Formation of a novel supraspinal-spinal connectome that relearns the same motor task after complete paralysis.

Having observed that electrical spinal cord stimulation and training enabled four patients with paraplegia with motor complete paralysis to regain voluntary leg movement, the underlying mechanisms involved in forming the newly established supraspinal-spinal functional connectivity have become of great interest. van den Brand et al. (Science 336: 1182-1185, 2012) subsequently, demonstrated the recovery, in response to spinal electro-neuromodulation and locomotor training, of voluntary stepping of the lower limbs in rats that received a lesion that is assumed to eliminate all long-descending cortical axons that project to lumbosacral segments. Here, we used a similar spinal lesion in rats to eliminate long-descending axons to determine whether a novel, trained motor behavior triggered by a unique auditory cue learned before a spinal lesion, could recover after the lesion. Hindlimb stepping recovered 1 mo after the spinal injury, but only after 2 mo, the novel and unique audio-triggered behavior was recovered, meaning that not only was a novel connectivity formed but also further evidence suggested that this highly unique behavioral response was independent of the recovery of the circuitry that generated stepping. The unique features of the newly formed supraspinal-spinal connections that mediated the recovery of the trained behavior is consistent with a guidance mechanism(s) that are highly use dependent.NEW & NOTEWORTHY Electrical spinal cord stimulation has enabled patients with paraplegia to regain voluntary leg movement, and so the underlying mechanisms involved in this recovery are of great interest. Here, we demonstrate in rodents the recovery of trained motor behavior after a spinal lesion. Rodents were trained to kick their right hindlimb in response to an auditory cue. This behavior recovered 2 mo after the paralyzing spinal cord injury but only with the assistance of electrical spinal cord stimulation.

Reelin dorsal horn neurons co-express Lmx1b and are mispositioned in disabled-1 mutant mice.

Mice missing either Reelin or Disabled-1 (Dab1) exhibit dorsal horn neuronal positioning errors and display heat hypersensitivity and mechanical insensitivity. Reelin binds its receptors, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, leading to the recruitment and phosphorylation of Dab1 and activation of downstream pathways that regulate neuronal migration. Previously, we reported that 70% of Dab1 laminae I-II neurons co-expressed LIM-homeobox transcription factor 1-beta (Lmx1b). Here, we asked whether Reelin-expressing dorsal horn neurons co-express Lmx1b, are mispositioned in dab1 mutants, and contribute to nociceptive abnormalities. About 90% of Reelin-labeled neurons are Lmx1b-positive in laminae I-II, confirming that most Reelin and Dab1 neurons are glutamatergic. We determined that Reelin-Lmx1b and Dab1-Lmx1b dorsal horn neurons are separate populations, and together, comprise 37% of Lmx1b-positive cells within and above the Isolectin B4 (IB4) layer in wild-type mice. Compared to wild-type mice, dab1 mutants have a reduced area of laminae I-II outer (above the IB4 layer), more Reelin-Lmx1b neurons within the IB4 layer, and fewer Reelin-Lmx1b neurons within the lateral reticulated area of lamina V and lateral spinal nucleus. Interestingly, both Reelin- and Dab1-labeled dorsal horn neurons sustain similar positioning errors in mutant mice. After noxious thermal and mechanical stimulation, Reelin, Lmx1b, and Reelin-Lmx1b neurons expressed Fos in laminae I-II and the lateral reticulated area in wild-type mice and, therefore, participate in nociceptive circuits. Together, our data suggest that disruption of the Reelin-signaling pathway results in neuroanatomical abnormalities that contribute to the nociceptive changes that characterize these mutant mice.

Olfactory Ensheathing Cell Transplantation after a Complete Spinal Cord Transection Mediates Neuroprotective and Immunomodulatory Mechanisms to Facilitate Regeneration.

UNLABELLED: Multiple neural and peripheral cell types rapidly respond to tissue damage after spinal cord injury to form a structurally and chemically inhibitory scar that limits axon regeneration. Astrocytes form an astroglial scar and produce chondroitin sulfate proteoglycans (CSPGs), activate microglia, and recruit blood-derived immune cells to the lesion for debris removal. One beneficial therapy, olfactory ensheathing cell (OEC) transplantation, results in functional improvements and promotes axon regeneration after spinal cord injury. The lack of an OEC-specific marker, however, has limited the investigation of mechanisms underlying their proregenerative effects. We compared the effects of enhanced green fluorescent protein-labeled fibroblast (FB) and OEC transplants acutely after a complete low-thoracic spinal cord transection in adult rats. We assessed the preservation of neurons and serotonergic axons, the levels of inhibitory CSPGs and myelin debris, and the extent of immune cell activation between 1 and 8 weeks postinjury. Our findings indicate that OECs survive longer than FBs post-transplantation, preserve axons and neurons, and reduce inhibitory molecules in the lesion core. Additionally, we show that OECs limit immune-cell activation and infiltration, whereas FBs alter astroglial scar formation and increase immune-cell infiltration and concomitant secondary tissue damage. Administration of cyclosporine-A to enhance graft survival demonstrated that immune suppression can augment OEC contact-mediated protection of axons and neurons during the first 2 weeks postinjury. Collectively, these data suggest that OECs have neuroprotective and immunomodulatory mechanisms that create a supportive environment for neuronal survival and axon regeneration after spinal cord injury. SIGNIFICANCE STATEMENT: Spinal cord injury creates physical and chemical barriers to axon regeneration. We used a complete spinal cord transection model and olfactory ensheathing cell (OEC) or fibroblast (FB; control) transplantation as a repair strategy. OECs, but not FBs, intermingled with astrocytes, facilitated astroglial scar border formation and sequestered invading peripheral cells. OECs attenuated immune cell infiltration, reduced secondary tissue damage, protected neurons and axons in the lesion core, and helped clear myelin debris. Immunosuppression enhanced survival of OECs and FBs, but only OEC transplantation promoted scaffold formation in the lesion site that facilitated axon regeneration and neuron preservation.

Disabled-1 dorsal horn spinal cord neurons co-express Lmx1b and function in nociceptive circuits.

The Reelin-signaling pathway is essential for correct neuronal positioning within the central nervous system. Mutant mice with a deletion of Reelin, its lipoprotein receptors, or its intracellular adaptor protein Disabled-1 (Dab1), exhibit nociceptive abnormalities: thermal (heat) hyperalgesia and reduced mechanical sensitivity. To determine dorsal horn alterations associated with these nociceptive abnormalities, we first characterized the correctly positioned Dab1 neurons in wild-type and mispositioned neurons in Reelin-signaling pathway mutant lumbar spinal cord. Using immunofluorescence, we found that 70% of the numerous Dab1 neurons in Reln+/+ laminae I-II and 67% of those in the lateral reticulated area and lateral spinal nucleus (LSN) co-express the LIM-homeobox transcription factor 1 beta (Lmx1b), an excitatory glutamatergic neuron marker. Evidence of Dab1- and Dab1-Lmx1b neuronal positioning errors was found within the isolectin B4 terminal region of Reln-/- lamina IIinner and in the lateral reticulated area and LSN, where about 50% of the Dab1-Lmx1b neurons are missing. Importantly, Dab1-Lmx1b neurons in laminae I-II and the lateral reticulated area express Fos after noxious thermal or mechanical stimulation and thus participate in these circuits. In another pain relevant locus - the lateral cervical nucleus (LCN), we also found about a 50% loss of Dab1-Lmx1b neurons in Reln-/- mice. We suggest that extensively mispositioned Dab1 projection neurons in the lateral reticulated area, LSN, and LCN and the more subtle positioning errors of Dab1 interneurons in laminae I-II contribute to the abnormalities in pain responses found in Reelin-signaling pathway mutants.

Novel Disabled-1-expressing neurons identified in adult brain and spinal cord.

Components of the Reelin-signaling pathway are highly expressed in embryos and regulate neuronal positioning, whereas these molecules are expressed at low levels in adults and modulate synaptic plasticity. Reelin binds to Apolipoprotein E receptor 2 and Very-low-density lipoprotein receptors, triggers the phosphorylation of Disabled-1 (Dab1), and initiates downstream signaling. The expression of Dab1 marks neurons that potentially respond to Reelin, yet phosphorylated Dab1 is difficult to detect due to its rapid ubiquitination and degradation. Here we used adult mice with a lacZ gene inserted into the dab1 locus to first verify the coexpression of ß-galactosidase (ß-gal) in established Dab1-immunoreactive neurons and then identify novel Dab1-expressing neurons. Both cerebellar Purkinje cells and spinal sympathetic preganglionic neurons have coincident Dab1 protein and ß-gal expression in dab1(lacZ/+) mice. Adult pyramidal neurons in cortical layers II-III and V are labeled with Dab1 and/or ß-gal and are inverted in the dab1(lacZ/lacZ) neocortex, but not in the somatosensory barrel fields. Novel Dab1 expression was identified in GABAergic medial septum/diagonal band projection neurons, cerebellar Golgi interneurons, and small neurons in the deep cerebellar nuclei. Adult somatic motor neurons also express Dab1 and show ventromedial positioning errors in dab1-null mice. These findings suggest that: (i) Reelin regulates the somatosensory barrel cortex differently than other neocortical areas, (ii) most Dab1 medial septum/diagonal band neurons are probably GABAergic projection neurons, and (iii) positioning errors in adult mutant Dab1-labeled neurons vary from subtle to extensive.

Olfactory ensheathing cells are hybrid glial cells that promote neural repair.

Olfactory ensheathing cells (OECs) are unique glial cells found in both the central and peripheral nervous systems where they support the continuous axonal outgrowth of immature olfactory sensory neurons to their targets. Here we show that following severe spinal cord injury, olfactory bulb-derived OECs transplanted near the injury site modify the normally inhibitory glial scar and facilitate axon regeneration past the scar border and into the lesion center. To understand the mechanisms underlying the reparative properties of such transplanted OECs, we used single-cell RNA-sequencing to study their gene expression programs. Our analyses revealed five diverse subtypes of OECs, each expressing novel marker genes and pathways indicative of progenitor, axonal regeneration and repair, secreted molecules, or microglia-like functions. As expected, we found substantial overlap of OEC genes with those of Schwann cells, but also with astrocytes, oligodendrocytes and microglia. We confirmed established markers on cultured OECs, and then localized select top genes of OEC subtypes in rat olfactory bulb tissue. In addition, we present evidence that OECs secrete both Reelin and Connective tissue growth factor, extracellular matrix molecules which are important for neural repair and axonal outgrowth. Our results support that adult OECs are a unique hybrid glia, some with progenitor characteristics, and that their gene expression patterns indicate diverse functions related to wound healing, injury repair and axonal regeneration.

Axon regeneration can facilitate or suppress hindlimb function after olfactory ensheathing glia transplantation.

Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.

OEG implantation and step training enhance hindlimb-stepping ability in adult spinal transected rats.

Numerous treatment strategies for spinal cord injury seek to maximize recovery of function and two strategies that show substantial promise are olfactory bulb-derived olfactory ensheathing glia (OEG) transplantation and treadmill step training. In this study we re-examined the issue of the effectiveness of OEG implantation but used objective, quantitative measures of motor performance to test if there is a complementary effect of long-term step training and olfactory bulb-derived OEG implantation. We studied complete mid-thoracic spinal cord transected adult female rats and compared four experimental groups: media-untrained, media-trained, OEG-untrained and OEG-trained. To assess the extent of hindlimb locomotor recovery at 4 and 7 months post-transection we used three quantitative measures of stepping ability: plantar stepping performance until failure, joint movement shape and movement frequency compared to sham controls. OEG transplantation alone significantly increased the number of plantar steps performed at 7 months post-transection, while training alone had no effect at either time point. Only OEG-injected rats plantar placed their hindpaws for more than two steps by the 7-month endpoint of the study. OEG transplantation combined with training resulted in the highest percentage of spinal rats per group that plantar stepped, and was the only group to significantly improve its stepping abilities between the 4- and 7-month evaluations. Additionally, OEG transplantation promoted tissue sparing at the transection site, regeneration of noradrenergic axons and serotonergic axons spanning the injury site. Interestingly, the caudal stump of media- and OEG-injected rats contained a similar density of serotonergic axons and occasional serotonin-labelled interneurons. These data demonstrate that olfactory bulb-derived OEG transplantation improves hindlimb stepping in paraplegic rats and further suggest that task-specific training may enhance this OEG effect.

Mispositioned Neurokinin-1 Receptor-Expressing Neurons Underlie Heat Hyperalgesia in Disabled-1 Mutant Mice.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Contribution of the Reelin signaling pathways to nociceptive processing.

The reeler gene encodes Reelin, a secreted glycoprotein that binds to the very-low-density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer 2), and induces Src- and Fyn-mediated tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This Reelin-Dab1 signaling pathway regulates neuronal positioning during development. A second Reelin pathway acts through Apoer 2-exon 19 to modulate synaptic plasticity in adult mice. We recently reported positioning errors in reeler dorsal horn laminae I-II and V, and the lateral spinal nucleus. Behavioral correlates of these positioning errors include a decreased mechanical and increased thermal sensitivity in reeler mice. Here we examined mice with deletions or modifications of both the Reelin-Dab1 signaling pathway and the Reelin-Apoer 2-exon 19 pathway on a Vldlr-deficient background. We detected reeler-like dorsal horn positioning errors only in Dab1 mutant and Apoer 2/Vldlr double mutant mice. Although Dab1 mutants, like reeler, showed decreased mechanical and increased thermal sensitivity, neither the single Vldlr or Apoer 2 knockouts, nor the Apoer 2-exon 19 mutants differed in their acute pain sensitivity from controls. However, despite the dramatic alterations in acute 'pain' processing in reeler and Dab1 mutants, the exacerbation of pain processing after tissue injury (hindpaw carrageenan injection) was preserved. Finally, we recapitulated the reeler dorsal horn positioning errors by inhibiting Dab1 phosphorylation in organotypic cultures. We conclude that the Reelin-Dab1 pathway differentially contributes to acute and persistent pain, and that the plasticity associated with the Reelin-Apoer 2-exon 19 pathway is distinct from that which contributes to injury-induced enhancement of 'pain' processing.

Evidence of axon connectivity across a spinal cord transection in rats treated with epidural stimulation and motor training combined with olfactory ensheathing cell transplantation.

Olfactory ensheathing cells (OECs) are unique glia that support axon outgrowth in the olfactory system, and when used as cellular therapy after spinal cord injury, improve recovery and axon regeneration. Here we assessed the effects of combining OEC transplantation with another promising therapy, epidural electrical stimulation during a rehabilitative motor task. Sprague-Dawley rats received a mid-thoracic transection and transplantation of OECs or fibroblasts (FBs) followed by lumbar stimulation while climbing an inclined grid. We injected pseudorabies virus (PRV) into hindlimb muscles 7 months post-injury to assess connectivity across the transection. Analyses showed that the number of serotonergic (5-HT) axons that crossed the rostral scar border and the area of neurofilament-positive axons in the injury site were both greater in OEC- than FB-treated rats. We detected PRV-labeled cells rostral to the transection and remarkable evidence of 5-HT and PRV axons crossing the injury site in 1 OEC- and 1 FB-treated rat. The axons that crossed suggested either axon regeneration (OEC) or small areas of probable tissue sparing (FB). Most PRV-labeled thoracic neurons were detected in laminae VII or X, and ~25% expressed Chx10, a marker for V2a interneurons. These findings suggest potential regeneration or sparing of circuits that connect thoracic interneurons to lumbar somatic motor neurons. Despite evidence of axonal connectivity, no behavioral changes were detected in this small-scale study. Together these data suggest that when supplemented with epidural stimulation and climbing, OEC transplantation can increase axonal growth across the injury site and may promote recovery of propriospinal circuitry.

Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin.

The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo.

L1 CAM expression in the superficial dorsal horn is derived from the dorsal root ganglion.

The cell adhesion molecule L1 is highly expressed on embryonic axons and may play a role in axonal outgrowth and fasciculation. Generally only low levels of L1 are found in adult spinal cord except for intense labeling in Lissauer's tract, in laminae I-II, and on dorsolateral funicular axons. In this study we determine the source of L1 immunoreactivity in the dorsal spinal cord, the presence of L1 expression on sprouting axons, and the effect of exercise on L1 expression. We determined the source of L1 immunoreactivity in the superficial dorsal horn by performing acute unilateral rhizotomies (T12-L4) in adult rats. This resulted in a marked decrease in L1 expression in Lissauer's tract and laminae I-II on the deafferented side. The peptidergic and nonpeptidergic small-diameter primary afferent markers, calcitonin gene-related peptide (CGRP) and the lectin IB4 respectively, closely correlated with L1 expression and also decreased dramatically after rhizotomy. Considering its developmental role, we asked whether L1 was expressed on sprouting axons following chronic rhizotomy. L1 and CGRP, but not IB4, were detected on sprouting axons. Lastly, we investigated the effect of exercise on L1 expression by giving animals with chronic rhizotomies free access to an exercise wheel. After extensive exercise, L1, CGRP, and IB4 expression levels were unchanged compared with those of sedentary chronic animals. Combined, these data demonstrate that the dorsal root ganglia is a major source of L1-positive axons in the superficial dorsal horn, that both L1 and CGRP identify sprouting axons following rhizotomy, and that exercise does not upregulate L1 expression.

Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures.

The regenerative capacity of adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth-inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC-neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core.

Expression of L1 decreases during postnatal development of rat spinal cord.

L1 is a cell adhesion molecule that is highly expressed on developing axons and is associated with neurite outgrowth, guidance, and fasciculation. In this study we systematically examined L1 expression at all spinal levels across eight postnatal ages to detect regional and developmental differences. We observed striking changes in the developmental pattern of L1 expression between birth (P0) and adult ages, with intense L1-immunopositive axons prevalent throughout the funiculi at P0 compared with predominantly L1-immunonegative funicular axons in adults. At all ages and spinal levels examined, some L1-positive dorsal root afferents entered the spinal cord, coursed in Lissauer's tract, and projected into the superficial dorsal horn and the dorsal columns, as well as across the dorsal commissure. Additional L1-positive axons were detected consistently around the perimeter of the spinal cord, in the dorsolateral funiculus, and adjacent to the central canal. While specific L1-labeled axons were detected at all ages, a pattern of segmental variation was observed within animals, with the highest levels of L1 expression detected in lumbar and sacral segments and the lowest in cervical spinal cord. The pattern of L1 immunoreactivity was compared to that of the growth-associated protein GAP-43 and the results indicated colabeling of most axons. These observations demonstrate that L1 is expressed on immature axons well into postnatal development, possibly until they have completed their differentiation. Furthermore, the L1-positive axons that continue to be detected in adults are likely to be either unmyelinated or sprouting axons.

Unique developmental patterns of GABAergic neurons in rat spinal cord.

Gamma-aminobutyric acid (GABA)ergic neurons have been postulated to compose an important component of local circuits in the adult spinal cord, yet their identity and axonal projections have not been well defined. We have found that, during early embryonic ages (E12-E16), both glutamic acid decarboxylase 65 (GAD65) and GABA were expressed in cell bodies and growing axons, whereas at older ages (E17-P28), they were localized primarily in terminal-like structures. To determine whether these developmental changes in GAD65 and GABA were due to an intracellular shift in the distribution pattern of GAD proteins, we used a spinal cord slice model. Initial experiments demonstrated that the pattern of GABAergic neurons within organotypic cultures mimicked the expression pattern seen in embryos. Sixteen-day-old embryonic slices grown 1 day in vitro contained many GAD65- and GAD67-labeled somata, whereas those grown 4 days in vitro contained primarily terminal-like varicosities. When isolated E14-E16 slices were grown for 4 days in vitro, the width of the GAD65-labeled ventral marginal zone decreased by 40-50%, a finding that suggests these GABAergic axons originated from sources both intrinsic and extrinsic to the slices. Finally, when axonal transport was blocked in vitro, the developmental subcellular localization of GAD65 and GAD67 was reversed, so that GABAergic cell bodies were detected at all ages examined. These data indicate that an intracellular redistribution of both forms of GAD underlie the developmental changes observed in GABAergic spinal cord neurons. Taken together, our findings suggest a rapid translocation of GAD proteins from cell bodies to synaptic terminals following axonal outgrowth and synaptogenesis.

Embryonic GABAergic spinal commissural neurons project rostrally to mesencephalic targets.

Although spinal commissural neurons serve as a model system for studying the mechanisms that underlie axonal pathfinding during development, little is known about their synaptic targets. Previously we identified a group of ventromedially located commissural neurons in rat spinal cord that are gamma-aminobutyric acid (GABA)-ergic and express L1 CAM on their axons. In this study, serial sagittal sections of embryos (E12-15) were processed for glutamic acid decarboxylase (GAD)-65 and L1 immunocytochemistry and showed labeled commissural axons coursing rostrally within the ventral marginal zone. Both GAD65- and L1-positive axons extended rostrally out of the spinal cord into the central part of the medulla and then into the midbrain. GAD65-positive axons branched and ended abruptly within the lateral midbrain. To determine the targets of these ventral commissural neurons, embryos (E13-15) were injected with DiI into the ventromedial spinal cord. At all three ages, DiI-labeled axons projected rostrally in the contralateral ventral marginal zone, turned into the central medulla, and then traveled to the midbrain. DiI-labeled axons appeared to terminate in the lateral midbrain by branching into small, punctate structures. In reciprocal experiments, DiI injected into the lateral midbrain identified an axon pathway that coursed through the brainstem, into the spinal cord ventral marginal zone, and then filled cell bodies in the contralateral ventromedial spinal cord. A spatial and temporal coincidence was apparent between the GAD65/L1- and the DiI-labeled pathways. Together these findings suggest that some GABAergic commissural neurons are early projection neurons to midbrain targets and most likely represent a spinomesencephalic pathway to the midbrain reticular formation.

Developmental distribution of reelin-positive cells and their secreted product in the rodent spinal cord.

To date, only sympathetic and parasympathetic preganglionic neurons are known to migrate abnormally in reeler mutant spinal cord. Reelin, the large extracellular matrix protein absent in reeler, is found in wild-type neurons bordering both groups of preganglionic neurons. To understand better Reelin's function in the spinal cord, we studied its developmental expression in both mice and rats. A remarkable conservation was found in the spatiotemporal pattern of Reelin in both species. Numerous Reelin-expressing cells were found in the intermediate zone, except for regions containing somatic and autonomic motor neurons. A band of Reelin-positive cells filled the superficial dorsal horn, whereas only a few immunoreactive cells populated the deep dorsal horn and dorsal commissure. High levels of diffuse Reelin product were detected in the lateral marginal and ventral ventricular zones in both rodent species. This expression pattern was detected at all segmental spinal cord levels during embryonic development and remained detectable at lower levels throughout the first postnatal month. To discriminate between the cellular and secreted forms of Reelin, brefeldin A was used to block secretion in organotypic cultures. Such perturbations revealed that the high levels of secreted Reelin in the lateral marginal zone were derived from varicose axons of more medially located Reelin-positive cells. Thus, the laterally located secreted Reelin product may normally prevent the preganglionic neurons from migrating too far medially. Based on the strong evolutionary conservation of Reelin expression and its postnatal detection, Reelin may have other important functions in addition to its role in neuronal migration.