Development changes in the distribution of gamma-aminobutyric acid-immunoreactive neurons in the embryonic chick lumbosacral spinal cord

The development of γ-aminobutyric acid (GABA)-immunoreactive neurons was investigated in the embryonic and posthatch chick lumbosacral spinal cord by using pre- and postembedding immunostaining with an anti-GABA antiserum. The first GABA-immunoreactive cells were detected in the ventral one-half of the spinal cord dorsal to the lateral motor exception of the lateral motor column, appeared throughout the entire extent of the ventral one-half of the spinal gray matter by E6. Thereafter, GABA-immunoreactive neurons extended from ventral to dorsal regions. Stained perikarya first appeared at E8 and then progressively accumulated in the dorsal horn, while immunoreactive neurons gradually declined in the ventral horn. The general pattern of GABA immunoreactivity characteristic of mature animals had been achieved by E12 and was only slightly altered afterwards. In the dorsal horn, most of the stained neurons were observed in laminae I–III, both at the upper (LS 1–3) and at the lower (LS 5–7) segments of the lumbosacral spinal cord. In the ventral horn, the upper and lower lumbosacral segments showed marked differences in the distribution of stained perikarya. GABAergic neurons were scattered in a relatively large region dorsomedial to the lateral motor column at the level of the upper lumbosacral segments, whereas they were confined to the dorsalmost region of lamina VII at the lower segments. The early expression of GABA immunoreactivity may indicate a trophic and synaptogenetic role for GABA in early phases of spinal cord development. The localization of GABAergic neurons in the ventral horn and their distribution along the rostrocaudal axis of the lumbosacral spinal cord coincide well with previous physiological findings, suggesting that some of these GABAergic neurons may be involved in neural circuits underlying alternating rhythmic motor activity of the embryonic chick spinal cord. © 1994 Wiley-Liss, Inc.     

Ontogenic changes of the GABAergic system in the embryonic mouse spinal cord

Numerous studies have demonstrated an excitatory action of GABA early in development, which is likely to play a neurotrophic role. In order to better understand the role of GABA in the mouse spinal cord, we followed the evolution of GABAergic neurons over the course of development. We investigated, in the present study, the ontogeny of GABA immunoreactive (GABA-ir) cell bodies and fibers in the embryonic mouse spinal cord at brachial and lumbar levels. GABA-ir somata were first detected at embryonic day 11.5 (E11.5) exclusively at brachial level in the marginal zone. By E13.5, the number of GABAergic neurons sharply increased throughout the extent of the ventral horn both at brachial and lumbar level. Stained perikarya first appeared in the future dorsal horn at E15.5 and progressively invaded this area while they decreased in number in the presumed ventral gray matter. At E12.5, E13.5 and E15.5, we checked the possibility that ventral GABA-ir cells could belong to the motoneuronal population. Using a GABA/Islet-1/2 double labeling, we did not detect any double-stained neurons indicating that spinal motoneurons do not synthesize GABA during the course of development. GABA-ir fibers also appeared at the E11.5 stage in the presumptive lateral white matter at brachial level. At E12.5 and E13.5, GABA-ir fibers progressively invaded the ventral marginal zone and by E15.5 reached the dorsal marginal zone. At E17.5 and postnatal day 0 (P0), the number of GABA-ir fibers declined in the white matter. Finally, by P0, GABA immunoreactivity that delineated somata was mainly restricted to the dorsal gray matter and declined in intensity and extent. The ventral gray matter exhibited very few GABA-ir cell bodies at this neonatal stage of development. The significance of the migration of somatic GABA immunoreactivity from ventral to the dorsal gray matter is discussed.     

Islet-1 expression in thoracic spinal motor neurons in prenatal mouse

The LIM homeodomain protein Islet-1, an embryonic marker for motoneurons in the spinal cord, has been reported to be heterogeneously expressed among motoneuron groups in mouse. In the present study, we examined Islet-1 expression in the thoracic and rostral lumbar spinal cord in prenatal mice. In the thoracic spinal cord at embryonic day 12.5 (E12.5) and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, whereas weaker immunoreactivity was observed in the ventral group. Strong Islet-1 immunoreactivity was also observed in the intermediolateral area and more medial part of the intermediate zone. In the rostral lumbar spinal cord at E12.5 and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, and in the intermediolateral nucleus, whereas weaker immunoreactivity was observed in the ventral, and dorsolateral groups. At E14.5, the number of Islet-1 immunoreactive neurons was reduced in the spinal cord, but the distribution pattern was similar to that at E12.5 and E13.5. At E15.5, Islet-1 immunoreactivity was almost completely confined to the intermediolateral area. Some weakly immunoreactive neurons were observed in the ventral horn. The findings of the present study indicated that Islet-1 expression at embryonic stages differs among the motoneuron groups in the thoracic and rostral lumbar spinal cord.     

Generation patterns of four groups of cholinergic neurons in rat cervical spinal cord: a combined tritiated thymidine autoradiographic and choline acetyltransferase immunocytochemical study

This report examines the generation of cholinergic neurons in the spinal cord in order to determine whether the transmitter phenotype of neurons is associated with specific patterns of neurogenesis. Previous immunocytochemical studies identified four groups of choline acetyltransferase (ChAT)-positive neurons in the cervical enlargement of the rat spinal cord. These cell groups vary in both somatic size and location along the previously described ventrodorsal neurogenic gradient of the spinal cord. Thus, large (and small) motoneurons are located in the ventral horn, medium-sized partition cells are found in the intermediate gray matter, small central canal cluster cells are situated within lamina X, and small dorsal horn neurons are scattered predominantly through laminae III-V. The relationships among the birthdays of these four subsets of cholinergic neurons have been examined by combining 3H-thymidine autoradiography and ChAT immunocytochemistry. Embryonic day 11 was the earliest time that neurons were generated within the cervical enlargement. Large and small ChAT-positive motoneurons were produced on E11 and 12, with 70% of both groups being born on E11. ChAT-positive partition cells were produced between E11 and 13, with their peak generation occurring on E12. Approximately 70% of the cholinergic central canal cluster and dorsal horn cells were born on E13, and the remainder of each of these groups was generated on E14. Other investigators have shown that all neurons within the rat cervical spinal cord are produced in a ventrodorsal sequence between E11 and E16. In contrast, ChAT-positive neurons are born only from E11 to E14 and are among the earliest cells generated in the ventral, intermediate, and dorsal subdivisions of the spinal cord. However, all cholinergic neurons are not generated simultaneously; rather their birthdays are correlated with their positions along the ventrodorsal gradient of neurogenesis. The fact that large motoneurons and medium-sized partition cells are born before small central canal cluster and dorsal horn cells would appear to support the generalization that large neurons are generated before small ones. However, the location of spinal cholinergic neurons within the neurogenic gradient seems to be more importantly associated with the time of cell generation than somal size. For example, when large and small motoneurons located at the same dorsoventral spinal level are compared, both sizes of cells are generated at the same time and in similar proportions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

Ontogeny of descending serotonergic innervation and evidence for intraspinal 5-HT neurons in the mouse spinal cord

Neuronal networks in the mouse spinal cord express serotonin (5-HT)-induced rhythmic motor activity at early developmental stages (embryonic day (E) 12.5). Later in development, by post-natal day (P) 10, the 5-HT-evoked rhythmic motor activity matures and acquires an adult locomotor-like pattern. With the view to establishing a relationship between the ontogeny of locomotor networks and the maturation of spinal 5-HT systems, we have traced 5-HT immunoreactivity in the mouse spinal cord from E12.5 to PN10. By E12.5, descending 5-HT immunoreactive (5-HT-ir) fibers that likely originate from raphe nuclei were detected in the ventral and lateral funiculi, at anterior cervical spinal levels, but not at more caudal levels. Descending 5-HT-ir axons reached thoracic levels at E14.5 and lumbar levels at E16.5. Some 5-HT-ir fibers could be detected in the ventral and intermediate gray matter by E16.5, whereas the dorsal gray matter was not invaded before PN0. At PN10, a dense serotonergic innervation was restricted to the gray matter with a high concentration of 5-HT-ir fibers in three areas: dorsal horn, ventral horn (where motoneurons are located) and intermediate area. Surprisingly, from E16.5 to PN10, 5-HT-ir intraspinal neurons were found, exclusively at sacral levels. Their somata lay in the gray matter around the central canal and preferentially in the ventro-median part of the ventral horn. The functional significance of these sacral 5-HT-ir neurons is discussed.     

Development of Calbindin-D28k Immunoreactive Neurons in the Embryonic Chick Lumbosacral Spinal Cord

The development of immunoreactivity for the calcium-binding protein calbindin-D28k (CaB) was investigated in the embryonic and hatched chick lumbosacral spinal cord. CaB-immunoreactive neurons were revealed in the dorsal and ventral horns as well as in the intermediate grey matter from early stages of neuronal development. CaB immunoreactivity was first detected in large neurons in the presumptive dorsal horn at embyronic day 5, while small neurons in the lateral dorsal horn were the last to appear, at embryonic day 10. We have identified and traced the morphological maturation of six CaB-immunoreactive cell groups, three in the dorsal horn and three in the ventral horn. In the dorsal horn these groups were (1) large neurons in the lateral dorsal horn (laminae I and IV), (2) small neurons in the lateral dorsal horn (lamina II), and (3) small neurons in the medial dorsal horn (lamina III). All three groups were present throughout the entire length of the lumbosacral spinal cord and showed persistent CaB immunoreactivity. In the ventral horn, CaB-immunoreactive neurons were classified into the following three categories: (1) Neurons dorsal to the lateral motor column (lamina VII). These neurons were present exclusively in the upper lumbosacral segments (LS1 – 3), and they showed steady CaB immunoreactivity during their maturation. (2) Neurons at the dorsomedial aspect of the lateral motor column (at the border of laminae VII and IX). This population of neurons was characteristic of the lower segments of the lumbosacral cord (LS5 – 7) and presented transient CaB expression. (3) Neurons within the lateral motor column (lamina IX). These neurons were dispersed throughout the length of the lumbosacral spinal cord. They were three to four times more numerous in the upper than in the lower lumbosacral segments, and their numbers declined throughout LS1 – 7 as the animal matured. The characteristic features of the development of neurons immunoreactive for CaB are discussed and correlated with previous neuroanatomical and physiological studies concerning sensory and motor functions of the developing chick spinal cord.     

Transient expression of calcitonin gene-related peptide immunoreactivity in the ventral horn of the post-natal rat cervical spinal cord.

In addition to the well known expression of calcitonin gene-related peptide (CGRP) immunoreactivity in primary afferent fibers in the dorsal horn and in motoneurons, this study has demonstrated, in rat, transient CGRP immunoreactivity in fine caliber varicose axons throughout the ventral horn and in a group of neuron cell bodies in the medial ventral horn. This was first observed at post-natal day 7 (P7) and had disappeared by P21. Physiological studies in chick embryonic spinal cord have shown that CGRP modulates spontaneous activity during development [Carr, P.A., Wenner, P., 1998. Calcitonin gene-related peptide and effects on spontaneous activity in embryonic chick spinal cord. Dev. Brain Res. 106, 47-55]. Neural activity increases post-natally in rat where it may play a role in refinement of sensorimotor synapses. This activity may also be modulated by CGRP.     

Changing pattern of expression of parvalbumin immunoreactivity during human fetal spinal cord development

Expression of the calcium binding protein parvalbumin (PV) by different classes of spinal neuron has been shown to be developmentally regulated in both rat and monkey. From postmortem studies of eight human cervical spinal cords ranging in age from 11 to 35 weeks postconceptional age, we report that parvalbumin immunoreactivity is similarly plastic in human lower cervical spinal cord development, with many changes occurring prenatally. At 11-14 weeks postconceptional age, there was prominent immunostaining of primary sensory afferents that could be seen coursing through the dorsal horn and extensively innervating the motoneuron pools. Motoneurons were also found to be clearly immunoreactive for choline acetyltransferase by this age. A few ventral horn neurons that were not motoneurons were also parvalbumin immunoreactive. By 24-27 weeks postconceptional age, sensory afferents were still immunoreactive, as were many other axons throughout the white matter. In addition, many ventral horn neurons were now immunoreactive as well as a few dorsal horn neurons. By 31-35 weeks postconceptional age, there was extensive immunostaining of neurons throughout the spinal cord, including a few moderately immunoreactive motoneurons. There were many immunopositive axons in all the white matter tracts except the corticospinal tracts; however, staining of sensory axons traversing the grey matter was less prominent by this age. In the rat, expression of PV by primary sensory neurons coincides with the onset of fetal limb movement. The onset of expression of PV in ventral horn neurons coincides with later developmental events after the arrival of corticospinal inputs, whereas widespread PV immunoreactivity in dorsal horn neurons marks the attainment of a mature pattern of PV expression. The extent to which expression of PV immunoreactivity can be taken to indicate landmarks in human development will be discussed.     

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.     

Neuroendocrine secretory protein 55 (NESP55) in the spinal cord of rat: an immunocytochemical study

The immunohistochemical expression of a novel chromogranin-like protein, neuroendocrine secretory protein 55 (NESP55), in the rat spinal cord was investigated. NESP55-immunoreactive cells were detected in the ventral horn, intermediate laminae, and deep dorsal horn, comprising motoneurons, autonomic neurons, and interneurons throughout all spinal segments. Within laminae I-II of the dorsal horn, one or two NESP55-positive cells were often seen. Nerve fibers also contained NESP55 immunoreactivity (IR) and were particularly prominent in the ventral horn. No nerve terminals/varicosities appeared to contain NESP55 in any spinal lamina. Double-staining experiments revealed that a high proportion of the NESP55-positive neurons were cholinergic. Moreover, NESP55-IR in the motoneurons was evenly distributed in the whole cytoplasm with a finely granular appearance. In contrast, the fluorescent material in the preganglionic neurons was concentrated in the perinuclear region and largely overlapped with the trans-Golgi network marker TGN38. Our data provide detailed morphological information on the distribution of NESP55-IR in the rat spinal cord. Also, the differential intracellular expression of NESP55-IR in the spinal motoneurons and autonomic neurons suggests that NESP55 may be processed into different secretory granules and may be involved in both constitutive and regulated pathways in these neurons.     

Early stages in the development of spinal motor neurons.

In order to identify early events in the differentiation of motor neurons, the expression of several developmentally regulated, neuronal molecules was investigated by immunohistochemistry on consecutive sections of cervical spinal cord. Motor neurons are among the first neurons to be born and to differentiate within the embryonic rat spinal cord. They undergo their terminal mitosis on embryonic days 10 and 11 (E10-11) and acquire detectable levels of the transmitter synthesizing enzyme, choline acetyltransferase, by E11.5. Staining with antibodies to the 68 kD neurofilament protein revealed motor neurons extending processes out the ventral root as early as E10.5. Monoclonal antibodies to two different epitopes on the cell adhesive molecule, NCAM, bound to myotomes on E10.5, and began to recognize ventral horn neurons by E11. Two other markers of developing neurons, the growth-associated protein, GAP-43, and the surface glycoprotein, TAG-1, were clearly detected on young motor neurons by E11.5. Thus, during the 36 hours following the final mitosis of their precursors, motor neurons acquire cytoskeletal, enzymatic, and cell surface components that distinguish them from other developing cells within the spinal cord. Not all of the newly acquired molecules continue to be expressed by motor neurons. Immunoreactivity for TAG-1 was lost by E12.5, followed by a gradual reduction of immunoreactivity for GAP-43 and the highly polysialylated form of NCAM. By E15, only antibodies to choline acetyltransferase (Phelps et al., J. Comp. Neurol. 307:1-10, 1990), and to neurofilaments, selectively stained motor neurons within the embryonic spinal cord. The transient presence of GAP-43, TAG-1, and the embryonic form of NCAM coincides with a period of vigorous axonal growth and declines when motor neurons reach their targets. This report describes the temporal sequence of early stages in the differentiation of the rodent motor neuronal phenotype. Some of these changes may be related to interactions with their synaptic partners.     

Immunocytochemical approach of GABAergic innervation of the mouse spinal cord using antibodies to GABA

The distribution of gamma-amino-butyric acid containing neurons in the Mouse spinal cord has been studied at both the light and electron microscope levels using antibodies against GABA and revelation by the Fab-peroxidase technique. At the light microscope level immunoreactive profiles of perikarya and neuronal processes were particularly abundant in the superficial laminae (I-IV) of the dorsal horn. Scattered soma profiles were found in the other layers and more particularly in the lamina X where Liquor contacting immuno-reactive neurons could be detected. GABAergic cell bodies were very sparse in the ventral horn. Electron microscopic observations confirmed the light microscope results: terminals constituted synaptic symmetrical contacts that provide a morphological basis for inhibition in the dorsal horn and for post-synaptic inhibition of motoneurons in the ventral horn.     

Three types of GABA-immunoreactive cells in the lamprey spinal cord

Polyclonal antisera raised against conjugated GABA were used to study the distribution of GABAergic neurons in the spinal cords of lampreys (Lampetra fluviatilis and Ichtyomyzon unicuspis) using immunofluorescence and peroxidase-antiperoxidase techniques. Three morphologically distinct types of GABA-immunoreactive (GABA-ir) cell bodies were observed, multipolar neurons in the lateral grey cell column, apparently bipolar cells in the ventral aspect of the dorsal horn, and small liquor-contacting cells surrounding the central canal. A high density of immunoreactive fibers of spinal origin were present in the lateral and ventral funiculi, whereas the dorsal column had a relatively low density. Dense GABA-ir plexuses were situated in the lateral spinal margin, and in the dorsal part of the dorsal horn. A chronic lesion of the rostral spinal cord did not result in any observable loss of GABA-ir fibers below or above the lesion, suggesting that the 3 types of segmental GABA-ir neurons are the main sources of the GABAergic innervation of the lamprey spinal cord.     

Neuroepithelial cells in the rat spinal cord express glutamate decarboxylase immunoreactivity in vivo and in vitro.

It is unknown whether neuroepithelial cells in the mammalian central nervous system express neurotransmitter-synthesizing enzymes. In this study, expression of glutamate decarboxylase (GAD), the gamma-aminobutyric acid (GABA)-synthesizing enzyme, was examined in proliferative cells and postmitotic neuroblasts in embryonic rat spinal cord. Immunostaining coronal sections of the embryonic spinal cord with K2 antiserum, which recognizes GAD proteins encoded by the GAD67 gene, revealed intensely stained neuroepithelial cells in the basal plate at embryonic day (E) 13, in the intermediate plate between E 13-16, and last seen in the alar plate at E 16. Nissl counterstaining demonstrated that a small number of these GAD-immunoreactive cells adjacent to the neural tube lumen were mitotic. The ventral-to-dorsal gradient of GAD expression in precursor cells and postmitotic neuroblasts correlates anatomically and temporally with the sequential generation of motoneurons, commissural neurons, and interneurons in the dorsal horn. Some of these GAD-immunoreactive neuroepithelial cells may re-enter the mitotic cycle, while others are postmitotic neuroblasts presumably migrating to the intermediate zone to differentiate into young neurons. Double-immunostaining cells acutely dissociated from E 11-18 spinal cords with K2 and anti-bromodeoxyuridine antisera, following a bromodeoxyuridine pulse in vivo, revealed considerable numbers of DNA-synthesizing cells immunoreactive for GAD. The absolute number of double-stained cells peaked during E 12-15, coinciding with terminal cell division in most spinal neurons. These observations suggest that spinal neuronal precursors can synthesize GAD-related proteins prior to, or during, the terminal cell cycle. Although GAD immunoreactivity revealed by K2 antiserum was detected in proliferative cells and in migrating postmitotic neuroblasts, GABA immunoreactivity was never detectable in these cells. These early embryonic GAD-immunoreactive neuroepithelial cells may either synthesize levels of GABA that cannot be detected immunocytochemically, and/or express enzymatically inactive GAD-related proteins.     

The Relationship Between Glycine and Gephyrin in Synapses of the Rat Spinal Cord

In order to examine the relationship between gephyrin (the peripheral membrane protein associated with glycine receptors) and glycinergic boutons, we have carried out a post-embedding immunogold study of glycine-like immunoreactivity on sections of rat lumbar spinal cord which had previously been reacted with monoclonal antibody to gephyrin. In all three areas examined (laminae I and II, lamina III and lamina IX) the majority of profiles which were presynaptic at gephyrin-immunoreactive synapses were enriched with glycine-like immunoreactivity. It was estimated that at least 83% of profiles presynaptic to gephyrin-immunoreactive synapses in the superficial dorsal horn (laminae I and II) were glycine-immunoreactive, while for lamina III and the ventral horn (lamina IX) the proportions were at least 91% and 98% respectively. This provides strong evidence that glycine is a transmitter at those synapses where gephyrin- and glycine-like immunoreactivities are both present, but suggests that gephyrin may sometimes be expressed at non-glycinergic synapses and indicates the need for caution in using gephyrin-immunoreactivity as a marker for glycinergic synapses within the spinal cord. By reacting serial sections of dorsal horn with antisera to glycine and GABA, we have shown that many boutons in laminae I-III of the dorsal horn show both types of immunoreactivity and are therefore likely to use both amino acids as inhibitory transmitters. Many of the boutons which were presynaptic at axoaxonic synapses in the ventral part of lamina II and in lamina III were glycine- and GABA-immunoreactive and in many cases the postsynaptic element was the central axon of a type II synaptic glomerulus. Taken together with pharmacological evidence, this suggests that inhibitory intemeurons in the dorsal horn which use both GABA and glycine may be important in controlling the flow of information from hair follicle afferents to other spinal neurons.     

Immunoreactivity for glutamic acid decarboxylase and several neuropeptides in the spinal cord of the raccoon.

The peroxidase-antiperoxidase method was used to examine major immunohistochemical features of the spinal cord of adult raccoons. The lateral portions of the ventral horn contained many large multipolar neurons that showed cholecystokinin-like immunoreactivity, suggesting the coexistence of cholecystokinin with acetylcholine in a subset of motoneurons. The dorsal horn revealed unique but overlapping patterns of immunoreactivity for glutamic acid decarboxylase, somatostatin, substance P, vasoactive intestinal polypeptide and cholecystokinin. The data imply that some of the peptides may coexist within the same dorsal root ganglion cells and their spinal cord processes.     

Ontological study of calbindin-D28k-like and parvalbumin-like immunoreactivities in rat spinal cord and dorsal root ganglia

The calcium ion plays an important role in some critical developmental events in the nervous system, such as neurulation and neurite elongation. Therefore, as the intracellular calcium-binding proteins calbindin-D28k (CaB) and parvalbumin (PV) may be expressed in these developmental events. Accordingly, the ontological expression of CaB and PV was examined immunocytochemically in the spinal cord and dorsal root ganglia (DRG) of the rat, in order to evaluate the relationship between CaB and PV expression, and other important developmental events. During the ontogenesis of the spinal cord, the CaB-like immunoreactivity was mainly observed in the cell somata. The immunoreactive cells in the ventral horn of the cervical and thoracic, lumbar, and sacral segments first appeared at embryonic day (E)-12, E-13, and E-14, respectively. However, these cells were not detected in the intermediate gray matter of the same segments at E-14, E-15, and E-16, respectively, and in the dorsal horn at E-14-E-15, E-16, and E-17, respectively. The peak of immunoreactive cells, both as to number and intensity, occurred in the perinatal period. However, from postnatal day (P)-14 on, the number and intensity of the positive cells decreased, the adult levels being reached at P-35. The PV-like immunoreactivity was mainly detected in the fibers and punctata during the ontogenesis of the spinal cord. The immunoreactive fibers first appeared on the surface of the dorsal horn in the cervical and thoracic segments at E-14, then entered the dorsal horn at E-15, and reached the intermediate gray matter and ventral horn at E-16. The first appearance of these fibers in the same areas of the lumbar and sacral segments occurred 1 day later than in the cervical and thoracic segments. During the perinatal period, the maximum content of PV-like immunoreactive fibers, together with many punctata, was seen in the gray matter. However, between P-14 and P-17, most of them lost immunoreactivity rapidly, with the exception of the medial region of the intermediate gray matter, where the PV-immunoreactive punctata remained up to the adult stage. In DRG neurons, both CaB and PV was expressed, but in different neurons. Neurons labeled with anti-CaB and anti-PV sera were first detected at E-16 and E-14, respectively. These neurons were large or medium-sized in the prenatal period.(ABSTRACT TRUNCATED AT 400 WORDS)     

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.