatory input, and the large temporal overlap between feedforward inhibition and evoked excitation, suggest that in situ any excitatory input in CA1 is effectively controlled by fast synaptic inhibition.

The effects of neurotrophin-3 (NT-3) and NT-4/5 on the function of axotomized group Ia afferents and motoneurons comprising the monosynaptic reflex pathway were investigated. The axotomized medial gastrocnemius (MG) nerve was provided with NT-3 or NT-4/5 for 8-35 d via an osmotic minipump attached to its central end at the time of axotomy. After this treatment, monosynaptic EPSPs were recorded intracellularly from MG or lateral gastrocnemius soleus (LGS) motoneurons in response to stimulation of the heteronymous nerve under pentobarbital anesthesia. Controls were preparations with axotomized nerves treated directly with vehicle; other axotomized controls were administered subcutaneous NT-3. Direct NT-3 administration (60 microgram/d) not only prevented the decline in EPSP amplitude from axotomized afferents (stimulate MG, record LGS) observed in axotomy controls but, after 5 weeks, led to EPSPs larger than those from intact afferents. These central changes were paralleled by recovery of group I afferent conduction velocity. Removal of NT-3 4-5 weeks after beginning treatment resulted in a decline of conduction velocity and EPSP amplitude within 1 week to values characteristic of axotomy. The increased synaptic efficacy after NT-3 treatment was associated with enhanced connectivity of single afferents to motoneurons. NT-4/5 induced modest recovery in group I afferent conduction velocity but not of the EPSPs they elicited. NT-3 or NT-4/5 had no effect on the properties of treated motoneurons or their monosynaptic EPSPs. We conclude that NT-3, and to a limited extent NT-4/5, promotes recovery of axotomized group Ia afferents but not axotomized motoneurons or the synapses on them.

Effects of calcium channel blocker flunarizine on spinal monosynaptic reflexes were investigated in spinal cats. Flunarizine was administered locally into the spinal cord (10, 50, 100 microM) and intraperitoneally (5, 10, 20 mg/kg). Adult cats (n = 10), weighing 1.5-3 kg were anesthetized with ketamine (50 mg/kg, i.m.) and artificially ventilated. Animals were spinalized at C1 level. A laminectomy was performed in the lumbosacral region. The ventral and dorsal roots of segment L5 were isolated and a pouch of skin was formed at the site of the dissection to allow the exposed tissues to be covered with liquid paraffin. The temperature was kept at 38.5 degrees C with a heating pad. A polyethylene cannula was introduced into the left carotid artery to monitor blood pressure, which was kept above 100 mmHg. The dorsal root of segment L5 was placed on a silver-silver chloride wire electrode for stimulation through an isolation unit. The reflex potentials were recorded from the ipsilateral L5 ventral root, mounted on a silver-silver chloride wire electrode. The systemic (5, 10, 20 mg/kg) and local (50 and 100 microM) dosages of cinnarizin derivative flunarizine significantly decreased the amplitude of reflex response (p < 0.05). Moreover, the latency of the monosynaptic reflexes was increased after administration of the drug (p < 0.05). Voltage-dependent calcium channels in the spinal cord may play an important role to regulate reflex respond.

Studies in rats have shown that the polysynaptic flexor reflex (FR) but not the monosynaptic reflexes are affected by N-methyl-D-aspartate (NMDA) receptor antagonists. Theoretically, the suppression of FR might be caused by an alteration of the spinal nociceptive neurons. To investigate, whether the non-competitive NMDA receptor antagonist memantine interferes with nociception in man, we studied both its effect on pain perception and on FR. In a double-blind study 14 male subjects were randomly assigned to either placebo or memantine (30 mg p.o.) treatment. H-reflex (HR) and FR as well as pain and tolerance threshold were determined prior to and 6 h after drug intake. Contrary to expectations, there were no differential treatment effects either on FR threshold and magnitude or on pain and tolerance thresholds or the HR amplitude.

1. The spinal volleys evoked by single transcranial magnetic or electric stimulation over the cerebral motor cortex were recorded from a bipolar electrode inserted into the cervical epidural space of three conscious human subjects. These volleys were termed direct (D) and indirect (I) waves according to their latency. 2. We measured the size and number of volleys elicited by magnetic stimulation at various intensities with subjects at rest and during 20 or 100 % maximum contraction of the contralateral first dorsal interosseous muscle (FDI). Surface EMG activity was also recorded. 3. Electrical stimulation evoked a D-wave volley. Magnetic stimulation at intensities up to about 15 % of stimulator output above threshold evoked only I-waves. At higher intensities, a D-wave could be seen in two of the three subjects. 4. At all intensities tested, voluntary contraction increased the number and size of the I-waves, particularly during maximum contractions. However, there was only a small effect on the threshold for evoking descending activity. Voluntary contraction produced large changes in the size of EMG responses recorded from FDI. 5. Because the recorded epidural activity is destined for muscles other than the FDI, it is impossible to say to what extent increased activity contributes to voluntary facilitation of EMG responses. Indeed, our results suggest that the main factor responsible for enhancing EMG responses in the transition from rest to activity is likely to be increased excitability of spinal motoneurones, rather than increases in the corticospinal volley. The latter may be more important in producing EMG facilitation at different levels of voluntary contraction.

The toxic lectin ricin was applied to the hamster chorda tympani (CT), producing anterograde degeneration of its terminal boutons within the gustatory zone of the nucleus of the solitary tract (NST). Immunocytochemistry was subsequently performed with antiserum against tyrosine hydroxylase (TH), and the synaptic relationships between degenerating CT terminal boutons and either TH-immunoreactive or unlabeled dendritic processes were examined at the electron microscopic level. Degenerating CT terminal boutons formed asymmetric axodendritic synapses and contained small, clear, spherical synaptic vesicles that were densely packed and evenly distributed throughout the ending, with no accumulation at the active synaptic. The degenerating CT terminated on the dendrites of TH-immunoreactive neurons in 36% (35/97) of the cases. The most frequent termination pattern involved the CT and two or three other inputs in synaptic contact with a single immunoreactive dendrite, resulting in a glomerular-like structure that was enclosed by glial processes. In 64% (62/97) of the cases, the degenerating CT was in synaptic contact with unlabeled dendrites, often forming a calyx-like synaptic profile that surrounded much of the perimeter of a single unlabeled dendrite. These results indicate that the TH-immunoreactive neurons of the gustatory NST receive direct input from the CT and taste receptors of the anterior tongue and that the termination patterns of the CT vary with its target neuron in the gustatory NST. The glomerular-like structure that characterizes many of the terminations of the CT provides an opportunity for the convergence of several functionally distinct inputs (both gustatory and somatosensory) onto putative dopaminergic neurons that may shape their responsiveness to the stimulation of the oral cavity.

1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg(2+)-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists--that is, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine > CPP > APV = 7-Clkyn > HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro.