Neonatal Mice Spinal Cord Interneurons Sending Axons in Dorsal Roots

Background: Spinal cord interneurons send their axons in the dorsal root. Their antidromic re could modulate peripheral receptors. Thus, it could control pain, other sensorial modality, or muscle spindle activity. In this study, we assessed a staining technique to analyze whether interneurons send axons in the neonate mouse’s dorsal roots. We conducted experiments in 10 Swiss-Webster mice, which ranged in age from 2 to 13 postnatal days. We dissected the spinal cord and studied it in vitro. Results: We observed interneurons in the spinal cord dorsal horn sending axons through dorsal roots. A mix of uorochromes applied in dorsal roots marked these interneurons. They have a different morphology than motoneurons. Primary afferent depolarization in afferent terminals produces antidromic action potentials (dorsal root reex; DRR). These reexes appeared by stimulation of adjacent dorsal roots. We found that in the presence of bicuculline, DRR recorded in the L4 dorsal root evoked by L5 dorsal root stimulation was reduced. Simultaneously, the monosynaptic reex (MR) in the L5 ventral root was not affected; nevertheless, a long-lasting after discharge appeared. The addition of 2-amino-5 phosphonovalric acid (AP5), an antagonist of NMDA receptors, abolished the MR without changing the after discharge. Action potentials persisted in dorsal roots even in low Ca2+ concentration. Conclusions: Thus, ring interneurons could send their axons by dorsal roots. Antidromic potentials may be characteristics of the neonatal mouse, probably disappearing in adulthood.


Introduction
Spinal cord interneurons send their axons in the dorsal root. Their antidromic re could modulate peripheral receptors. Spontaneous ring and occasional bursting in dorsal roots (DR) occurred after elevating the extracellular potassium concentration in isolated spinal cords of neonatal rats (1). The increase in potassium concentrations is also associated with seizures episodes. They occurred with primary afferent depolarizations and antidromic discharges of nerve impulses in DR bers (1,2). Antidromic activity occurred in the dorsal root ganglia in chronically axotomized rats (3). They can block or affect orthodromic impulses colliding with incoming afferent volleys (1,4). This mechanism would require high ring frequencies in the antidromic discharge (5). Interneurons sending axons via DR in the spinal cord produce antidromic action potential regulating different types of peripheral receptors (6).
Ventral funiculus stimulation also evoked antidromic discharges in dorsal roots in a Petri dish brain stem-spinal cord preparation of neonatal (0-5-day old) rats. These discharges occurred by the underlying afferent terminal depolarization reaching ring threshold (7). Spontaneous interneuron activities play a critical role in the development of neuronal networks. Their discharges were conducted antidromically along the DR preceding those in the ventral root (VR) lumbar motoneurons. The action potential propagates centrally and triggers EPSPs in motoneurons. An indication of axons in dorsal roots coming from spinal cord interneurons is staining neurons with uorochromes.

Neuron Labeling
We analyzed the uorescent marker patterns in all spinal cords (n = 10) used for this study. The RDA application in the L4 dorsal root produced the red uorescent staining in afferent bers. It also stained rst-order interneurons by RDA leakage due to lower MW. They were marked close to terminal branches ( Fig. 2A). Application of FDA (green) in dorsal root L5 only marked afferent bers. Some interneurons were marked in red only when they were close to RDA afferent bers (Fig. 2B, the afferent ber are indicated by arrows). Application of a mixture of RDA and FDA in DR produced yellow staining. We only considered interneurons sending their axon by the dorsal roots when stained in yellow. We found some interneurons marked in yellow ( Fig. 2C-D, indicated by arrows) at the dorsal horn or in the intermediate nucleus.
To determinate the interneuron localization, we marked some spinal cord DR's exclusively with FDA, and most of the afferent ber ends in the dorsal horn (Fig. 3A). In other cases, we retrogradely marked some interneurons with FDA in L5 and RDA in L4 dorsal root and localized interneurons in the dorsal horn ( Fig. 3B). FDA marked interneurons seem to indicate that they project their axons through the dorsal roots. In contrast, the RDA application in L5 and FDA in L4 dorsal root did not stain interneurons located in this region. With an FDA and RDA mixture applied in L4 and FDA mainly in L5 dorsal roots, we found marked yellow interneurons close to the intermediate nuclei region (Fig. 3C). Their morphology is different from motoneurons stained by the mixture of uorochromes applied in the L5 ventral root (Fig. 4B).
The afferent bers arriving in the motor nucleus exhibited a bulb-like terminal when we applied the uorochrome mixture in dorsal roots (Fig. 4A). With FDA, we marked some bers green (Fig. 4B). We did not see any RDA leakage. The mixture applied in L5 VR stained neurons, revealed the motoneuron morphology. However, no interneurons showed marks in this ventral motor region. In addition, some marked cells resembled neurons traveling in rafts on the spinal cord dorsal surface when the uorochrome mixture penetrated the dorsal roots (Fig. 4C). Then, they began to penetrate the deep layers of the spinal cord (Fig. 4D).
In Fig. 5A-B, the spinal cord cut exhibits one and two nuclei at different stages. We found only one nucleus with small-sized neurons at P2, and we observed two nuclei at P13. We counted the number of neurons at P2 and P13 and measured the cell size ( Fig. 5C-F). The graphs in Figs. 5C and D illustrate the size of all neurons stained in P2 and P13. We performed a linear regression to determine the mean value. In P2, most neurons were less than 2000 µm. In P13, the size and number of neurons in both nuclei increased; even the smallest neurons were more extensive than in P2 (Fig. 5E). The difference in the mean values of the two groups (P2 and P13 < average value) are higher than those occurred by chance; there is a statistically signi cant difference between the two groups (P < 0.05). In P13, the mean value of the size of the neurons in both nuclei was approximately 8000 µm (Fig. 4F).

Discharges in the dorsal and ventral roots
In 10 day old mice (n = 4), we stimulated the L5 dorsal root to produce a monosynaptic re ex. It registered in the L5 ventral root and the DRR in the L4 dorsal root. We took control of the monosynaptic re ex, the dorsal, and ventral re ex activity in normal aCSF (Fig. 6A). Recordings were obtained and were similar in all animals (n = 4 ) in these experiments.
Bathing with bicuculine (10-20 µM) eliminated DRR but not the monosynaptic re ex (Fig. 6B). Interestingly, a long latency re ex occurred after the bicuculline application. Bicuculline has already described inducing locomotion episodes after rhythmic activity recorded in the ventral roots (not illustrated) (Duenas SH & Eidelberg, 1979). Similar activity has been observed in spinal cord motor neurons in the turtle in the presence of bicuculine (9).
AP5 and bicuculline application decreased MR and DRR ( Fig. 6C-D); after a few minutes, it eliminated, but not after discharge. We recorded sporadic action potentials in DR ( Fig. 6C-D).
After washing out bicuculline and AP5, the normal MR and DRR were reestablished (Fig. 6E). A low Ca + solution was then applied; MR, DRR, and after discharge disappeared; interestingly action potentials were observed in DR (Fig. 6F).

Discussion
In our experiments, spinal interneurons send axons through dorsal roots. We localized most of these interneurons close to the intermediate nucleus. They have several shapes that differ from motoneurons.
In our study, we did not study dendritic arborizations nor their changes with age, as assessed in previous studies. In previous studies, Westerga & Gramsbergen observed a considerable increase in motoneuron soma size in rats, but with different distribution and arborizations patterns in a developing stage, which are longer and more extensive at rst in cervical than in the lumbar region (10). This temporal and spatial differences may in uence the motor development in a rostrocaudal manner (11). Dendrite bundles appeared relatively late in the Soleus' motoneuron compared to the Tibial anterior; this is related to the ne-tuning of neuronal activity, rather than patterning of motor activity (10). These observations will be studied in neonatal mice.
Developing serotoninergic motoneuron innervation is related to the postnatal development of motor function already recognized in the second postnatal week (11). In our study, we found a signi cant neuronal soma size increase at a similar postnatal age. Marked neurons are not of the same type or from a speci c neuron group. That could be related to a different organization of the activation pattern.
We found some cells traveling in the spinal cord dorsal surface. We did not know if these cells are neurons or glia. In a developmental study of kittens, the volume of the lateral cervical nucleus and the glial cells increased sixfold during 120-day observation, as did both the volumes of myelinated axons (12).
As we noticed cells traveling in rafts in the dorsal horn surface of the spinal cord in the mouse spinal cord, further immunohistological studies could reveal the type of cells and clarify if some of them are progenitor neurons (13)(14)(15).
We cannot con rm whether the recorded interneurons produce activity (action potentials) traveling antidromically in dorsal roots. However, we found antidromic activity in dorsal roots, even in bicuculline, AP5, and low calcium. In another study, 2-4 postnatal day mice presented depression curves unexplained by presynaptic activation failure (suppressed by AP5). Low calcium concentration reduced average amplitude and depression, and a higher calcium concentration increased average amplitude and depression. Increasing the bath temperature from 24 to 32 Celsius produced little change in amplitudes, but the depression was noticeably reduced at most frequencies (16). Therefore, these AP could be generated by these interneurons when their axons are su ciently depolarized.
5HT, DA, and NA produced no change in the compound antidromic potentials evoked by intraspinal microstimulation, indicating that DRP depression is unrelated to direct changes in the excitability of intraspinal afferent bers (17). Thus, antidromic activity could have an origin other than PAD, and consequently, other functions. Ephaptic interaction in afferent bers could also produce antidromic ring (18).
Antidromic spike function in dorsal roots could participate in regulating activity in the afferent in ow of information related to in ammation and pain. DRR in afferent ber raises the hypothesis that mediated antidromic activity contributes to neurogenic in ammation (19). Sectioning the sciatic nerve of neonatal rat's triggers growth of afferent ber in VR, and stimulation in the L5 spinal cord evoked long latency antidromic potentials in the L5 ventral root. However, in normal rats, such potentials rarely appeared (20). Several experimental conditions, such as axotomy of sensory afferents, produced ectopic antidromic activity in their respective DRG, due to branched sensory afferents ber (3).
In our experiments, the antidromic activity in DR, even in low calcium concentration, is indicative of axons in dorsal roots. We cannot assert their functional signi cance or action in the neonatal mouse. It would be essential to nd out whether these antidromic potentials in dorsal afferent bers are favoring some spinal circuit formation which remain in adulthood or are only part of a development process.
Sympathetic preganglionic neurons (PGNs) in the neonatal rat's isolated spinal cord could be synaptically activated either by the dorsal root or spinal pathway stimulation. Dorsal root projections already appeared mature in the neonatal rat, and primary afferents did not appear to project directly to PGNs (21).

Conclusions
In our experiments, spinal interneurons send axons by dorsal roots. Thus, the AP comes to the interneurons sending axons in dorsal roots. Some spikes also occurred in ventral roots. In neonatal mice, spinal cord bipolar neurons could exist, sending axons through ventral and dorsal roots. Thus, AP could be produced by neurons with axons in ventral and dorsal roots. The presence of these interneurons in their maturity and their functional role in neonatal mice should be analyzed.
We used the double labeling technique, which to our knowledge, is the rst time that it has been employed to identify interneurons with axons in dorsal roots. The nal location of these interneurons in adult mice spinal cords and their function will be investigated to elucidate the functional connections in adulthood.

Materials And Methods
The rst purpose in this study was to assess the presence of spinal cord interneurons sending axons in dorsal roots. The second aim was to evaluate whether there are antidromic potentials in the neonatal mouse spinal cord dorsal roots. For studying dorsal root functionality, we also analyzed DRR in the L5 dorsal root. Likewise, we studied MR modulation produced by electrical stimulation on the L5 DR and recorded this re ex in L5 VR. We also added bicuculline, a GABA antagonist drug, and the glutamic antagonist AP5 for analysing neural transmission implied in these re exes.

Subjects
We did experiments in 10 Swiss-Webster mice isolated spinal cords in vitro preparations at 2 to 13 postnatal days. They were housed one single mouse per cage at room temperature. Experimental protocols and animal care were under the NIH guidelines (USA) and approved by the Institutional Ethics Animals were anesthetized by inhalation with methoxy-urane. When fully anesthetized, they were decapitated. After ventral laminectomy, we used a tungsten needle to perform a longitudinal hemisection and kept ventral and dorsal roots between the T6 and sacral spinal cord segments. Other researchers followed this procedure in previous studies (22)(23)(24). One hemicord was placed in a Sylgard silicone elastomer tube at the bottom of a recording chamber. The hemicord was perfused with oxygenated ACSF owing at 10-14 ml/min. The bath solutions in owed aCSF through a servo-controlled heater (TC-324B, Warner instruments) for temperature monitoring. The bath solution recirculated at all times, even during wash out.
In most cases, we used RDA and FDA in 50%. By mixing the markers we assured that the interneurons were marked correctly, thereby avoiding an RDA transsynaptic ow leak or insu cient FDA antidromic traveling distally to afferent ber terminals. Lower RDA molecular weight could produce to leakage, whereas the higher molecular weight could not even travel deep enough.
In some experiments, we labeled DR afferent bers by applying FDA, RDA, or the mixture of both uorochromes to the cut L4 or L5 or both DR's for marking the afferent ber ending in the motor nuclei (Fig. 1B). We also retrogradely labeled motoneurons by applying RDA and FDA to the L4-L5 ventral root (n = 7).
We used negative pressure to introduce the roots in the tubes producing a tight seal, avoiding any uorescent marker leakage. We used the markers diluted in a aCSF ten mmol/L solution, with 0.2% TritonX-100 (Sigma Chemical Co.). We employed ne suction electrodes pulled from polyethylene tubing (PE-190, Clay Adams, Parsippany, NJ.). After 18-24 hours, the spinal cord was xed by immersion in 4% of PFA in a 0.1% phosphate buffer (7.4 pH) overnight. After ascending sucrose cryoprotecter concentrations, we cut the spinal cords in coronal slices on a freezing microtome. Tissue sections placed on slides, dehydrated in ascending alcohol concentrations, cleared with Xylene and covered with an antifade mounting medium (Vectashield, Vector Laboratories Inc. Burlingame, CA). We examined tissue sections with an inverted Zeiss microscope and a laser scanning confocal imaging System (LSM 510). We analyzed images containing several optical sections in the Z plane and saved them from evaluating the morphology and synaptology of interneurons, motoneurons, and afferent bers. We reconstructed three-dimensional arrangements with Zeiss LSM 510 software.

Stimulation and recording
We placed the dorsal and ventral roots of segments L4 and L5 into the polyethylene suction electrodes for either stimulation or recordings.
We produced MR and DRR by stimulating the dorsal root lament at the L5 segment in the afferent bers. To continue, we applied ten pulse trains (0.5 ms pulse duration with 2-min intervals) ranging from 16 Hz to 0.125 Hz. We recorded the MR at the L5 ventral root segment, and the DRR at the L4 dorsal root (Fig. 1A). Ca2 + concentration was zero in some experiments. We labeled these experiments as low calcium concentrations.

Data Acquisition
The signals obtained from the recording suction electrodes on DR and VR were ampli ed with Cyberamp 380 ampli ers (axon instruments: band 10-10 kHz) and digitized at 10 kHz with 16 bits resolution A/D converter (National Instruments NBIO-16) and then stored in the computer. We did data analysis off-line using NIH institute software packages.

Statistical analysis.
In some experiments, we measured the ventral horn neuron soma size. We studied them at 2 and 13 postnatal days (P2 and P13). We carried out a linear regression analysis to establish the average soma size value at the respective age, using the Sigma-Plot software v11. We applied Normality tests (Shapiro-Wilk) to the three groups (P2, P13 < average value, and P13). We performed a t-test to compare the soma size among different groups.

Declarations Ethical Approval
We carried out experiments in full compliance with ethical standards approved by the NIH guidelines (USA) and approved by the Institutional Ethics Committee, according to the Mexican O cial Norm (NOM-062-ZOO-1999).
Consent for publication I Judith Marcela Duenas Jimenez hereby declare that I participated in the study and in the development of the manuscript titled. I have read the nal version and give my consent for the article to be published in BMC Neuroscience.

Availability of data and material
The datasets in this study are available on request to the corresponding author.
Competing interests I declare that I have no signi cant competing nancial, professional, or personal interests that might have in uenced the performance or presentation of the work described in this manuscript.

Funding
Not applicable.
Author's contribution All authors contributed to the study design and performed experiments. Sergio Horacio Dueñas Jiménez developed the concept and performed the material preparation, data collection, and analysis. Luis Castillo Hernandez wrote the rst draft of the manuscript. All authors commented on previous versions of the paper and approved the nal manuscript.

Figure 1
Mouse spinal cord drawing illustrating ventral and dorsal roots in thoracic and hemisected spinal cord lumbar segments. A) The stimulation suction electrode (SSE) did administer at the L5 dorsal segment: the recordings electrodes applied in the L4 dorsal segment for dorsal root re ex (DRR), and monosynaptic re ex in the L5 ventral root (MR-VR). B) For neurons with axon in DR uorescent dextran amines, and a mixture of both were added in suction electrodes in the dorsal roots L4 or /and L5 for orthograde labeling (OL). For motoneuron retrograde labeling (RL), ventral roots L4 or L5 were lled with uorochromes.     DRR and MR Recordings. DRR and MR control recording (indicated by arrows, upper and lower traces in A). They were recorded in L4 dorsal and L5 ventral roots, respectively. A: aCSF control, B: aCSF with bicuculline (10-20 µM). C and D illustrating MR and DRR in the presence of bicuculline plus AP5 (100 µM). Bicuculline eliminated DRR, and long-latency re exes observed in VR. MR depression began 2-4 min after applying AP5. Note that most of the MR were almost fully eliminated, but DR action potentials still appeared. E) Dorsal and ventral root re exes recovered after drug washout. F: VR and DR recording under low Ca2+ environment; the ventral and dorsal re exes were eliminated, but spiking persisted in both ventral and dorsal roots (indicated by arrows).

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