Co-induction of nitric oxide synthase, bcl-2 and growth-associated protein-43 in spinal motoneurons during axon regeneration in the lizard tail

In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons. We previously ascertained that these events involve nitric oxide synthase induction in the axotomized motoneurons, suggesting a correlation of this enzyme with regeneration-associated gene expression. Here we investigated, in lizards, whether the cell death repressor Bcl-2 protein and growth-associated protein-43 (GAP-43) were also induced in motoneurons that innervate the regenerated tail in the first month post-caudotomy. Single and multiple immunocytochemical techniques, and quantitative image analysis, were performed. Nitric oxide synthase, GAP-43 or Bcl-2 immunoreactivity was very low or absent in spinal motoneurons of control lizards with intact tail. Nitric oxide synthase and GAP-43 were induced during the first month post-caudotomy in more than 75% of motoneurons which innnervate the regenerate. Bcl-2 was induced in approximately 95% of these motoneurons at five and 15days, and in about 35% at one month. The intensity of Bcl-2 and GAP-43 immunostaining peaked at five days, and nitric oxide synthase at 15days; immunoreactivity to these proteins was still significantly high at one month. Immunofluorescence revealed co-localization of nitric oxide synthase, GAP-43 and Bcl-2 in the vast majority of motoneurons at five and 15days post-caudotomy.These findings demonstrate that co-induction of nitric oxide synthase, Bcl-2 and GAP-43 may be part of the molecular repertoire of injured motoneurons committed to survival and axon regeneration, and strongly favor a role of nitric oxide synthase in motoneuron plasticity.     

Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig

Summary This investigation attempted to determine the mode of distribution and synaptic targets of the cartwheel cell axon in the guinea pig dorsal cochlear nucleus (DCoN). Antiserum against PEP-19, a putative calcium-binding neuropeptide, was employed at the light and electron microscopic levels. We show that in the hindbrain of the guinea pig, cerebellar Purkinje cells and DCoN cartwheel cells are the most densely immunoreactive neurons. The PEP-19 immunoreaction product is localized to all neuronal compartments of these cells. Primary targets of cartwheel cell axons are the DCoN pyramidal cells, the large efferent neurons of layer 2. These neurons receive numerous immunoreactive synaptic boutons on their cell bodies and apical and basal dendritic arbors. A PEP-19-immunoreactive axonal plexus, largely formed by cartwheel cell axons, highlights layer 3, co-extensively with the basal arbors of pyramidal cells. This plexus is oriented predominantly in the transstrial plane of the DCoN, in parallel with the sheetlike basal dendritic arbor of pyramidal neurons and with the isofrequency bands of primary cochlear nerve fibers. PEP-19-positive boutons contain pleomorphic synaptic vesicles and form symmetric synaptic junctions, indicative of inhibitory innervation. In addition, immunoreactive boutons, similar to those synapsing on pyramidal neurons, were observed on the cell bodies and main dendritic trunks of cartwheel neurons, indicating a system of recurrent collaterals. Furthermore, a small number of PEP-19-positive axons of unknown origin reach the caudal rim of the posteroventral cochlear nucleus. Within the territory of distribution of the cartwheel cell axon are the dendrites of at least two other types of DCoN neuron, the vertical cells of Lorente de Nó and the giant cells. These neurons may represent additional targets of the cartwheel cell axon, but this remains to be ascertained with specific methods. Our data demonstrate that the cartwheel neurons modulate the activity of pyramidal neurons and, therefore, play a key role in shaping the output of the DCoN superficial layers.     

Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43

The purpose of the present study was to test changes in the expression of growth-associated protein (GAP-43) after chronic treatment with two different psychotomimetic drugs: amphetamine and phencyclidine. Rats were treated chronically for 7 days (twice daily) with 5 mg/kg of amphetamine and phencyclidine and sacrificed after 2, 5 or 7 days of treatment, and following 7, 14 or 21 days of recovery after full treatment (7 days). Separate groups of rats were treated on the same regiment with haloperidol, and control group was treated with vehicle. To determine the effects of different psychotomimetic drugs on the expression of GAP-43 we have used Northern blotting and quantitative in situ hybridization. Treatment with amphetamine induced decrease of GAP-43 mRNA expression, that was detected also during recovery period, up to 14 days after the last day of 7 days treatments. On the contrary, PCP induced increase of GAP-43 mRNA expression, that was detectable from the first days of treatment until 21 days after the last day of treatment. Treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. It can be suggested that GAP-43 upregulation upon phencyclidine treatment occurs as a result of functional activation of pathways able to participate in remodeling, while amphetamine showed neurotoxic effect, decreasing expression of GAP-43 mRNA.     

Immunohistochemical distribution of growth-associated protein 43 (GAP-43) in developing rat nasoincisor papilla

We employed immunohistochemistry of growth-associated protein 43 (GAP-43) to trace the early development of gustatory nerves and alpha-gustducin to demonstrate mature taste buds in the rat nasoincisor papilla (NP). The sequential changes of gustatory structures revealed eight characteristic stages. One, at embryonic day 16 (E16), GAP-43-immunoreactive (IR) nerve fibers were observed in close relation with presumptive taste buds in the lateral apical epithelium on each side of NP; meanwhile, no immunoreactivity could be observed in the papillary epithelium. Two, at E17, fine GAP-43-IR nerve fibers first invaded the apical epithelium of the papilla. Three, at E19, GAP-43-IR nerve fibers were extensive in apical epithelium and colonized in immature taste buds. Four, at E20, GAP-43-IR nerve fibers were first observed in ductal epithelium (lining the medial wall of nasoincisor ducts). Five, at postnatal day 1 (P1), immunoreactive nerve fibers first coincided with immature taste buds in the ductal epithelium. Six, at P3, alpha-gustducin-IR cells identical for mature taste buds were simultaneously demonstrated in both apical and ductal epithelium. Seven, at P14, progressive taste bud proliferation and maturation as well as neural invasion were demonstrated in all regions of the epithelium. Eight, during advanced stage in adult animals, extensive innervation was traced especially in close relation with taste buds. The sequential topographic patterns of NP gustatory structures seem very specific as compared to those in other locations of mammalian gustatory system. The present study reveals that gustatory nerves preceded the development of taste buds. However, further investigations are required to examine such a characteristic model for the neurogenic theory of taste induction.     

Colocalization of neuronal nitric oxide synthase with GABA in rat cuneate nucleus

Summary Pre- and post-embedding immunocytochemistry were employed in this electron microscopic investigation of cuneate neurons that are enriched in GABA and in nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide. GABAergic neurons are local circuit interneurons; 10–20% of them also contain nitric oxide synthase. These are among the smallest GABA-positive perikarya. We describe a network of processes in the rat cuneate nucleus that are immunopositive for nitric oxide synthase. Axon terminals positive for nitric oxide synthase are small and make synapses mainly onto dendrites; they make only occasional axo-axonic contacts. Double-labelling immunocytochemistry verified that the large majority of terminals positive for nitric oxide synthase also contained GABA. However, most GABA-positive profiles were negative for nitric oxide synthase and GABA-positive terminals that are negative for nitric oxide synthase frequently made axo-axonic contacts. These results suggest that nitric oxide synthase is within a specialized subpopulation of interneurons in the cuneate nucleus.     

Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.     

Changes in mRNA of protein inhibitor of neuronal nitric oxide synthase following facial nerve transection

Protein inhibitor of neuronal nitric oxide synthase (PIN) is reported as the protein inhibiting neuronal nitric oxide synthase (nNOS) activity by preventing dimerization of nNOS. It was also reported that PIN inhibits the activity of all nitric oxide synthase (NOS) isozymes. We examined the effects of facial nerve transection on PIN mRNA and NOS expression by in situ hybridization for PIN mRNA and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining. PIN mRNA was initially expressed and transiently increased from 3 to 5 days and returned to the basal level at 7 days after axotomy in the motoneurons of the facial nucleus. NADPH-d-positive motoneurons were found from 7 days post-operation in the facial nucleus. These results suggest that PIN may interact with NOS from 7 days post-operation.     

Effects of contralateral sound stimulation on unit activity of ventral cochlear nucleus neurons

The cochlear nucleus (CN) commissural connection represents the first opportunity for convergence of binaural information in the auditory brainstem. All major neuron types in the ventral CN (VCN) are innervated by a diverse population of cells in the contralateral VCN. This study examined the effect of contralateral sound stimulation on the spontaneous rates (SRs) of neurons in the VCN. Unit activity was recorded with silicon-substrate multichannel probes which allowed recordings from up to 16 sites simultaneously. On average, 30% of units showed short-latency (often only 2 ms greater than the latencies of ipsilateral sound-evoked responses) inhibition of SR by wideband contralateral noise bursts. Fewer units (4.5%) were excited by contralateral noise at sound levels low enough to exclude excitation by acoustic crossover. Both regular and irregular units in the anterior VCN (AVCN) and posterior VCN (PVCN) were inhibited by contralateral sound. Decrements in SR followed a monotonic function with increases in contralateral sound level, except where responses could be attributed to acoustic crossover. Restricting the contralateral noise bandwidth resulted in a frequency-specific inhibition, dominated by frequencies at and below the ipsilateral BF of the unit, consistent with anatomical findings of the tonotopic organization of the CN commissural pathway. The latencies of these effects are compatible with mono, di and tri-synaptic connections reflecting CN commissural pathway effects.     

GABA can improve acoustic contrast in the rat ventral cochlear nucleus

The effect of microiontophoretically applied -aminobutyric acid (GABA) and its agonists and antagonists on the response pattern of single units in the ventral cochlear nucleus (VCN) of the rat was examined in order to study GABA's physiological function in auditory processing. The effects of the drugs were judged by changes of spontaneous and sound-evoked activity in peristimulus-time histograms (PSTHs) of at least 20 consecutive presentations of acoustic stimuli. GABA inhibited the discharge activity of the majority of neurons. All response types found in the VCN except onset-I responders were sensitive to GABA. The GABAergic inhibition is most probably mediated by GABA_A receptors, since the GABA_A-receptor agonist muscimol, but not the GABA_B-receptor agonist baclofen, mimicked the effect of GABA. The GABA_A-receptor antagonists, bicuculline and picrotoxin, had an excitatory effect on the neurons' spontaneous activity, suggesting a tonic endogeneous release of GABA which exerts a permanent inhibition on VCN neurons. Although inhibitory, iontophoresis of GABA emphasized the response to stimulus onset in the PSTHs by means of a stronger inhibition of spontaneous activity. When using iontophoretical currents which did not suppress the neuronal activity completely, a strong inhibition of spontaneous activity was accompanied by only a small inhibition of tone-evoked activity. Under these conditions, the response to tone onset was frequently not inhibited at all. Therefore, GABA's physiological function is possibly to improve the contrast between transient acoustic signals and ongoing background activity. In order to test this hypothesis, the test tone was masked by continuous background noise. Indeed, GABA reduced the noise-evoked discharge more than the toneevoked discharge, leaving the onset peak in the PSTHs almost unchanged. Thus, GABAergic input improves the signal-to-noise ratio for acoustic transients in VCN neurons. Our data suggest that a functional role of GABA in the VCN is to act as a transmitter within a descending inhibitory feedback loop of the auditory brainstem which serves to improve the transmission of relevant acoustic signals in constant background noise.     

Soluble guanylate cyclase and neuronal nitric oxide synthase colocalize in rat nucleus tractus solitarii

Nitric oxide has been implicated in transmission of cardiovascular signals in the nucleus tractus solitarii (NTS). Pharmacological studies suggest that activation of neurons by nitric oxide in the NTS may involve soluble guanylate cyclase (sGC). However, anatomical data supporting this suggestion have not been available. In this study, we tested the hypothesis that neurons and fibers containing neuronal nitric oxide synthase (nNOS) lie in close proximity to those containing sGC and the two enzymes colocalize in some neurons and fibers in the NTS. We perfused six rats and obtained brain stem sections for double immunofluorescent staining utilizing antibodies selective for sGC and for nNOS combined with confocal microscopy. The distribution and staining intensity of nNOS-immunoreactivity (IR) was similar to our earlier reports. IR of sGC was present in cell bodies, proximal dendrites and fibers of many brain stem regions. Strong sGC-IR was noted in the hypoglossal, dorsal motor nucleus of vagus and gracilis nuclei. The NTS exhibited moderate sGC-IR. Superimposed images showed that many NTS neurons contained both nNOS-IR and sGC-IR. The percentage of sGC-IR positive cells that were also nNOS-IR positive differed among NTS subnuclei. Similarly, the percentage of nNOS-IR positive cells that were also sGC positive differed among NTS subnuclei. Fibers stained for both nNOS-IR and sGC-IR were also present in NTS subnuclei. In addition, we identified fibers that were stained for nNOS-IR or sGC-IR alone and often found such singly labeled fibers apposed to each other. These data support our hypothesis and provide anatomical support for the suggestion that nitroxidergic activation of the NTS involves sGC.     

Localization of vesicular glutamate transporters and neuronal nitric oxide synthase in rat nucleus tractus solitarii

Previously we reported that glutamate and neuronal nitric oxide synthase (nNOS) colocalize in neurons of the nucleus tractus solitarii (NTS). That finding provided anatomical support for the suggestion that nitric oxide and glutamate interact in cardiovascular regulation by the NTS. Here we test the hypothesis that nNOS colocalizes with vesicular glutamate transporters (VGluT1 and VGluT2) in the NTS. Immunoreactivity (IR) for VGluT better identifies glutamatergic terminals than does glutamate-IR, which may label metabolic as well as transmitter stores of the amino acid. We used fluorescent immunohistochemistry combined with confocal laser scanning microscopy to study IR for VGluT1, VGluT2 and nNOS in rat NTS. A high density of VGluT1-IR positive fibers was present in the gracilis and cuneatus nuclei while in the NTS we found a moderate density in the lateral and interstitial subnuclei and a low density in the dorsolateral, ventral and intermediate subnuclei. The medial, central, commissural and gelatinosus subnuclei contained few VGluT1-IR containing fibers. Thus, VGluT1 containing fibers are not prominent in portions of the NTS where cardiovascular afferent fibers terminate. In contrast, we found a high density of VGluT2-IR containing fibers in the gelatinosus subnucleus and subpostremal area and a moderate density in cardiovascular regions such as the dorsolateral and medial subnuclei as well as in the central and lateral subnuclei. We found a low density in the ventral, intermediate, interstitial and commissural subnuclei. VGluT1-IR and VGluT2-IR rarely colocalized in fibers within the NTS. VGluT1-IR did not colocalize with nNOS, but VGluT2-IR and nNOS-IR colocalized in fibers in all NTS subnuclei. When compared with the other NTS subnuclei, the dorsolateral, gelatinosus and subpostremal subnuclei had higher frequencies of colocalization of VGluT2-IR and nNOS-IR. VGluT2-IR positive fibers were also apposed to nNOS-IR positive fibers throughout the NTS. These data support our hypothesis and confirm that glutamatergic fibers in the NTS contain nNOS.     

Distribution of growth-associated protein, B-50 (GAP-43) in the mammalian enteric nervous system

The presence of the growth-associated protein, B-50 (also known as GAP-43) was investigated in the adult mammalian enteric nervous system. The small intestine of rat, ferret and human was examined by immunohistochemistry. Dense B-50-like immunoreactivity was localized in nerves throughout the wall of the rat, ferret and human small intestine, notably in the myenteric and submucous plexuses, where in the ferret ileum it co-localized with vasoactive intestinal polypeptide-immunoreactive fibre groups. Material with the biochemical and immunological characteristics of rat B-50 was extracted from the rat ileum. In-situ hybridization demonstrated that enteric neurons express B-50. These findings are consistent with a role for B-50 in the documented plasticity of the adult enteric nervous system.     

Colocalization of GluR1 and neuronal nitric oxide synthase in rat nucleus tractus solitarii neurons.

Previously we demonstrated that glutamate and neuronal nitric oxide synthase (nNOS) containing neuronal elements are frequently apposed in subnuclei of the rat nucleus tractus solitarii. It is known that glutamate receptors (GluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype participate in cardiovascular regulation by the nucleus tractus solitarii and that responses to AMPA receptor activation may be linked to NO. Therefore, in the present study, we further tested the hypothesis that the calcium-permeable subunit GluR1 of AMPA type GluRs and nNOS are colocalized in neurons of the nucleus tractus solitarii. Distribution of GluR1 and nNOS in rat nucleus tractus solitarii was investigated by double fluorescent immunohistochemistry combined with confocal laser scanning microscopy.Numerous GluR1 immunoreactive cells and fibers were present in subnuclei of the nucleus tractus solitarii. The staining intensity of GluR1 immunoreactive cells varied among subnuclei. Cells in the interstitial subnucleus contained the highest GluR1 staining intensity. A moderate intensity of staining was present in the intermediate, dorsolateral, ventral, and commissural subnuclei. A slightly lower level of GluR1 immunoreactivity was present in cells of the medial subnucleus. Cells in the central subnucleus contained a low level of GluR1 immunoreactivity. The staining intensity of GluR1 immunoreactive fibers also varied among subnuclei. Distribution of nNOS immunoreactivity in the nucleus tractus solitarii and other brain stem areas was the same as in our earlier reports. Superimposition of confocal images of nNOS immunoreactivity and GluR1 immunoreactivity allowed us to identify double-labeled structures. Nearly all neurons that were immunoreactive for nNOS contained GluR1 immunoreactivity, but only a proportion of GluR1 immunoreactive cells contained nNOS immunoreactivity. Double-labeled neurons were present in all subnuclei of the nucleus tractus solitarii. The percentages of GluR1 immunoreactive cells that also contained nNOS immunoreactivity differed among subnuclei of the nucleus tractus solitarii. Fibers that labeled for nNOS alone, GluR1 alone or both were present among labeled cells in these subnuclei.These data support the hypothesis that GluR1 and nNOS are colocalized in neurons of nucleus tractus solitarii. The demonstration of this anatomical relationship provides further anatomical support for the hypothesis that activation of AMPA receptors on neurons that synthesize NO in the nucleus tractus solitarii contributes to autonomic regulation.     

糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元的变化

目的：观察糖尿病大鼠下丘脑视上核神经元型一氧化氮合酶(nNOS)免疫阳性神经元数量的变化。方法：用链脲佐菌素诱导建立糖尿病大鼠模型;免疫细胞化学染色显示nNOS免疫阳性神经元,并进行定量分析。结果：糖尿病视上核nNOS免疫阳性神经元着色深浅不一,着色较深的阳性神经元散在分布,神经元的形态多样,突起较少。对照组大鼠视上核nNOS免疫阳性神经元较稀疏,各时期无明显改变。糖尿病2w,nNOS免疫阳性神经元数量与对照组无显著差异;7w,nNOS阳性神经元较密集,明显多于对照组;12w,nNOS免疫阳性神经元数量略低于7w,但仍多于对照组。结论：糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元数量明显增多。     

Hypobaric hypoxia induces fos and neuronal nitric oxide synthase expression in the paraventricular and supraoptic nucleus in rats

This study examined the effects of high altitude exposure on neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus in adult and neonatal rats. In adult control rats, occasional Fos-like immunoreactive neurons were localized in both the hypothalamic nuclei. A marked increase in Fos positive cells was induced at 1-4 h following altitude exposure but it was reduced to levels comparable to the controls at 24 h. The expression of neuronal nitric oxide synthase (nNOS) immunoreactivity in the PVN and SON followed a similar temporal pattern. The nNOS immunoreactivity, which was constitutively expressed in the hypothalamic neurons in the control rats, was noticeably augmented at 1-4 h, but it was comparable to the controls at 24 h following altitude exposure. In postnatal rats, Fos expression was not detected in the hypothalamic neurons of the controls. Induction of Fos expression was observed in some neurons at 1-4 h following altitude exposure but it was diminished at 24 h. There was no noticeable change in nNOS expression in both the control and altitude exposed postnatal rats; in both instances, it was barely detectable. It is concluded that both the PVN and SON of the adult rats are activated at high altitude exposure and that they may be involved in the regulation of neuroendocrine, cardiovascular and respiratory functions in hypobaric hypoxia. This study has also shown the differential response of the hypothalamus neurons between the two age groups to the hypoxic insult. Our results suggest that the adult neurons are probably more sensitive to the reduced oxygen levels in hypobaric hypoxia, as reflected by the upregulated NOS expression in this age group but not in the postnatal rats.     

Organization of the disynaptic pathway from the anteroventral cochlear nucleus to the lateral superior olivary nucleus in the ferret

 The medial nucleus of the trapezoid body (MNTB) is one of three major nuclei of the superior olivary complex and provides an important inhibitory input from the contralateral ear to the lateral superior olivary nucleus (LSO) in the initial binaural pathway for coding interaural intensity differences. The major input to the MNTB from the contralateral anteroventral cochlear nucleus (AVCN) involves giant, calyx-like endings that have a one-to-one relationship with cells in the MNTB as confirmed in the ferret in this study. The main objective of the present study was to define the subsequent organization of projections from cells receiving these calyx-like endings. Several anatomical tracers (Phaseolus vulgaris leucoagglutinin, dextran-biotin, and biocytin) were used that are transported both anterogradely and retrogradely within neuronal projections in order to define the organization of MNTB connections with the LSO in the adult ferret. Analysis focused on determining the topography in both the transverse and longitudinal planes of the projections. Focal tracer injections in the LSO resulted in retrograde labeling of a long, narrow column of cells in the MNTB. The orientation and location of labeled cells was dependent on the medial-lateral position of the injection site. In the rostral-caudal dimension of MNTB, there was no such topographic relation between the injection site and the position of labeled cells. Labeled cells in the MNTB were distributed more or less evenly in a longitudinal column regardless of whether the injection site was restricted to the rostral, middle or caudal part of the LSO. In keeping with this pattern, tracer injections in the MNTB resulted in bands of labeled axons that distributed endings throughout the rostral-caudal axis of the LSO. These bands or sheets varied in medial-lateral position relative to the location of the injection site, but lacked any such rostral-caudal gradient. Thus, overall the MNTB-LSO projections have a convergent-divergent pattern of organization. While MNTB cells receive singular calyx-like endings from the AVCN, LSO cells receive projections from a long column of cells in the MNTB. Implications for processing interaural intensity differences are discussed. Accepted: 23 July 1998     

The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury

When adult dorsal root ganglion cells are dissociated and maintained in vitro, both the small dark and the large light neurons show increases in the growth-associated protein GAP-43, a membrane phosphoprotein associated with neuronal development and plasticity. Immunoreactivity for GAP-43 appears in the cytoplasm of the cell bodies as early as 3.5 h post axotomy and is present in neurites and growth cones as soon as they develop. At early stages of culture (4 h to eight days) satellite/Schwann cells are also immunoreactive for GAP-43. Neurons in isolated whole dorsal root ganglion maintained in vitro become GAP-43-immunoreactive between 2 and 3 h after axotomy. It takes three days however, after cutting or crushing the sciatic nerve in adult rats in vivo, for GAP-43 immunoreactivity to appear in the axotomized dorsal root ganglion cells. GAP-43 immunoreactivity can be detected in the central terminals of primary afferent neurons in the superficial laminae of the dorsal horn of the lumbar enlargement four days after sciatic cut or crush. The intensity of the GAP-43 staining reaches a peak at 21 days and becomes undetectable nine weeks following crush injury and 36 weeks following sciatic nerve cut. The pattern of GAP-43 staining is identical to the distribution of sciatic small-calibre afferent terminals. Little or no staining is present in the deep dorsal horn, but GAP-43 does appear in the ipsilateral gracile nucleus 22 days after sciatic injury. In investigating the mechanism of GAP-43 regulation, blockade of axon transport in the sciatic nerve with vinblastine (10(-5) M-10(-4) M) or capsaicin (1.5%) was found to produce a pattern of GAP-43 immunoreactivity in the dorsal horn identical to that found with crush, while electrical stimulation of the sciatic nerve had no effect. Axotomy of primary sensory neurons or the interruption of axon transport in the periphery therefore acts to trigger GAP-43 production in the cell body. The GAP-43 is transported to both the peripheral and the central terminals of the afferents. In the CNS the elevated GAP-43 levels may contribute to an inappropriate synaptic reorganization of afferent terminals that could play a role in the sensory disorders that follow nerve injury.     

Development of sensory innervation in rat tibia: co-localization of CGRP and substance P with growth-associated protein 43 (GAP-43)

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.     

An atlas of glycine- and GABA-like immunoreactivity and colocalization in the cochlear nuclear complex of the guinea pig

Summary The distribution and colocalization of -aminobutyric acid (GABA)- and glycine-like immunoreactivity in the cochlear nuclear complex of the guinea pig have been studied to produce a light microscopic atlas. The method used was based on post-embedding immunocytochemistry in pairs of 0.5-m-thick plastic sections treated with polyclonal antibodies against conjugated GABA and glycine respectively. Immunoreactive cells, presumably short axon neurones, predominated in the dorsal cochlear nucleus, with mostly single-GABA-labelled cells in the superficial layer, double-labelled in the middle, and single-glycine-labelled in the deep layers. A few large single-glycine-labelled cells, interpreted as commissural neurons, occurred in the ventral nucleus. Scattered double-labelled cells, probably Golgi cells, were seen in the granule cell domain. Immunolabelled puncta of all three staining categories occurred in large numbers throughout the complex, apposed to somata and in the neuropil, showing a differential distribution onto different types of neuron. Three immunolabelled tracts were noted: the tuberculoventral tract, the commissural acoustic stria, and the trapezoidal descending fibres. Most of the fibres in these tracts were single-labelled for glycine, although in the last mentioned tract single-GABA- and double-labelled fibres were also found. Some of the immunolabelled cell types described here are proposed as the origins of the similarly labelled puncta and fibres on the basis of known intrinsic connections.Abbreviations 1-4 DCN layers 1 to 4 - as acoustic stria - AVCN anteroventral cochlear nucleus - C caudal - cap cap area - cas commissural acoustic stria - cnr cochlear nerve root - co commissural cell - CRVCN central region of the VCN - cw cartwheel cell - CZ confluence zone - d dendrite - D dorsal - das dorsal acoustic stria - DCN dorsal cochlear nucleus - df descending fibres - ep ependyma - flocc flocculus - g glial cell - GABA -aminobutyric acid - GLY glycine - gi giant cell - gl/gla globular cell/area - Go Golgi cell - gr granule cell - ias intermediate acoustic stria - icp inferior cerebellar peduncle - lam granule cell lamina - mp/mpa multipolar cell/area - oc/oca octopus cell/area - PVCN posteroventral cochlear nucleus - py pyramidal cell - R rostral - sgl superficial granule cell layer - spcg subpeduncular corner of granule cells - sph/spha spherical cell/area - st stellate cell - tb trapezoid body - tv tuberculoventral cell - TVT tuberculoventral tract - V ventral - VCN ventral cochlear nucleus - vn vestibular nerve