NMDA receptor-mediated oscillatory activity in the neonatal rat spinal cord is serotonin dependent

The effect of serotonin (5-HT) receptor blockade on rhythmic network activity and on N-methyl--aspartate (NMDA) receptor-induced membrane voltage oscillations was examined using an in vitro neonatal rat spinal cord preparation. Pharmacologically induced rhythmic hindlimb activity, monitored via flexor and extensor electroneurograms or ventral root recordings, was abolished by 5-HT receptor antagonists. Intrinsic motoneuronal voltage oscillations, induced by NMDA in the presence of tetrodotoxin (TTX), either were abolished completely or transformed to long-lasting voltage shifts by 5-HT receptor antagonists. Conversely, 5-HT application facilitated the expression of NMDA-receptor-mediated rhythmic voltage oscillations. The results suggest that an interplay between 5-HT and NMDA receptor actions may be critical for the production of rhythmic motor behavior in the mammalian spinal cord, both at the network and single cell level.     

Whole cell recordings of lumbar motoneurons during locomotor-like activity in the in vitro neonatal rat spinal cord

Whole cell current- and voltage-clamp recordings were obtained from lumbar motoneurons in the isolated neonatal rat spinal cord to characterize the behavior of motoneurons during neurochemically induced locomotor-like activity. Bath application of serotonin (10-100 muM) in combination with N-methyl-D-aspartate (1-12 muM) initially produced tonic membrane depolarization (mean = 26 mV), increased input resistance, decreased rheobase, and increased spike inactivation in response to depolarizing current pulse injections. After the initial tonic depolarization, rhythmic fluctuations of the motoneuron membrane potential (locomotor drive potentials; LDPs) developed that were modulated phasically in association with ventral root discharge. The peak and trough voltage levels of the LDP fluctuated above and below the membrane potential recorded immediately before the onset of rhythmic activity. Similarly, firing frequency was modulated above and below prelocomotion firing rates (in those motoneurons that displayed neurochemically induced tonic firing immediately before the onset of rhythmic activity). These observations are consistent with an alternation between phasic excitatory and inhibitory synaptic drives. The amplitude of LDPs and rhythmic excitatory drive current increased with membrane depolarization from -80 to -40 mV and then decreased with further depolarization, thus displaying nonlinear voltage-dependence. Faster frequency, small amplitude voltage fluctuations were observed superimposed on the depolarized phase of LDPs. In some motoneurons, the trajectory of these superimposed fluctuations was consistent with a synaptic origin, whereas in other cells, the regular sinusoidal appearance of the fluctuations and the occurrence of superimposed plateau potentials were more compatible with the activation of an intrinsic membrane property. One motoneuron displayed exclusively excitatory phasic drive, and another motoneuron was characterized by inhibitory phasic drive alone, during rhythmic activity. These findings are compatible with the concept of a central pattern generator that is capable of delivering both excitatory and inhibitory drive to motoneurons during locomotion. The data also suggest that the rhythmic excitatory and inhibitory outputs of the hypothetical half-center model can be dissociated and operate in isolation.     

GABA-receptor-independent dorsal root afferents depolarization in the neonatal rat spinal cord

Dorsal root afferent depolarization and antidromic firing were studied in isolated spinal cords of neonatal rats. Spontaneous firing accompanied by occasional bursts could be recorded from most dorsal roots in the majority of the cords. The afferent bursts were enhanced after elevation of the extracellular potassium concentration ([K+]e) by 1-2 mM. More substantial afferent bursts were produced when the cords were isolated with intact brain stems. Rhythmic afferent bursts could be recorded from dorsal roots in some of the cords during motor rhythm induced by bath-applied serotonin and N-methyl--aspartate (NMDA). Bilaterally synchronous afferent bursts were produced in pairs of dorsal roots after replacing the NaCl in the perfusate with sodium-2-hydroxyethansulfonate or after application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline with or without serotonin (5-HT) and NMDA. Antidromic afferent bursts also could be elicited under these conditions by stimulation of adjacent dorsal roots, ventrolateral funiculus axons, or ventral white commissural (VWC) fibers. The antidromic bursts were superimposed on prolonged dorsal root potentials (DRPs) and accompanied by a prolonged increase in intraspinal afferent excitability. Surgical manipulations of the cord revealed that afferent firing in the presence of bicuculline persisted in the hemicords after hemisection and still was observed after removal of their ventral horns. Cutting the VWC throughout its length did not perturb the bilateral synchronicity of the discharge. These findings suggest that the activity of dorsal horn neurons is sufficient to produce the discharge and that the bilateral synchronicity can be maintained by cross connectivity that is relayed from side to side dorsal to the VWC. Antagonists of GABAB, 5-HT2/5-HT1C, or glutamate metabotropic group II and III receptors could not abolish afferent depolarization in the presence of bicuculline. Depolarization comparable in amplitude to DRPs, could be produced in tetrodotoxin-treated cords by elevation of [K+]e to the levels reported to develop in the neonatal rat spinal cord in response to dorsal root stimulation. A mechanism involving potassium transients produced by neuronal activity therefore is suggested to be the major cause of the GABA-independent afferent depolarization reported in our study. Possible implications of potassium transients in the developing and the adult mammalian spinal cord are discussed.     

In vivo echo-planar imaging of rat spinal cord

An integrated approach to echo-planar imaging of rat spinal cord in vivo with a small field of view (FOV) is presented. This protocol is based on a multishot interleaved echo-planar imaging (EPI) sequence and includes: 1) use of an inductively coupled implantable coil for improved signal-to-noise ratio (SNR); 2) three-dimensional (3D) automatic shimming of the magnetic field over the spinal cord; and 3) post-acquisition data processing using a multireference scan for minimizing image artifacts. Some of the practical issues in implementing this protocol are discussed. This imaging protocol will be useful in characterizing the spinal cord pathology using techniques that are otherwise time-consuming, such as diffusion tensor imaging.     

Characterization of commissural interneurons in the lumbar region of the neonatal rat spinal cord

Neurons with axons that extend to the contralateral side of the spinal cord--commissural interneurons (CINs)--coordinate left/right alternation during locomotion. Little is known about the organization of CINs in the mammalian spinal cord. To determine the numbers, distribution, dendritic morphologies, axonal trajectories, and termination patterns of CINs located in the lumbar spinal cord of the neonatal rat, several different retrograde and anterograde axonal tracing paradigms were performed with fluorescent dextran amines and the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). CINs with ascending (aCINs) and descending (dCINs) axons were labeled independently. The aCINs and dCINs occupied different but overlapping domains within the transverse plane. The aCINs were clustered into four recognizable groups, and the dCINs were clustered into two recognizable groups. All dCINs and most aCINs were located within the gray matter, with somata ranging from 10-30 microm in diameter and with large, multipolar dendritic trees. One group of aCINs was located outside the gray matter along the dorsal and dorsolateral margin and had dendrites that were nearly confined to the dorsolateral surface. All CIN axons traversed the ventral commissure at right angles to the midline. CIN axons coursed up to six or seven segments rostrally and/or caudally in the ventral and ventrolateral white matter and gave off collaterals over a shorter range, predominantly to the ventral gray matter. These findings show that the lumbar spinal cord of the neonatal rat contains substantial numbers of CINs with axon projections and collateral ranges spanning several segments and that CINs projecting rostrally vs. caudally have different distributions in the transverse plane. The study provides an anatomical framework for future electrophysiological studies of the spinal neuronal circuits underlying locomotion in mammals.     

Glutamate receptor-mediated hyperexcitability after ethanol exposure in isolated neonatal rat spinal cord

The influence of maternal opioid receptor blockade (50 mg/kg naltrexone, NTX) or saline (controls) throughout pregnancy on nociception and brain opioid receptor characteristics of rat offspring were examined; all animals were crossfostered to untreated mothers at birth. At 21 and 30 days, NTX-exposed pups weighed 8.2-24.3% more than controls, but both NTX and control groups were of similar body weights at 48, 60, and 80 days. Rats in the NTX and control groups displayed comparable baseline reactions to the hotplate. Morphine challenge tests and nociceptive measures revealed that NTX-subjected offspring examined at 21, 30, 48, and 60 days did not react to dosages that invoked 42-132% decreases from baseline levels in controls. Animals exposed prenatally to NTX were analgesic when injected with the opioid butorphanol or the nonopioid xylazine. The binding affinity (Kd) and capacity (Bmax) of delta and kappa opioid receptors were similar in NTX and control groups at 21 and 80 days. However, the Bmax, but not the Kd, of mu opioid receptors was subnormal in NTX offspring by about 20% in contrast to control rats at 21 and 80 days. The results imply that the interactions of some endogenous opioids with opioid receptors during development are determinants of certain aspects of pain sensitivity as well as the density of particular opioid receptors in the postnatal period.     

Modulation of neonatal rat hypoglossal motoneuron excitability by serotonin

The effects of 5-HT on neonatal rat hypoglossal motoneurons (HMs) were studied in two in vitro slice preparations. Serotonin caused either reversible depolarization or the generation of an inward current (I5-HT) in every cell tested. I5-HT persisted after synaptic blockade. In most of the cells tested, the magnitude of I5-HT was independent of membrane potential (-50 to -120 mV), and 5-HT had little effect on input resistance or slope conductance. In addition, 5-HT significantly reduced the amplitude of the post-spike medium-duration afterhyperpolarization. This reduction probably contributed to the resulting increase in the slope of the relationship describing the steady-state firing frequency response to injected current (f-I) observed in the presence of 5-HT. Thus, 5-HT increases the excitability of neonatal HMs via at least two different postsynaptic mechanisms.     

Receptors of monoamine in sympathetic preganglionic neurons of neonatal rat spinal cord in vitro

By means of intracellular recordings from spinal cord slices of neonatal rats in vitro, the effects of 5-hydroxytryptamine (5-HT), nor-adrenaline (NA) and adrenaline (AD) on membrane potential in sympathetic preganglionic neurons (SPN) were observed, in order to clarify whether these neuron contain a single type of the monoamine receptor or in combination with more than one type of receptors. The results showed that: (1) 5-HT, NA and AD induced membrane depolarization respectively in 57.1% (16/28), 60% (15/25) and 52.4% (11/21) of SPN. (2) According to the reactions of SPN to the three monoamines, several subtypes of SPN could be divided: those sensitive to all the three monoamines (3/19), those sensitive to two of them (9/19), those only sensitive to one type of monoamines (4/19) and those insensitive at all (3/19). The significance of coexistence of more than one type of the three monoamines in a single neuron remains to be elucidated.     

Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord

The motor programme executed by the spinal cord to generate locomotion involves glutamate-mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5-hydroxytryptamine (5-HT), we investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5-HT (2.5-30 microM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5-HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min-1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5-HT doses were increased (range 20-40 microM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non-NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5-HT, albeit with doses larger than in the control solution (15-40 microM). When NMDA and non-NMDA receptors were simultaneously blocked, 5-HT (5-120 microM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non-NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.     

Evidence for age-dependent changes in motoneuron dendritic morphology following neonatal nerve-crush in the rat

Motoneurons supplying the extensor hallucis longus muscle of the rat were temporarily separated from the muscle by sciatic nerve-crush at five days postnatally. Such treatment permanently alters the reflex response of the affected motoneurons without the large-scale cell death associated with nerve-crush at birth. After reinnervation, the motoneurons were retrogradely labelled with cholera toxin subunit-B conjugated to horseradish peroxidase and the dendritic tree of each labelled cell was analysed. When compared to normal data, significantly higher levels of dendritic density were observed in the rostrodorsally orientated parts of the dendritic field. This was similar to that found previously for the same motor pool after nerve-crush at birth. However, in other parts of the field where a lower dendritic density was found after nerve-crush at birth, no change was seen after nerve-crush at five days. These data present evidence for the influence of sensory afferents on the development of motoneuron dendrites. Taken together with the previous findings after nerve-crush at birth, we suggest that the differential dendritic changes caused by neonatal nerve lesion contribute to an imbalance in the pattern of excitatory and inhibitory inputs to the motoneuron, which results either in cell death, or the abnormal activity seen in those motoneurons which survive.     

Analysis of EPSCs and IPSCs carrying rhythmic, locomotor-related information in the isolated spinal cord of the neonatal rat

To understand better the synaptic language used by neurons in active networks, we have analyzed postsynaptic currents (PSCs) received by interneurons in the isolated spinal cord from neonatal rats during 5-hydroxytryptamine- and N-methyl--aspartate-induced fictive locomotion. Using a computer algorithm, we identified PSCs in rhythmically active interneurons in laminae VII and X. To test whether the PSCs actually participated in the transmission of the cyclic, locomotor-related signal, we constructed an analytic current trace based on only the identified events. Each identified PSC was fitted by a mathematical function, and the shape of this function was added to a baseline with time delays given by the time positions of the identified PSCs. By averaging the resulting analytic current trace over several cycles, we showed that the identified PSCs built a cyclic signal locked to the rhythmic activity recorded from the ventral roots. Furthermore, subtraction of the analytic from the original current trace reduced the amplitude of the cyclic signal received by these cells. Thus the identified PSCs contributed to the cyclic information, allowing us to analyze how they built the compound cyclic signal. Most often there was an inverse relationship between the contribution from excitatory and inhibitory PSCs during the cyclic modulation, indicating that there was a reciprocal regulation of the presynaptic inhibitory and excitatory cells. Comparing the most inhibitory and most excitatory halves of the locomotor related cycle, there was a considerably larger modulation of the frequency of PSCs than of their amplitude. The small and sometimes insignificant modulation of PSC amplitude suggests that facilitation and depression had little importance for the information transfer. The modest amplitude modification also suggests that the large range of available PSC amplitudes seen in these neurons was not used very efficiently to code the cyclic information.     

Magnetic resonance imaging in cervical spinal cord compression

In patients with cervical spondylotic myelopathy MRI sometimes shows increased signal intensity zones on the T2-weighted images. It has been suggested that these findings carry prognostic significance. We studied 56 subjects with cervical spinal cord compression. Twelve patients showed an increased signal intensity (21.4%) and a prevalence of narrowing of the AP-diameter (62% vs 24%). Furthermore, in this group, there was evidence of a longer mean duration of the symptoms and, in most of the patients, of more serious clinical conditions. The importance of these predisposing factors remains, however, to be clarified since they are also present in some patients without the increased signal intensity.     

The number of postsynaptic currents necessary to produce locomotor-related cyclic information in neurons in the neonatal rat spinal cord

To understand better how synaptic signaling contributes to network activity, we analyzed the potential contribution of putative unitary postsynaptic currents (PSCs) to locomotor-related information received by spinal interneurons in neonatal rats. The average cyclic modulation of the whole-cell current in 13 neurons was quantified as the difference between the current integral (charge) during the first and second halves of the cyclic locomotor network output. Between 7.6 and 303 average unitary PSCs per second were needed to produce the cyclic modulation. This number is so low that very few (1-5) of the synapses contributing to the cyclic information need to be active simultaneously. This suggests that individual presynaptic cells in a central locomotor network can have a powerful influence on other neurons.     

Development of spontaneous synaptic transmission in the rat spinal cord

Dorsal root afferents form synaptic connections on motoneurons a few days after motoneuron clustering in the rat lumbar spinal cord, but frequent spontaneous synaptic potentials are detected only after birth. To increase our understanding of the mechanisms underlying the differentiation of synaptic transmission, we examined the developmental changes in properties of spontaneous synaptic transmission at early stages of synapse formation. Spontaneous postsynaptic currents (PSCs) and tetrodotoxin (TTX)-resistant miniature PSCs (mPSCs) were measured in spinal motoneurons of embryonic and postnatal rats using whole cell patch-clamp recordings. Spontaneous PSC frequencies were higher than mPSC frequencies in both embryonic and postnatal motoneurons, suggesting that even at embryonic stages, when action-potential firing rate was low, presynaptic action potentials played an important role in triggering spontaneous PSCs. After birth, the twofold increase in spontaneous PSC frequency was attributed to an increase in action-potential-independent quantal release rather than to a higher rate of action-potential firing. In embryonic motoneurons, the fluctuations in peak amplitude of spontaneous PSCs were normally distributed around single peaks with modal values similar to those of mPSCs. These data indicated that early in synapse differentiation spontaneous PSCs were primarily composed of currents generated by quantal release. After birth, mean mPSC amplitude increased by 50% but mean quantal current amplitude did not change. Synchronous, multiquantal release was apparent in postnatal motoneurons only in high-K+ extracellular solution. Comparison of the properties of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) demonstrated that mean mEPSC frequency was higher than mIPSC frequency, suggesting that either excitatory synapses outnumbered inhibitory synapses or that the probability of excitatory transmitter release was higher than the release of inhibitory neurotransmitters. The finding that mIPSC duration was several-fold longer than mEPSC duration implied that despite their lower frequency, inhibitory currents could modulate motoneuron synaptic integration by shunting incoming excitatory inputs for prolonged time intervals.     

Behavioral effects of spinal cord transection in the developing rat

Albino rats, 0, 9, 12, 15, 18, 21 or greater than 90 days of age, were given a mid-thoracic spinal cord transection. Evaluation of responses of the hindlimbs to a variety of behavioral tasks was begun on the day of surgery and at intervals throughout the postoperative survival period (up to 300 days). Two investigators, independently and without knowledge of the animals' ages or survival times, rated the response data. Histological study showed all transections to be complete. Large differences in behavior are observed when animals trasected at the neonatal stage (0-4 days of age) are compared with animals transected at the weanling stage (21-26 days of age)37. Results of the present investigation indicate a critical period near 15 days of age; animals lesioned prior to this age (0, 9, 12 days of age) show response development and recovery similar to the neonatally lesioned animal, whereas those animals lesioned at a later age (18, 21, greater than 90 days of age) show little recovery and are behaviorally similar to the weanling transected animal. In animals lesioned prior to the fifteenth postnatal day, postural responses appear depressed for a brief period but recover rapidly while most responses of animals in the older groups are depressed for longer periods and never attain the degree of recovery characteristic of the neonatally transected animal. Finally, like the neonatally transected animal, rats lesioned on the ninth and twelfth postnatal day develop certain responses at appropriate times relative to normal response development. If, however, these responses are mature and supraspinal control is present at the time of lesioning, they appear to be permanently depressed and fail to recover.     

An in vitro functionally mature mouse spinal cord preparation for the study of spinal motor networks

An in vitro isolated whole spinal cord preparation has been developed in 'motor functionally mature' mice; that is mice of developmental maturity sufficient to weight-bear and walk. In balb/c mice this stage occurs at around postnatal day 10 (P10). Administration of strychnine elicited synchronous activity bilaterally in lumbar ventral roots. Rhythmic alternating locomotor-like activity could be produced by application of a combination of serotonin (5-HT), N-methyl-d-aspartate (NMDA), and dopamine in animals up to P12. Using a live cell-dead cell assay, it is demonstrated that there are primarily viable cells throughout the lumbar spinal cord. The viability of descending pathways was demonstrated with stimulation of the mid-thoracic white matter tracts. In addition, polysynaptic segmental reflexes could be elicited. Although usually absent in whole cord preparations, monosynaptic reflexes could invariably be elicited following longitudinal midline hemisection, leading to the possible explanation that there might be an active crossed pathway producing presynaptic inhibition of primary afferent terminals. The data demonstrate that this functionally mature spinal cord preparation can be used for the study of spinal cord physiology including locomotion.     

Effect of sciatic nerve transection or TTX application on enzyme activity in rat spinal cord

BACKGROUND/PURPOSE: Treatment of several congenital anomalies is frequently hindered by lack of enough tissue for surgical reconstruction in the neonatal period. The purposes of this study were (1) introduction of a novel concept in perinatal surgery, involving minimally invasive harvest of fetal tissue, which is then processed through tissue engineering techniques in vitro while pregnancy is allowed to continue, so that, at delivery, the newborn can benefit from having autologous, expanded tissue promptly available for surgical implantation at birth; (2) analysis of the progress of an engineered fetal skin graft with time, after implantation in the neonate; and (3) study of the effects of current tissue engineering techniques on fetal keratinocytes and fetal dermal fibroblasts. METHODS: Ten 90- to 95-day-gestation fetal lambs underwent surgical creation of two large paramedian excisional skin defects on the posterior body wall. Subsequently, fetal skin specimens no larger than 1.5 x 1.5 cm were videofetoscopically harvested. Fetal keratinocytes and dermal fibroblasts were then separately cultivated and expanded in vitro for 45 to 50 days, resulting in a total of approximately 250 to 300 million cells. Seven to 10 days before fetal delivery, all cells were seeded in two layers on a 16 to 20-cm2, 3-mm thick biodegradable polyglycolic acid polymer matrix. One to 4 days after delivery, the autologous engineered skin was implanted over one of two previously created skin defects. The second skin defect region received an absorbable polymer scaffold without cells as a control. If necessary, the original skin wounds were further amplified before implantation. Each animal provided at least one time-point for histological analysis of both types of repair through excisional biopsies performed at weekly intervals, up to 8 weeks postimplantation. Normal skin specimens were also used as controls. RESULTS: Fetal and neonatal survival rates were 100%. Based on previous postnatal skin engineering studies, fetal dermal fibroblasts multiplied significantly faster in vitro (approximately fivefold) than expected. Fetal keratinocytes multiplied at expected postnatal rates. The engineered grafts induced faster epithelization of the wound (partial at 1 week and complete between 2 and 3 weeks postoperatively) than did the acellular ones (partial at 3 weeks and complete between 3 and 4 weeks postoperatively). Analysis of skin architecture showed a higher level of epidermal organization and less dermal scarring in the wounds that received the engineered, cell-implanted polymer scaffold. CONCLUSIONS: (1) Videofetoscopically assisted fetal tissue engineering is a viable method for obtaining expanded autologous tissue for prompt surgical reconstruction at birth. (2) Fetal skin can be expanded and engineered in vitro at faster rates than expected postnatally, with current tissue engineering techniques. (3) Engineered autologous fetal skin induces a faster and more organized healing of neonatal skin defects than that observed with second intention. This concept may prove useful for the treatment of certain human neonatal conditions such as giant neoplasias, ectopia cordis, and other body wall defects.     

Calcium and magnesium deficiency-induced atrophy of muscle and calcium accumulation in the spinal cord

Four consecutive patients with mycosis fungoides received cyclic chemotherapy using cyclophosphamide, vincristine, and prednisone (COP). In one case, there was complete remission of disease for 15 months. In another, there was 50% regression of tumors with healing of the ulcerated surfaces. The third patient showed complete clearing of scaling and redness of the skin. In the fourth patient, the lesions progressed.     

Modulation of lamprey fictive swimming and motoneuron physiology by dopamine, and its immunocytochemical localization in the spinal cord

The antiseptic activity of Listerine and Cool Mint Listerine against methicillin-resistant Staphylococcus aureus (MRSA), Candida albicans, human immuno deficiency virus (HIV) and oral bacteria was examined in this study. Exposure for 30 seconds to Listerine killed MRSA completely. Exposure for 30 seconds significantly decreased viable cells of C. albicans. More than 60% of HIV was inactivated by a 30 second exposure to 50% Listerine. Listerine exhibited a potent bactericidal effect against cariogenic and periodontopathic bacteria. Cool Mint Listerine had almost the same antiseptic effect against tested microorganisms. Listerine appears to be effective for killing etiologic microorganisms of opportunistic infection in the oral cavity.