Adult Opossums Research Articles

Ontogeny of the immune system of the brushtail possum, Trichosurus vulpecula.

The numbers and distribution of T and B cells in the thoracic thymus, spleen and intestinal tissue and the proliferation of T lymphocytes were examined during pouch life and in the adult to determine when the developing brushtail possum reaches immunological maturity. CD3-positive cells were observed in the thoracic thymus at day 2 post-partum indicating that the thymus produces T lymphocytes at or soon after birth. By day 25 the thymus was fully populated with CD3-positive T lymphocytes and they were observed in distinct regions of the cortex and medulla. By day 48 post-partum, B and T lymphocytes were identified in the follicles and parafollicular areas of the spleen. Although the numbers of T and B cells in the spleen increased significantly from day 25 to day 100 post-partum (P < 0.005), fewer cells were present at day 150 post-partum than in the adult (P < 0.05). Peyer's patches were not observed in the intestines up to day 73 post-partum. However, both T and B cells were observed in the intestinal lymph nodes. Although the T lymphocytes at weaning showed a proliferative response, the response was not as great as that observed in the adult possum. Thus, the immune system of the possum is not fully developed at weaning but continues its development after pouch life.

The pathogenesis of experimental endo-bronchial Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula)

Aim. To study the nature and development of experimentally induced respiratory tuberculosis in possums and compare the lesions observed with those seen in the natural disease. Methods. Thirty-three adult possums were inoculated via the endo-bronchial route with 20–100 colony forming units of Mycobacterium bovis. The possums were killed at 1, 2, 3 and 4 weeks after inoculation and the nature and distribution of the lesions studied in detail histopatbologically. Alveolar macrophages recovered from the infected possums were also studied ultrastructurally. Results. Macroscopic lesions were largely confined to the respiratory tract, increasing in size and number with time. Histology greatly increased the detection of the total number of lesions. The most common sites affected outside the respiratory tract were the liver and hepatic lymph nodes, but lesions were less common in peripheral lymph nodes than is observed in the natural disease. Intra-pulmonary lesions were centred on blood vessels and their associated lymphatics. Peripheral blood lymphocyte blastogenic responses to M. bovis antigens were first detected at 3 weeks after inoculation, which was 1 week after lymphocyte infiltrations were detected in the lungs, but 1 week before the majority of infections became generalised. Conclusions. Differences in the nature of pulmonary lesions and the distribution of lesions were observed between experimentally induced and the natural disease. Rapid haematogenous and lymphatic spread occurs early in the experimentally induced disease.

Injection of a boar sperm factor causes calcium oscillations in oocytes of the marsupial opossum, Monodelphis domestica

At fertilisation, the sperm triggers an abrupt and transient increase in intracellular calcium ([Ca2+]i) in the oocyte cytoplasm. In eutherian mammals, oocytes exhibit multiple [Ca2+]i transients which are necessary for egg activation. We investigated [Ca2+]i in the marsupial opossum, Monodelphis domestica. Embryo development in this therian mammal is quite distinct from that in most Eutheria. Oestrus was induced in an adult female opossum by introduction of a male into her cage. Injection of a boar sperm extract induced repetitive increases in [Ca2+]i. Each oscillation travelled across and around the periphery of the egg in a wave-like manner. A control injection of KCl elicited no change in [Ca2+]i. This is the first observation of [Ca2+]i oscillations in the oocyte of a marsupial. The repetitive nature of the [Ca2+]i changes were more similar to those in oocytes of Eutheria than those in oocytes of non-mammalian vertebrates.

Physiological changes in the brushtail possum (Trichosurus vulpecula) following relocation from Armidale to Brisbane, Australia.

To determine the effect of relocation on the health of possums the body weights and hormone and immune responses of six male and nine female brushtail possums were monitored for 20 weeks following transfer from the environs of Armidale into enclosures in Brisbane. Over the first 6 weeks of captivity, male possums lost 11.0% of their original body weight and females lost 16.8%. The mean concentrations of plasma cortisol in the male and female possums were 14.5 and 29.4 ng/mL, respectively, and did not change over the 20-week period. Male and female possums displayed a similar pattern of thyroxine secretion over the 20 weeks, with low concentrations up to week seven (2.1 and 2.7 ng/mL, respectively) increasing to 6.9 and 5.8 ng/mL in weeks 7-12 (P < 0.005). This increase in the concentration of thyroxine corresponded with the increase in body weight. The number of white blood cells (WBCs) and the percentage of neutrophils increased from the capture to week 6-10. However, during the last 10 weeks of captivity the number of WBCs and the percentage of neutrophils decreased, indicating recovery of the immune system. This was in accord with the proliferative response of lymphocytes to the T cell mitogen PHA that increased from weeks 11-15 to weeks 16-20 in both male and female possums. The results above suggest that the Armidale possums, like the Brisbane possums, were stressed following their relocation; however, their immune systems were able to gradually recover as they adjusted to their new environment in Brisbane. The death rate of pouch young and of adult female possums after relocation was considerably higher in the Armidale possums compared to Brisbane possums. The mortality rate of Brisbane possums over the first 20 weeks of captivity was 8.3% and 19.6% for male and female possums, respectively, and for Armidale possums 16.6% and 47.1%, respectively. The possums transferred from the environs of Armidale into captivity in Brisbane were under greater stress than possums captured in Brisbane and placed in captivity in Brisbane.

Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum).

We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N-linked glycosylation sites, a potential N-terminal signal peptide and a potential C-terminal trans-membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N-terminal signal peptide sequences and C-terminal trans-membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae.

Marsupial Light Chains: Complexity and Conservation of λ in the OpossumMonodelphis domestica

AbstractThe Igλ chains in the South American opossum, Monodelphis domestica, were analyzed at the expressed cDNA and genomic organization level, the first described for a nonplacental mammal. The Vλ segment repertoire in the opossum was found to be comprised of at least three diverse Vλ families. Each of these families appears to be related to distinct Vλ families present in placental mammals, suggesting the divergence of these genes before the separation of metatherians and eutherians more than 100 million years ago. Based on framework and constant region sequences from full-length cDNAs and intron sequences from genomic clones, it appears that there are multiple functional Jλ-Cλ pairs in the opossum locus. The opossum Jλ-Cλ sequences are phylogenetically clustered, suggesting that these gene duplications are more recent and species specific. Sequence analysis of a large set of functional, expressed Vλ-Jλ recombinations is consistent with an unbiased, highly diverse λ light chain repertoire in the adult opossum. Overall, the complexity of the Igλ locus appears to be greater than that found in the Ig heavy chain locus in the opossum, and light chains are therefore likely to contribute significantly to Ig diversity in this species.

Neurogenesis and migration of receptor neurons in the vomeronasal sensory epithelium in the opossum, Monodelphis domestica.

The sensory epithelium of the vomeronasal organ (VNO) contains primary chemosensory receptor neurons that project to the accessory olfactory bulb (AOB). In the present study, neurogenesis and cell migration in the sensory epithelium of the VNO were analyzed in opossums (Monodelphis domestica) by using bromodeoxyuridine (BrdU) labeling. 1) In the VNO of normal adult opossums, BrdU labeled a small number of cells localized in the basal region of the sensory epithelium. After 1 or 2 weeks of survival, the labeled cells appeared in the receptor cell layers and became receptor neurons, as indicated by coexpression of the G proteins G(i alpha2) or G(o alpha). 2) In the VNO in which the receptor neurons had been destroyed by removing the AOB, the number of BrdU-labeled cells in the reconstituting sensory epithelium was greatly increased compared with that in the intact VNO. The labeled cells were also located in the basal region of the sensory epithelium. 3) In the developing VNO (at postnatal day 10), more cells in the basal region of the sensory epithelium were labeled than in the adult VNO, indicating rapid cell proliferation; and there appeared to be more labeled cells in the basal region near the margins of the sensory epithelium where it meets the nonsensory epithelium. These observations demonstrate that, in the opossum VNO, there is a population of proliferating cells in the basal region close to the basal lamina in the sensory epithelium. The newly generated neurons in the basal region migrate vertically into the receptor cell layer.

CDNA sequence analysis, gene expression and protein localisation of the inhibin alpha-subunit of Australian brushtail possum (Trichosurus vulpecula).

An inhibin alpha-subunit cDNA sequence from the Australian brushtail possum (Trichosurus vulpecula) has been identified and analysed. The cDNA includes an open reading frame encoding a predicted precursor protein of 361 amino acids. The predicted protein sequence includes four possible proteolytic cleavage sites, 12 evolutionarily conserved cysteine residues and three potential N-linked glycosylation sites. The mature alpha-subunit is the carboxyl terminal fragment (alphaC) consisting of 131 amino acids. The full-length precursor protein shows a mean identity with eutherian homologues of 69.8%. The homology is not evenly distributed, with the putative alphaC fragment showing the highest level (79.7%). Using Northern hybridisation, an alpha-subunit transcript of approximately 1.6 kb was detected in adult possum ovary. Using in situ hybridisation and immunocytochemistry, inhibin alpha-subunit was localised exclusively to the granulosa cell layers of follicles. Hybridisation and immunostaining for the inhibin alpha-subunit were first observed in granulosa cells of primary follicles and the expression continued throughout all stages of follicular growth. Inhibin alpha-subunit mRNA and protein were also detected in cells of the corpus luteum. In summary, results indicate considerable conservation of the structure and possible function of the inhibin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages. The expression data suggest that, in the adult possum, inhibin may have a role in ovarian follicular growth from the primary stage of development.

Ultrastructural pattern of glucagon producing-cells in the gastric mucosa of the developing opossum Didelphis albiventris (Marsupialia)

Gastric glucagon-producing cells of the pouch young, weaned and adult opossum Didelphis albiventris were investigated by immunocytochemical and ultrastructural methods. In sections of the oxyntic mucosa stained by immunoperoxidase it was observed that glucagon-immunoreactive cells increased in number during pouch development (from 7.9 +/- 4.7 to 35.4 +/- 5.9 cells/mm2), decreasing progressively to weaned (14.2 +/- 4.8 cells/mm2) and adult animals (10.4 +/- 3.0 cells/mm2). The glucagon-immunoreactive cells presented numerous electrondense, round cytoplasmic granules surrounded by a membrane. Frequently, the dense core of the granules was separated from the enveloping membrane by a delicate electronlucent halo or by a less electrondense peripheral halo. This pattern of the granular component was similar to the ultrastructural morphology observed in pancreatic islet glucagon-producing cells and A-like cells of the gastric mucosa of those eutherian mammals studied so far.

Adult Opossums (Didelphis virginiana) Demonstrate Near Normal Locomotion after Spinal Cord Transection as Neonates

When the thoracic spinal cord of the North American opossum (Didelphis virginiana) is transected on postnatal day (PD) 5, the site of injury becomes bridged by histologically recognizable spinal cord and axons which form major long tracts grow through the lesion. In the present study we asked whether opossums lesioned on PD5 have normal use of the hindlimbs as adults and, if so, whether that use is dependent upon axons which grow through the lesion site. The thoracic spinal cord was transected on PD5 and 6 months later, hindlimb function was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. All animals supported their weight with the hindlimbs and used their hindlimbs normally during overground locomotion. In some cases, the spinal cord was retransected at the original lesion site or just caudal to it 6 months after the original transection and paralysis of the hindlimbs ensued. Surprisingly, however, these animals gradually recovered some ability to support their weight and to step with the hindlimbs. Similar recovery was not seen in animals transected only as adults. In order to verify that descending axons which grew through the lesion during development were still present in the adult animal, opossums subjected to transection of the thoracic cord on PD5 were reoperated and Fast blue was injected several segments caudal to the lesion. In all cases, neurons were labeled rostral to the lesion in each of the spinal and supraspinal nuclei labeled by comparable injections in unlesioned, age-matched controls. The results of orthograde tracing studies indicated that axons which grew through the lesion innervated areas that were appropriate for them.

Development of Walking, Swimming and Neuronal Connections after Complete Spinal Cord Transection in the Neonatal Opossum,Monodelphis domestica

Development of coordinated movements was quantitatively assessed in adult opossums (Monodelphis domestica) with thoracic spinal cords transected by (1) crushing 7-8 d after birth [postnatal days 7-8 (P7-P8)]; at 2-3 years of age, systematic behavioral tests (e.g., climbing, footprint analysis, and swimming) showed only minor differences between control (n = 5) and operated (n = 10) animals; and (2) cutting on P4-P6; at 1 month these opossums exhibited coordinated walking movements but were unable to right themselves from a supine position, unlike controls (n = 6). When tested at 2 or 6 months, they could right themselves and showed remarkable coordination, albeit with more differences from controls than after a crush. No animals with spinal cords that were crushed at P14-18 survived because of cannibalism by the mother. Morphological studies (n = 10) 3 months-3 years after crush at 1 week showed restoration of structural continuity and normal appearance at the lesion site. Animals with cut rather than crushed cords showed continuity but greater morphological deficits. That lesions were complete was demonstrated by examining morphology and nerve impulse conduction immediately after crushing or cutting the spinal cord in controls. After lumbar spinal cord injection of 10 kDa dextran amine, retrogradely labeled cells were found rostral to the lesion in hindbrain and midbrain nuclei. Conduction was restored across the site of the lesion. Thus complete spinal cord transection in neonatal Monodelphis was followed by development of coordinated movements and repair of the spinal cord, a process that included development of functional connections by axons that crossed the lesion.

Possums (Trichosurus vulpecula) on Hawke's Bay farmland: Spatial distribution and population structure before and after a control operation

Over 6.5 years, 723 possums were live‐trapped and marked in swamp, willow, and farmland habitats on approx. 350 ha of two adjoining sheep farms in Hawke's Bay, New Zealand. About equal numbers of males and females were trapped in each habitat, but males were recaptured more often than females. Eighty‐five percent of the possums were trapped along the margins of swamp and willow habitats that comprised only 6% of the study area. The swamp habitat contained at least 8.8 possums ha‐1, the willow habitat 16.7 possums ha‐1, and the farmland habitat 0.13 possums ha‐1. Possums generally moved less than 200 m between successive recaptures, and there were few trap‐revealed movements between habitats. Possums on part of one farm comprising 23 ha of swamp and willows were then poisoned with cyanide in bait stations. Two hundred and fifty‐eight possums—including 142 previously marked animals—were found dead, an estimated 90% of the population. Fifty‐nine percent of the marked possums were poisoned within 100 m of their most recent trapping point, and 85% within 200 m. Proportionately more adult possums were killed on the first night's poisoning and more juveniles on subsequent nights. After the poisoning, the surviving possums showed very varied responses to the reduced population density, ranging from no change in location or range of movements to changes in location, range of movements, or both.

Trypanosoma cruziin the OpossumDidelphis marsupialis:A Study of the Correlations and Kinetics of the Systemic and Scent Gland Infections in Naturally and Experimentally Infected Animals

The genusDidelphis(Marsupialia, Didelphidae) has the unique capacity of supporting both multiplication cycles ofTrypanosoma cruzisimultaneously; besides the intracellular forms, the epimastigotes can be found multiplying and differentiating abundantly in the lumen of the scent glands. The biological significance of the life cycle ofT. cruziwithin the scent glands ofDidelphis marsupialis,as well as its contribution to the epidemiology of the disease, is presently unclear. In order to clarify the mechanisms involved in the colonization of this singular habitat byT. cruzi,as well as to understand its biological role, we have carried out a serological and parasitological follow-up of both natural and experimental infections of young and adult opossums. Although all natural infections were stable and long lasting, no infected scent glands were found, indicating that the stability of the systemic infections does not depend on the presence of flagellates in the scent gland. In 84% of the experimentally infected animals the colonization of the scent glands was preceded by a period of patent parasitemia. Parasitism of the scent glands was essentially permanent and bilateral, and its maintenance was independent of circulating parasites. Moreover, the course of the infection differed depending on the source (scent glands versus axenic culture-derived) of the metacyclic forms. Our results suggest that parasitism of the SG ofD. marsupialisis most likely a secondary acquisition, a step toward independence from the insect vector, similarly to what is accepted forTrypanosoma equiperdum.

Ontogeny of Androgen Receptor-Like Immunoreactivity in the Reproductive Tract of Male Monodelphis domestica 1

The distribution of nuclear androgen receptor-like immunoreactivity was studied in the reproductive tract of the developing and adult gray short-tailed Brazilian opossum (Monodelphis domestica), using the well-characterized rabbit polyclonal androgen receptor antibody, PG21. Androgen receptor-like immunoreactivity was first detected on the fifth day of postnatal age, in the mesenchymal tissues of the ductus deferens, gubernaculum testis, inguinal, and scrotal areas; the urogenital sinus; and the genital tubercle. Androgen receptor-like immunoreactivity was first seen in the interstitial cells of the epididymis at 45 days of age; the testes developed androgen receptor-like immunoreactivity at 60 days of age. The epithelium of prostatic glands contained androgen receptor-like immunoreactivity only in the adult. The presence of androgen receptor-like immunoreactivity during development correlated well with the known androgen dependence of the differentiation of most reproductive organs.

Transthyretin distribution in the developing choroid plexus of the South American opossum (Monodelphis domestica).

The distributions of transthyretin and albumin in the choroid plexus during brain development have been compared. The South American opossum was chosen because the young are born around the time of choroid plexus formation. Previous work showed that in the adult opossum, transthyretin is expressed in the choroid plexus cells. However, systematic studies of transthyretin in the choroid plexus during development have not been carried out before. Transthyretin was present in 90-95% of the choroidal cells form birth to adulthood. In most cells, transthyretin immunoreactivity was concentrated in the apical region of the cytoplasm. Double labelling of choroid plexus sections with antibodies to albumin and transthyretin showed that 1-2% of cells were positive for both proteins. These findings suggest that from the very earliest stage of choroid plexus formation most epithelial cells both synthesize and contain transthyretin, and a few of these transthyretin-synthesizing cells also contain albumin that is probably being transferred form blood to the cerebrospinal fluid.

The follicle-stimulating hormone beta-subunit gene of the common brushtail possum (Trichosurus vulpecula): analysis of cDNA sequence and expression.

Reverse transcription-PCR has been used to obtain a cDNA sequence from the follicle-stimulating hormone (FSH) beta-subunit gene of the Australian brushtail possum (Trichosurus vulpecula). Comparisons of the possum FSHbeta-mRNA coding region nucleotide sequence with that of six eutherian mammal homologues reveals a mean percent identity of 77.3% and 76.8% at the nucleotide and predicted amino acid-sequence levels respectively. Furthermore, the predicted amino acid sequence of the possum FSHbeta mature protein shows evolutionary conservation of twelve cysteine residues and two potential N-linked glycosylation sites. The protein lacks the CAGY motif present in most reported glycoprotein beta-subunit sequences. The translation termination codon and consensus polyadenylation sequence overlap, a feature observed in other mammalian FSHbeta genes. Northern hybridization of total RNA from adult female possum pituitary revealed three hybridizing transcripts of approximately 2.8, 1.2 and 0.5 kb which may arise from utilizing alternative polyadenylation signals. In situ hybridization localized the FSHbeta transcripts to a sub-population of anterior pituitary cells interpreted as being gonadotropes. In summary the results indicate considerable evolutionary conservation of the structure of the FSH beta-subunit gene between the marsupial and eutherian mammalian lineages.

Autoradiographic localization of arginine vasopressin binding sites in the brain of adult and developing Brazilian opossums.

We are utilizing the Brazilian short-tailed opossum, Monodelphis domestica, to study the development of the vasopressinergic system. Earlier studies demonstrated that arginine vasopressin-like immunoreactivity was present very early in the Brazilian opossum brain, suggesting a role for vasopressin in the developing central nervous system of mammals. In this study, we have utilized [3H]arginine vasopressin autoradiography to describe the distribution of arginine vasopressin binding sites in adult and developing Brazilian opossum brains. In general, arginine vasopressin binding patterns in adult opossum brains resembled those of other species. However, we found very few labelled areas in neonatal Brazilian opossum brains. At birth, only the ventral tegmental area and the nucleus of the solitary tract were labelled. Binding was not evident in the forebrain until 25 days of postnatal age. The anterior pituitary was heavily labelled from birth onward, but binding in the brain itself remained at low levels until 35 days postnatal. Heavy binding was observed in only a few areas of the brain in adults, including the dorsal part of the lateral septal nucleus, the suprachiasmatic nucleus, the dorsal and median raphe, the nucleus of the solitary tract, and the caudal part of the spinal trigeminal nucleus. Surprisingly, arginine vasopressin binding sites in the Brazilian opossum appeared much later than arginine vasopressin immunoreactivity and, in many cases, after neurogenesis was complete. These findings suggest that the arginine vasopressin binding sites are not playing a developmental role in opossums, although the peptide is present at an early age.

The vestibular primary afferents and the vestibulospinal projections in the developing and adult opossum, Monodelphis domestica.

The gray short-tailed opossum Monodelphis domestica is born in a very immature state (eyes and ears closed, budlike hindlimbs, etc.) 14 days post-coitum, but it can locomote with its forelimbs from the mother's genital aperture to a nipple to which it attaches. The forelimb movements allowing this behavior may be the expression of central pattern generators in the spinal cord, but sensory clues must guide it. One such system may be the vestibular system, which senses linear and angular acceleration and has a strong influence on posture and balance at rest and during locomotion in adult animals. Using the neuronal tracer DiI, we have looked at the vestibular primary afferents and the vestibular nuclear projections to the cervical spinal cord in newborn and postnatal opossums, as well as in the adult animal. The projections in the adult opossum conform to those described for other mammals. Fibers of the vestibular portion of the eighth nerve distribute to all four vestibular nuclei and toward the cerebellar primordium on the day of birth. In addition, some of the fibers project to the contralateral vestibular ganglion, a projection that is not found in the adult opossum. Projections from the lateral, medial and inferior vestibular nuclei to the cervical cord are also present in the newborn. Although we cannot exclude the possibility that chemical and/or tactile guidance is used for directing the movements of the newborn opossum, our results support the hypothesis that the vestibular system may be directly involved in the control of these movements. However, not all components of the system are equally developed at birth, and the circuit from the utricle to the lateral vestibular nucleus and from the latter to the cervical cord may be better formed than that from the semicircular canals to the medial and inferior vestibular nuclei to the cervical cord.

Ontogeny of suckling mechanisms in opossums (Didelphis virginiana).

Although adult mammals have several different mechanisms for drinking liquids, relatively little is known about the variation in mechanisms used by infants for suckling and how the mechanics of suckling change as a function of ontogeny. We examined suckling in infant opossums, animals that as adults use a lapping mechanism to drink. The youngest infants (45 days old) used a suction, pumping mechanism, based on dorsal/ventral tongue movements, similar to what has been documented in infant primates and pigs. However, slightly older infants (66 days old), when removed from their mother, used a lapping or licking mechanism that is closer to the anterior/posterior tongue movements that adult opossums use during drinking. Littermates remaining with the mother continued to use the pumping mechanism observed in the youngest animals. These older animals lacked teeth and were not physiologically mature enough to live independently of their mothers. We conclude that the neural controls for two distinct feeding mechanisms exist simultaneously in infant opossums and that the change from a suckling, infantile motor pattern to an adult lapping one is not strictly a temporal change.

Evidence for GAP-43 within descending spinal axons in the North American opossum, Didelphis virginiana.

We have shown previously that GAP-43, a growth associated protein characteristically present in growing and regenerating axons, is relatively abundant in the spinal cord of adult opossums. In the present study, we combined the orthograde transport of the fluorescent marker Fluoro-Ruby with immunofluorescence for GAP-43 to determine if any of it is present within descending spinal axons. When Fluoro-Ruby was injected into the red nucleus and midbrain tegmentum, the medial pontine or medullary reticular formation, the medullary raphe or the lateral vestibular nucleus, axons were labeled in the expected areas of the spinal cord, but in most cases none showed evidence for GAP-43. In two of the four cases with rubral injections, however, a few labeled axons within the rubrospinal tract showed GAP-43 immunofluorescence, and in one case with an injection of the gigantocellular reticular nucleus and adjacent raphe, labeled axons within lamina IX immunostained for the protein. Since serotoninergic neurons are present within the gigantocellular reticular nucleus and adjacent raphe, and axons of the same phenotype are abundant within lamina IX, we asked whether serotoninergic axons contain GAP-43. When sections of the spinal cord were immunostained for both serotonin and GAP-43, many axons within lamina IX showed evidence for both substances. Such axons appeared to contact presumptive motoneurons. In cases with Fluoro-Ruby injections of the forelimb motor cortex, labeled axons were present within the pyramidal tract, and some of them showed evidence for GAP-43.