Characteristic development of the GABA-removal system in the mouse spinal cord.

GABA is a predominant inhibitory neurotransmitter in the CNS. Released GABA is removed from the synaptic cleft by two GABA transporters (GATs), GAT-1 and GAT-3, and their dysfunction affects brain functions. The present study aimed to reveal the ontogeny of the GABA-removal system by examining the immunohistochemical localization of GAT-1 and GAT-3 in the embryonic and postnatal mouse cervical spinal cord. In the dorsal horn, GAT-1 was localized within the presynapses of inhibitory axons after embryonic day 15 (E15), a little prior to GABAergic synapse formation. The GAT-1-positive dots increased in density until postnatal day 21 (P21). By contrast, in the ventral horn, GAT-1-positive dots were sparse during development, although many transient GABAergic synapses were formed before birth. GAT-3 was first localized within the radial processes of radial glia in the ventral part on E12 and the dorsal part on E15. The initial localization of the GAT-3 was almost concomitant with the dispersal of GABAergic neurons. GAT-3 continued to be localized within the processes of astrocytes, and increased in expression until P21. These results suggested the following: (1) before synapse formation, GABA may be transported into the processes of radial glia or immature astrocytes by GAT-3. (2) At the transient GABAergic synapses in the ventral horn, GABA may not be reuptaken into the presynapses. (3) In the dorsal horn, GABA may start to be reuptaken by GAT-1 a little prior to synapse formation. (4) After synapse formation, GAT-3 may continue to remove GABA from immature and mature synaptic clefts into the processes of astrocytes. (5) Development of the GABA-removal system may be completed by P21.

Birth-date dependent arrangement of spinal enkephalinergic neurons: evidence from the preproenkephalin-green fluorescent protein transgenic mice.

Enkephalin (ENK) has been postulated to play important roles in modulating nociceptive transmission, and it has been proved that ENKergic neurons acted as a critical component of sensory circuit in the adult spinal cord. Revealing the developmental characteristics of spinal ENKergic neurons will be helpful for understanding the formation and alteration of the sensory circuit under pain status. However, the relationship between the embryonic birth date and the adult distribution of ENKergic neurons has remained largely unknown due to the difficulties in visualizing the ENKergic neurons clearly. Taking advantage of the preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice in identifying ENKergic neurons, we performed the current birth-dating study and examined the spinal ENKergic neurogenesis. The ENKergic neurons born on different developmental stages and their final location during adulthood were investigated by combining bromodeoxyuridine (BrdU) incorporation and GFP labeling. The spinal ENKergic neurogenesis was restricted at E9.5 to E14.5, and fitted in the same pattern of spinal neurogenesis. Further comparative analysis revealed that spinal ENKergic neurons underwent heterogeneous characteristics. Our study also indicated that the laminar arrangement of ENKergic neurons in the superficial spinal dorsal horn depended on the neurogenesis stages. Taken together, the present study suggested that the birth date of ENKergic neurons is one determinant for their arrangement and function.

Distinct development of GABA system in the ventral and dorsal horns in the embryonic mouse spinal cord.

In the adult brain, γ-amino butyric acid (GABA) is an inhibitory neurotransmitter, whereas it acts as an excitatory transmitter in the immature brain, and may be involved in morphogenesis. In the present study, we immunohistochemically examined the developmental changes in GABA signaling in the embryonic mouse cervical spinal cord. Glutamic acid decarboxylase and GABA were markers for GABA neurons. Vesicular GABA transporter was a marker for GABAergic and glycinergic terminals. Potassium chloride cotransporter 2 was a marker for GABAergic inhibition. We found five points: (1) In the ventral part, GABA neurons were divided into three groups. The first differentiated group sent commissural axons after embryonic day 11 (E11), but disappeared or changed their transmitter by E15. The second and third differentiated groups were localized in the ventral horn after E12, and sent axons to the ipsilateral marginal zone. There was a distal-to-proximal gradient in varicosity formation in GABAergic axons and a superficial-to-deep gradient in GABAergic synapse formation in the ventral horn; (2) In the dorsal horn, GABA neurons were localized after E13, and synapses were diffusely formed after E15; (3) GABA may be excitatory for several days before synapses formation; (4) There was a ventral-to-dorsal gradient in the development of GABA signaling. The GABAergic inhibitory network may develop in the ventral horn between E15 and E17, and GABA may transiently play crucial roles in inhibitory regulation of the motor system in the mouse fetus; (5) GABA signaling continued to develop after birth, and GABAergic system diminished in the ventral horn.

Bone morphogenetic protein signaling is required in the dorsal neural folds before neurulation for the induction of spinal neural crest cells and dorsal neurons.

Bone Morphogenetic Protein (BMP) activity has been implicated as a key regulator of multiple aspects of dorsal neural tube development. BMP signaling in the dorsal-most neuroepithelial cells presumably plays a critical role. We use tissue-specific gene ablation to probe the roles of BMPR1A, the type 1 BMP receptor that is seemingly the best candidate to mediate the activities of BMPs on early dorsal neural development. We use two different Cre lines expressed in the dorsal neural folds, one prior to spinal neurulation and one shortly afterward, together with a Bmpr1a conditional null mutation. Our findings indicate that BMPR1A signaling in the dorsal neural folds is important for hindbrain neural tube closure, but suggest it is dispensable for spinal neurulation. Our results also demonstrate a requirement for BMP signaling in patterning of dorsal neural tube cell fate and in neural crest cell formation, and imply a critical period shortly before neural tube closure.

Identification of cerebellin2 in chick and its preferential expression by subsets of developing sensory neurons and their targets in the dorsal horn.

The cerebellins are a family of four secreted proteins, two of which, Cbln1 and Cbln3, play an important role in the formation and maintenance of parallel fiber-Purkinje cell synapses. We have identified the chicken homologue of Cbln2 and, through the use of in situ hybridization, shown that it is expressed by specific subsets of neurons in the dorsal root ganglia (DRGs) and spinal cord starting shortly after those neurons are generated. In the developing spinal cord, Cbln2 is highly expressed by dI1, dI3, dI5, and dILB dorsal interneurons and to a lesser extent by dI2, dI4, dI6, and dILA dorsal interneurons, but not by ventral (v0-v3) interneurons. After the spinal cord has matured and neurons have migrated to their final destinations, Cbln2 is abundant in the dorsal horn. In the DRGs, Cbln2 is expressed by TrkB+ and TrkC+ sensory neurons, but not by TrkA+ sensory neurons. Interestingly, regions of the spinal cord where TrkB+ and TrkC+ afferents terminate (i.e., laminae II, III, IV, and VI) exhibit the highest levels of Cbln2 expression. Cbln2 is also expressed by preganglionic sympathetic neurons and their targets in the sympathetic chain ganglia. Thus, the results show that Cbln2 is frequently expressed by synaptically connected neuronal populations. This, in turn, raises the possibility that if Cbln2, like Cbln1, plays a role in the formation and maintenance of synapses, it may somehow mediate bi-directional communication between discrete populations of neurons and their appropriate neuronal targets.

RETouching upon mechanoreceptors.

The rapidly adapting (RA) low-threshold mechanoreceptors respond to movement of the skin and vibration and are critical for the perception of texture and shape. In this issue of Neuron, two papers (Bourane et al. and Luo et al.) demonstrate that early-born Ret+ sensory neurons are RA mechanoreceptors, whose peripheral nerve terminals are associated with Meissner corpuscles, longitudinal lanceolate endings, and Pacinian corpuscles. The studies further show that Ret signaling is essential for the development of these mechanoreceptors.

Long-term actions of interleukin-1beta on delay and tonic firing neurons in rat superficial dorsal horn and their relevance to central sensitization.

BACKGROUND: Cytokines such as interleukin 1beta (IL-1beta) have been implicated in the development of central sensitization that is characteristic of neuropathic pain. To examine its long-term effect on nociceptive processing, defined medium organotypic cultures of rat spinal cord were exposed to 100 pM IL-1beta for 6-8 d. Interleukin effects in the dorsal horn were examined by whole-cell patch-clamp recording and Ca(2+) imaging techniques. RESULTS: Examination of the cultures with confocal Fluo-4 AM imaging showed that IL-1beta increased the change in intracellular Ca(2+) produced by exposure to 35-50 mM K+. This is consistent with a modest increase in overall dorsal horn excitability. Despite this, IL-1beta did not have a direct effect on rheobase or resting membrane potential nor did it selectively destroy any specific neuronal population. All effects were instead confined to changes in synaptic transmission. A variety of pre- and postsynaptic actions of IL-1beta were seen in five different electrophysiologically-defined neuronal phenotypes. In putative excitatory 'delay' neurons, cytokine treatment increased the amplitude of spontaneous EPSC's (sEPSC) and decreased the frequency of spontaneous IPSC's (sIPSC). These effects would be expected to increase dorsal horn excitability and to facilitate the transfer of nociceptive information. However, other actions of IL-1beta included disinhibition of putative inhibitory 'tonic' neurons and an increase in the amplitude of sIPSC's in 'delay' neurons. CONCLUSION: Since spinal microglial activation peaks between 3 and 7 days after the initiation of chronic peripheral nerve injury and these cells release IL-1beta at this time, our findings define some of the neurophysiological mechanisms whereby nerve-injury induced release of IL-1beta may contribute to the central sensitization associated with chronic neuropathic pain.

A transcriptional network coordinately determines transmitter and peptidergic fate in the dorsal spinal cord.

Dorsal horn neurons express many different neuropeptides that modulate sensory perception like the sensation of pain. Inhibitory neurons of the dorsal horn derive from postmitotic neurons that express Pax2, Lbx1 and Lhx1/5, and diversify during maturation. In particular, fractions of maturing inhibitory neurons express various neuropeptides. We demonstrate here that a coordinate molecular mechanism determines inhibitory and peptidergic fate in the developing dorsal horn. A bHLH factor complex that contains Ptf1a acts as upstream regulator and initiates the expression of several downstream transcription factors in the future inhibitory neurons, of which Pax2 is known to determine the neurotransmitter phenotype. We demonstrate here that dynorphin, galanin, NPY, nociceptin and enkephalin expression depends on Ptf1a, indicating that these neuropeptides are expressed in inhibitory neurons. Furthermore, we show that Neurod1/2/6 and Lhx1/5, which act downstream of Ptf1a, control distinct aspects of peptidergic differentiation. In particular, the Neurod1/2/6 factors are essential for dynorphin and galanin expression, whereas the Lhx1/5 factors are essential for NPY expression. We conclude that a transcriptional network operates in maturing dorsal horn neurons that coordinately determines transmitter and peptidergic fate.

Ptf1a, Lbx1 and Pax2 coordinate glycinergic and peptidergic transmitter phenotypes in dorsal spinal inhibitory neurons.

Inhibitory neurons in the dorsal horn synthesize a variety of neurotransmitters, including GABA, glycine and a set of peptides. Here we show that three transcription factors, Ptf1a, Pax2, and Lbx1, which have been reported to promote a GABAergic cell fate, also specify glycinergic and peptidergic transmitter phenotypes. First, Ptf1a appears to be a master regulator, as indicated by a requirement of Ptf1a for the expression of glycinergic marker GlyT2 and a set of peptides, including neuropeptide Y (NPY), nociceptin/orphanin FQ (N/OFQ), somatostatin (SOM), enkephalin (ENK), dynorphin (DYN) and galanin (GAL). Second, Pax2 is a downstream target of Ptf1a and controls subsets of transmitter phenotypes, including the expression of GlyT2, NPY, N/OFQ, DYN, and GAL, but is dispensable for SOM or ENK expression. Third, for Lbx1, due to neuronal cell loss at late stages, our analyses focused on early embryonic stages, and we found that Lbx1 is required for the expression of GlyT2, NPY, N/OFQ and is partially responsible for SOM expression. Our studies therefore suggest a coordinated and hierarchical specification of a variety of neurotransmitters in dorsal spinal inhibitory neurons.

Pbx3 is required for normal locomotion and dorsal horn development.

The transcription cofactor Pbx3 is critical for the function of hindbrain circuits controlling respiration in mammals, but the perinatal lethality caused by constitutively null mutations has hampered investigation of other roles it may play in neural development and function. Here we report that the conditional loss of Pbx3 function in most tissues caudal to the hindbrain resulted in progressive deficits of posture, locomotion, and sensation that became apparent during adolescence. In adult mutants, the size of the dorsal horn of the spinal cord and the numbers of calbindin-, PKC-gamma, and calretinin-expressing neurons in laminae I-III were markedly reduced, but the ventral cord and peripheral nervous system appeared normal. In the embryonic dorsal horn, Pbx3 expression was restricted to a subset of glutamatergic neurons, but its absence did not affect the initial balance of excitatory and inhibitory interneuron phenotypes. By embryonic day 15 a subset of Meis(+) glutamatergic neurons assumed abnormally superficial positions and the number of calbindin(+) neurons was increased three-fold in the mutants. Loss of Pbx3 function thus leads to the incorrect specification of some glutamatergic neurons in the dorsal horn and alters the integration of peripheral sensation into the spinal circuitry regulating locomotion.

Neuron type-specific effects of brain-derived neurotrophic factor in rat superficial dorsal horn and their relevance to 'central sensitization'.

Chronic constriction injury (CCI) of the rat sciatic nerve increases the excitability of the spinal dorsal horn. This 'central sensitization' leads to pain behaviours analogous to human neuropathic pain. We have established that CCI increases excitatory synaptic drive to putative excitatory, 'delay' firing neurons in the substantia gelatinosa but attenuates that to putative inhibitory, 'tonic' firing neurons. Here, we use a defined-medium organotypic culture (DMOTC) system to investigate the long-term actions of brain-derived neurotrophic factor (BDNF) as a possible instigator of these changes. The age of the cultures and their 5-6 day exposure to BDNF paralleled the protocol used for CCI in vivo. Effects of BDNF (200 ng ml(-1)) in DMOTC were reminiscent of those seen with CCI in vivo. These included decreased synaptic drive to 'tonic' neurons and increased synaptic drive to 'delay' neurons with only small effects on their membrane excitability. Actions of BDNF on 'delay' neurons were exclusively presynaptic and involved increased mEPSC frequency and amplitude without changes in the function of postsynaptic AMPA receptors. By contrast, BDNF exerted both pre- and postsynaptic actions on 'tonic' cells; mEPSC frequency and amplitude were decreased and the decay time constant reduced by 35%. These selective and differential actions of BDNF on excitatory and inhibitory neurons contributed to a global increase in dorsal horn network excitability as assessed by the amplitude of depolarization-induced increases in intracellular Ca(2+). Such changes and their underlying cellular mechanisms are likely to contribute to CCI-induced 'central sensitization' and hence to the onset of neuropathic pain.

Estrogen receptor beta is essential for sprouting of nociceptive primary afferents and for morphogenesis and maintenance of the dorsal horn interneurons.

Estrogen is known to influence pain, but the specific roles of the two estrogen receptors (ERs) in the spinal cord are unknown. In the present study, we have examined the expression of ERalpha and ERbeta in the spinal cord and have looked for defects in pain pathways in ERbeta knockout (ERbeta(-/-)) mice. In the spinal cords of 10-month-old WT mice, ERbeta-positive cells were localized in lamina II, whereas ERalpha-positive cells were mainly localized in lamina I. In ERbeta(-/-) mice, there were higher levels of calcitonin gene-regulated peptide and substance P in spinal cord dorsal horn and isolectin B4 in the dorsal root ganglion. In the superficial layers of the spinal cord, there was a decrease in the number of calretinin (CR)-positive neurons, and in the outer layer II, there was a loss of calbindin-positive interneurons. During embryogenesis, ERbeta was first detectable in the spinal cord at embryonic day 13.5 (E13.5), and ERalpha was first detectable at E15.5. During middle and later embryonic stages, ERbeta was abundantly expressed in the superficial layers of the dorsal horn. ERalpha was also expressed in the dorsal horn but was limited to fewer neurons. Double staining for ERbeta and CR showed that, in the superficial dorsal horn of WT neonates [postnatal day 0 (P0)], most CR neurons also expressed ERbeta. At this stage, few CR-positive cells were detected in the dorsal horn of ERbeta(-/-) mice. Taken together, these findings suggest that, early in embryogenesis, ERbeta is involved in dorsal horn morphogenesis and in sensory afferent fiber projections to the dorsal horn and that ERbeta is essential for survival of dorsal horn interneurons throughout life.

Potentiation of glutamatergic synaptic transmission by protein kinase C-mediated sensitization of TRPV1 at the first sensory synapse.

Sensory input from the periphery to the CNS is critically dependent on the strength of synaptic transmission at the first sensory synapse formed between primary afferent dorsal root ganglion (DRG) and superficial dorsal horn (DH) neurons of the spinal cord. Transient receptor potential vanilloid 1 (TRPV1) expressed on a subset of sensory neurons plays an important role in chronic inflammatory thermal nociception. Activation of protein kinase C (PKC) sensitizes TRPV1, which may contribute to the pathophysiology of chronic pain conditions. In this study, we have examined the modulation of TRPV1-mediated enhancement of excitatory synaptic transmission in response to PKC activation. Miniature excitatory postsynaptic currents (mEPSCs) from embryonic rat DRG-DH neuronal cocultures were recorded by patch clamping DH neurons. Capsaicin potently increased the frequency but not the amplitude of mEPSCs in a calcium-dependent manner, suggesting TRPV1-mediated glutamate release from presynaptic terminals of sensory neurons. Continued or repeated applications of capsaicin reduced the frequency of mEPSCs over time. The PKC activator phorbol 12,13-dibutyrate (PDBu) alone increased mEPSC events to a certain extent in a reversible manner but capsaicin further synergistically enhanced the frequency of mEPSCs. The PKC inhibitor bisindolylmaleimide (BIM) abolished PDBu-mediated potentiation of TRPV1-dependent increases in mEPSC frequency, suggesting modulation of TRPV1 by PKC-induced phosphorylation. In addition, at normal body temperatures ( approximately 37 degrees C) PKC-mediated enhancement of mEPSC frequency is significantly decreased by a specific TRPV1 antagonist, suggesting a physiological role of TRPV1 at the central terminals. Furthermore, bradykinin (BK) significantly potentiated TRPV1-modulated synaptic responses by activating the PLC-PKC pathway. Our results indicate that TRPV1 activation can modulate excitatory synaptic transmission at the first sensory synapse and its effects can further be augmented by activation of PKC. Increased gain of sensory input by TRPV1-induced enhancement of glutamate release and its potentiation by various inflammatory mediators may contribute to persistent pain conditions. Selective targeting of TRPV1 expressed on the central terminals of sensory neurons may serve as a strategy to alleviate chronic intractable pain conditions.

mig-5/Dsh controls cell fate determination and cell migration in C. elegans.

Cell fate determination and cell migration are two essential events in the development of an organism. We identify mig-5, a Dishevelled family member, as a gene that regulates several cell fate decisions and cell migrations that are important during C. elegans embryonic and larval development. In offspring from mig-5 mutants, cell migrations are defective during hypodermal morphogenesis, QL neuroblast migration, and the gonad arm migration led by the distal tip cells (DTCs). In addition to abnormal migration, DTC fate is affected, resulting in either an absent or an extra DTC. The cell fates of the anchor cell in hermaphrodites and the linker cells in the male gonad are also defective, often resulting in the cells adopting the fates of their sister lineage. Moreover, 2 degrees vulval precursor cells occasionally adopt the 3 degrees vulval cell fate, resulting in a deformed vulva, and the P12 hypodermal precursor often differentiates into a second P11 cell. These defects demonstrate that MIG-5 is essential in determining proper cell fate and cell migration throughout C. elegans development.

Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal sensory interneurons.

Sensory information from the periphery is integrated and transduced by excitatory and inhibitory interneurons in the dorsal spinal cord. Recent studies have identified a number of postmitotic factors that control the generation of these sensory interneurons. We show that Gsh1/2 and Ascl1 (Mash1), which are expressed in sensory interneuron progenitors, control the choice between excitatory and inhibitory cell fates in the developing mouse spinal cord. During the early phase of neurogenesis, Gsh1/2 and Ascl1 coordinately regulate the expression of Tlx3, which is a critical postmitotic determinant for dorsal glutamatergic sensory interneurons. However, at later developmental times, Ascl1 controls the expression of Ptf1a in dIL(A) progenitors to promote inhibitory neuron differentiation while at the same time upregulating Notch signaling to ensure the proper generation of dIL(B) excitatory neurons. We propose that this switch in Ascl1 function enables the cogeneration of inhibitory and excitatory sensory interneurons from a common pool of dorsal progenitors.

Functional and selective RNA interference in developing axons and growth cones.

Developing axons and growth cones contain "local" mRNAs that are translated in response to various extracellular signaling molecules and have roles in several processes during axonal development, including axonal pathfinding, orientation of axons in chemotactic gradients, and in the regulation of neurotransmitter release. The molecular mechanisms that regulate mRNA translation within axons and growth cones are unknown. Here we show that proteins involved in RNA interference (RNAi), including argonaute-3 and argonaute-4, Dicer, and the fragile X mental retardation protein, are found in developing axons and growth cones. These proteins assemble into functional RNA-induced silencing complexes as transfection of small interfering RNAs selectively into distal axons results in distal axon-specific mRNA knock-down, without reducing transcript levels in proximal axons or associated diffusion of small interfering RNA into proximal axons or cell bodies. RhoA mRNA is localized to axons and growth cones, and intra-axonal translation of RhoA is required for growth cone collapse elicited by Semaphorin 3A (Sema3A), an axonal guidance cue. Selective knock-down of axonal RhoA mRNA abolishes Sema3A-dependent growth cone collapse. Our results demonstrate functional and potent RNAi in axons and identify an approach to spatially regulate mRNA transcripts at a subcellular level in neurons.

Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn.

Mutations in the human and mouse PTF1A/Ptf1a genes result in permanent diabetes mellitus and cerebellar agenesis. We show that Ptf1a is present in precursors to GABAergic neurons in spinal cord dorsal horn as well as the cerebellum. A null mutation in Ptf1a reveals its requirement for the dorsal horn GABAergic neurons. Specifically, Ptf1a is required for the generation of early-born (dI4, E10.5) and late-born (dIL(A), E12.5) dorsal interneuron populations identified by homeodomain factors Lhx1/5 and Pax2. Furthermore, in the absence of Ptf1a, the dI4 dorsal interneurons trans-fate to dI5 (Lmx1b(+)), and the dIL(A) to dIL(B) (Lmx1b(+);Tlx3(+)). This mis-specification of neurons results in a complete loss of inhibitory GABAergic neurons and an increase in the excitatory glutamatergic neurons in the dorsal horn of the spinal cord by E16.5. Thus, Ptf1a function is essential for GABAergic over glutamatergic neuronal cell fates in the developing spinal cord, and provides an important genetic link between inhibitory and excitatory interneuron development.

Ectopic HOXA5 expression results in abnormal differentiation, migration and p53-independent cell death of superficial dorsal horn neurons.

Previously, we reported a line of mice (Hoxa5SV2) that ectopically expresses HOXA5 in the developing cervical and brachial dorsal spinal cord. Animals from this line exhibited a clear loss of cells in the outer lamina of the mature dorsal horn that coincided with an adult phenotype of sensory and motor defects of the forelimb. In this report, we examined the etiology of lost dorsal horn cells. Cells normally fated to populate the outer laminae I-III of the dorsal horn migrated inappropriately, as the percentage of laterally positioned cells in the dorsal horn was significantly reduced in Hoxa5SV2 transgenics. Apoptosis was a major cause of cell loss while proliferation of neurons was not affected in Hoxa5SV2 animals. Although Hoxa5 has been shown in vitro to regulate p53 expression and cause p53-dependent apoptosis, p53 was not required in vivo for the inappropriate apoptosis seen in Hoxa5SV2 mice, or for the normal death of motor neurons. Normal apoptosis is not dependent on Hoxa5, as the level of ventral horn motor neuron apoptosis was not changed in Hoxa5 null animals. As a possible cause of aberrant migration and/or apoptosis of dorsal neurons, misexpression of cell type markers was demonstrated. Further, the expression pattern of laminar markers was altered and sensory fibers aberrantly penetrated the outer lamina of mutants. Our evidence suggests that the loss of dorsal horn neurons in Hoxa5SV2 mutants was due to misexpression of dorsal horn neuronal markers, aberrant migration, and inappropriate apoptosis.

Embryonic development of choline acetyltransferase and nitric oxide synthase in the spinal cord of pigeons and chickens with special reference to the superficial dorsal horn.

The superficial dorsal horn of birds as well as mammals contains both cholinergic and nitrergic neuronal structures as evident from the presence of the synthesizing enzymes such as choline acetyltransferase and nitric oxide synthase, which is an NADPH diaphorase. In the rat, both systems develop only postnatally. Rats are altricial at birth whereas pigeons and chickens are semiprecocial or precocial, respectively, at the time of hatching. Immunocytochemical studies of choline acetyltransferase and nitric oxide synthase in the developing avian spinal cord (starting with embryonic day 12 of 18 in the pigeon and 14 of 21 in the chicken) showed that both systems are well developed in the superficial dorsal horn at the time of hatching in both avian species. In the pigeon, choline acetyltransferase-positive superficial dorsal horn neurons appear only on the day of hatching (E18), whereas nitric oxide synthase-positive neurons can be first detected at stage E14. In the chicken, nitric oxide synthase-positive neurons are present already at stage E14, whereas choline acetyltransferase-positive neurons appear at stage E20. Autonomic and somatic motor neurons show adult-like choline acetyltransferase-immunoreactivity and/or nitric oxide synthase-immunoreactivity at the earliest stages investigated. It is concluded that the stage of maturation at birth or hatching plays an important role in the development of superficial dorsal horn cholinergic and nitrergic systems.

The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord.

Neurons of the dorsal horn integrate and relay sensory information and arise during development in the dorsal spinal cord, the alar plate. Class A and B neurons emerge in the dorsal and ventral alar plate, differ in their dependence on roof plate signals for specification, and settle in the deep and superficial dorsal horn, respectively. We show here that the basic helix-loop-helix (bHLH) gene Olig3 is expressed in progenitor cells that generate class A (dI1-dI3) neurons and that Olig3 is an important factor in the development of these neuronal cell types. In Olig3 mutant mice, the development of class A neurons is impaired; dI1 neurons are generated in reduced numbers, whereas dI2 and dI3 neurons are misspecified and assume the identity of class B neurons. Conversely, Olig3 represses the emergence of class B neurons in the chick spinal cord. We conclude that Olig3 expression distinguishes the two major classes of progenitors in the dorsal spinal cord and determines the distinct specification program of class A neurons.