Characterization of the RFamide-like neuropeptides in the nervus terminalis of the goldfish (Carassius auratus).

FMRFamide-immunoreactivity has been demonstrated in the CNS of many vertebrate species. We sought to further characterize this immunoreactivity in nervus terminalis retinal efferents of the goldfish using an antiserum raised against a bovine morphine modulating peptide (A18Famide). This antiserum robustly labels nervus terminalis efferents to the retina, as well as a sub-population of retinal amacrine cells. Under immunocytochemical conditions the antiserum cross-reacted with neuropeptide Y-like as well as A18Famide-like peptides, but under conditions of radioimmunoassay it was highly specific for A18Famide-like peptides. High pressure liquid chromatography, gel permeation chromatography and radioimmunoassay showed that at least two different RFamide-like peptides, approximately the same size as the bovine RFamide-like peptides, are present in the goldfish nervus terminalis.

Substance P-, F8Famide-, and A18Famide-like immunoreactivity in the nervus terminalis and retina of the goldfish Carassius auratus.

We re-investigated the occurrence of substance P-like immunoreactivity in the retina of the goldfish Carassius auratus using antisera to substance P and other tachykinins. Most antisera labelled a previously described single class of mono-stratified amacrine cells arborizing in layer 3 of the inner plexiform layer. Preabsorption experiments showed that these amacrine cells contained at least one tachykinin-like peptide. One antiserum (INC 353) to substance P labelled not only these amacrine cells but also fibres in layer 1 of the inner plexiform layer and fibres in the optic nerve. These fibres were identified as retinopetal projections of the nervus terminalis, in part because of colocalized labelling with antisera against gonadotropin-releasing hormone and FMRFamide. Preabsorption experiments showed that the substance P-immunoreactive material in the nervus terminalis was not substance P or any other typical tachykinin. Labelling of the nervus terminalis with INC 353 was blocked by preabsorption with two bovine FMRF-amide-like peptides, F8Famide and A18Famide, which contain a substance P(4-7)-like region. Antisera to F8Famide and A18Famide strongly labelled ganglia of the nervus terminalis and retinopetal fibres. We suggest that labelling of the nervus terminalis by antisera to substance P and FMRFamide occurs because of homologies between these antigens and a non-tachykinin, endogenous peptide that is similar to F8Famide and A18Famide.

Direct sequencing of terminal regions of genomic P1 clones. A general strategy for the design of sequence-tagged site markers.

A method for the preparation of P1 DNA is presented, which allows the direct sequencing of ends of inserts in genomic P1 clones using the Applied Biosystems 373A DNA Sequencer and the Dye Terminator sequencing methodology. We surveyed several common methods of DNA preparation including alkaline lysis, Triton-lysozyme lysis, CsCl density-gradient purification, and a commercial column matrix DNA purification kit manufactured by Qiagen. We found that a modified alkaline lysis preparation of P1 DNA was most successful for generating P1 DNA that could be sequenced directly. We also noted that the host bacterial strain from which the P1 DNA was purified dramatically affected the quality of sequencing templates. The bacterial strains NS3145 and NS3529, in which the Drosophila melanogaster and human P1 genomic libraries are harbored, routinely yielded poor-quality sequencing templates. However, the bacterial strain DH10B routinely yielded P1 DNA that was sequenced successfully. A bacterial mating scheme is presented that exploits gamma delta transposition events to allow the transfer of P1 clones from the library host strain to DH10B. Using either an SP6 or a T7 primer, an average of 350 base pairs of DNA sequence was obtained with an uncalled base frequency of approximately 2%. About 4% of P1 end sequences generated corresponded to unique Drosophila loci present in the Genbank database. These single-pass DNA sequences were used to design sequence-tagged site markers for physical mapping studies in both humans and Drosophila.

Effects of lesions of the optic nerve, optic tectum and nervus terminalis on rod precursor proliferation in the goldfish retina.

Teleost retinas grow throughout life by proliferation of neuroblasts at the retinal margin and dedicated rod precursors in the outer nuclear layer. Mechanisms regulating this proliferation are largely unknown. Previous investigators observed that rod precursor replication, as detected by incorporation of radioactive thymidine into cells of the outer nuclear layer, is enhanced after optic nerve crush. We attempted to determine whether this was due to severing of the retinopetal (nervus terminalis, n.t.) or retinofugal (retinal ganglion cell) axons in the optic nerve of the goldfish, Carassius auratus. In the first series of experiments, we ablated unilaterally the optic nerve, olfactory bulb (containing n.t. ganglia), or optic tectum (containing retinal ganglion cell axons and n.t. collaterals). Rod precursor proliferation increased dramatically in both retinas as soon as 5 days after surgery; in addition, the numbers of dividing cells were greater in the ipsilateral retina 10-15 days after optic nerve crush or tectal ablation and in the contralateral retina 20-25 days after olfactory bulb ablation. These observations are not accounted for by the known projections of retinal ganglion cells, but are consistent with the projections of the n.t. In the second series of experiments, n.t. projections to the brain and retina were severed bilaterally 7-8 weeks before the unilateral optic nerve crush or hemitectal ablation. Rod precursor proliferation increased as before, but the quantities of dividing cells were always equal in both retinas. We conclude that the n.t. may modulate rod proliferation locally and that injury to (some) brain regions may cause release of mitogens that affect rod precursors in both retinas.

Etiologic agents, incidence, and improved diagnostic methods of cantharidin toxicosis in horses.

In addition to the 3-striped blister beetles (Epicauta temexa and E occidentalis), other sources of equine cantharidin toxicosis were identified at the Texas Veterinary Medical Diagnostic Laboratory and included E albida and E attrivittata and the previously incriminated E pardalis and E pennsylvanica. Improved methods for diagnosing cantharidin or blister beetle toxicosis involve partial purification of urine and gastric content extracts, using silica cartridges, followed by analysis, using capillary gas chromatography/mass spectrometry. During a 26-month period, 53 episodes of cantharidin toxicosis in horses were confirmed at our diagnostic laboratory. Concentrations of cantharidin in urine and gastric contents ranged from 0.0003 to 3.50 micrograms/g. Peak incidences were observed in late summer and early fall.

Elevations in gonadotrophin concentrations and milt volumes as a result of spawning behavior in the goldfish.

In many male mammals and birds, exposure to sexual stimuli results in acute elevations of circulating luteinizing hormone (LH) and testosterone (T); a similar phenomemon has now been observed in the male goldfish (Carassius auratus). Mature males placed with either a receptive female or stimulus pairs of spawning goldfish had gonadotrophin (GtH) concentrations and expressible milt (sperm) volumes that were significantly greater than those of males kept in all-male groups. This stimulatory effect lasted from 20 min to at least 2 hr for GtH (20 degrees) and from less than or equal to 1 hr to greater than or equal to 24 hr for milt (14 degrees). When males were separated from the spawning pair by either a solid or perforated clear partition, no elevations of GtH or milt levels occurred. In contrast, these values increased in males placed in contact with a spawning pair, even when that pair contained no female, but a male induced to perform female sexual behavior by treatment with prostaglandin. These results suggest that, in goldfish, access to a spawning situation is necessary for rapid elevations in GtH and milt. Furthermore, it appears that the males must be sexually active in order for these physiological changes to occur, as males that failed to engage in courtship behavior with a spawning pair had GtH and milt values not different from isolated fish. This suggests that male sexual behavior and elevations in milt and GtH are concurrent events that share a common activation pathway in the brain. The increase in milt may be due to both neurally and hormonally mediated events that ensure milt availability for imminent spawning activity.

Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. I. Gonadotropin, growth hormone, and thyroid hormones.

White suckers (Catostomus commersoni; Cypriniformes, Teleosteii) spawning in a small stream in central Alberta were captured during different stages of their spawning migrations in 1981 and 1982, blood was sampled, and the fish were examined to determine their reproductive condition. Blood samples were analyzed for gonadotropin (GtH), growth hormone (GH), triiodothyronine (T3), and thyroxine (T4) by radioimmunoassay. GtH levels in both sexes were lowest prior to the onset of spawning, increased significantly in spawning males, females in which germinal vesicle migration had begun, and ovulated females and then dropped significantly in spent fish of both sexes. GH was lowest in prespawning females, increased significantly at ovulation, and remained high in spent females. In contrast, GH levels in males were relatively constant throughout spawning. In both sexes, highest T4 levels were found in prespawning fish, and T4 decreased significantly in spent fish. Although a similar decline was seen in T3 in 1981, in 1982 there were no T3 changes associated with changes in reproductive condition. No significant diurnal variations were detected in the levels of GtH or T3; T4 levels appeared to vary on a diurnal basis in prespawning males only. Spawning activity in both sexes therefore appears to be associated with increases in GtH occurring at ovulation in females and at the initiation of spawning in males. GH levels may also be related to reproductive condition in females, but not in males. The relationship of thyroid hormone levels to reproductive condition is less clear, however, and these levels may reflect both endocrine and environmental influences on thyroid function.

Effects of olfactory tract lesions on sexual and feeding behavior in the goldfish.

The paired olfactory tracts (OTs) of goldfish each have a lateral (LOT) and medial (MOT) olfactory tract, the latter with medial (mMOT) and lateral (lMOT) subdivisions. Bilateral OT section (OTX) reduced, while unilateral OT section (UNI) did not impair, male sexual behavior and feeding responses to a complex food odor; in contrast, female sexual behavior, induced in both sexes by prostaglandin treatment, was little affected by OTX. Experiments in which control fish were given UNI and treatment groups received UNI plus selective section of the remaining OT assessed the role of the OT subdivisions in the two olfactory-influenced behaviors (male sexual behavior and feeding). LOT section did not affect courtship, while MOT section reduced courtship to the low levels seen in OTX males; within the MOT, the mMOT may be more important than the lMOT for courtship expression. In contrast to male sexual behavior, feeding responses were less affected by MOT than LOT section. With respect to male sexual behavior, these findings demonstrate differential functions for the anatomically distinct subdivisions of the goldfish olfactory tracts, possibly related to their distinct terminal fields within the telencephalon.

Effects of forebrain lesions on spawning behaviour in the male goldfish.

Lesions were stereotaxically placed in medial nuclei of the ventral telencephalon and preoptic area of male goldfish. Only lesions in the area ventralis telencephali pars supracommissuralis (Vs) and posterior pars ventralis (pVv) were effective in reducing the proportion of males spawning, as compared to sham groups, both 5 days (Experiment 1) and up to 4 weeks (Experiment 2) postoperatively. Spawning consistency over 7 weekly tests was negatively correlated with the volume of Vs-pVv destruction. Two-thirds of Vs-pVv lesioned males spawned on at least one of their weekly tests, with latencies for the onset of each courtship behaviour similar to those of control fish, partial performance of the spawning sequence was rare. These results suggest that lesion of the supracommissural telencephalon (Vs-pVv region) blocks the initiation of spawning behaviour in the male goldfish, perhaps by lowering reproductive motivation specifically or by interfering with the perception of sexual, particularly olfactory, cues.

Brain lesions and short-term endocrine effects of monosodium L-glutamate in goldfish, Carassius auratus.

Monosodium L-glutamate (MSG) was injected intraperitoneally into goldfish at a dosage of 2.5 mg/g body weight. At 24 h post-injection there was a marked hypertrophy and edema in the region of the nucleus lateralis tuberis (NLT) from the anterior margin of the pituitary stalk through to the posterior end of the NLT, irrespective of the sex of the goldfish. A similar hypertrophy and edema occurred ventral to the anterior commissure in the preoptic region in the anterior-ventral nucleus preopticus periventricularis (NPP). At 6 h post-injection a slight vacuolization was evident in these two regions, and at two days the hypertrophy and edema had abated from the extent observed at 24 h post-injection. At five and eight days post-injection only necrotic cells were found in the affected NLT region, but only a small band of necrotic cells was evident in the anterior-ventral preoptic region. No other brain lesions were evident. Serum levels of gonadotropin (GtH) were increased at 6 h, 24 h, and two days after treatment with MSG, but were similar to control values at five, seven and eight days after MSG in male and female goldfish. Exocytosis of small dark secretory granules in gonadotrophs was evident at 24 h after MSG in a fish with a somewhat greater increase in serum GtH than usually found. The time course of increased serum GtH levels postinjection of MSG is consistent with the observed time course of hypertrophy and atrophy of NLT neurons; the increase in serum levels of GtH is interpreted to reflect a stimulation of release of GtH-releasing factor from neurons in the NLT. Electron microscope investigation indicates that prolactin cells have increased secretory and synthetic activity from 24 h through to seven days post-injection of MSG. The mechanism for stimulation of the prolactin cells by MSG is not known. No other changes in activity of adenohypophysial secretory cells were found.