IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.     

Functional repair of transected spinal cord in embryonic chick

The purpose of this study was to determine the developmental stage of the chick embryo when descending spinal tracts lose the capacity for anatomical and functional repair after complete transection of the thoracic spinal cord. Previous studies have demonstrated that the first reticulospinal projections descend to the lumbar cord by embryonic day (E) 5. A comparison of the distribution and density of retrogradely labelled brainstem-spinal neurons in embryos versus hatchling chicks suggests that the descent of all brainstem-spinal projections is essentially complete to lumbar levels between E10 and El2. Transections and control sham operations were performed on different embryos from E3 through E14 of development. After a recovery period of 5-18 days, the extent of anatomical repair was assessed by injecting a small volume of a retrograde tract-tracing chemical into the upper lumbar spinal cord, caudal to the transection site. The brainstem nuclei were then examined for the number and distribution of retrogradely labelled brainstem-spinal neurons. In comparison to control animals, anatomical recovery appeared to be complete for embryos transected as late as E12, whereas thoracic cord transections conducted on E13-E14 resulted in reduced labelling of most brainstem-spinal nuclei. In addition, a number of E3-E6 transected embryos were allowed to hatch and with some assistance a few E7-E14 transected embryos also hatched. Functional recovery was assessed by behavioral observations and by focal electrical stimulation of brainstem locomotor regions (known to have direct projections to the lumbar spinal cord). Brainstem stimulation experiments were undertaken on transected and control embryos, either in ovo on E18-E20 or after hatching. Leg and wing muscle electromyographic recordings were used to monitor any brainstem evoked motor activity. Voluntary open-field locomotion (hatchling chicks) or brainstem evoked locomotion (embryonic or hatchling) in animals transected on or before E12 was indistinguishable from that observed in control (i.e. sham-operated or unoperated) chicks, indicating that complete functional recovery had occurred. In contrast, chicks transected on or after El3 showed reduced functional recovery. Since a previous study has shown that neurogenesis in chick brainstem-spinal neurons is complete prior to E5, the possible intrinsic neuronal mechanisms underlying the repair of descending supraspinal pathways are: (1) subsequent projections from later developing (undamaged) neurons, or (2) regrowth of previously axotomized projections (regeneration). For the E5-E12 chick embryos examined in this study, significant descending supraspinal fibers are present within the thoracic cord at the time of transection. Even if the transection is made at E12, when descending projections have completed their development to the lumbar cord, there is still a similar number and distribution of brainstem-spinal neurons labelled afterward (when compared to controls). This suggests that regeneration of previously axotomized projections may account for some of the observed anatomical and functional repair of brainstem-spinal pathways.     

Fibroblast growth factor treatment produces differential effects on survival and neurite outgrowth from identified bulbospinal neurons in vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

Fibroblast Growth Factor Treatment Produces Differential Effects on Survival and Neurite Outgrowth from Identified Bulbospinal Neurons in Vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

Growth inhibitory effects of a mu opioid on cultured cholinergic neurons from fetal rat ventral forebrain, brainstem, and spinal cord

Cholinergic pathways play a role in respiration in the mammalian brain, and agents that affect respiratory function such as opioid peptides might have positive or negative neurotrophic effects during the development of these cholinergic connections. Rat fetal nerve cell cultures from developmental stages E14-E18 were established in 96-well plates from ventral forebrain (VFB), an area rich in cholinergic neurons, and from brainstem and rostral spinal cord, areas where respiratory control systems and cholinergic neurons co-exist. High affinity 3H-choline uptake was highest in E14 VFB cultures and decreased to 20% of this value by E16 and E18. Choline uptakes in E14 brainstem and spinal cord were only 20% and 13%, respectively, of E14 VFB uptake. A mu opioid receptor agonist, d-ala2-mePhe4-gly(ol)5]-enkephalin (DAMGO), was tested for its effect on somal area and neurite outgrowth in E16 cultures. Cholinergic neurons were identified by immunostaining with choline acetyltransferase antibody. DAMGO (10(-8) M) significantly decreased somal area in VFB cultures and spinal cord, but had no effect on somal area in brainstem. Naltrexone (10(-6) M) reversed this inhibition. Spinal cord cell neurite outgrowth was inhibited by DAMGO, and this inhibition was reversed by naltrexone. DAMGO had no significant effect on neurite length in VFB. Brainstem neurite length was paradoxically increased by both DAMGO and naltrexone. It was concluded that mu-selective opioid peptides inhibit growth of cultured cholinergic neurons in VFB and spinal cord, but not in the brainstem. There was no evidence for endogenous opioid activity in either VFB or spinal cord cultures.     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

Developmental patterns of neurite outgrowth from chick embryo spinal cord and retinal neurons on laminin substrates

It has been previously found that neurite outgrowth on collagen substrates decreases with increasing gestational age of chick embryo spinal cord and retinal neurons in tissue culture. In the current study, laminin, polylysine and collagen were compared in their efficacy in promoting neurite extension from chick embryo spinal cord neurons aged 6-16 days or retinal neurons aged 8-16 days in ovo. The percentage of neurons with neurites and the length of the neurites were determined at 1 and 3 days in culture. There was a significant increase in neuritogenesis by laminin and polylysine compared to collagen for both spinal cord and retinal neurons. Further, in spinal cord cultures grown on a laminin substrate, there was no decline in neurite outgrowth with increasing developmental age of the neurons as was seen on collagen and polylysine. Neurite length measurements also demonstrated a significant stimulation of neuritogenesis for spinal cord, but not retinal, neurons by laminin compared to polylysine or collagen in 1-day cultures. The results demonstrate tissue-specific differences in the developmental patterns of neurite outgrowth. Retinal neurons appear to have intrinsic changes in their ability to respond to extracellular promoting factors or substrates, while spinal cord neurite outgrowth can be regulated by these extrinsic factors.     

A high-throughput screen to identify novel compounds to promote neurite outgrowth

Following spinal cord injury, a variety of inhibitory molecules hinder the success of axon regeneration. The motile tip of the axon, the growth cone, shares a similar cytoskeletal array as a migrating cell, and in general the cytoskeleton is regulated by a conserved set of signaling pathways that act downstream of guidance cue and growth factor receptors. We exploit these similarities by using migrating cells as a model system to screen for extracts that promote axon outgrowth. The screen is a high-throughput wound-healing assay performed by a 96-pin tool Biogrid robot where positive candidates are identified as extracts that stimulate complete wound healing. Testing of positive candidates on chick DRG explants has lead to the identification of extracts that promote neurite outgrowth on permissive and inhibitory substrates. Extracts can be fractionated to purity, identifying novel compounds that promote neurite outgrowth on inhibitory substrates.     

Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Reorganization of descending motor tracts in the rat spinal cord

Following lesion of the central nervous system (CNS), reinnervation of denervated areas may occur via two distinct processes: regeneration of the lesioned fibres or/and sprouting from adjacent intact fibres into the deafferented zone. Both regeneration and axonal sprouting are very limited in the fully mature CNS of higher vertebrates, but can be enhanced by neutralizing the neurite outgrowth inhibitory protein Nogo-A. This study takes advantage of the distinct spinal projection pattern of two descending tracts, the corticospinal tract (CST) and the rubrospinal tract (RST), to investigate if re-innervation of denervated targets can occur by sprouting of anatomically separate, undamaged tracts in the adult rat spinal cord. The CST was transected bilaterally at its entry into the pyramidal decussation. Anatomical studies of the RST in IN-1 antibody-treated rats showed a reorganization of the RST projection pattern after neutralization of the myelin associated neurite growth inhibitor Nogo-A. The terminal arborizations of the rubrospinal fibres, which are normally restricted to the intermediate layers of the spinal cord, invaded the ventral horn but not the dorsal horn of the cervical spinal cord. Moreover, new close appositions were observed, in the ventral horn, onto motoneurons normally receiving CST projections. Red nucleus microstimulation experiments confirmed the reorganization of the RST system. These observations indicate that mature descending motor tracts are capable of significant intraspinal reorganization following lesion and suggests the expression of cues guiding and/or stabilizing newly formed sprouts in the adult, denervated spinal cord.     

Tissue sections from the mature rat brain and spinal cord as substrates for neurite outgrowth in vitro: extensive growth on gray matter but little growth on white matter

The failure of axons to regenerate within the brain and spinal cord of mature mammals has been attributed to the absence of growth-promoting substances, especially extracellular matrix components, or to the presence of growth-inhibiting substances, particularly components associated with CNS myelin. The ability of mature mammalian CNS tissue to support neurite regeneration was tested by growing explants of embryonic chick lumbar sympathetic ganglia on fresh frozen sections of the mature rat brain and spinal cord. The extent of neurite outgrowth was quantified using morphometric analysis for explants grown on sections that included most of the major anatomical divisions of the CNS. Extensive, but variable, regeneration was present on gray matter regions, whereas major white matter tracts showed poor support, if any, for neurite growth. The results are consistent with the presence of growth-inhibiting factors associated with CNS white matter but also indicate that most gray matter regions of the mature mammalian brain and spinal cord will support axonal regeneration in tissue culture in spite of the absence of known extracellular matrix components.     

Neurite branching on deformable substrates

The mechanical properties of substrates underlying cells can have profound effects on cell structure and function. To examine the effect of substrate deformability on neuronal cell growth, protein-laminated polyacrylamide gels were prepared with differing amounts of bisacrylamide to generate substrates of varying deformability with elastic moduli ranging from 500 to 5500 dyne/cm. Mouse spinal cord primary neuronal cells were plated on the gels and allowed to grow and extend neurites for several weeks in culture. While neurons grew well on the gels, glia, which are normally co-cultured with the neurons, did not survive on these deformable substrates even though the chemical environment was permissive for their growth. Substrate flexibility also had a significant effect on neurite branching. Neurons grown on softer substrates formed more than three times as many branches as those grown on stiffer gels. These results show that mechanical properties of the substrate specifically direct the formation of neurite branches, which are critical for appropriate synaptic connections during development and regeneration.     

Early axon and dendritic outgrowth of spinal accessory motor neurons studied with DiI in fixed tissues

We have utilized lateral diffusion of DiI in fixed tissues (Godement et al., '87: Development 101: 697-713) to study early axon and dendritic outgrowth of spinal accessory motor neurons in embryonic rats. Crystals were placed in the central canal of the cervical spinal cord near the ventral commissure in order to label growing accessory axons anterogradely and on the spinal accessory nerve to label somata and dendrites retrogradely. Animals were studied on E11-E13. We show here that it is possible to stain axonal and dendritic processes from the earliest stages of motor neuron differentiation by using DiI. Our results demonstrate that, unlike axons of other cervical motor neurons, accessory axons traverse the lateral region of the embryonic cord, which consists of neuroepithelial endfeet. Thus an affinity for neuroepithelial endfeet could partially explain their unusual intraspinal trajectory. We also show that morphology of the spinal accessory growth cones differs according to position along the accessory nerve pathway. Finally, we show that accessory motor neuron axons are in the region of their target precursors prior to the initiation of dendritic arborization. Use of DiI in fixed tissue allows study of process outgrowth in mammalian spinal cord with detail previously obtainable only in nonmammalian vertebrates.     

Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study

Olfactory ensheathing cells (OECs) are cells that display Schwann cell or astrocyte-like properties. They are a source of growth factors and adhesion molecules which play a very important role as neuronal support enhancing cellular survival. Over the past 10 years, OECs have emerged as a leading reparative candidate, when transplanted into the injured spinal cord, having shown significant promise in the regeneration of spinal cord lesions. In this study we assessed the efficacy of OECs on the survival and neurite outgrowth of hippocampal neurons in vitro. Co-cultures of OECs and hippocampal of postnatal rats were successfully established and cells were immunocytochemically characterized. Some hippocampal cultures were added with growth factors, as bFGF, NGF and GDNF. Furthermore, conditioned medium from OECs cultures was used to feed some hippocampal neurons coverslips. Our results show that in co-cultures of hippocampal neurons and OECs the number of neurons and their neurite outgrowth were significantly increased in comparison with controls. Moreover, we showed that NGF and GDNF promoted a more positive effect in both neuronal survival and neurite outgrowth than bFGF. OEC-conditioned media stimulated both the neuronal survival and dense neurite outgrowth. These data indicate that OECs, as a source of growth factors, can promote the survival and the neurite outgrowth of hippocampal neurons in vitro and that bFGF, NGF and GDNF support them differently. Therefore, as OECs and their secreted growth factors appear to exert a neuroprotective effect for functional restoration and for neural plasticity in neurodegenerative disorders, they might be considered an approach for functional recovery.     

Degradation of Chondroitin Sulfate Proteoglycan Enhances the Neurite-Promoting Potential of Spinal Cord Tissue

The contribution of chondroitin sulfate proteoglycan (CSPG) in the suppression of axonal growth in rat spinal cord has been examined by means of anin vitrobioassay in which regenerating neurons are grown on tissue section substrata. Dissociated embryonic chick dorsal root ganglionic neurons were grown on normal and injured adult spinal cord tissue sections treated with chondroitinases. Neuritic growth on normal spinal cord tissue was meager. However, both the percentage of neurons with neurites and the average neurite length were substantially greater on sections treated with chondroitinase ABC. Enzymes that specifically degraded dermatan sulfate or hyaluronan were ineffective. Neuritic growth was significantly greater on injured (compared to normal) spinal cord and a further dramatic increase resulted from chondroitinase ABC treatment. Neurites grew equally within white and gray matter regions after chondroitinase treatment. Observed increases in neurite outgrowth on chondroitinase-treated tissues were largely inhibited in the presence of function-blocking laminin antibodies. These findings indicate that inhibitory CSPG is widely distributed and predominant in both normal and injured spinal cord tissues. Additionally, inhibitory CSPG is implicated in negating the potential stimulatory effects of laminin that might otherwise support spinal cord regeneration.     

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expressionin vitro

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.     

Epac mediates cyclic AMP-dependent axon growth, guidance and regeneration

A decline in developing neuronal cAMP levels appears to render mammalian axons susceptible to growth inhibitory factors in the damaged CNS. cAMP elevation enhances axon regeneration, but the cellular mechanisms involved have yet to be fully elucidated. Epac has been identified as a signaling protein that can be activated by cAMP independently of PKA, but little is known of its expression or role in the nervous system. We report that Epac expression is developmentally regulated in the rat nervous system, and that activation of Epac promotes DRG neurite outgrowth and is as effective as cAMP elevation in promoting neurite regeneration on spinal cord tissue. Additionally, siRNA mediated knockdown of Epac reduces DRG neurite outgrowth, prevents the increased growth promoted by cAMP elevation and also diminishes the ability of embryonic neurons to grow processes on spinal cord tissue. Furthermore, we show that asymmetric activation of Epac promotes attractive growth cone turning in a similar manner to cAMP activation. We propose that Epac plays a role in mediating cAMP-dependent axon growth and guidance, and may provide an important target for inducing axon regeneration in vivo.     

Regeneration of adult rat spinal cord is promoted by the soluble KDI domain of gamma1 laminin

Regeneration in the central nervous system (CNS) of adult mammals is hampered by formation of a glial scar and by proteins released from the myelin sheaths of injured neuronal pathways. Our recent data indicate that the KDI (Lys-Asp-Ile) domain of gamma1 laminin neutralizes both glial- and myelin-derived inhibitory signals and promotes survival and neurite outgrowth of cultured human spinal cord neurons. We show that after complete transection of the adult rat spinal cord, animals receiving onsite infusion of the KDI domain via osmotic mini-pumps recover and are able to sustain their body weights and walk with their hindlimbs. Animals treated with placebo suffer from irreversible hindlimb paralysis. Microscopic and molecular analyses of the spinal cords indicate that the KDI domain reduces tissue damage at the lesion site and enables neurite outgrowth through the injured area to effect functional recovery of the initially paralyzed animals. That the KDI domain enhances regeneration of acute spinal cord injuries in the adult rat suggests that it may be used to promote regeneration of spinal cord injuries in humans.     

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.