The distribution of neurons which express the gene for the growth-associated protein, GAP-43, in the adult carp central nervous system (CNS) was studied by in situ hybridization using newly formed RNA probes for carp GAP-43 mRNA. A great number of neurons heavily labeled by the 35S-labeled antisense probe were found in the telencephalon, diencephalon, mesencephalon, optic tectum, pontine area, medulla oblongata and spinal cord. Motoneurons of the cranial nerves, i.e., the oculomotor, trochlear, trigeminal and spinal motor nerves, also strongly expressed GAP-43 mRNA, in contrast to the low level of GAP-43 signals in the motoneurons in the adult mammalian CNS. These results suggested that synaptogenesis and continuous synaptic reorganization might normally occur in the adult carp nervous system, since GAP-43 protein is generally accepted to be essential for the dynamic growth of axonal processes which leads to synaptogenesis.
In the mature skeletal muscle of the adult carp, a number of small-sized neuromuscular junctions (NMJs), which were visualized with acetylcholinesterase (AchE) histochemistry, were detected on each muscle fiber. This polyinnervation pattern was similar to that of the immature muscle of mammalian embryos. These findings indicate that, unlike mammalian muscles, maturation of carp muscles is not accompanied by the synapse elimination which is thought to be coupled with the down-regulation of motoneuron GAP-43. NMJs of the adult carp muscle are supposed to be continuously reorganized, keeping the motoneurons expressing GAP-43.
The expression of GAP-43 under physiological conditions in the adult carp CNS may facilitate axonal regeneration in various kinds of carp CNS neurons.
The growth-associated protein, GAP-43, is a neuron-specific and membrane-associated phosphoprotein. GAP-43 is highly conserved in its amino acid sequences among vertebrates including mammals (Skene, 1989). This protein is rapidly transported to distal axonal segments and is localized in the growth cone membrane (Benowitz et al., 1989; Skene, 1989). The expression of GAP-43 is known to be closely correlated with axonal growth under developmental or regenerative conditions in mammals, amphibians and fishes (Benowitz et al., 1981, 1983; Skene and Willard, 1981; Jacobson et al., 1986; Basi et al., 1987; Moya et al., 1989; Skene, 1989; Dani et al., 1991; Tetzlaff et al., 1991; Chong et al., 1992; Lindå et al., 1992; Reh et al., 1993; Palacios et al., 1994).
In the central nervous system (CNS) of mammals, the expression of this protein and mRNA declines markedly to unde-tectable levels in most neurons after their synapse connections have been completed at the end stage of the development (Moya et al., 1989; Caroni and Becker, 1992). Exceptionally, in some brain regions of adult mammals such as the pyramidal layer of the hippocampus and monoaminagic neurons, GAP-43 expression continues throughout their lives. This persistence of GAP-43 mRNA synthesis in the adult brain has been thought to play a significant role in physiological synaptic plasticity and remodeling (Meberg and Routtenberg, 1991; Kruger et al., 1992,1993). In the case of axonal injuries, GAP-43 mRNA expression is up-regulated in the damaged neurons of both the peripheral nervous system (PNS) and CNS (Redshaw and Bisby, 1984; Hoffman, 1989; Verge et al.,1990). Certain CNS neurons such as cortical pyramidal neurons do not exhibit up-regulation of GAP-43 following axotomy and axonal regeneration does not occur (Elliott et al.,1997). Thus, the reactive re-expression of GAP-43 may be important for successful axonal regeneration (Tetzlaff et al.,1991; Elliott et al.,1997).
In fish also, the synthesis of GAP-43 has been reported to be induced in the retinal ganglion cells during optic nerve regeneration (Benowitz et al., 1981,1983; Benowitz and Schmidt, 1987; Perry et al.,1987). However, the distribution of the neurons which express GAP-43 in the normal brain of adult fish has not been fully elucidated.
In the present study, we demonstrate by in situ hybridization that many neurons of the brain and spinal cord of the adult carp express GAP-43 mRNA and we discuss the possible association between the expression of GAP-43 in moto-neurons and the construction of neuromuscular junctions (NMJs) in adult carp.
MATERIALS AND METHODS
Adult carp (Cyprirus carpio), 21–23 cm in body length, which were capable of reproduction, were kept at 21–22°C in 70-litter aquarium. Fish were used for in situ hybridization and acetylcholinesterase (AchE) staining. Adult male Sprague-Dawley rats (10 weeks old) were used for AchE staining.
For in situ hybridization, carp were anesthetized with 0.02% tricaine methanesulfonate, and their brains were removed and frozen in OCT compound (Miles Inc.) on the dry ice.
For AchE staining, carp were anesthetized with the same anesthetic as above, and rats were anesthetized with pentobarbital sodium (i.p., 60 mg/kg body weight). They were then fixed by transcardinal perfusion with 2% paraformaldehyde and 2.2% glutaral-dehyde in 0.1 M phosphate buffer (pH 7.4). Skeletal muscles were removed from the dorsal part of carp or from the femoral region of rats and were further immersed in the same fixatives for 4 hr at 4°C.
Synthesis of carp cDNA probes
Total cellular RNA was extracted from the carp whole brain as described elsewhere (Chomczynski and Sacchi, 1987). A cDNA library was constructed using Superscript cDNA synthesis Kit (GIBCO-BRL, Gainthersberg, MD, USA). To isolate carp cDNAs encording GAP-43, PCR was carried out with oligomers F and R (5'-ATGCTGT-GCTGTATCAGGAG-3' and 5'-TTAAACATTCTGGTCTTTGC-3') using part of the cDNA library as a template. The oligomers were designed from the previously reported nucleotide sequence of goldfish GAP-43 (Michael and Skene, 1989). PCR was performed according to the instruction of AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT, USA) using a thermal cycler 480 (Perkin Elmer Cetus) under the following thermal cycling conditions: denaturation, 94°C for 30 sec; 55°C for 30 sec; extension, 72°C for 60 sec; the last extension, 72°C for 5 min; cycle number, 30. PCR products were ligated into the EcoRV site of P-Bluescript KS-vector (Stratagene, La Jolla, CA, USA), where a dideoxythymidine monophosphate had been added. Carp GAP-43 cDNA was thus cloned and sequenced, and it showed 90% homology with goldfish GAP-43 cDNA.
Northern blot analysis
Twenty μg of RNA was separated on a 1 % agarose gel containing 2.2 M formaldehyde. The RNA was transferred to nylon membrane (Pall Biosupport, NY, USA) and was UV cross-linked. The membrane was probed with carp GAP-43 cDNA which was labeled with 32P-dCTP (6000 Ci/mmol) using a labeling kit Prime I Ml (Stratagene). Hybridization was performed in hybridization buffer (0.5 M sodium hydrogenphosphate, 1% bovine serum albmin (BSA), 1 mM sodium dodecyl sulfate (SDS)) containing the labeled probe (1 × 106 cpm/ml) at 65°C overnight. The membrane was washed with 0.1 × SSC, 0.1 % SDS at 65°C for 20 mim and exposed to X-ray film.
In situ hybridization
Antisense RNA probe was synthesized with T3 or T7 RNA polymerase (GIBCO-BRL) in the presence of 35S-UTP (800 Ci/mmol) using selected carp GAP-43 cDNAs as templates. Sense probe was synthesized in a similar manner and used for the control study.
Coronal, frozen sections of fresh carp brains were cut at 6 μm with a cryostat, mounted onto glass-slides coated with Vectabond reagent (Vector Lab., Inc., USA) and stored at –80°C until use. After being dried in air, sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min, and rinsed in 2 × SSC (0.3 M NaCl, 0.03 M Na citrate, pH 7.0). They were placed in acetic anhydride solution (0.25% acetic anhydride, 0.1 M triethanolamine HCl, pH 8.0) for 10 min, and then rinsed in 2 × SSC. Finally, sections were dehydrated through a graded ethanol series and air dried. RNA probes were heated to 60°C in hybridization buffer (50% formamide, 0.5 M NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 × Denhardt's solution, 0.1% SDS, 10% dextran sulfate, 20 mM DTT, 500 μg/ml yeast tRNA, 5 ng/μl UTPαs) just prior to hybridization. The probe concentration was 1 × 106 cpm/ml. Covered with silicone-coated cov-erslips, sections were incubated with RNA probes overnight at 55°C in a humidified chamber. After incubation, coverslips were removed in 4 × SSC containing 10 mM DTT, and sections were washed in the same solution for 15 min at 40°C. They were then incubated at 37°C for 30 min in RNaseA (Sigma, St. Louis, MO, USA) (20 μg/ml) dissolved in RNase buffer (0.5 M NaCl, 10 mM Tris-HCl pH 8.0), and washed in 0.1 × SSC containing 20 mM DTT at 55°C for 30 min. After wash in 0.1 × SSC at room temperature, sections were dehydrated in ethanol and air-dried. They were then dipped in NTB-2 emulsion (Eastman Kodak, USA) and exposed for 3 to 4 days at 4°C. After development with D-19 (Eastman Kodak), sections were counter-stained with hematoxylin and eosin and were observed under a microscope (Zeiss Axiophoto, Karl-Zeiss, Germany).
AchE staining procedure
AchE histochemistry was carried out according to the method of Tago et al. (1986). Twenty to thirty μm-thick sections of sketetal muscles were cut with a cryostat and air-dried. They were washed in 0.1 M maleate buffer (pH 6.0) and were incubated in the reaction medium for AchE containing 1.8 mM acetylthiocholine iodide, 0.5 mM K3Fe(CN)4, 3 mM CuSO4 and 5 mM sodium citrate in 65 mM maleate buffer (pH 6.0) for 1 hr at room temperature. After washing in 50 mM Tris-HCl (pH 7.6) several times, sections were incubated in a solution containing 0.04% 3,3'-diaminobenzidine tetrahydrocholide and 0.3% nickel ammonium sulfate in 50 mM Tris-HCl (pH 7.6) for 5 mim. Subsequently, 0.003% H2O2 was added to the solution and sections were further incubated for 5 min. Sections were observed under a light microscope (Optiphoto, Nikon, Tokyo, Japan).
By in situ hybridization, we could detect the signals for GAP-43 mRNA on the perikaryal cytoplasm of nerve cells in various regions of the carp CNS. Control sections to which the sense probe had been hybridized showed no significant signals. The findings are described in detail below.
Northern blot analysis of the carp brain mRNA
We used the carp GAP-43 cDNA to probe the RNA derived from the adult carp hole brain. The specific single band of carp GAP-43 mRNA was found at approximately 1.35 kb long (Fig. 1).
Telencephalon and preoptic area
In the telencephalon which consists of the area ventralis and the area dorsalis, strong signals for GAP-43 mRNA were found in almost all nerve cells (data not shown).
The preoptic area extends from the caudal end of the telencephalon to the rostral of the nucleus habenularis (NH) in the diencephalon. In the nucleus preopticus periventricularis (NPP), which is located around the third ventricle, many neurons displayed GAP-43 mRNA signals. The nucleus preopticus (NPO) existing around the third ventricle ventral to NPP was also positive for GAP-43 mRNA (data not shown).
The diencephalon is composed of the epithalamus, dorsal thalamus, ventral thalamus and hypothalamus.
The epithalamus consists of only the nucleus habenularis (NH), which showed a very low level of mRNA signals for GAP-43 on small neurons (data not shown). The dorsal thalamus is divided into two thalamic nuclei, i.e., dorsomedial thalamic nucleus (NDM) facing the third ventricle and dorsolateral thalamic nucleus (NDL) located lateral to NDM. These dorsal thalamic nuclei displayed strong mRNA signals for GAP-43 (Fig. 2a). The ventral thalamus is also separated into two thalamic nuclei: the ventromedial thalamic nucleus (NVM) and the ventrolateral thalamic nucleus (NVL). These ventral thalamic nuclei showed mRNA signals at the same levels as NDM and NDL (Fig. 2a). In the hypothalamus, some nuclei are located around the third ventricle and others in the medial-ventral part or the inferior lobe (LI). Strong mRNA signals for GAP-43 were found in almost all hypothalamic nuclei, i.e., nucleus anterior tuberis (NAT) (data not shown), nucleus lateralistuberis (NLT), nucleus recessus lateralis (NRL), nucleus posterior tuberis (NPT) (data not shown) and nucleus centralis (NC) (Fig. 2a). In NAT, NLT, NRL and NPT located around the ventricle, the cells heavily labeled with the GAP-43 mRNA probe were not ependymal cells but nerve cells (Fig. 2c).
The diencephalic nuclei other than those described above also showed mRNA signals for GAP-43. In the medial and lateral part of the hypothalamus, strong signals were found in the mammillary body (MB), tertiary gustatory nucleus (TGN), nucleus preglomerulosus par lateralis (PGL), nucleus cere-bellosus hypothalami (NCH) and posterior thalamic nucleus (NTP) (Fig. 2a,b).
The torus semicircularis (TS) is a pair of longitudinal ridges and forms the wall of the mesencephalic ventricle, and the nucleus isthmi (IS) lies more ventricle to the TS. Both TS and IS were found to show strong signals (Fig. 3a). The nucleus lateralis valvulae (NLV) is located medial to TS and ventral to the valvula cerebelli, and extending widely in a rostrocaudal direction. The central part of NLV showed the mRNA signals for GAP-43 at the same levels as TS and IS (Fig. 3a, b). The superium reticular formation (SRF), located medial to the central NLV, also showed strong signals (Fig. 3a). Torus longitu-dinalis (TL) is another pair of longitudinal ridges and extends from the medial border of the optic tectum into the mesencephalic ventricle. In TL, neurons in the dorsal part are larger in size than in the ventral part. The dorsal neurons had stronger mRNA signals for GAP-43 than the ventral neurons (Fig. 3b). Nuclei of the cranial nerves, i.e., nuclei of the oculomotor nerve (NIII) and trochlear nerve (NIV), are located in the mesencephalon. Although it is difficult to distinguish the NIII nucleus from the NIV nucleus, many neurons of this area were found to have strong signals for GAP-43 (Fig. 3a, c).
The optic tectum is composed of six layers. Among them, the stratum fibrosum et griseum superficiale (SFGC) and stratum griseum centrale (SGC) contained many large-sized neurons which were found to show GAP-43 mRNA signals. In the stratum griseum periventriculare (SPV), strong signals were found in small-sized neurons (Fig. 4).
Granular cells (G) located in the valvula cerebelli and the corpus cerebelli showed weak signals for GAP-43 mRNA. The granular eminence (EG) also displyed weak signals. In the purkinje cell layer (P), purkinje cells and eurodendroid cells, which are difficult to distinguish from each other, were positive for GAP-43 mRNA signals. In the molecular layer (M), many neurons thought to be satellite cells were positive for GAP-43 mRNA (Fig. 5).
In the secondary gustatory nucleus (SGN), the nucleus of the lateral longitudinal fascicle (NFLL) and the reticular formation (RF), many neurons showed mRNA signals for GAP-43. In the pontine area, NLV is situated medial and lateral to the SGN. Almost all the neurons in NLV were labeled clearly by the GAP-43 mRNA probe (Fig. 6a). Both the sensory and motor nuclei of the trigeminal nerve (NMT) and Mauthner cells (data not shown) showed strong signals (Fig. 6b).
In carp, the dorsal medulla is composed of three lobes, i.e., the facial, glossopharyngeal and vagal lobes. These lobes showed weak GAP-43 mRNA signals. In the ventral medulla oblongata, the ventral motoneuron group of the vagus nerve (NXM) showed strong GAP-43 mRNA signals. In the most ventral part of the brain stem, the inferior olivary nucleus (IO) also showed strong signals (Fig. 7).
GAP-43 mRNA signals were found in the gray matter of the spinal cord at the anterior and middle levels of the dorsal fin. In the dorsal horn (DH), most of the neurons appeared to have clear signals. In the ventral horn (VH) on the other hand, strong signals were detected in the motoneurons, and the small neurons which were thought to be interneurons were also labeled by the probe (Fig. 8a, b).
Neuromuscular junctions (NMJs) of the skeletal muscle
This study disclosed that the adult carp motoneurons show strong GAP-43 mRNA signals. On the other hand, it has been reported that, in the cranial and spinal motoneurons of adult rats, expression of GAP-43 mRNA and its protein was very low or undetectable. To explain this species difference, we investigated the morphology of NMJs in the carp and rat skeletal muscles with acetylcholinesterase histochemistry to compare the innervation pattern of skeletal muscles on these animals.
In the white skeletal muscle at the dorsal part of adult carp, which should be innervated by spinal motor neurons, a number of small round or oval-shaped junctional plaques were found over the entire length of the individual muscle fibers. Many long extended endplates were also detected around the muscle fibers (Fig. 9a).
On the other hand, in the skeletal muscle of the femoral region of adult rat, the endplates were oval-shaped and larger in size in comparison to those of the carp. Only a single endplate was found on each muscle fiber (Fig. 9b).
GAP-43 is one of the major components of growth cones (Skene, 1989; Van Hoff et al., 1989; Strittmatter et al., 1990), and the level of its mRNA expression increases during axonal elongation in the developmental stage (Skene and Willard, 1981; Jacobson et al., 1986; Skene and Virag, 1989). After development, in the mammalian CNS, most neurons down-regulate the GAP-43 gene to undetectable levels (Oestreicher and Gispen, 1986; Benowitz et al., 1988, 1989; Mc Guire et al., 1988; Neve et al., 1988; De la Monte et al., 1989; Dani et al., 1991; Meberg and Routtenberg, 1991). However, in some regions of the adult rat brain, such as the pars conpacta of substantia nigra, the locus coeruleus and raphé nuclei, which have long and extensively dispersed axons and many synaptic connections, neurons continue to express GAP-43 mRNA and its protein (Kruger et al.,1993). Those neurons which continue to express GAP-43 mRNA and protein may possess a propensity for continuous axonal growth and remodeling of synaptic connections.
Neurogenesis continues in adulthood in the optic tectum of the adult goldfish (Raymond and Easter, 1983) and in the cerebellum of the adult gymnoiform fish (Zupanc et al.,1996). In the present study using in situ hybridization, we demonstrated that the brain and spinal cord of the adult carp contain many neurons which strongly express GAP-43 mRNA. This result may indicate that neurons of the adult carp CNS have a potential to continue axonal growth and synapse remodeling. On the other hand, granular cells of the cerebellum and granular eminence (EG) and small-sized neurons of the nucleus habenularis (NH) and torus longitudinalis (TL) showed only weak signals. These types of neurons might not perform active synaptogenesis and/or axonal elongation in the adult carp brain.
In the mature rat brain, all cranial motoneurons lack expression of GAP-43 mRNA (Kruger et al., 1993), and its protein is difficult to detect by immunohistochemistry (Hassan et al., 1994; Johnson et al., 1995). On the other hand, our present study revealed that the cranial and spinal motoneurons of adult carp which project to the skeletal muscle are strongly positive for GAP-43 mRNA. This species difference in the motoneuron GAP-43 expression may depend on the development and plasticity of neuromuscular junctions (NMJs) for the following reasons.
In the CNS of mammals including rats, the down-regulation of GAP-43 in the spinal motoneurons are associated with synaptic elimination at the skeletal muscle. In rat embryos, axonal processes of motoneurons reach the developing skeletal muscle in a manner of polyinnervation at E11 and multiple synapses are formed on the muscle fibers from E12. The increase in the number of muscle fibers stops shortly after birth, and hypertrophy is responsible for the postnatal growth of muscle (Goldspink, 1972,1974; Campion, 1984). Synapse elimination of the skeletal muscle begins at P8 and is completed at P14, so that one NMJ can be formed upon each muscle fiber (Brown et al., 1976; Dennis, 1981; Bennet, 1983). Caroni and Becker (1992) reported that the level of GAP-43 mRNA expression declined rapidly in the spinal motoneurons of the postnatal rat at the time corresponding to the onset of synapse elimination. In contrast, in fish, the skeletal muscle remains polyneuronally innervated through adulthood and motor axons form multiple terminals on each muscle fiber (Akster, 1983; Eisen et al.,1986; Westerfield et al.,1986). We also observed by acetylcholinesterase staining that there are many small NMJs on each white muscle fiber of the dorsal part of adult carp. Thus, synapse elimination, which is closely associated with the down-regulating of motoneuron GAP-43, does not significantly occur in fish. In the carp, muscle fibers keep increasing in number throughout adulthood (Koumans et al.,1993). This hyperplasia of the skeletal muscle fibers should be physiologically accompanied by an increase in the number of NMJs so that the neuromuscular system can work well as a functional unit. The strong expression of GAP-43 mRNA in the adult carp spinal motoneurons as revealed in our study is likely to reflect the increase in number of NMJs, i.e., the physiological neoformation of synaptic terminals during adulthood.
Under the pathological conditions caused by axotomy of the peripheral motor nerves of rats, GAP-43 mRNA increases in the damaged motoneurons (Chong et al.,1992; Lindå et al.,1992; Palacios et al.,1994; Kitamuraet al.,1995), and seems to contribute to axonal regeneration. Similarly, following injury of the ratCNS, such as by transection of the rubrospinal tract, up-regulation of GAP-43 mRNA occurs in the damaged rubrospinal neurons (Tetzlaff et al., 1991). In the carp CNS, axonal regeneration of large neurons in the reticular formation takes place after complete spinal cord transection (Yamada et al.,1995). We investigated the expression GAP-43 mRNA in the brainstem of carp 7 days after transection of the spinal cord, but could not detect significant increase for the signals of GAP-43 mRNA on the level of microautoradiography (data not shown). Since a large number of neurons of the adult carp brain including those of the reticular formation normally express GAP-43 mRNA strongly, further up-regulation of GAP-43 may not be needed by the damaged neurons of the carp CNS.
Our in situ hybridization made it clear that many neurons in the normal carp brain and spinal cord strongly express GAP-43 mRNA. This might suggests that synaptogenesis and continuous synaptic reorganization normally occur in the adult fish CNS which is considered to be ever-growing throughout life.
inferior olivary nucleus
nucleus cerebellosus hypothalami
dor-solateral thalamic nucleus
dorsomedial thalamic nucleus
nucleus of the lateral longitudinal fascicle
nucleus lateralis tuberis
nucleus lateralis valvulae
motor nucleus of the trigeminal nerve
nucleus recessus lateralis
posterior thalamic nucleus
ventrolateral thalamic nucleus
ventrome-dial thalamic nucleus
nucleus of the oculomotor nerve
nucleus of the trochlear nerve
motor nucleus of the vagus nerve
nucleus preglomerulosus pars lateralis
stratum album centrale
stratum fibrosum etgriseum superficiale
stratum griseum centrale
secondary gustatory nucleus
superium reticular formation
tertiary gustatory nucleus
lateral lobe of the valvula cerebelli.
We thank Dr. Hisao Hujita for his helpful discussion and Drs. Hironobu Ito and Masami Yoshimoto for their helpful comments on the identification of the nucleus of the carp brain. This work was partly supported by Japan Health Science Foundation.